Distinct effects of PKC- and PKA-dependent signaling cascades on subcellular TRPV4 localization in distal nephron cells. Shown are representative confocal plane micrographs (axes are shown) and corresponding cross-sections (indicated by arrows ) demonstrating TRPV4 localization (anti-TRPV4, pseudocolor green ) in split-opened murine distal nephrons in the control ( A ) and after a 15-min pretreatment with 200 n M PMA ( B ), a 15-min pretreatment with 20 ␮ M forskolin ( C ), and a 15-min pretreatment with 20 ␮ M forskolin and 20 ␮ M H-89 ( D ). Nuclear DAPI staining is shown by pseudocolor blue . a and b indicate 

Contexts in source publication

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... 4. Acute activation of the PKA signaling cascade promotes apical TRPV4 translocation. A , distribution of averaged relative fluorescent signals representing TRPV4 localization along a line on z -axis in individual cells from distal nephrons similar to that shown in Fig. 3 in the control ( black ) and after a 15-min pretreatment with 200 n M PMA ( green ), a 15-min pretreatment with 20 ␮ M forskolin ( red ), and a 15-min pretreatment with 20 ␮ M forskolin and 20 ␮ M  ...

Context 2

... dehydrated and mounted with permanent mounting medium (Thermo Sci- entific). Labeled tissue samples were examined with an Nikon Eclipse Ti inverted confocal fluorescence microscope using a 40 ϫ Plan Fluor oil immersion (1.3 numerical aperture) objective. Samples were excited with 405 and 488 nm laser diodes, and emission was captured with a 16-bit CoolSNAP HQ 2 cam- era (Photometrics) interfaced to a PC running NIS-Elements version 4.00 software. Three-dimensional stacks of split- opened distal nephrons were generated from a series of confocal plane images with 0.25- ␮ m steps. Solutions —The typical bath solution was 150 m M NaCl, 5 m M KCl, 1 m M CaCl 2 , 2 m M MgCl 2 , 5 m M glucose, and 10 m M HEPES (pH 7.4). All reagents were applied by perfusing the experimental chamber at 1.5 ml/min. To test the effect of elevated flow on [Ca 2 ϩ ] i , the rate of perfusion was instantly increased from 1.5 ml/min ( ϳ 15 mm H 2 O) to 15 ml/min ( ϳ 80 mm H O). Using a parallel plate chamber, we recently esti- mated that this maneuver produces shear stress of 3 dynes/ cm 2 (12). This value fits well within the physiological range of shear stress present in the rat and mouse collecting duct as was assessed previously (10, 24). Prior termination of a respective cell-permeable activator/inhibitor of PKC- and PKA-dependent pathways did not affect the magnitude of the flow-mediated [Ca 2 ϩ ] i response due to poor reversibility of the agent. Data Analysis —All summarized data are reported as means Ϯ S.E. Data were compared using a t test or one-way analysis of variance as appropriate. p Յ 0.05 was considered significant. [Ca ] i Responses in Distal Nephron Cells —We have recently documented that the Ca 2 ϩ -permeable TRPV4 channel is a crit- ical determinant of mechanosensitive properties in distal nephron cells (10, 12). Genetic deletion of TRPV4 abolishes [Ca 2 ϩ ] i elevations in response to elevated flow in murine distal nephrons (12). PKC and PKA can directly phosphorylate TRPV4 in expression systems (19). Here, we probed whether these signaling cascades are involved in controlling mechanosensitive [Ca 2 ϩ ] i elevations by affecting TRPV4 activity and expression patterns in freshly isolated split-opened distal nephrons. Fig. 1 A documents the average time course of changes in [Ca 2 ϩ ] i levels in individual cells within a split- opened area of freshly isolated distal nephrons in response to an abrupt 10-fold elevation in flow over the apical surface. Acute stimulation of PKC with 200 n M phorbol 12-myristate 13-ace- tate (PMA) greatly potentiated flow-mediated elevations in [Ca 2 ϩ ] i . Of note, PMA treatment also had a mild stimulatory effect on the basal levels of [Ca 2 ϩ ] i (Fig. 1 A ). As summarized in Fig. 1 B , the responses to flow were similarly increased from 31 Ϯ 3 n M in the control to 49 Ϯ 4 n M and 58 Ϯ 5 n M when PMA was applied for 5 and 15 min, respectively. Administration of a highly selective, cell-permeable PKC inhibitor, bisindolylmaleimide I (BIM-I; 200 n M ), for 10 min significantly decreased the amplitude of the flow-mediated [Ca 2 ϩ ] i response from 32 Ϯ 2 n M to 12 Ϯ 2 n M (Fig. 1, C and D ). Pharmacological PKC inhibition also had a tendency to decrease basal [Ca 2 ϩ ] i levels (Fig. 1 C ). Overall, we conclude that [Ca 2 ϩ ] i responses to elevated flow in distal nephron cells are positively regulated by the PKC signaling cascade. We next probed the involvement of the PKA signaling cascade in the regulation of flow-dependent Ca 2 ϩ responses in distal nephron cells. Fig. 2 A documents the average time course of changes in [Ca 2 ϩ ] levels in response to elevated flow under the control conditions and after 15 min treatment with forskolin (20 ␮ M ) to elevate intracellular cAMP levels. However, this maneuver failed to affect flow-induced Ca 2 ϩ responses in distal nephron cells. The amplitudes of the response were 28 Ϯ 3 n M and 29 Ϯ 3 n M in the control and after forskolin treatment, respectively (Fig. 2 B ). These results suggest that acute activation of the PKA signaling cascade alone has no appreciable role in the regulation of TRPV4 functional activity and, subse- quently, flow-dependent [Ca 2 ϩ ] i elevations in distal nephron cells. TRPV4 Trafficking Is Regulated by PKA but Not PKC —We next used immunofluorescence microscopy in split-opened distal nephrons to examine whether stimulation of the PKC and PKA cascades alters subcellular TRPV4 localization to promote trafficking to the apical plasma membrane. Consistent with our previous report (12), TRPV4 expression was dominant in the apical/subapical regions under the control conditions, as apparent from a representative confocal fluorescent image in Fig. 3 A . Pretreatment with the PKC activator PMA (200 n M ) for 15 min had no apparent effect on TRPV4 subcellular localization (Fig. 3 B ). In contrast, TRPV4 localized to the apical plasma membrane when split-opened distal nephrons were pretreated with 20 ␮ M forskolin for 15 min (Fig. 3 C ). Forskolin-induced redistribution was precluded by the PKA inhibitor H-89 (20 ␮ M ) (Fig. 3 D ). To perform a quantitative estimation of the observed changes in subcellular TRPV4 localization, we employed line- scan analysis of the fluorescent signal distribution along the z -axis in cross-sections of three-dimensional stacks similar to those shown in Fig. 3. Fig. 4 A shows the averaged distribution pattern of fluorescence intensity representing TRPV4 localization in the control and after pretreatment with PMA, forskolin, and forskolin and H-89. As is clear, stimulation of the PKA pathway with forskolin shifted the maximum of the fluorescent signal toward the apical region. Furthermore, forskolin also caused sharpening of the fluorescence intensity profile. As summarized in Fig. 4 B , the average half-width of the fluorescence intensity was significantly reduced from 3.06 Ϯ 0.07 ␮ m ( n ϭ 108) in the control to 1.34 Ϯ 0.04 ␮ m ( n ϭ 123) after forskolin treatment. At the same time, the half-width was 2.92 0.17 m ( n 105) after treatment with PMA and 2.99 0.18 ␮ m ( n ϭ 95) after treatment with H-89 and forskolin. These values were not significantly different from the control. Overall, the results in Figs. 3 and 4 suggest that activation of PKA but not PKC signaling cascades promotes TRPV4 trafficking to the apical plasma membrane. The apparent lack of forskolin-mediated augmentation of the flow-induced [Ca 2 ϩ ] i response (Fig. 2), despite the prominent trafficking of TRPV4 to the apical compartment (Figs. 3 and 4), may indicate that translocated channels were not yet inserted into the plasma membrane. In this case, [Ca 2 ϩ ] i stimulation was required to incorporate TRPV4 into the apical membrane and augment cellular responses to elevated flow. To probe this, we treated split-opened distal nephrons with 20 ␮ M forskolin and quantified the amplitudes of two consequent flow-induced [Ca 2 ϩ ] i responses in the continued presence of the PKA cascade activator (Fig. 5 A ). However, [Ca 2 ϩ ] i elevations induced by the first application of increased flow did not result in appreciable potentiation of the second flow-mediated [Ca 2 ϩ ] i response. As summarized in Fig. 5 B , the amplitudes of the first and second responses during forskolin treatment were 27 Ϯ 1 and 25 Ϯ 1 n M , respectively, and were not different from the amplitude of the flow-mediated [Ca 2 ϩ ] i response in the control (29 Ϯ 2 n M ). Therefore, it appears that activation of the PKA-dependent pathway likely results in translocation of silent TRPV4 to the apical membrane and that lack of augmentation of flow-dependent [Ca 2 ϩ ] i responses is not associated with inability of the channels to be inserted. mediated [Ca ] i Elevations in the Distal Nephron —Our results point to distinct modes of TRPV4 regulation by PKC and PKA signaling cascades. Whereas the PKC-dependent pathway stimulated TRPV4 and enhanced mechanosensitive [Ca 2 ϩ ] i responses (Fig. 1) without affecting subcellular TRPV4 distribution (Fig. 3 B ), the PKA-dependent pathway promoted apical TRPV4 trafficking (Fig. 3 C ) but failed to augment functional TRPV4 status (Figs. 2 and 5). Thus, we next tested whether PKA- and PKC-dependent pathways are cooperative in augmenting TRPV4-mediated [Ca 2 ϩ ] i responses to flow. Concomitant stimulation of both pathways with 200 n M PMA and 20 M forskolin drastically augmented the amplitude of flow-induced [Ca 2 ϩ ] i responses (Fig. 6 A , black trace ). This elevation was significantly greater than that in response to application of PMA alone (Fig. 6 A , gray trace ). The absolute eleva- tions in [Ca ] i in response to flow were 34 2, 77 4, and 55 Ϯ 3 n M in the control, after treatment with PMA ϩ forskolin, and after treatment with PMA alone, respectively (Fig. 6 B ). Interestingly, we observed a prominent gradual increase of ϳ 80 n M in basal [Ca ] i levels in distal nephron cells upon concom- itant treatment with 200 n M PMA and 20 ␮ M forskolin (Fig. 6 A ). Therefore, the absolute elevation of [Ca 2 ϩ ] i from the values under unstimulated conditions reflects the total level of TRPV4 activation by mechanical stimuli ( i.e. flow) in the presence of simultaneous stimulation of the PKA and PKC cascades. As summarized in Fig. 6 C , the absolute amplitude of the [Ca 2 ϩ ] i response to flow was 162 Ϯ 5 n M , which was significantly greater than the response in the presence of PKC stimulation alone (68 Ϯ 5 n M ). Finally, to probe whether the additive stimulation of the flow-dependent [Ca 2 ϩ ] i response and gradual increases in the basal [Ca 2 ϩ ] i levels in response to simultaneous activation of the PKC and PKA cascades occur in a TRPV4-dependent manner, we repeated the treatment with PMA and forskolin in the presence of the selective TRPV4 inhibitor HC-067047 (4 ␮ M ). As is clear from the average time course (Fig. 7 A ), TRPV4 blockade abolished the progressive increase in [Ca 2 ϩ ] i levels. Moreover, HC-067047 precluded ...

Context 3

... cam- era (Photometrics) interfaced to a PC running NIS-Elements version 4.00 software. Three-dimensional stacks of split- opened distal nephrons were generated from a series of confocal plane images with 0.25- ␮ m steps. Solutions —The typical bath solution was 150 m M NaCl, 5 m M KCl, 1 m M CaCl 2 , 2 m M MgCl 2 , 5 m M glucose, and 10 m M HEPES (pH 7.4). All reagents were applied by perfusing the experimental chamber at 1.5 ml/min. To test the effect of elevated flow on [Ca 2 ϩ ] i , the rate of perfusion was instantly increased from 1.5 ml/min ( ϳ 15 mm H 2 O) to 15 ml/min ( ϳ 80 mm H O). Using a parallel plate chamber, we recently esti- mated that this maneuver produces shear stress of 3 dynes/ cm 2 (12). This value fits well within the physiological range of shear stress present in the rat and mouse collecting duct as was assessed previously (10, 24). Prior termination of a respective cell-permeable activator/inhibitor of PKC- and PKA-dependent pathways did not affect the magnitude of the flow-mediated [Ca 2 ϩ ] i response due to poor reversibility of the agent. Data Analysis —All summarized data are reported as means Ϯ S.E. Data were compared using a t test or one-way analysis of variance as appropriate. p Յ 0.05 was considered significant. [Ca ] i Responses in Distal Nephron Cells —We have recently documented that the Ca 2 ϩ -permeable TRPV4 channel is a crit- ical determinant of mechanosensitive properties in distal nephron cells (10, 12). Genetic deletion of TRPV4 abolishes [Ca 2 ϩ ] i elevations in response to elevated flow in murine distal nephrons (12). PKC and PKA can directly phosphorylate TRPV4 in expression systems (19). Here, we probed whether these signaling cascades are involved in controlling mechanosensitive [Ca 2 ϩ ] i elevations by affecting TRPV4 activity and expression patterns in freshly isolated split-opened distal nephrons. Fig. 1 A documents the average time course of changes in [Ca 2 ϩ ] i levels in individual cells within a split- opened area of freshly isolated distal nephrons in response to an abrupt 10-fold elevation in flow over the apical surface. Acute stimulation of PKC with 200 n M phorbol 12-myristate 13-ace- tate (PMA) greatly potentiated flow-mediated elevations in [Ca 2 ϩ ] i . Of note, PMA treatment also had a mild stimulatory effect on the basal levels of [Ca 2 ϩ ] i (Fig. 1 A ). As summarized in Fig. 1 B , the responses to flow were similarly increased from 31 Ϯ 3 n M in the control to 49 Ϯ 4 n M and 58 Ϯ 5 n M when PMA was applied for 5 and 15 min, respectively. Administration of a highly selective, cell-permeable PKC inhibitor, bisindolylmaleimide I (BIM-I; 200 n M ), for 10 min significantly decreased the amplitude of the flow-mediated [Ca 2 ϩ ] i response from 32 Ϯ 2 n M to 12 Ϯ 2 n M (Fig. 1, C and D ). Pharmacological PKC inhibition also had a tendency to decrease basal [Ca 2 ϩ ] i levels (Fig. 1 C ). Overall, we conclude that [Ca 2 ϩ ] i responses to elevated flow in distal nephron cells are positively regulated by the PKC signaling cascade. We next probed the involvement of the PKA signaling cascade in the regulation of flow-dependent Ca 2 ϩ responses in distal nephron cells. Fig. 2 A documents the average time course of changes in [Ca 2 ϩ ] levels in response to elevated flow under the control conditions and after 15 min treatment with forskolin (20 ␮ M ) to elevate intracellular cAMP levels. However, this maneuver failed to affect flow-induced Ca 2 ϩ responses in distal nephron cells. The amplitudes of the response were 28 Ϯ 3 n M and 29 Ϯ 3 n M in the control and after forskolin treatment, respectively (Fig. 2 B ). These results suggest that acute activation of the PKA signaling cascade alone has no appreciable role in the regulation of TRPV4 functional activity and, subse- quently, flow-dependent [Ca 2 ϩ ] i elevations in distal nephron cells. TRPV4 Trafficking Is Regulated by PKA but Not PKC —We next used immunofluorescence microscopy in split-opened distal nephrons to examine whether stimulation of the PKC and PKA cascades alters subcellular TRPV4 localization to promote trafficking to the apical plasma membrane. Consistent with our previous report (12), TRPV4 expression was dominant in the apical/subapical regions under the control conditions, as apparent from a representative confocal fluorescent image in Fig. 3 A . Pretreatment with the PKC activator PMA (200 n M ) for 15 min had no apparent effect on TRPV4 subcellular localization (Fig. 3 B ). In contrast, TRPV4 localized to the apical plasma membrane when split-opened distal nephrons were pretreated with 20 ␮ M forskolin for 15 min (Fig. 3 C ). Forskolin-induced redistribution was precluded by the PKA inhibitor H-89 (20 ␮ M ) (Fig. 3 D ). To perform a quantitative estimation of the observed changes in subcellular TRPV4 localization, we employed line- scan analysis of the fluorescent signal distribution along the z -axis in cross-sections of three-dimensional stacks similar to those shown in Fig. 3. Fig. 4 A shows the averaged distribution pattern of fluorescence intensity representing TRPV4 localization in the control and after pretreatment with PMA, forskolin, and forskolin and H-89. As is clear, stimulation of the PKA pathway with forskolin shifted the maximum of the fluorescent signal toward the apical region. Furthermore, forskolin also caused sharpening of the fluorescence intensity profile. As summarized in Fig. 4 B , the average half-width of the fluorescence intensity was significantly reduced from 3.06 Ϯ 0.07 ␮ m ( n ϭ 108) in the control to 1.34 Ϯ 0.04 ␮ m ( n ϭ 123) after forskolin treatment. At the same time, the half-width was 2.92 0.17 m ( n 105) after treatment with PMA and 2.99 0.18 ␮ m ( n ϭ 95) after treatment with H-89 and forskolin. These values were not significantly different from the control. Overall, the results in Figs. 3 and 4 suggest that activation of PKA but not PKC signaling cascades promotes TRPV4 trafficking to the apical plasma membrane. The apparent lack of forskolin-mediated augmentation of the flow-induced [Ca 2 ϩ ] i response (Fig. 2), despite the prominent trafficking of TRPV4 to the apical compartment (Figs. 3 and 4), may indicate that translocated channels were not yet inserted into the plasma membrane. In this case, [Ca 2 ϩ ] i stimulation was required to incorporate TRPV4 into the apical membrane and augment cellular responses to elevated flow. To probe this, we treated split-opened distal nephrons with 20 ␮ M forskolin and quantified the amplitudes of two consequent flow-induced [Ca 2 ϩ ] i responses in the continued presence of the PKA cascade activator (Fig. 5 A ). However, [Ca 2 ϩ ] i elevations induced by the first application of increased flow did not result in appreciable potentiation of the second flow-mediated [Ca 2 ϩ ] i response. As summarized in Fig. 5 B , the amplitudes of the first and second responses during forskolin treatment were 27 Ϯ 1 and 25 Ϯ 1 n M , respectively, and were not different from the amplitude of the flow-mediated [Ca 2 ϩ ] i response in the control (29 Ϯ 2 n M ). Therefore, it appears that activation of the PKA-dependent pathway likely results in translocation of silent TRPV4 to the apical membrane and that lack of augmentation of flow-dependent [Ca 2 ϩ ] i responses is not associated with inability of the channels to be inserted. mediated [Ca ] i Elevations in the Distal Nephron —Our results point to distinct modes of TRPV4 regulation by PKC and PKA signaling cascades. Whereas the PKC-dependent pathway stimulated TRPV4 and enhanced mechanosensitive [Ca 2 ϩ ] i responses (Fig. 1) without affecting subcellular TRPV4 distribution (Fig. 3 B ), the PKA-dependent pathway promoted apical TRPV4 trafficking (Fig. 3 C ) but failed to augment functional TRPV4 status (Figs. 2 and 5). Thus, we next tested whether PKA- and PKC-dependent pathways are cooperative in augmenting TRPV4-mediated [Ca 2 ϩ ] i responses to flow. Concomitant stimulation of both pathways with 200 n M PMA and 20 M forskolin drastically augmented the amplitude of flow-induced [Ca 2 ϩ ] i responses (Fig. 6 A , black trace ). This elevation was significantly greater than that in response to application of PMA alone (Fig. 6 A , gray trace ). The absolute eleva- tions in [Ca ] i in response to flow were 34 2, 77 4, and 55 Ϯ 3 n M in the control, after treatment with PMA ϩ forskolin, and after treatment with PMA alone, respectively (Fig. 6 B ). Interestingly, we observed a prominent gradual increase of ϳ 80 n M in basal [Ca ] i levels in distal nephron cells upon concom- itant treatment with 200 n M PMA and 20 ␮ M forskolin (Fig. 6 A ). Therefore, the absolute elevation of [Ca 2 ϩ ] i from the values under unstimulated conditions reflects the total level of TRPV4 activation by mechanical stimuli ( i.e. flow) in the presence of simultaneous stimulation of the PKA and PKC cascades. As summarized in Fig. 6 C , the absolute amplitude of the [Ca 2 ϩ ] i response to flow was 162 Ϯ 5 n M , which was significantly greater than the response in the presence of PKC stimulation alone (68 Ϯ 5 n M ). Finally, to probe whether the additive stimulation of the flow-dependent [Ca 2 ϩ ] i response and gradual increases in the basal [Ca 2 ϩ ] i levels in response to simultaneous activation of the PKC and PKA cascades occur in a TRPV4-dependent manner, we repeated the treatment with PMA and forskolin in the presence of the selective TRPV4 inhibitor HC-067047 (4 ␮ M ). As is clear from the average time course (Fig. 7 A ), TRPV4 blockade abolished the progressive increase in [Ca 2 ϩ ] i levels. Moreover, HC-067047 precluded flow-mediated [Ca 2 ϩ ] i responses even in the presence of the activated PKC and PKA cascades (Fig. 7 B ). The amplitudes of the flow-mediated [Ca 2 ϩ ] i response were 33 Ϯ 2 n M in the control and 5 Ϯ 2 n M after treatment with forskolin, PMA, and HC-067047. Overall, we conclude that the coordinated stimulation of both PKC and PKA cascades additively increases ...

Context 4

... 10 min significantly decreased the amplitude of the flow-mediated [Ca 2 ϩ ] i response from 32 Ϯ 2 n M to 12 Ϯ 2 n M (Fig. 1, C and D ). Pharmacological PKC inhibition also had a tendency to decrease basal [Ca 2 ϩ ] i levels (Fig. 1 C ). Overall, we conclude that [Ca 2 ϩ ] i responses to elevated flow in distal nephron cells are positively regulated by the PKC signaling cascade. We next probed the involvement of the PKA signaling cascade in the regulation of flow-dependent Ca 2 ϩ responses in distal nephron cells. Fig. 2 A documents the average time course of changes in [Ca 2 ϩ ] levels in response to elevated flow under the control conditions and after 15 min treatment with forskolin (20 ␮ M ) to elevate intracellular cAMP levels. However, this maneuver failed to affect flow-induced Ca 2 ϩ responses in distal nephron cells. The amplitudes of the response were 28 Ϯ 3 n M and 29 Ϯ 3 n M in the control and after forskolin treatment, respectively (Fig. 2 B ). These results suggest that acute activation of the PKA signaling cascade alone has no appreciable role in the regulation of TRPV4 functional activity and, subse- quently, flow-dependent [Ca 2 ϩ ] i elevations in distal nephron cells. TRPV4 Trafficking Is Regulated by PKA but Not PKC —We next used immunofluorescence microscopy in split-opened distal nephrons to examine whether stimulation of the PKC and PKA cascades alters subcellular TRPV4 localization to promote trafficking to the apical plasma membrane. Consistent with our previous report (12), TRPV4 expression was dominant in the apical/subapical regions under the control conditions, as apparent from a representative confocal fluorescent image in Fig. 3 A . Pretreatment with the PKC activator PMA (200 n M ) for 15 min had no apparent effect on TRPV4 subcellular localization (Fig. 3 B ). In contrast, TRPV4 localized to the apical plasma membrane when split-opened distal nephrons were pretreated with 20 ␮ M forskolin for 15 min (Fig. 3 C ). Forskolin-induced redistribution was precluded by the PKA inhibitor H-89 (20 ␮ M ) (Fig. 3 D ). To perform a quantitative estimation of the observed changes in subcellular TRPV4 localization, we employed line- scan analysis of the fluorescent signal distribution along the z -axis in cross-sections of three-dimensional stacks similar to those shown in Fig. 3. Fig. 4 A shows the averaged distribution pattern of fluorescence intensity representing TRPV4 localization in the control and after pretreatment with PMA, forskolin, and forskolin and H-89. As is clear, stimulation of the PKA pathway with forskolin shifted the maximum of the fluorescent signal toward the apical region. Furthermore, forskolin also caused sharpening of the fluorescence intensity profile. As summarized in Fig. 4 B , the average half-width of the fluorescence intensity was significantly reduced from 3.06 Ϯ 0.07 ␮ m ( n ϭ 108) in the control to 1.34 Ϯ 0.04 ␮ m ( n ϭ 123) after forskolin treatment. At the same time, the half-width was 2.92 0.17 m ( n 105) after treatment with PMA and 2.99 0.18 ␮ m ( n ϭ 95) after treatment with H-89 and forskolin. These values were not significantly different from the control. Overall, the results in Figs. 3 and 4 suggest that activation of PKA but not PKC signaling cascades promotes TRPV4 trafficking to the apical plasma membrane. The apparent lack of forskolin-mediated augmentation of the flow-induced [Ca 2 ϩ ] i response (Fig. 2), despite the prominent trafficking of TRPV4 to the apical compartment (Figs. 3 and 4), may indicate that translocated channels were not yet inserted into the plasma membrane. In this case, [Ca 2 ϩ ] i stimulation was required to incorporate TRPV4 into the apical membrane and augment cellular responses to elevated flow. To probe this, we treated split-opened distal nephrons with 20 ␮ M forskolin and quantified the amplitudes of two consequent flow-induced [Ca 2 ϩ ] i responses in the continued presence of the PKA cascade activator (Fig. 5 A ). However, [Ca 2 ϩ ] i elevations induced by the first application of increased flow did not result in appreciable potentiation of the second flow-mediated [Ca 2 ϩ ] i response. As summarized in Fig. 5 B , the amplitudes of the first and second responses during forskolin treatment were 27 Ϯ 1 and 25 Ϯ 1 n M , respectively, and were not different from the amplitude of the flow-mediated [Ca 2 ϩ ] i response in the control (29 Ϯ 2 n M ). Therefore, it appears that activation of the PKA-dependent pathway likely results in translocation of silent TRPV4 to the apical membrane and that lack of augmentation of flow-dependent [Ca 2 ϩ ] i responses is not associated with inability of the channels to be inserted. mediated [Ca ] i Elevations in the Distal Nephron —Our results point to distinct modes of TRPV4 regulation by PKC and PKA signaling cascades. Whereas the PKC-dependent pathway stimulated TRPV4 and enhanced mechanosensitive [Ca 2 ϩ ] i responses (Fig. 1) without affecting subcellular TRPV4 distribution (Fig. 3 B ), the PKA-dependent pathway promoted apical TRPV4 trafficking (Fig. 3 C ) but failed to augment functional TRPV4 status (Figs. 2 and 5). Thus, we next tested whether PKA- and PKC-dependent pathways are cooperative in augmenting TRPV4-mediated [Ca 2 ϩ ] i responses to flow. Concomitant stimulation of both pathways with 200 n M PMA and 20 M forskolin drastically augmented the amplitude of flow-induced [Ca 2 ϩ ] i responses (Fig. 6 A , black trace ). This elevation was significantly greater than that in response to application of PMA alone (Fig. 6 A , gray trace ). The absolute eleva- tions in [Ca ] i in response to flow were 34 2, 77 4, and 55 Ϯ 3 n M in the control, after treatment with PMA ϩ forskolin, and after treatment with PMA alone, respectively (Fig. 6 B ). Interestingly, we observed a prominent gradual increase of ϳ 80 n M in basal [Ca ] i levels in distal nephron cells upon concom- itant treatment with 200 n M PMA and 20 ␮ M forskolin (Fig. 6 A ). Therefore, the absolute elevation of [Ca 2 ϩ ] i from the values under unstimulated conditions reflects the total level of TRPV4 activation by mechanical stimuli ( i.e. flow) in the presence of simultaneous stimulation of the PKA and PKC cascades. As summarized in Fig. 6 C , the absolute amplitude of the [Ca 2 ϩ ] i response to flow was 162 Ϯ 5 n M , which was significantly greater than the response in the presence of PKC stimulation alone (68 Ϯ 5 n M ). Finally, to probe whether the additive stimulation of the flow-dependent [Ca 2 ϩ ] i response and gradual increases in the basal [Ca 2 ϩ ] i levels in response to simultaneous activation of the PKC and PKA cascades occur in a TRPV4-dependent manner, we repeated the treatment with PMA and forskolin in the presence of the selective TRPV4 inhibitor HC-067047 (4 ␮ M ). As is clear from the average time course (Fig. 7 A ), TRPV4 blockade abolished the progressive increase in [Ca 2 ϩ ] i levels. Moreover, HC-067047 precluded flow-mediated [Ca 2 ϩ ] i responses even in the presence of the activated PKC and PKA cascades (Fig. 7 B ). The amplitudes of the flow-mediated [Ca 2 ϩ ] i response were 33 Ϯ 2 n M in the control and 5 Ϯ 2 n M after treatment with forskolin, PMA, and HC-067047. Overall, we conclude that the coordinated stimulation of both PKC and PKA cascades additively increases the amplitude of the TRPV4- mediated [Ca 2 ϩ ] i response to flow and, importantly, augments the basal TRPV4 activity, resulting in a progressive increase in the resting [Ca 2 ϩ ] levels. It has been recently demonstrated that the activity of the Ca 2 ϩ -permeable TRPV4 channel is central for [Ca 2 ϩ ] i elevations in distal nephron cells in response to dynamic changes in tubular fluid flow (11, 12, 16, 20). Adequate mechanosensitive [Ca 2 ϩ ] i responses are important determinants of many physiological processes in late nephron segments, including flow-dependent K ϩ secretion (15, 25), regulatory volume decreases (26), etc. Furthermore, we and others have recently demonstrated that pharmacological stimulation of TRPV4 activity is instrumental for blunting renal cystogenesis in ARPKD models (18, 27). In this study, we defined two distinct intracellular signaling cascades separately controlling TRPV4 trafficking and functional activity in murine distal nephrons. We found that the PKC-dependent signaling pathway is responsible for augmented TRPV4 activation by elevated flow over the apical plasma membrane. In contrast, TRPV4 translocation to the apical plasma membrane is a PKA-dependent process. We have provided substantial experimental evidence that TRPV4 serves as a route of Ca 2 ϩ influx into distal nephron cells in response to elevated luminal flow. First, we documented that silencing of TRPV4 expression in cultured collecting duct cells disrupts Ca 2 ϩ responses to shear stress (10). Second, genetic ablation of TRPV4 in mice abolishes flow-induced [Ca 2 ϩ ] i elevations in the connecting tubule and cortical collecting duct (12). Consistently, in this study, we have demonstrated that pharmacological inhibition of TRPV4 with the highly selective antagonist HC-067047 precludes changes in [Ca 2 ϩ ] i during elevations in flow (Fig. 7). Finally, we and others found that the presence of extracellular Ca 2 ϩ is mandatory for flow-induced [Ca 2 ϩ ] i elevations in renal cells (10, 28). Therefore, we are con- fident that monitoring changes in [Ca 2 ϩ ] i in freshly isolated split-opened murine distal nephrons is a reliable way to assess the rate of TRPV4 activation in native tissue by a physiologically relevant stimulus, i.e. elevated flow. In this study, we did not identify principal and intercalated cells. As we reported previously (12), principal cells exhibit a mildly increased amplitude of flow-mediated [Ca 2 ϩ ] i responses compared with intercalated cells, and this correlates with higher levels of TRPV4 expression in the former. A similar amplitude of flow-induced increases in [Ca 2 ϩ ] i in principal and ...

Context 5

... anti-rabbit IgG labeled with Alexa Fluor 488 (1:1000 dilution; Invitrogen) in 1% normal goat serum and 0.1% Triton X-100 in PBS. After washing three times with PBS for 5 min, the samples were stained with DAPI (1.5 ␮ M ; Calbiochem) to visualize nuclei. Subsequently, the samples were dehydrated and mounted with permanent mounting medium (Thermo Sci- entific). Labeled tissue samples were examined with an Nikon Eclipse Ti inverted confocal fluorescence microscope using a 40 ϫ Plan Fluor oil immersion (1.3 numerical aperture) objective. Samples were excited with 405 and 488 nm laser diodes, and emission was captured with a 16-bit CoolSNAP HQ 2 cam- era (Photometrics) interfaced to a PC running NIS-Elements version 4.00 software. Three-dimensional stacks of split- opened distal nephrons were generated from a series of confocal plane images with 0.25- ␮ m steps. Solutions —The typical bath solution was 150 m M NaCl, 5 m M KCl, 1 m M CaCl 2 , 2 m M MgCl 2 , 5 m M glucose, and 10 m M HEPES (pH 7.4). All reagents were applied by perfusing the experimental chamber at 1.5 ml/min. To test the effect of elevated flow on [Ca 2 ϩ ] i , the rate of perfusion was instantly increased from 1.5 ml/min ( ϳ 15 mm H 2 O) to 15 ml/min ( ϳ 80 mm H O). Using a parallel plate chamber, we recently esti- mated that this maneuver produces shear stress of 3 dynes/ cm 2 (12). This value fits well within the physiological range of shear stress present in the rat and mouse collecting duct as was assessed previously (10, 24). Prior termination of a respective cell-permeable activator/inhibitor of PKC- and PKA-dependent pathways did not affect the magnitude of the flow-mediated [Ca 2 ϩ ] i response due to poor reversibility of the agent. Data Analysis —All summarized data are reported as means Ϯ S.E. Data were compared using a t test or one-way analysis of variance as appropriate. p Յ 0.05 was considered significant. [Ca ] i Responses in Distal Nephron Cells —We have recently documented that the Ca 2 ϩ -permeable TRPV4 channel is a crit- ical determinant of mechanosensitive properties in distal nephron cells (10, 12). Genetic deletion of TRPV4 abolishes [Ca 2 ϩ ] i elevations in response to elevated flow in murine distal nephrons (12). PKC and PKA can directly phosphorylate TRPV4 in expression systems (19). Here, we probed whether these signaling cascades are involved in controlling mechanosensitive [Ca 2 ϩ ] i elevations by affecting TRPV4 activity and expression patterns in freshly isolated split-opened distal nephrons. Fig. 1 A documents the average time course of changes in [Ca 2 ϩ ] i levels in individual cells within a split- opened area of freshly isolated distal nephrons in response to an abrupt 10-fold elevation in flow over the apical surface. Acute stimulation of PKC with 200 n M phorbol 12-myristate 13-ace- tate (PMA) greatly potentiated flow-mediated elevations in [Ca 2 ϩ ] i . Of note, PMA treatment also had a mild stimulatory effect on the basal levels of [Ca 2 ϩ ] i (Fig. 1 A ). As summarized in Fig. 1 B , the responses to flow were similarly increased from 31 Ϯ 3 n M in the control to 49 Ϯ 4 n M and 58 Ϯ 5 n M when PMA was applied for 5 and 15 min, respectively. Administration of a highly selective, cell-permeable PKC inhibitor, bisindolylmaleimide I (BIM-I; 200 n M ), for 10 min significantly decreased the amplitude of the flow-mediated [Ca 2 ϩ ] i response from 32 Ϯ 2 n M to 12 Ϯ 2 n M (Fig. 1, C and D ). Pharmacological PKC inhibition also had a tendency to decrease basal [Ca 2 ϩ ] i levels (Fig. 1 C ). Overall, we conclude that [Ca 2 ϩ ] i responses to elevated flow in distal nephron cells are positively regulated by the PKC signaling cascade. We next probed the involvement of the PKA signaling cascade in the regulation of flow-dependent Ca 2 ϩ responses in distal nephron cells. Fig. 2 A documents the average time course of changes in [Ca 2 ϩ ] levels in response to elevated flow under the control conditions and after 15 min treatment with forskolin (20 ␮ M ) to elevate intracellular cAMP levels. However, this maneuver failed to affect flow-induced Ca 2 ϩ responses in distal nephron cells. The amplitudes of the response were 28 Ϯ 3 n M and 29 Ϯ 3 n M in the control and after forskolin treatment, respectively (Fig. 2 B ). These results suggest that acute activation of the PKA signaling cascade alone has no appreciable role in the regulation of TRPV4 functional activity and, subse- quently, flow-dependent [Ca 2 ϩ ] i elevations in distal nephron cells. TRPV4 Trafficking Is Regulated by PKA but Not PKC —We next used immunofluorescence microscopy in split-opened distal nephrons to examine whether stimulation of the PKC and PKA cascades alters subcellular TRPV4 localization to promote trafficking to the apical plasma membrane. Consistent with our previous report (12), TRPV4 expression was dominant in the apical/subapical regions under the control conditions, as apparent from a representative confocal fluorescent image in Fig. 3 A . Pretreatment with the PKC activator PMA (200 n M ) for 15 min had no apparent effect on TRPV4 subcellular localization (Fig. 3 B ). In contrast, TRPV4 localized to the apical plasma membrane when split-opened distal nephrons were pretreated with 20 ␮ M forskolin for 15 min (Fig. 3 C ). Forskolin-induced redistribution was precluded by the PKA inhibitor H-89 (20 ␮ M ) (Fig. 3 D ). To perform a quantitative estimation of the observed changes in subcellular TRPV4 localization, we employed line- scan analysis of the fluorescent signal distribution along the z -axis in cross-sections of three-dimensional stacks similar to those shown in Fig. 3. Fig. 4 A shows the averaged distribution pattern of fluorescence intensity representing TRPV4 localization in the control and after pretreatment with PMA, forskolin, and forskolin and H-89. As is clear, stimulation of the PKA pathway with forskolin shifted the maximum of the fluorescent signal toward the apical region. Furthermore, forskolin also caused sharpening of the fluorescence intensity profile. As summarized in Fig. 4 B , the average half-width of the fluorescence intensity was significantly reduced from 3.06 Ϯ 0.07 ␮ m ( n ϭ 108) in the control to 1.34 Ϯ 0.04 ␮ m ( n ϭ 123) after forskolin treatment. At the same time, the half-width was 2.92 0.17 m ( n 105) after treatment with PMA and 2.99 0.18 ␮ m ( n ϭ 95) after treatment with H-89 and forskolin. These values were not significantly different from the control. Overall, the results in Figs. 3 and 4 suggest that activation of PKA but not PKC signaling cascades promotes TRPV4 trafficking to the apical plasma membrane. The apparent lack of forskolin-mediated augmentation of the flow-induced [Ca 2 ϩ ] i response (Fig. 2), despite the prominent trafficking of TRPV4 to the apical compartment (Figs. 3 and 4), may indicate that translocated channels were not yet inserted into the plasma membrane. In this case, [Ca 2 ϩ ] i stimulation was required to incorporate TRPV4 into the apical membrane and augment cellular responses to elevated flow. To probe this, we treated split-opened distal nephrons with 20 ␮ M forskolin and quantified the amplitudes of two consequent flow-induced [Ca 2 ϩ ] i responses in the continued presence of the PKA cascade activator (Fig. 5 A ). However, [Ca 2 ϩ ] i elevations induced by the first application of increased flow did not result in appreciable potentiation of the second flow-mediated [Ca 2 ϩ ] i response. As summarized in Fig. 5 B , the amplitudes of the first and second responses during forskolin treatment were 27 Ϯ 1 and 25 Ϯ 1 n M , respectively, and were not different from the amplitude of the flow-mediated [Ca 2 ϩ ] i response in the control (29 Ϯ 2 n M ). Therefore, it appears that activation of the PKA-dependent pathway likely results in translocation of silent TRPV4 to the apical membrane and that lack of augmentation of flow-dependent [Ca 2 ϩ ] i responses is not associated with inability of the channels to be inserted. mediated [Ca ] i Elevations in the Distal Nephron —Our results point to distinct modes of TRPV4 regulation by PKC and PKA signaling cascades. Whereas the PKC-dependent pathway stimulated TRPV4 and enhanced mechanosensitive [Ca 2 ϩ ] i responses (Fig. 1) without affecting subcellular TRPV4 distribution (Fig. 3 B ), the PKA-dependent pathway promoted apical TRPV4 trafficking (Fig. 3 C ) but failed to augment functional TRPV4 status (Figs. 2 and 5). Thus, we next tested whether PKA- and PKC-dependent pathways are cooperative in augmenting TRPV4-mediated [Ca 2 ϩ ] i responses to flow. Concomitant stimulation of both pathways with 200 n M PMA and 20 M forskolin drastically augmented the amplitude of flow-induced [Ca 2 ϩ ] i responses (Fig. 6 A , black trace ). This elevation was significantly greater than that in response to application of PMA alone (Fig. 6 A , gray trace ). The absolute eleva- tions in [Ca ] i in response to flow were 34 2, 77 4, and 55 Ϯ 3 n M in the control, after treatment with PMA ϩ forskolin, and after treatment with PMA alone, respectively (Fig. 6 B ). Interestingly, we observed a prominent gradual increase of ϳ 80 n M in basal [Ca ] i levels in distal nephron cells upon concom- itant treatment with 200 n M PMA and 20 ␮ M forskolin (Fig. 6 A ). Therefore, the absolute elevation of [Ca 2 ϩ ] i from the values under unstimulated conditions reflects the total level of TRPV4 activation by mechanical stimuli ( i.e. flow) in the presence of simultaneous stimulation of the PKA and PKC cascades. As summarized in Fig. 6 C , the absolute amplitude of the [Ca 2 ϩ ] i response to flow was 162 Ϯ 5 n M , which was significantly greater than the response in the presence of PKC stimulation alone (68 Ϯ 5 n M ). Finally, to probe whether the additive stimulation of the flow-dependent [Ca 2 ϩ ] i response and gradual increases in the basal [Ca 2 ϩ ] i levels in response to simultaneous activation of the PKC and PKA cascades occur in a ...

Context 6

... ). This elevation was significantly greater than that in response to application of PMA alone (Fig. 6 A , gray trace ). The absolute eleva- tions in [Ca ] i in response to flow were 34 2, 77 4, and 55 Ϯ 3 n M in the control, after treatment with PMA ϩ forskolin, and after treatment with PMA alone, respectively (Fig. 6 B ). Interestingly, we observed a prominent gradual increase of ϳ 80 n M in basal [Ca ] i levels in distal nephron cells upon concom- itant treatment with 200 n M PMA and 20 ␮ M forskolin (Fig. 6 A ). Therefore, the absolute elevation of [Ca 2 ϩ ] i from the values under unstimulated conditions reflects the total level of TRPV4 activation by mechanical stimuli ( i.e. flow) in the presence of simultaneous stimulation of the PKA and PKC cascades. As summarized in Fig. 6 C , the absolute amplitude of the [Ca 2 ϩ ] i response to flow was 162 Ϯ 5 n M , which was significantly greater than the response in the presence of PKC stimulation alone (68 Ϯ 5 n M ). Finally, to probe whether the additive stimulation of the flow-dependent [Ca 2 ϩ ] i response and gradual increases in the basal [Ca 2 ϩ ] i levels in response to simultaneous activation of the PKC and PKA cascades occur in a TRPV4-dependent manner, we repeated the treatment with PMA and forskolin in the presence of the selective TRPV4 inhibitor HC-067047 (4 ␮ M ). As is clear from the average time course (Fig. 7 A ), TRPV4 blockade abolished the progressive increase in [Ca 2 ϩ ] i levels. Moreover, HC-067047 precluded flow-mediated [Ca 2 ϩ ] i responses even in the presence of the activated PKC and PKA cascades (Fig. 7 B ). The amplitudes of the flow-mediated [Ca 2 ϩ ] i response were 33 Ϯ 2 n M in the control and 5 Ϯ 2 n M after treatment with forskolin, PMA, and HC-067047. Overall, we conclude that the coordinated stimulation of both PKC and PKA cascades additively increases the amplitude of the TRPV4- mediated [Ca 2 ϩ ] i response to flow and, importantly, augments the basal TRPV4 activity, resulting in a progressive increase in the resting [Ca 2 ϩ ] levels. It has been recently demonstrated that the activity of the Ca 2 ϩ -permeable TRPV4 channel is central for [Ca 2 ϩ ] i elevations in distal nephron cells in response to dynamic changes in tubular fluid flow (11, 12, 16, 20). Adequate mechanosensitive [Ca 2 ϩ ] i responses are important determinants of many physiological processes in late nephron segments, including flow-dependent K ϩ secretion (15, 25), regulatory volume decreases (26), etc. Furthermore, we and others have recently demonstrated that pharmacological stimulation of TRPV4 activity is instrumental for blunting renal cystogenesis in ARPKD models (18, 27). In this study, we defined two distinct intracellular signaling cascades separately controlling TRPV4 trafficking and functional activity in murine distal nephrons. We found that the PKC-dependent signaling pathway is responsible for augmented TRPV4 activation by elevated flow over the apical plasma membrane. In contrast, TRPV4 translocation to the apical plasma membrane is a PKA-dependent process. We have provided substantial experimental evidence that TRPV4 serves as a route of Ca 2 ϩ influx into distal nephron cells in response to elevated luminal flow. First, we documented that silencing of TRPV4 expression in cultured collecting duct cells disrupts Ca 2 ϩ responses to shear stress (10). Second, genetic ablation of TRPV4 in mice abolishes flow-induced [Ca 2 ϩ ] i elevations in the connecting tubule and cortical collecting duct (12). Consistently, in this study, we have demonstrated that pharmacological inhibition of TRPV4 with the highly selective antagonist HC-067047 precludes changes in [Ca 2 ϩ ] i during elevations in flow (Fig. 7). Finally, we and others found that the presence of extracellular Ca 2 ϩ is mandatory for flow-induced [Ca 2 ϩ ] i elevations in renal cells (10, 28). Therefore, we are con- fident that monitoring changes in [Ca 2 ϩ ] i in freshly isolated split-opened murine distal nephrons is a reliable way to assess the rate of TRPV4 activation in native tissue by a physiologically relevant stimulus, i.e. elevated flow. In this study, we did not identify principal and intercalated cells. As we reported previously (12), principal cells exhibit a mildly increased amplitude of flow-mediated [Ca 2 ϩ ] i responses compared with intercalated cells, and this correlates with higher levels of TRPV4 expression in the former. A similar amplitude of flow-induced increases in [Ca 2 ϩ ] i in principal and intercalated cells was also reported in microperfused rabbit cortical collecting ducts (29, 30). The detailed analysis did not reveal noticeable heterogene- ity in the rate of potentiation of flow-dependent [Ca 2 ϩ ] i responses in individual cells during activation of PKC signaling with PMA (Fig. 1). Furthermore, we have also observed similar translocation of TRPV4 to the apical membrane in “low TRPV4-expressing” intercalated cells after treatment with for- skolin (Fig. 3). Thus, it is reasonable to suggest that the mechanisms of TRPV4 regulation by PKA and PKC in principal and intercalated cells are the same. We have shown that flow-induced [Ca 2 ϩ ] i elevations are under dynamic regulation of the PKC-dependent pathway. Stimulation of PKC led to an acute augmentation of [Ca 2 ϩ ] i responses to elevated flow (Fig. 1, A and B ), whereas inhibition of PKC with BIM-I greatly diminished mechanosensitive [Ca 2 ϩ ] i elevations (Fig. 1, C and D ). Importantly, we demonstrated that this regulation occurred in a TRPV4-dependent manner because inhibition of TRPV4 with HC-067047 abolished cellular responses to elevated flow even upon activation of PKC (Fig. 7). Interestingly, inhibition of PKC with BIM-I and Go6976 was shown to preclude transient flow-mediated [Ca 2 ϩ ] i elevations and flow-dependent potassium secretion in perfused rabbit cortical collecting ducts (30). In contrast, we did not observe complete inhibition of flow-mediated [Ca 2 ϩ ] i responses during PKC blockade. However, we used a 5-fold lower concentration of BIM-I, which is more selective. PKC was shown to directly phosphorylate multiple Ser/Thr residues within the N terminus of TRPV4 overexpressed in HEK293 cells to augment channel activation by hypotonicity (19). It remains to be determined whether PKC-mediated regulation of TRPV4 function in distal nephron cells involves direct channel phosphorylation. Of note, PMA can also directly interact with transmembrane domains 3 and 4 of TRPV4 (31, 32). However, this can lead to activation of the channel only at 37 °C and has a minor direct effect on TRPV4 activity at room temperature as used here. The important observation of this study is that stimulation of TRPV4 trafficking to the apical plasma membrane is not associated with augmentation of TRPV4-mediated [Ca 2 ϩ ] i responses to elevated flow (Figs. 2– 4). Furthermore, we have demonstrated that translocation of TRPV4 to the apical plasma membrane is regulated by PKA-dependent mechanisms (Fig. 4). This indicates that TRPV4 trafficking in distal nephron cells might be at least partially under the control of antidiuretic hor- mone (vasopressin). TRPV4 was recently shown to functionally interact with AQP2, the well known end effector of vasopressin, and the trafficking of TRPV4 to the plasma membrane in M1 collecting duct cells occurs only in the presence of AQP2 (26). Although this was associated with enhanced responses to hypotonicity, we failed to observe an augmented response to flow in forskolin-treated distal nephrons (Fig. 2). The nature of this discrepancy requires further investigation. Importantly, lack of mechanosensitive [Ca 2 ϩ ] i responses (33–35) and elevated cAMP levels (36, 37) have been consistently reported for cyst cells of many polycystic kidney disease models. Using a novel preparation to isolate collecting duct-derived cyst monolayers from a rat model of ARPKD, we recently demonstrated drastic decreases in TRPV4 activity despite prominent apical localization of the channel in cyst cells, which is likely a consequence of elevated cAMP levels (18). This is consistent with our conclu- sions that trafficking and activation of TRPV4 require distinct intracellular signaling inputs. Interestingly, another Ca 2 ϩ -permeable channel, TRPC3, which is natively expressed in distal nephron cells (38), is also translocated to the apical plasma membrane in response to vasopressin treatment via stimulation of the cAMP/PKA pathway (39, 40). However, it remains unclear whether this redistribution is associated with augmented TRPC3-dependent [Ca 2 ϩ ] i elevations. In this study, we have also provided evidence that TRPV4 activity is an important determinant of basal [Ca 2 ϩ ] i levels in distal nephron cells. Stimulation of PKC led not only to augmentation of TRPV4-mediated [Ca 2 ϩ ] i responses to flow but also to a gradual elevation of the [Ca 2 ϩ ] i base line (Fig. 1 A ). This elevation was greatly potentiated after stimulation of apical TRPV4 trafficking with PKA (Fig. 6 A ). In contrast, we observed a tendency to reduce basal [Ca 2 ϩ ] i levels when PKC was inhib- ited by BIM-I (Fig. 1 C ). These observations support the concep- tion that basal TRPV4 activity under unstimulated conditions ( i.e. in the absence of external mechanical inputs) is sufficient to adjust resting [Ca 2 ϩ ] i levels in murine distal nephron cells. We recently reported that impaired TRPV4 activity is associated with reduced resting [Ca 2 ϩ ] i levels in collecting duct-derived cyst cells during ARPKD and, vice versa, that restoration of TRPV4 activity increases [Ca 2 ϩ ] i levels to the values seen in normal rat distal nephron cells (18). In summary, in this study, we have identified the signaling determinants of TRPV4 function in murine native distal nephron cells. We have reported that stimulation of TRPV4 activity and TRPV4 trafficking is under discrete but synergistic control of the PKC- and PKA-dependent pathways. ...

Context 7

... After washing three times with PBS for 5 min, the samples were stained with DAPI (1.5 ␮ M ; Calbiochem) to visualize nuclei. Subsequently, the samples were dehydrated and mounted with permanent mounting medium (Thermo Sci- entific). Labeled tissue samples were examined with an Nikon Eclipse Ti inverted confocal fluorescence microscope using a 40 ϫ Plan Fluor oil immersion (1.3 numerical aperture) objective. Samples were excited with 405 and 488 nm laser diodes, and emission was captured with a 16-bit CoolSNAP HQ 2 cam- era (Photometrics) interfaced to a PC running NIS-Elements version 4.00 software. Three-dimensional stacks of split- opened distal nephrons were generated from a series of confocal plane images with 0.25- ␮ m steps. Solutions —The typical bath solution was 150 m M NaCl, 5 m M KCl, 1 m M CaCl 2 , 2 m M MgCl 2 , 5 m M glucose, and 10 m M HEPES (pH 7.4). All reagents were applied by perfusing the experimental chamber at 1.5 ml/min. To test the effect of elevated flow on [Ca 2 ϩ ] i , the rate of perfusion was instantly increased from 1.5 ml/min ( ϳ 15 mm H 2 O) to 15 ml/min ( ϳ 80 mm H O). Using a parallel plate chamber, we recently esti- mated that this maneuver produces shear stress of 3 dynes/ cm 2 (12). This value fits well within the physiological range of shear stress present in the rat and mouse collecting duct as was assessed previously (10, 24). Prior termination of a respective cell-permeable activator/inhibitor of PKC- and PKA-dependent pathways did not affect the magnitude of the flow-mediated [Ca 2 ϩ ] i response due to poor reversibility of the agent. Data Analysis —All summarized data are reported as means Ϯ S.E. Data were compared using a t test or one-way analysis of variance as appropriate. p Յ 0.05 was considered significant. [Ca ] i Responses in Distal Nephron Cells —We have recently documented that the Ca 2 ϩ -permeable TRPV4 channel is a crit- ical determinant of mechanosensitive properties in distal nephron cells (10, 12). Genetic deletion of TRPV4 abolishes [Ca 2 ϩ ] i elevations in response to elevated flow in murine distal nephrons (12). PKC and PKA can directly phosphorylate TRPV4 in expression systems (19). Here, we probed whether these signaling cascades are involved in controlling mechanosensitive [Ca 2 ϩ ] i elevations by affecting TRPV4 activity and expression patterns in freshly isolated split-opened distal nephrons. Fig. 1 A documents the average time course of changes in [Ca 2 ϩ ] i levels in individual cells within a split- opened area of freshly isolated distal nephrons in response to an abrupt 10-fold elevation in flow over the apical surface. Acute stimulation of PKC with 200 n M phorbol 12-myristate 13-ace- tate (PMA) greatly potentiated flow-mediated elevations in [Ca 2 ϩ ] i . Of note, PMA treatment also had a mild stimulatory effect on the basal levels of [Ca 2 ϩ ] i (Fig. 1 A ). As summarized in Fig. 1 B , the responses to flow were similarly increased from 31 Ϯ 3 n M in the control to 49 Ϯ 4 n M and 58 Ϯ 5 n M when PMA was applied for 5 and 15 min, respectively. Administration of a highly selective, cell-permeable PKC inhibitor, bisindolylmaleimide I (BIM-I; 200 n M ), for 10 min significantly decreased the amplitude of the flow-mediated [Ca 2 ϩ ] i response from 32 Ϯ 2 n M to 12 Ϯ 2 n M (Fig. 1, C and D ). Pharmacological PKC inhibition also had a tendency to decrease basal [Ca 2 ϩ ] i levels (Fig. 1 C ). Overall, we conclude that [Ca 2 ϩ ] i responses to elevated flow in distal nephron cells are positively regulated by the PKC signaling cascade. We next probed the involvement of the PKA signaling cascade in the regulation of flow-dependent Ca 2 ϩ responses in distal nephron cells. Fig. 2 A documents the average time course of changes in [Ca 2 ϩ ] levels in response to elevated flow under the control conditions and after 15 min treatment with forskolin (20 ␮ M ) to elevate intracellular cAMP levels. However, this maneuver failed to affect flow-induced Ca 2 ϩ responses in distal nephron cells. The amplitudes of the response were 28 Ϯ 3 n M and 29 Ϯ 3 n M in the control and after forskolin treatment, respectively (Fig. 2 B ). These results suggest that acute activation of the PKA signaling cascade alone has no appreciable role in the regulation of TRPV4 functional activity and, subse- quently, flow-dependent [Ca 2 ϩ ] i elevations in distal nephron cells. TRPV4 Trafficking Is Regulated by PKA but Not PKC —We next used immunofluorescence microscopy in split-opened distal nephrons to examine whether stimulation of the PKC and PKA cascades alters subcellular TRPV4 localization to promote trafficking to the apical plasma membrane. Consistent with our previous report (12), TRPV4 expression was dominant in the apical/subapical regions under the control conditions, as apparent from a representative confocal fluorescent image in Fig. 3 A . Pretreatment with the PKC activator PMA (200 n M ) for 15 min had no apparent effect on TRPV4 subcellular localization (Fig. 3 B ). In contrast, TRPV4 localized to the apical plasma membrane when split-opened distal nephrons were pretreated with 20 ␮ M forskolin for 15 min (Fig. 3 C ). Forskolin-induced redistribution was precluded by the PKA inhibitor H-89 (20 ␮ M ) (Fig. 3 D ). To perform a quantitative estimation of the observed changes in subcellular TRPV4 localization, we employed line- scan analysis of the fluorescent signal distribution along the z -axis in cross-sections of three-dimensional stacks similar to those shown in Fig. 3. Fig. 4 A shows the averaged distribution pattern of fluorescence intensity representing TRPV4 localization in the control and after pretreatment with PMA, forskolin, and forskolin and H-89. As is clear, stimulation of the PKA pathway with forskolin shifted the maximum of the fluorescent signal toward the apical region. Furthermore, forskolin also caused sharpening of the fluorescence intensity profile. As summarized in Fig. 4 B , the average half-width of the fluorescence intensity was significantly reduced from 3.06 Ϯ 0.07 ␮ m ( n ϭ 108) in the control to 1.34 Ϯ 0.04 ␮ m ( n ϭ 123) after forskolin treatment. At the same time, the half-width was 2.92 0.17 m ( n 105) after treatment with PMA and 2.99 0.18 ␮ m ( n ϭ 95) after treatment with H-89 and forskolin. These values were not significantly different from the control. Overall, the results in Figs. 3 and 4 suggest that activation of PKA but not PKC signaling cascades promotes TRPV4 trafficking to the apical plasma membrane. The apparent lack of forskolin-mediated augmentation of the flow-induced [Ca 2 ϩ ] i response (Fig. 2), despite the prominent trafficking of TRPV4 to the apical compartment (Figs. 3 and 4), may indicate that translocated channels were not yet inserted into the plasma membrane. In this case, [Ca 2 ϩ ] i stimulation was required to incorporate TRPV4 into the apical membrane and augment cellular responses to elevated flow. To probe this, we treated split-opened distal nephrons with 20 ␮ M forskolin and quantified the amplitudes of two consequent flow-induced [Ca 2 ϩ ] i responses in the continued presence of the PKA cascade activator (Fig. 5 A ). However, [Ca 2 ϩ ] i elevations induced by the first application of increased flow did not result in appreciable potentiation of the second flow-mediated [Ca 2 ϩ ] i response. As summarized in Fig. 5 B , the amplitudes of the first and second responses during forskolin treatment were 27 Ϯ 1 and 25 Ϯ 1 n M , respectively, and were not different from the amplitude of the flow-mediated [Ca 2 ϩ ] i response in the control (29 Ϯ 2 n M ). Therefore, it appears that activation of the PKA-dependent pathway likely results in translocation of silent TRPV4 to the apical membrane and that lack of augmentation of flow-dependent [Ca 2 ϩ ] i responses is not associated with inability of the channels to be inserted. mediated [Ca ] i Elevations in the Distal Nephron —Our results point to distinct modes of TRPV4 regulation by PKC and PKA signaling cascades. Whereas the PKC-dependent pathway stimulated TRPV4 and enhanced mechanosensitive [Ca 2 ϩ ] i responses (Fig. 1) without affecting subcellular TRPV4 distribution (Fig. 3 B ), the PKA-dependent pathway promoted apical TRPV4 trafficking (Fig. 3 C ) but failed to augment functional TRPV4 status (Figs. 2 and 5). Thus, we next tested whether PKA- and PKC-dependent pathways are cooperative in augmenting TRPV4-mediated [Ca 2 ϩ ] i responses to flow. Concomitant stimulation of both pathways with 200 n M PMA and 20 M forskolin drastically augmented the amplitude of flow-induced [Ca 2 ϩ ] i responses (Fig. 6 A , black trace ). This elevation was significantly greater than that in response to application of PMA alone (Fig. 6 A , gray trace ). The absolute eleva- tions in [Ca ] i in response to flow were 34 2, 77 4, and 55 Ϯ 3 n M in the control, after treatment with PMA ϩ forskolin, and after treatment with PMA alone, respectively (Fig. 6 B ). Interestingly, we observed a prominent gradual increase of ϳ 80 n M in basal [Ca ] i levels in distal nephron cells upon concom- itant treatment with 200 n M PMA and 20 ␮ M forskolin (Fig. 6 A ). Therefore, the absolute elevation of [Ca 2 ϩ ] i from the values under unstimulated conditions reflects the total level of TRPV4 activation by mechanical stimuli ( i.e. flow) in the presence of simultaneous stimulation of the PKA and PKC cascades. As summarized in Fig. 6 C , the absolute amplitude of the [Ca 2 ϩ ] i response to flow was 162 Ϯ 5 n M , which was significantly greater than the response in the presence of PKC stimulation alone (68 Ϯ 5 n M ). Finally, to probe whether the additive stimulation of the flow-dependent [Ca 2 ϩ ] i response and gradual increases in the basal [Ca 2 ϩ ] i levels in response to simultaneous activation of the PKC and PKA cascades occur in a TRPV4-dependent manner, we repeated the treatment with PMA and forskolin in the presence of the selective TRPV4 inhibitor HC-067047 (4 ␮ M ). As ...

Context 8

... were examined with an Nikon Eclipse Ti inverted confocal fluorescence microscope using a 40 ϫ Plan Fluor oil immersion (1.3 numerical aperture) objective. Samples were excited with 405 and 488 nm laser diodes, and emission was captured with a 16-bit CoolSNAP HQ 2 cam- era (Photometrics) interfaced to a PC running NIS-Elements version 4.00 software. Three-dimensional stacks of split- opened distal nephrons were generated from a series of confocal plane images with 0.25- ␮ m steps. Solutions —The typical bath solution was 150 m M NaCl, 5 m M KCl, 1 m M CaCl 2 , 2 m M MgCl 2 , 5 m M glucose, and 10 m M HEPES (pH 7.4). All reagents were applied by perfusing the experimental chamber at 1.5 ml/min. To test the effect of elevated flow on [Ca 2 ϩ ] i , the rate of perfusion was instantly increased from 1.5 ml/min ( ϳ 15 mm H 2 O) to 15 ml/min ( ϳ 80 mm H O). Using a parallel plate chamber, we recently esti- mated that this maneuver produces shear stress of 3 dynes/ cm 2 (12). This value fits well within the physiological range of shear stress present in the rat and mouse collecting duct as was assessed previously (10, 24). Prior termination of a respective cell-permeable activator/inhibitor of PKC- and PKA-dependent pathways did not affect the magnitude of the flow-mediated [Ca 2 ϩ ] i response due to poor reversibility of the agent. Data Analysis —All summarized data are reported as means Ϯ S.E. Data were compared using a t test or one-way analysis of variance as appropriate. p Յ 0.05 was considered significant. [Ca ] i Responses in Distal Nephron Cells —We have recently documented that the Ca 2 ϩ -permeable TRPV4 channel is a crit- ical determinant of mechanosensitive properties in distal nephron cells (10, 12). Genetic deletion of TRPV4 abolishes [Ca 2 ϩ ] i elevations in response to elevated flow in murine distal nephrons (12). PKC and PKA can directly phosphorylate TRPV4 in expression systems (19). Here, we probed whether these signaling cascades are involved in controlling mechanosensitive [Ca 2 ϩ ] i elevations by affecting TRPV4 activity and expression patterns in freshly isolated split-opened distal nephrons. Fig. 1 A documents the average time course of changes in [Ca 2 ϩ ] i levels in individual cells within a split- opened area of freshly isolated distal nephrons in response to an abrupt 10-fold elevation in flow over the apical surface. Acute stimulation of PKC with 200 n M phorbol 12-myristate 13-ace- tate (PMA) greatly potentiated flow-mediated elevations in [Ca 2 ϩ ] i . Of note, PMA treatment also had a mild stimulatory effect on the basal levels of [Ca 2 ϩ ] i (Fig. 1 A ). As summarized in Fig. 1 B , the responses to flow were similarly increased from 31 Ϯ 3 n M in the control to 49 Ϯ 4 n M and 58 Ϯ 5 n M when PMA was applied for 5 and 15 min, respectively. Administration of a highly selective, cell-permeable PKC inhibitor, bisindolylmaleimide I (BIM-I; 200 n M ), for 10 min significantly decreased the amplitude of the flow-mediated [Ca 2 ϩ ] i response from 32 Ϯ 2 n M to 12 Ϯ 2 n M (Fig. 1, C and D ). Pharmacological PKC inhibition also had a tendency to decrease basal [Ca 2 ϩ ] i levels (Fig. 1 C ). Overall, we conclude that [Ca 2 ϩ ] i responses to elevated flow in distal nephron cells are positively regulated by the PKC signaling cascade. We next probed the involvement of the PKA signaling cascade in the regulation of flow-dependent Ca 2 ϩ responses in distal nephron cells. Fig. 2 A documents the average time course of changes in [Ca 2 ϩ ] levels in response to elevated flow under the control conditions and after 15 min treatment with forskolin (20 ␮ M ) to elevate intracellular cAMP levels. However, this maneuver failed to affect flow-induced Ca 2 ϩ responses in distal nephron cells. The amplitudes of the response were 28 Ϯ 3 n M and 29 Ϯ 3 n M in the control and after forskolin treatment, respectively (Fig. 2 B ). These results suggest that acute activation of the PKA signaling cascade alone has no appreciable role in the regulation of TRPV4 functional activity and, subse- quently, flow-dependent [Ca 2 ϩ ] i elevations in distal nephron cells. TRPV4 Trafficking Is Regulated by PKA but Not PKC —We next used immunofluorescence microscopy in split-opened distal nephrons to examine whether stimulation of the PKC and PKA cascades alters subcellular TRPV4 localization to promote trafficking to the apical plasma membrane. Consistent with our previous report (12), TRPV4 expression was dominant in the apical/subapical regions under the control conditions, as apparent from a representative confocal fluorescent image in Fig. 3 A . Pretreatment with the PKC activator PMA (200 n M ) for 15 min had no apparent effect on TRPV4 subcellular localization (Fig. 3 B ). In contrast, TRPV4 localized to the apical plasma membrane when split-opened distal nephrons were pretreated with 20 ␮ M forskolin for 15 min (Fig. 3 C ). Forskolin-induced redistribution was precluded by the PKA inhibitor H-89 (20 ␮ M ) (Fig. 3 D ). To perform a quantitative estimation of the observed changes in subcellular TRPV4 localization, we employed line- scan analysis of the fluorescent signal distribution along the z -axis in cross-sections of three-dimensional stacks similar to those shown in Fig. 3. Fig. 4 A shows the averaged distribution pattern of fluorescence intensity representing TRPV4 localization in the control and after pretreatment with PMA, forskolin, and forskolin and H-89. As is clear, stimulation of the PKA pathway with forskolin shifted the maximum of the fluorescent signal toward the apical region. Furthermore, forskolin also caused sharpening of the fluorescence intensity profile. As summarized in Fig. 4 B , the average half-width of the fluorescence intensity was significantly reduced from 3.06 Ϯ 0.07 ␮ m ( n ϭ 108) in the control to 1.34 Ϯ 0.04 ␮ m ( n ϭ 123) after forskolin treatment. At the same time, the half-width was 2.92 0.17 m ( n 105) after treatment with PMA and 2.99 0.18 ␮ m ( n ϭ 95) after treatment with H-89 and forskolin. These values were not significantly different from the control. Overall, the results in Figs. 3 and 4 suggest that activation of PKA but not PKC signaling cascades promotes TRPV4 trafficking to the apical plasma membrane. The apparent lack of forskolin-mediated augmentation of the flow-induced [Ca 2 ϩ ] i response (Fig. 2), despite the prominent trafficking of TRPV4 to the apical compartment (Figs. 3 and 4), may indicate that translocated channels were not yet inserted into the plasma membrane. In this case, [Ca 2 ϩ ] i stimulation was required to incorporate TRPV4 into the apical membrane and augment cellular responses to elevated flow. To probe this, we treated split-opened distal nephrons with 20 ␮ M forskolin and quantified the amplitudes of two consequent flow-induced [Ca 2 ϩ ] i responses in the continued presence of the PKA cascade activator (Fig. 5 A ). However, [Ca 2 ϩ ] i elevations induced by the first application of increased flow did not result in appreciable potentiation of the second flow-mediated [Ca 2 ϩ ] i response. As summarized in Fig. 5 B , the amplitudes of the first and second responses during forskolin treatment were 27 Ϯ 1 and 25 Ϯ 1 n M , respectively, and were not different from the amplitude of the flow-mediated [Ca 2 ϩ ] i response in the control (29 Ϯ 2 n M ). Therefore, it appears that activation of the PKA-dependent pathway likely results in translocation of silent TRPV4 to the apical membrane and that lack of augmentation of flow-dependent [Ca 2 ϩ ] i responses is not associated with inability of the channels to be inserted. mediated [Ca ] i Elevations in the Distal Nephron —Our results point to distinct modes of TRPV4 regulation by PKC and PKA signaling cascades. Whereas the PKC-dependent pathway stimulated TRPV4 and enhanced mechanosensitive [Ca 2 ϩ ] i responses (Fig. 1) without affecting subcellular TRPV4 distribution (Fig. 3 B ), the PKA-dependent pathway promoted apical TRPV4 trafficking (Fig. 3 C ) but failed to augment functional TRPV4 status (Figs. 2 and 5). Thus, we next tested whether PKA- and PKC-dependent pathways are cooperative in augmenting TRPV4-mediated [Ca 2 ϩ ] i responses to flow. Concomitant stimulation of both pathways with 200 n M PMA and 20 M forskolin drastically augmented the amplitude of flow-induced [Ca 2 ϩ ] i responses (Fig. 6 A , black trace ). This elevation was significantly greater than that in response to application of PMA alone (Fig. 6 A , gray trace ). The absolute eleva- tions in [Ca ] i in response to flow were 34 2, 77 4, and 55 Ϯ 3 n M in the control, after treatment with PMA ϩ forskolin, and after treatment with PMA alone, respectively (Fig. 6 B ). Interestingly, we observed a prominent gradual increase of ϳ 80 n M in basal [Ca ] i levels in distal nephron cells upon concom- itant treatment with 200 n M PMA and 20 ␮ M forskolin (Fig. 6 A ). Therefore, the absolute elevation of [Ca 2 ϩ ] i from the values under unstimulated conditions reflects the total level of TRPV4 activation by mechanical stimuli ( i.e. flow) in the presence of simultaneous stimulation of the PKA and PKC cascades. As summarized in Fig. 6 C , the absolute amplitude of the [Ca 2 ϩ ] i response to flow was 162 Ϯ 5 n M , which was significantly greater than the response in the presence of PKC stimulation alone (68 Ϯ 5 n M ). Finally, to probe whether the additive stimulation of the flow-dependent [Ca 2 ϩ ] i response and gradual increases in the basal [Ca 2 ϩ ] i levels in response to simultaneous activation of the PKC and PKA cascades occur in a TRPV4-dependent manner, we repeated the treatment with PMA and forskolin in the presence of the selective TRPV4 inhibitor HC-067047 (4 ␮ M ). As is clear from the average time course (Fig. 7 A ), TRPV4 blockade abolished the progressive increase in [Ca 2 ϩ ] i levels. Moreover, HC-067047 precluded flow-mediated [Ca 2 ϩ ] i responses even in the presence of the activated PKC and PKA cascades ...

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... 2 ϩ ] i response from 32 Ϯ 2 n M to 12 Ϯ 2 n M (Fig. 1, C and D ). Pharmacological PKC inhibition also had a tendency to decrease basal [Ca 2 ϩ ] i levels (Fig. 1 C ). Overall, we conclude that [Ca 2 ϩ ] i responses to elevated flow in distal nephron cells are positively regulated by the PKC signaling cascade. We next probed the involvement of the PKA signaling cascade in the regulation of flow-dependent Ca 2 ϩ responses in distal nephron cells. Fig. 2 A documents the average time course of changes in [Ca 2 ϩ ] levels in response to elevated flow under the control conditions and after 15 min treatment with forskolin (20 ␮ M ) to elevate intracellular cAMP levels. However, this maneuver failed to affect flow-induced Ca 2 ϩ responses in distal nephron cells. The amplitudes of the response were 28 Ϯ 3 n M and 29 Ϯ 3 n M in the control and after forskolin treatment, respectively (Fig. 2 B ). These results suggest that acute activation of the PKA signaling cascade alone has no appreciable role in the regulation of TRPV4 functional activity and, subse- quently, flow-dependent [Ca 2 ϩ ] i elevations in distal nephron cells. TRPV4 Trafficking Is Regulated by PKA but Not PKC —We next used immunofluorescence microscopy in split-opened distal nephrons to examine whether stimulation of the PKC and PKA cascades alters subcellular TRPV4 localization to promote trafficking to the apical plasma membrane. Consistent with our previous report (12), TRPV4 expression was dominant in the apical/subapical regions under the control conditions, as apparent from a representative confocal fluorescent image in Fig. 3 A . Pretreatment with the PKC activator PMA (200 n M ) for 15 min had no apparent effect on TRPV4 subcellular localization (Fig. 3 B ). In contrast, TRPV4 localized to the apical plasma membrane when split-opened distal nephrons were pretreated with 20 ␮ M forskolin for 15 min (Fig. 3 C ). Forskolin-induced redistribution was precluded by the PKA inhibitor H-89 (20 ␮ M ) (Fig. 3 D ). To perform a quantitative estimation of the observed changes in subcellular TRPV4 localization, we employed line- scan analysis of the fluorescent signal distribution along the z -axis in cross-sections of three-dimensional stacks similar to those shown in Fig. 3. Fig. 4 A shows the averaged distribution pattern of fluorescence intensity representing TRPV4 localization in the control and after pretreatment with PMA, forskolin, and forskolin and H-89. As is clear, stimulation of the PKA pathway with forskolin shifted the maximum of the fluorescent signal toward the apical region. Furthermore, forskolin also caused sharpening of the fluorescence intensity profile. As summarized in Fig. 4 B , the average half-width of the fluorescence intensity was significantly reduced from 3.06 Ϯ 0.07 ␮ m ( n ϭ 108) in the control to 1.34 Ϯ 0.04 ␮ m ( n ϭ 123) after forskolin treatment. At the same time, the half-width was 2.92 0.17 m ( n 105) after treatment with PMA and 2.99 0.18 ␮ m ( n ϭ 95) after treatment with H-89 and forskolin. These values were not significantly different from the control. Overall, the results in Figs. 3 and 4 suggest that activation of PKA but not PKC signaling cascades promotes TRPV4 trafficking to the apical plasma membrane. The apparent lack of forskolin-mediated augmentation of the flow-induced [Ca 2 ϩ ] i response (Fig. 2), despite the prominent trafficking of TRPV4 to the apical compartment (Figs. 3 and 4), may indicate that translocated channels were not yet inserted into the plasma membrane. In this case, [Ca 2 ϩ ] i stimulation was required to incorporate TRPV4 into the apical membrane and augment cellular responses to elevated flow. To probe this, we treated split-opened distal nephrons with 20 ␮ M forskolin and quantified the amplitudes of two consequent flow-induced [Ca 2 ϩ ] i responses in the continued presence of the PKA cascade activator (Fig. 5 A ). However, [Ca 2 ϩ ] i elevations induced by the first application of increased flow did not result in appreciable potentiation of the second flow-mediated [Ca 2 ϩ ] i response. As summarized in Fig. 5 B , the amplitudes of the first and second responses during forskolin treatment were 27 Ϯ 1 and 25 Ϯ 1 n M , respectively, and were not different from the amplitude of the flow-mediated [Ca 2 ϩ ] i response in the control (29 Ϯ 2 n M ). Therefore, it appears that activation of the PKA-dependent pathway likely results in translocation of silent TRPV4 to the apical membrane and that lack of augmentation of flow-dependent [Ca 2 ϩ ] i responses is not associated with inability of the channels to be inserted. mediated [Ca ] i Elevations in the Distal Nephron —Our results point to distinct modes of TRPV4 regulation by PKC and PKA signaling cascades. Whereas the PKC-dependent pathway stimulated TRPV4 and enhanced mechanosensitive [Ca 2 ϩ ] i responses (Fig. 1) without affecting subcellular TRPV4 distribution (Fig. 3 B ), the PKA-dependent pathway promoted apical TRPV4 trafficking (Fig. 3 C ) but failed to augment functional TRPV4 status (Figs. 2 and 5). Thus, we next tested whether PKA- and PKC-dependent pathways are cooperative in augmenting TRPV4-mediated [Ca 2 ϩ ] i responses to flow. Concomitant stimulation of both pathways with 200 n M PMA and 20 M forskolin drastically augmented the amplitude of flow-induced [Ca 2 ϩ ] i responses (Fig. 6 A , black trace ). This elevation was significantly greater than that in response to application of PMA alone (Fig. 6 A , gray trace ). The absolute eleva- tions in [Ca ] i in response to flow were 34 2, 77 4, and 55 Ϯ 3 n M in the control, after treatment with PMA ϩ forskolin, and after treatment with PMA alone, respectively (Fig. 6 B ). Interestingly, we observed a prominent gradual increase of ϳ 80 n M in basal [Ca ] i levels in distal nephron cells upon concom- itant treatment with 200 n M PMA and 20 ␮ M forskolin (Fig. 6 A ). Therefore, the absolute elevation of [Ca 2 ϩ ] i from the values under unstimulated conditions reflects the total level of TRPV4 activation by mechanical stimuli ( i.e. flow) in the presence of simultaneous stimulation of the PKA and PKC cascades. As summarized in Fig. 6 C , the absolute amplitude of the [Ca 2 ϩ ] i response to flow was 162 Ϯ 5 n M , which was significantly greater than the response in the presence of PKC stimulation alone (68 Ϯ 5 n M ). Finally, to probe whether the additive stimulation of the flow-dependent [Ca 2 ϩ ] i response and gradual increases in the basal [Ca 2 ϩ ] i levels in response to simultaneous activation of the PKC and PKA cascades occur in a TRPV4-dependent manner, we repeated the treatment with PMA and forskolin in the presence of the selective TRPV4 inhibitor HC-067047 (4 ␮ M ). As is clear from the average time course (Fig. 7 A ), TRPV4 blockade abolished the progressive increase in [Ca 2 ϩ ] i levels. Moreover, HC-067047 precluded flow-mediated [Ca 2 ϩ ] i responses even in the presence of the activated PKC and PKA cascades (Fig. 7 B ). The amplitudes of the flow-mediated [Ca 2 ϩ ] i response were 33 Ϯ 2 n M in the control and 5 Ϯ 2 n M after treatment with forskolin, PMA, and HC-067047. Overall, we conclude that the coordinated stimulation of both PKC and PKA cascades additively increases the amplitude of the TRPV4- mediated [Ca 2 ϩ ] i response to flow and, importantly, augments the basal TRPV4 activity, resulting in a progressive increase in the resting [Ca 2 ϩ ] levels. It has been recently demonstrated that the activity of the Ca 2 ϩ -permeable TRPV4 channel is central for [Ca 2 ϩ ] i elevations in distal nephron cells in response to dynamic changes in tubular fluid flow (11, 12, 16, 20). Adequate mechanosensitive [Ca 2 ϩ ] i responses are important determinants of many physiological processes in late nephron segments, including flow-dependent K ϩ secretion (15, 25), regulatory volume decreases (26), etc. Furthermore, we and others have recently demonstrated that pharmacological stimulation of TRPV4 activity is instrumental for blunting renal cystogenesis in ARPKD models (18, 27). In this study, we defined two distinct intracellular signaling cascades separately controlling TRPV4 trafficking and functional activity in murine distal nephrons. We found that the PKC-dependent signaling pathway is responsible for augmented TRPV4 activation by elevated flow over the apical plasma membrane. In contrast, TRPV4 translocation to the apical plasma membrane is a PKA-dependent process. We have provided substantial experimental evidence that TRPV4 serves as a route of Ca 2 ϩ influx into distal nephron cells in response to elevated luminal flow. First, we documented that silencing of TRPV4 expression in cultured collecting duct cells disrupts Ca 2 ϩ responses to shear stress (10). Second, genetic ablation of TRPV4 in mice abolishes flow-induced [Ca 2 ϩ ] i elevations in the connecting tubule and cortical collecting duct (12). Consistently, in this study, we have demonstrated that pharmacological inhibition of TRPV4 with the highly selective antagonist HC-067047 precludes changes in [Ca 2 ϩ ] i during elevations in flow (Fig. 7). Finally, we and others found that the presence of extracellular Ca 2 ϩ is mandatory for flow-induced [Ca 2 ϩ ] i elevations in renal cells (10, 28). Therefore, we are con- fident that monitoring changes in [Ca 2 ϩ ] i in freshly isolated split-opened murine distal nephrons is a reliable way to assess the rate of TRPV4 activation in native tissue by a physiologically relevant stimulus, i.e. elevated flow. In this study, we did not identify principal and intercalated cells. As we reported previously (12), principal cells exhibit a mildly increased amplitude of flow-mediated [Ca 2 ϩ ] i responses compared with intercalated cells, and this correlates with higher levels of TRPV4 expression in the former. A similar amplitude of flow-induced increases in [Ca 2 ϩ ] i in principal and intercalated cells was also reported in microperfused rabbit cortical collecting ...