Comparison of 5 deiodinase (D) activity in different tissues from virgin and lactating rats. Control animals included intact virgin animals and lactating mothers in which suckling was continuous (SC), the rest of the lactating rats were separated from their pups for 12 h. One group was killed after 12 h of non-suckling (12 hNS), a second group was resuckled by their pups for 15 min and killed 4 h later (resuckled group; RS), the last two groups were treated with 40 μ g/100 g body weight norepinephrine (NE) and 100 μ g/100 g body weight isoproterenol (ISO) respectively, and killed 4 h later. Virgin animals received the same doses of both substances. Data are expressed as the means S.D. ( n = 6). Means with different letters are signi fi cantly different ( P < 0 · 05). 

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... and norepinephrine (NE) and isoproterenol (ISO) from Sigma Chemical (St Louis, MO, USA). Oligonucleotides were synthesized by GIBCO-BRL (Gaithersburg, MD, USA). All other reagents were of the highest purity commercially available. Virgin (250 g) and primiparous (300 g) Sprague – Dawley rats were housed in individual cages in a room with a 14 h light:10 h darkness cycle. On postpartum day 1, the size of the litter was adjusted to ten pups per mother and all the experimental procedures were conducted on postpartum day 10 1. All animals were fed Purina Lab Chow and tap water ad libitum . Procedures regarding care, administration of treatment and euthanasia of animals were reviewed and approved by the supporting Direccion general de asuntos del personal academico (DGAPA)/UNAM Committee. All animals were killed by decapitation and two samples of liver, heart, BAT and MG of each individual were dissected. One sample of each tissue was immediately frozen on liquid nitrogen and the other sample was homogenized in a guanidine thiocyanate solution. Deiodinase activity was determined by a modi fi cation of the radiolabeled iodide release method as previously standardized for each tissue (Aceves et al. 1994). All tissues were homogenized in a solution containing 10 mM HEPES (pH 7 · 0), 0 · 32 M sucrose, 1 · 0 mM EDTA and 20 mM DTT, and centrifuged at 2800 g for 30 min at 4 C. Assay conditions for tissues containing D1 activity (liver, heart and MG) were 0 · 2 – 200 μg protein, 2 nM 125 I-rT3, 0 · 5 μM non-radiolabeled rT3 and 5 mM DTT. For BAT (which expresses D2 enzyme) the assay was carried out with 200 μg protein, 2 nM 125 I-rT4 and 20 mM DTT. After 3 h of incubation, released acid-soluble radioiodide was isolated by chromatography in Dowex 50W-X2 columns (Bio-Rad, Richmond, CA, USA). Proteins were measured by the Bradford method (Bio-Rad protein assay; Bio-Rad). Results are expressed as fmol or pmol radioiodide released/mg protein/h. D1 or D2 mRNAs were identi fi ed by using a previously standardized semi-quantitative PCR procedure in which an amplicon of the structural protein cyclophilin (Cyc) was simultaneously ampli fi ed (Aceves et al. 1999 a ). Brie fl y, the reverse transcription (RT) reaction was primed with oligo(dT) and subscripted with 5 μg total RNA. The PCR reaction was carried out using 5 μl RT mixture and the following primers: for D1, M2s and M6as, for D2, D22s and D26as and for Cyc, Cyc1s and Cyc7as (see Fig. 1). Ampli fi cation was carried out for 32 cycles. Each cycle consisted of melting at 94 C 45 s, annealing at 52 C 45 s, and an extension at 72 C 1 min. As a control, a reaction mixture containing an RNA sample with the appropriate oligonucleotide primers, but without the RT, was included in every experiment. The sizes of the resultant PCR fragments were 251 bp for D1, 805 bp for D2 and 521 bp for Cyc, and were resolved on a 2% agarose gel and visualized using ethidium bromide. The sizes of the bands were con fi rmed by a restriction digested pUC plasmid (1 kb DNA ladder; GIBCO-BRL). After a Polaroid picture had been taken, the pictures were digi- tized using a Hewlett Packard Scanner Jet 11CX, and the signals were analyzed by using an editing version of the NIH-image program. Values obtained were normalized according to the Cyc mRNA levels detected in each sample. The length of 3 UTR D1 mRNA was assessed by using a previously standardized 3 -rapid ampli fi cation cDNA ends (RACE) procedure (Aceves et al. 1999 a ) and 5 μg total RNA from liver, heart and MG. The RT reaction was primed with oligo(dT) containing a terminal speci fi c sequence, AP (see Fig. 1). Ampli fi cation was carried out for twenty cycles consisting of melting at 94 C 45 s, annealing at 50 C 45 s, and an extension at 72 C 1 min, with J3s and UAP oligos (see Fig. 1) and a second tandem of 40 cycles of melting at 94 C 45 s, annealing at 52 C 45 s, and extension at 72 C 1 min with the same oligos. The resultant PCR fragments were 956 and 506 bp for large and short mRNA forms respectively. To verify the presence of the long form of D1 mRNA in each tissue a PCR ampli fi cation was carried out with oligos whose sequence is only present in the 3 -terminal region of the long mRNA form (As and Bas), with a resultant fragment of 136 pb (see Fig. 1). Ampli fi ed fragments were electrophoresed on 2% agarose gel and visualized using ethidium bromide. Data are expressed as the means . . Di erences between experimental groups were analyzed using a one-way ANOVA and Tukey ’ s HSD test. Di ff erences with a P < 0 · 05 were considered statistically signi fi cant. All experiments had parallel controls, which consisted of intact virgin rats and mothers with continuous suckling. Whenever the glands were not resuckled by pups, accumulated milk was removed by administration of OT (30 mU; i.p.) 1 min before they were killed. This procedure ensures that accumulated milk will not act as a dilution factor when tissue proteins are quanti fi ed. Figure 2 shows the changes exhibited by 5 D activity in all groups. Enzymatic values di ff ered between virgin and lactating rats in all tissues analyzed. Lactation was accompanied by signi fi cantly higher 5 D activity values in heart and MG, and lower activity in liver and BAT. After 12 h of non-suckling, the mothers exhibited a signi fi cant ( ] 50%) decrease in heart and MG D1 activity, whereas in liver and BAT the enzymatic activity remained unchanged. Within the initial 12-h non-suckling interval, resuckling (4 h) could restore the enzymatic activities to control values in heart and MG. Adrenergic stimulation exerted a di ff erential e ff ect on 5 D activities. In virgin animals, NE and ISO administration increased heart D1 and BAT D2 activities. In lactating animals, both NE and ISO elicited a signi fi cant 5 D stimulatory response in heart and MG, but not in BAT. In contrast, in neither virgin nor lactating animals were these adrenergic agents able to evoke a detectable response in liver D1 activity. As depicted in Fig. 3 these changes in enzyme activity were parallel with those exhibited by D1 and D2 mRNAs, thus suggesting that these treatments exert their action at transcriptional level. The PCR-RACE method was employed to analyze the 3 UTR D1 mRNA from liver, heart and MG. Figure 4 shows that two fragments were ampli fi ed from heart and liver cDNAs, indicating the presence of two D1 mRNA forms in these tissues. In previous work, we have ampli fi ed and sequenced these fragments and showed that the longer form uses the second polyadenylation site in the 3 UTR region of the mRNA (Navarro et al. 1997). A more intense signal was evident for the short fragment than the larger one, suggesting that the short mRNA is more abundant. Nevertheless, it is di ffi cult to interpret the relative quantities of two isoforms considering that a co-ampli fi cation took place, and the replication e ffi ciency is dependent on the fragment size (Stolovitzky & Cacchi 1996). In the case of MG, only the short fragment was evident in lactating animals. When we corroborated the presence of the long mRNA form using PCR ampli fi cation for the last portion of the 3 UTR, fragments of the expected size were obtained in liver and heart from virgin and lactating animals in all experimental conditions. The quantity of the large fragment was increased in virgin animals treated with adrenergic agonists (NE and ISO), whereas in lactating rats this increase was exhibited in continuous suckling, resuckling and adrenergic stimulation (NE and ISO). In MG, this fragment was ampli fi ed in cDNAs from resuckled and adrenergic-stimulated lactating animals. The present results con fi rm and extend our previous analysis of the thyroid – SNS interaction during lactation. Besides corroborating that lactating rats exhibit an organ- speci fi c thyroid hormone-deiodinative rearrangement that resembles hypothyroidism (Valverde-R & Aceves 1989, Aceves et al. 1994), the present data show for the fi rst time that suckling concurrently stimulates heart and MG D1. This simultaneous activation is mediated by -adrenergic receptors and suggests that this SNS – D1 interaction is part of a physiological mechanism co-ordinating the metabolic demands imposed by milk production. This notion agrees with previous observations showing that (1) the characteristic homeorhetic adjustments of lactation are modulated by the lactational intensity (litter size, number and fre- quency of suckling, etc.) (Prentice & Prentice 1988, Valverde-R & Aceves 1989); (2) mammary D1 is stimulated in vivo by the NE released in the sympathetic nerve endings (Aceves et al. 1999 b ); and (3) in the heart, a classic noradrenergic target tissue, lactation increases the NE turnover rate (Bauman & Currie 1980). Although it is well documented that in several organs D2 is regulated by the SNS (Silva 1996, Kohrle 1996, 1999), in the case of D1 the evidence is scarce and contradictory. The possibility of an important e ff ect of catecholamines on extrathyroidal conversion of T4 to T3 was suggested by clinical studies where -receptor antagonists decreased the plasma concentration of T3 in thyro- toxic or hypothyroid patients maintained on a fi xed dose of T4 (Murchison et al. 1979, Sulkin et al. 1984). Subse- quently, in vitro studies in liver and kidney demonstrated that D1 inhibitory e ff ects of propranolol were related to its lipid solubility and not to its -adrenergic blocking activity (Heyma et al. 1978, Sulkin et al. 1984). Moreover, when kidney preparations were treated with NE and -agonists, the expected stimulatory D1 response did not appear (Heyma et al. 1980). Besides con fi rming the lack of -adrenergic in fl uence upon liver D1, our fi ndings demonstrate that -adrenergic mechanisms do stimulate, in a concerted manner, D1 enzyme in heart and lactating MG. This fi nding indicates that catecholamines need to be added to the ample variety of regulatory in fl uences known to a ff ect ...