Effects of papaverine on carbachol- and high K(+)-induced contraction in the bovine abomasum

Abstract

The effects of papaverine on carbachol (CCh)- and high K(+)- induced contraction in the bovine abomasum were investigated. Papaverine inhibited CCh (1 µM) - and KCl (65 mM)-induced contractions in a concentration-dependent manner. Forskolin or sodium nitroprusside inhibited CCh-induced contractions in a concentration-dependent manner in association with increases in the cAMP or cGMP contents, whereas papaverine increased cGMP contents only at 30 µM. Changes in the extracellular Ca(2+) from 1.5 mM to 7.5 mM reduced verapamil-induced relaxation in high K(+)-depolarized muscles, but papaverine-induced relaxation did not change. Futhermore, papaverine (30 µM) and NaCN (300 µM) decreased the creatine phosphate contents. These results suggest that the relaxing effects of papaverine on the bovine abomasum are mainly due to the inhibition of aerobic energy metabolism.

Full text

FULL PAPER Pharmacology
Effects of papaverine on carbachol- and high K+-induced contraction in the bovine
abomasum
Takeharu KANEDA1)*, Erika SAITO1,2), Hidenori KANDA1), Norimoto URAKAWA1) and Kazumasa SHIMIZU1)
1)Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, Nippon Veterinary and Life Science University, 7–1 Kyonan-cho
1-chome, Musashino, Tokyo 180–8602, Japan
2)School of Veterinary Nursing and Technology, Nippon Veterinary and Life Science University, 7–1 Kyonan-cho 1-chome, Musashino,
Tokyo 180–8602, Japan
(Received 5 February 2015/Accepted 15 May 2015/Published online in J-STAGE 28 May 2015)
ABSTRACT. The effects of papaverine on carbachol (CCh) -and high K+- induced contraction in the bovine abomasum were investigated.
Papaverine inhibited CCh (1 µM) -and KCl (65 mM) -induced contractions in a concentration-dependent manner. Forskolin or sodium
nitroprusside inhibited CCh-induced contractions in a concentration-dependent manner in association with increases in the cAMP or cGMP
contents, whereas papaverine increased cGMP contents only at 30 µM. Changes in the extracellular Ca2+ from 1.5 mM to 7.5 mM reduced
verapamil-induced relaxation in high K+-depolarized muscles, but papaverine-induced relaxation did not change. Futhermore, papaverine
(30 µM) and NaCN (300 µM) decreased the creatine phosphate contents. These results suggest that the relaxing effects of papaverine on the
bovine abomasum are mainly due to the inhibition of aerobic energy metabolism.
KEY WORDS: abomasum, cAMP, cGMP, creatine phosphate, papaverine
doi: 10 .1292/jvm s.15- 0 080; J. Vet. Med. Sci. 77(10): 12 69 –1274, 2015
Based on the electrophysiological and mechanical behav-
iors, smooth muscles are classied as either phasic or tonic
muscles [12, 19, 30]. Phasic smooth muscles are electrically
quiescent into the resting condition and reveal spontaneous
electrical spikes. Depolarization with high K+ induces their
initial phasic contraction, which is then followed by a de-
cline to a low steady-state level of spike activity [11]. Phasic
muscles include the ileum, urinary bladder, uterus and vas
deferens. In contrast, tonic smooth muscles show depolar-
ization with or without low-level continuous spike activities.
Their high K+-induced depolarization typically evokes a
slowly developing sustained contraction as found in tonic
muscles, such as the aorta and trachea. The contractile dif-
ferences among different smooth muscles appear to be at-
tributed to various pathways found in oxidative metabolism
[7, 15], as well as differences in the protein expression [1,
2, 9, 20, 24, 31] and electrophysiological responses [6, 37].
The relaxing mechanism of papaverine, a non-selective
smooth muscle relaxant, has been previously explained
as the follows: 1) it inhibits phosphodiesterase (PDE) and
results in an intracellular accumulation of cAMP and/or
cGMP [22, 25, 32], 2) it inhibits mitochondrial respiration
[26, 27, 33–35] and 3) it results in the effects on Ca2+ move-
ment [13]. Although these different mechanisms have been
proposed by respected researchers, there were no previous
studies comparing and evaluating these three mechanisms
in various smooth muscles. We have recently suggested that
papaverine inhibited smooth muscle contraction primarily
through the accumulation of cAMP and/or cGMP by inhibit-
ing PDE in tonic muscles, such as rat aorta [16] and bovine
trachea [17]. However, we found that papaverine worked to
inhibit mitochondrial respiration in phasic muscles, such as
guinea pig ileum [16], urinary bladder [28] and rat uterine
[29]. These studies provided the rst evidence that relaxation
mechanisms of papaverine differ in phasic and tonic smooth
muscles. In cattle, the function of abomasums, but not fore-
stomach, is similar to that of the stomach of non-ruminant.
Motility of the gastrointestinal (GI) tract, which includes
abomasums in cattle, is controlled by the autonomic nervous
system [4, 10]. With the exception of sphincter muscles, the
motility of the GI tract is stimulated by vagal cholinergic
nerves and depressed by adrenergic nerves in mammalians.
However, it has been reported that the expression of adren-
ergic receptors mRNA in bovine GI tract is low [23]. On the
other hand, the presence of nitrergic neurons in bovine GI
tract has been shown by histochemical and immunouores-
cence analyses [36]. However, to our knowledge, there are
few reports investigating the effects of adrenergic agonists
and/or nitrergic agents on the motility of bovine abomasums.
Displacement of the abomasum is a common disease in dairy
cattle, and one possible cause has been reported [8]. When a
cow is in diagnosed with abomasum displacement, the tonus
of the abomasum decreases. Therefore, examining the relax-
ing mechanisms of smooth muscle in the abomasum is im-
portant for understanding the pathophysiology of abomasum
displacement. We applied papaverine, and the inhibitory
responses are evaluated to clarify the inhibitory mechanisms
in the abomasum.
In the present study, we characterized the inhibitory
*CorrespondenCe to: Kaneda, T., Laboratory of Veterinary Phar-
macology, School of Veterinary Medicine, Nippon Veterinary and
Life Science University, 7–1 Kyonan-cho 1-chome, Musashino,
Tokyo 180–8602, Japan. e-mail: t-kaneda@nvlu.ac.jp
©2015 The Japanese Society of Veterinary Science
This is an open-access article distributed under the terms of the Creative
Commons Attribution Non-Commercial No Derivatives (by-nc-nd)
License <http://creativecommons.org/licenses/by-nc-nd/3.0/>.

T. K ANEDA ET A L.
1270
mechanisms of papaverine in the bovine abomasum (phasic
muscle). This investigation was performed by measuring
muscle tension and levels of cAMP, cGMP, phosphocreatine
(PCr) and adenosine triphosphate (ATP) contents in the bo-
vine abomasum.
MATERIALS AND METHODS
Muscle preparations and tension measurement: Aboma-
sums from adult Japanese Black cattle of either sex were
obtained from a local abattoir. The mucosal layers were
removed by cutting with ne scissors, and smooth muscle
tissues were isolated from the fundic region. The circular
muscle strips were incubated with physiological salt solution
(PSS) containing (in mM) 136.8 NaCl, 5.4 KCl, 1.5 CaCl2,
1.0 MgCl2, 23.8 NaHCO3 and 5.6 glucose. PSS was aerated
with 95% O2 and 5% CO2 to adjust the pH to 7.4 at 37°C. In
some experiments, PSS included 7.5 mM CaCl2. Muscle ten-
sion was recorded isometrically. One end of each strip was
bound to a glass holder, and the other end was connected by
a silk thread to a strain-gauge transducer (TB-611T; Nihon
Kohden, Tokyo, Japan) in an organ bath containing PSS with
a resting tension of 2g. The muscle strips were equilibrated
for 30 min to obtain a stable contractility induced by hyper-
osmotically added 65 mM KCl (high K+). When the contrac-
tile response induced by 1 µM carbachol (CCh) or high K+
reached a steady level about 15–20 min after addition to a
muscle strip, papaverine, forskolin, nitroprusside, verapamil
or NaCN was added cumulatively.
Assay of cAMP or cGMP content: The cAMP or cGMP
content in the muscle strips was measured by enzyme im-
munoassay. After incubation of the strips with papaverine,
forskolin or sodium nitroprusside for 10 min in the presence
of CCh (1 µM), the strips were rapidly frozen in liquid ni-
trogen and stored at −80°C until homogenized in 6% trichlo-
roacetic acid (0.4 ml). The homogenate was centrifuged at
3,000 × g for 15 min, and the supernatant was washed with
1.5 ml of water-saturated dietylether four times; the cGMP
or cAMP content of the strips was assayed using an enzyme
immunoassay kit (GE Healthcare, Buckinghamshire, U.K.).
The cAMP or cGMP content was expressed in picomole per
gram wet weight of the tissue.
Assay of PCr and ATP: PCr and ATP contents in muscle
strips were measured by high-performance liquid chroma-
tography (HPLC), as reported previously [17]. After the
muscles were electrically stimulated for 5 min, papaverine
or sodium cyanide (NaCN) was added to the organ bath for
20 min. After incubation, the muscles were rapidly frozen
in liquid nitrogen and stored at −80°C until homogenization
in 0.3 ml of 9% perchloric acid. The homogenate was cen-
trifuged at 15,000 × g for 5 min, and then, the supernatant
was neutralized with 0.25 ml of 2 M KHCO3. Neutralized
extracts were spun once more, and 20 µl of supernatant ap-
plied to HPLC.
The HPLC system (Shimadzu, Kyoto, Japan) comprised a
pump (LC-10AT), system controller (SCL-10A), auto injec-
tor (SIL-10AF), column oven (CTO-10A) and wavelength-
selectable detector (SPD-10Ai) set at 216 nm.
Chromatography was performed using a µRPC C2/C18
ST system (4.6-mm internal diameter and 100-mm length;
GE Healthcare) with mobile phase solutions of 50 mM
KH2PO4, and 5 mM terabutylammonium hydrogen sulfate
(TBAHS) (pH 6.0, buffer A), and 50 mM KH2PO4, 5 mM
TBAHS and 40% methanol (pH 6.0, buffer B). The ow rate
was 1.0 ml/min, and the elution started with 65% buffer A.
In the rst 14 min, buffer B increased at a rate of 2.5%/min.
This was followed by elution with 70% buffer B for 20 min,
then with 100% buffer A for 10 min. These procedures were
programmed using a system controller. The sensitivity of the
detector was usually set at 1.0 absorbance units full scale,
and the oven temperature was set at 40°C. PCr and ATP con-
tents are expressed as micromoles per gram of wet weight.
Chemicals: Chemicals used were papaverine, forskolin,
sodium nitroprusside, verapamil, carbachol (Sigma-Aldrich,
St. Louis, MO, U.S.A.) and NaCN (Wako Pure Chemical,
Osaka, Japan). Stock solution of forskolin was prepared in
ethanol, and all other drugs were prepared in distilled deion-
ized water.
Statistics: The values are expressed as mean ± SEM, and
the IC50 values (concentration producing 50% relaxation)
were determined by linear regression analysis. Statistical
analyses were performed by the Student’s t-test. Statistical
signicance was established at P-values lower than 0.05.
These calculations and statistical analysis were performed
using GraphPad Prism4 and Excel 2010 for Windows.
RESULTS
Effects of papaverine, forskolin or sodium nitroprus-
side on the CCh- or high K+-induced contraction: When a
contractile response induced by 65 mM KCl (high K+) or 1
µM CCh reached a steady level about 15–20 min after ap-
plication, papaverine (1–100 µM) was added cumulatively.
Papaverine inhibited the CCh- or high K+-induced contrac-
tion in a concentration-dependent manner (Fig. 1A).
Forskolin (0.1–100µM), an adenylyl cyclase activator,
or sodium nitroprusside (SNP), a soluble guanylyl cyclase
activator (0.003–30 µM), also inhibited the CCh- or high
K+-induced contraction in a concentration-dependent man-
ner (Fig. 1B and C). The values for IC50 and the maximum
relaxation for these agents of CCh- or high K+- induced
contraction are presented in Table 1.
Effects of Papaverine, forskolin and SNP on cAMP and
cGMP contents: In the presence of CCh (1 µM), forskolin (3
and 10 µM) increased the cAMP contents in a concentration-
dependent manner, but papaverine (10 and 30 µM) did not
change cAMP contents (Fig. 2A). By contrast, SNP (1 and 10
µM) increased cGMP contents in a concentration-dependent
manner, but only the higher concentration of papaverine (30
µM) increased the cGMP contents, and this dose induced
maximum relaxation (Fig. 2B).
Effects of extracellular calcium on papaverine- and
verapamil-induced inhibitions of high K+-induced contrac-
tion: Verapamil (0.01–30 µM) inhibited high K+-induced
contraction in a concentration-dependent manner. Increases
of extracellular Ca2+ from 1.5 to 7.5 mM recovered the inhi-

EFFECTS OF PAPAVERINE IN BOVI NE ABOMASUM 1271
bition of high K+-induced contraction caused by verapamil
(Fig. 3B), but the same increases in extracellular Ca2+ did
not recover the inhibition caused by papaverine (Fig. 3A).
Effects of papaverine and NaCN on a muscle contraction,
and PCr and ATP contents: Figure 4A shows the effects
of NaCN on CCh- or high K+-induced contraction. NaCN
(10–1,000 µM) inhibited CCh- or high K+-induced contrac-
tion in a concentration-dependent manner.
Addition of papaverine (30 µM) and NaCN (300 µM),
which showed maximum or sub-maximum inhibition of
CCh-induced contraction, signicantly decreased PCr, but
not ATP contents in abomasum (Fig. 4B).
DISCUSSION
Based on the following observations, we have concluded
that the inhibitory mechanisms of papaverine on CCh- or
high K+-induced contraction in the bovine abomasum may
be due to the inhibition of mitochondrial respiration. 1) In
the presence of CCh, papaverine increased cGMP content
only at 30 µM and did not alter cAMP content. 2) Increases
in extracellular Ca2+ from 1.5 to 7.5 mM attenuated the inhi-
bition of high K+-induced contraction caused by verapamil,
but not that caused by papaverine. 3) Papaverine and NaCN
decreased PCr contents in the bovine abomasum.
PDEs are currently classied into 11 families [3], and vari-
ous selective PDE inhibitors have been found [5]. In our pre-
vious study, vardenal (a selective PDE 5 inhibitor) induced
relaxation in association with the increases in the cGMP
content and was the most potent relaxing agent involved in
inhibition of PDE5 [18]. This suggests that PDE 5 functions
to hydrolyze cGMP in bovine abomasum. As reported previ-
ously, papaverine-induced relaxation was associated with
increases in cAMP and/or cGMP in rat aorta [16] and bovine
trachea [17]. In the present study, papaverine increased
cGMP only at 30 µM, which induced maximum relaxation
and did not affect cAMP content. Moreover, the papaverine-
induced increases of cGMP contents were smaller than SNP-
induced increases of cGMP contents. These results suggest
that relaxing mechanism of papaverine may not involve with
accumulation of cGMP due to the inhibition of PDE in the
bovine abomasum.
Fig. 1. Effects of papaverine, forskolin and sodium nitroprus-
side (SNP) on the contraction induced by 1 µM carbachol
(CCh) and 65 mM KCl (high K+) in bovine abomasum.
Preparations were precontracted with CCh or high K+, and
then, the specied agents were added to the bath solution.
The maximum contractions induced by high K+ and CCh
in the absence of these agents were taken as 100%. Each
point represents the mean of 4–7 preparations. Vertical bars
indicate SEM.
Table 1. IC50 and maximum relaxation values for papaverine, forskolin, sodium nitroprusside
and NaCN in the bovine abomasum treated with 1 µM carbachol or 65 mM KCl
Agent Stimulant IC50 (µM) Maximum relaxation
(%) n
Papaverine 1 µM carbachol 8.1 (7.0–9.1) 81.4 ± 9.2 7
65 mM KCl 16.5 (13.6–20.0) 73.0 ± 8.2 6
Forskolin 1 µM carbachol 12.9 (10–15.8) 88.6 ± 1.9 4
65 mM KCl >100 20.8 ± 11.2 6
Nitroprusside 1 µM carbachol 0.3 (0.2–0.5) 94.4 ± 5.6 6
65 mM KCl >30 36.5 ± 3.5 7
NaCN 1 µM carbachol 150.0 (121.3–190.1) 81.7 ± 6.1 4
65 mM KCl >1,000 46.7 ± 7.0 6
Numbers in parentheses indicate 95% condence interval. The maximum relaxation represents the
resting tension after washing and was considered to be 100%.

T. K ANEDA ET A L.
1272
In guinea pig trachea, papaverine has been shown to in-
hibit a Ba2+ inward current in a manner independent of the
intracellular cAMP levels [13]. This suggests that papaverine
directly inhibits Ca2+ inux. In the present study, verapamil
inhibited high K+-induced contraction in the abomasum,
and increasing levels of extracellular Ca2+ from 1.5 to 7.5
mM attenuated the inhibition of the contraction caused by
verapamil. However, increases in extracellular Ca2+ did not
affect the inhibition of high K+-induced contraction by pa-
paverine. These results suggest that relaxation of papaverine
is not related to blockade of Ca2+ movement.
Some papers reported the possibility that papaverine is
involved in the inhibition of mitochondrial respiration [26,
27, 33–35]. Tsuda et al. [33–35] showed that papaverine
inhibited high K+-induced contraction and O2 consumption
in guinea pig taenia coli. They further found that papaverine
inhibited mitochondrial respiration by blocking the trans-
duction of an electron between NADH and coenzyme Q and
by inhibiting NADH, NADHP-diaphorase. Moreover, it has
been thought that PCr / creatine kinase system plays a role
in the transport of high energy phosphates from the mito-
chondrial compartment to the sites of energy utilization, cor-
relating with oxidative metabolism in mammalian smooth
muscle [15, 21]. Ishida and Takagi [14] demonstrated that
papaverine decreased the content of PCr and ATP in guinea
pig taenia coli in a concentration-dependent manner. In the
present study, NaCN and papaverine inhibited CCh- and high
K+-induced contraction with similar potency, and papaverine
and NaCN decreased PCr contents in the abomasums. On
the other hand, the effects of forskolin and sodium nitroprus-
side were more potently CCh-induced contraction than the
high K+-induced contraction. These data indicate that the
inhibitory mechanisms of papaverine on both CCh- and high
K+-induced contraction in bovine abomasum are probably
similar as that in other muscles studied [28, 29]. However,
in our previous study, papaverine-induced relaxation in the
bovine trachea was not related to changes of PCr contents
[17]. These studies suggest that the relaxing mechanism of
papaverine on CCh- and high K+-induced contraction in
the bovine abomasum is closely related to the inhibition of
mitochondrial respiration in guinea pig taenia coli, but not
Fig. 2. The effects of papaverine and forskolin on the cAMP (A)
or papaverine and SNP on the cGMP (B) contents of the bovine
abomasum. Preparations were precontracted with 1 µM CCh and
then treated with the specied agents for 10 min. The control was
treated with vehicle instead of these agents. Each point represents
the mean of 4 experiments. Vertical bars indicate the SEM. * and
**: Signicant difference from each respective control, with P<0.05
and P<0.01, respectively.
Fig. 3. The effects of extracellular Ca2+ on papaverine- or
verapamil-induced inhibition of high K+-enhanced contractions.
Effects of papaverine (A) and verapamil (B) on contraction in-
duced by high K+ in abomasum in PSS including 1.5 or 7.5 mM
CaCl2. Ordinate: Contractions induced by high K+ just before the
application of each agent were considered as 100%. Each point
represents the mean of 4–8 preparations. Vertical bars indicate
SEM. **P<0.01 versus respective controls.

EFFECTS OF PAPAVERINE IN BOVI NE ABOMASUM 1273
bovine trachea.
In many cases, abomasal displacement occurs in the early
postpartum period. In the abdominal cavity, the increased
uterine volume caused by pregnancy may press on nearby
abdominal organs, such as the abomasum, and induce isch-
emic state, resulting in atony. However, we have not any data
that indicate an association between the abdominal hypoxia
in bovine abomasum. Further studies are needed to clarify
the potential involvement of abdominal hypoxia in bovine
displaced abomasum.
In conclusion, it is suggested that papaverine inhibited
CCh- and high K+-induced muscle contraction primarily via
the inhibition of mitochondrial respiration in bovine aboma-
sum which was classied as a phasic muscle.
ACKNOWLEDGMENT. This work was supported in part
by the Academic Frontier Project for Private Universities
of the Ministry of Education, Culture, Sports, Science and
Technology of Japan (2005–2009).
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