Effects of heptanol and carbenoxolone on noradrenaline induced
contractions in guinea pig vas deferens
D. Palani, P. Ghildyal, Rohit Manchanda ⁎
Biomedical Engineering Group, School of Biosciences and Bioengineering, Indian Institute of Technology-Bombay, Mumbai-400076, India
Abstract
We examined the effects of two putative gap junction blockers, heptanol and carbenoxolone, on noradrenaline-induced contractions in
guinea pig vas deferens. The force generated due to the exogenously added noradrenaline (20 μM) consisted of two components: the tonic
and the oscillatory. 2 mM heptanol abolished the oscillatory contractions and drastically suppressed both the maximum force (by 85.4 ±
18.2%) as well as the tonic component (by 28.8 ± 5.1%) (P b 0.01, n = 7). However, the effects of carbenoxolone (50 μM) were strikingly
different, with the spikes of the oscillatory component being merged into a steady, “fused” contraction, without affecting the maximum force
developed. The L-type Ca2+ channel blocker nifedipine (2 μM) abolished the oscillatory component of the contractions and significantly
reduced the maximum force and tonic component (by 82.4 ± 6.8% and 19.7 ± 6.4% respectively; P b 0.01, n = 4), in a manner similar to that
elicited by heptanol. Our results indicate that (i) while carbenoxolone specifically blocks gap junctions, heptanol appears to exert its actions
through non-gap junctional mechanisms, possibly by blocking VGCCs in smooth muscle; (ii) gap junctions play a significant modulatory
role in the generation of noradrenaline-induced contractions in guinea pig vas deferens, particularly in the emergence of oscillatory
contractions, while the maximum force developed may be independent of gap junctional contribution.
Keywords: Noradrenaline; Guineapig vas deferens; Gap junction; Heptanol; Carbenoxolone
1. Introduction
Gap junctions, which are thought to mediate cell-to-cell
coupling in smooth muscles, have been suggested to be
responsible for the synchronization of the final physiological
response of the muscles, i.e. the contractions, possibly via
synchronization of secondary messengers spread, such as
propagating Ca2+ waves across smooth muscle cells (Hennig
et al., 2002; Haefliger et al., 2004). However, the precise
contribution of gap junctions to smooth muscle contractility
remains poorly understood. Using presumptive gap junction
blockers such as heptanol (Tsai et al., 1995) the role of gap
junctions in coordinating neurogenic contractions of rodent
vas deferens has received some attention in the past, since
these contractions have been pharmacologically well characterized (Venkateswarlu et al., 1999; Palani and Manchanda,
2006). However, these studies have yielded equivocal
findings, primarily because it is not possible to completely
exclude presynaptic mechanisms operating during neurotransmission, on which the putative gap junction blockers
may exert non-selective actions. In this respect, contractions
induced by exogenously added agonists can help clarify some
of these issues since the involvement of prejunctional
mechanisms is greatly reduced or eliminated.
In the guinea pig vas deferens, exogenously added noradrenaline has been shown to evoke contractions with two
distinct components — the tonic and oscillatory components (Tsunobuchi and Gomi, 1990; Kato et al., 1995). The
tonic component has been shown to be primarily dependent on Ca 2+ release from intracellular stores (Tsunobuchi
and Gomi, 1990), whereas the oscillatory component of
the calcium waves reportedly relies on entry of extracellular
57
Ca2+ and on calcium-induced calcium release (CICR)
(Lopez-Lopez et al., 1994; Sham et al., 1995). Although
these Ca2+ mediated mechanisms have been delineated, the
role of gap junctions in the generation of the two components of noradrenaline-induced contractions has not yet
been explored.
In this study, we have investigated the role of gap junctions
in determining the contractile response of the guinea pig
vas deferens (GPVD) to exogenously added noradrenaline
by examining the effects of the two putative gap junction
blockers that are widely used to address the question of
syncytial function, in both smooth and cardiac muscle (Sell
et al., 2002; Chen et al., 2005; Fanchaouy et al., 2005) i.e.
1-heptanol and carbenoxolone. While both heptanol and
carbenoxolone have been reported to be relatively specific
gap junction blockers (Pietro et al., 2004; Matsue et al.,
2006; Palani et al., 2006; Perez Velazquez et al., 2006),
others have reported non-selective actions of these agents.
One of these is that they may modulate the activity of voltage
gated Ca2+ channels (VGCCs) (Verrecchia and Herve, 1997).
We have therefore compared the effects of these compounds
with the effect of nifedipine, an established L-type Ca2+
channel blocker, to evaluate their effects as against those
resulting from VGCC block.
Comparison of our findings with heptanol and carbenoxolone in GPVD suggests that of these two agents, heptanol
may have significant L-type Ca2+ channel blocking effects
under the conditions of our experiments, while carbenoxolone
may be a more specific gap junction blocker. Evaluation of the
effects of carbenoxolone implies that gap junctional communication may be critical for development of oscillatory
contractions but may not effect maximum force developed
which may be determined principally by the direct action of the
agonist on the cells independent of gap junctions.
glucose 11.1) equilibrated with carbogen (95% O2, 5% CO2) to
maintain the pH at 7.4–7.5. The tissue was allowed to rest for
45 min in the organ bath at a resting tension of 2 mN and all the
experiments are conducted at room temperature (25–30 °C).
The output of the strain gauge force transducer was fed to a DC
amplifier (Recorders and Medicare Systems, Chandigarh,
India). Signal was displayed on a digital storage oscilloscope
(TDS 210, Tektronix, USA) and simultaneously recorded on a
digital tape recorder (DTR-1204, Biologic, France). The data
were later digitized and collected on a PC using a National
Instruments, AT-MIO-16XE data acquisition card and analyzed using the software Whole Cell Program (WCP, courtesy
Dr. J. Dempster, and Stracthclyde University, Scotland).
20 μM noradrenaline was used to elicit contraction in the
GPVD. The total volume of noradrenaline stock solution
added to the organ bath was less than 10 μl, so as to affect the
composition of the Krebs solution minimally. Noradrenaline
was directly added to the organ bath and it was left in contact
with the tissue for 2 min before washing out. During this
period flow of Krebs solution was stopped. The tissue was
allowed to recover for 30 min between successive periods of
stimulations. Heptanol and carbenoxolone solutions were
prepared in Krebs solution before every experiment and were
introduced by replacing the normal Krebs solution in the
organ bath. To study the effect of heptanol, carbenoxolone
and nifedipine, tissue was incubated with these chemicals for
5 min, 30 min and 25 min, respectively. Experiments with
nifedipine were conducted under semi dark conditions.
Exogenously added noradrenaline has been shown to
induce release of ATP from non-neuronal sites (Katsuragi
et al., 1991), which could activate post-junctional purinergic
P2X receptors. To eliminate the possibility of a contribution
from purinergic mechanisms, all experiments were conducted in presence of 300 μM suramin, a purinergic blocker
(Nicholas et al., 1992; Bültmann and Starke, 2001).
2. Methods
2.3. Chemicals
2.1. Animals
Male Dunkin–Hartley guinea pigs (350–500 gm),
obtained from the National Institute of Nutrition, Hyderabad,
were stunned, and exsanguinated and the vasa differentia
were carefully removed and cleared of the surrounding
connective tissue. The experimental procedures used in this
study were approved by Animal Experimentation Ethics
Committee of the Indian Institute of Technology — Bombay.
2.2. Contraction studies
The isolated vas was mounted vertically in an organ bath
(volume 25 ml). The prostatic end of the vas was tied to an
isometric force transducer and the epididymal end was affixed
firmly to the bottom of the organ bath using surgical sutures.
The tissue was continuously superfused with physiological
Kreb's solution (composition in mM: NaCl 118.4, KCl 4.7,
MgCl2 1.2, CaCl2 2.5, NaHCO3 25.0, NaH2PO4 0.4 and
Noradrenaline (Arterenol Bitartarate), carbenoxolone and
nifedipine were obtained from Sigma-Aldrich, Mumbai,
India. Suramin was obtained from Tocris-Cookson Ltd., UK.
Stock solution for noradrenaline was prepared in 100 mM
ascorbic acid solution. Salts used for the Krebs solution were
obtained from S.D. Fine Chemicals Ltd., Mumbai, India.
2.4. Statistics
All values have been indicated as mean ± S.D. The
Student's t-test was used to determine statistical significance
between groups of data, with P b 0.01 being taken to indicate
a statistically significant difference.
3. Results
The effects of 1-heptanol (0.05–2 mM) and carbenoxolone
(10–200 μM) on contractions induced by 20 μM noradrenaline
58
3.1. Effect of carbenoxolone on noradrenaline-induced
contraction
Fig. 1. Contractile response of guinea pig vas deferens to exogenously added
20 μM noradrenaline in presence of carbenoxolone (A) and heptanol (B).
The concentration of carbenoxolone or heptanol added is indicated
alongside each trace. The total force was measured from the resting tension
to peak of the contraction as shown in B: (control). The amplitude of the
tonic component was measured as the height between the resting tension and
the base of the overlying oscillations, as shown in A: control. The average
peak value of the spikes in the oscillatory component was taken as the height
of the oscillatory component. Duration of noradrenaline incubation is
indicated at the bottom by dashed horizontal line. The time to reach peak of
the tonic component is delayed in presence of carbenoxolone which was
marked with 7 in both control and carbenoxolone traces.
At 10 to 200 μM, concentrations, carbenoxolone radically
altered the profile of noradrenaline-induced contractions in
GPVD (Fig. 1A) (n = 7). While the spikes in the oscillatory
component were replaced by a fused contraction at all
concentrations tested.
The maximum force generated in the presence of 200 μM
carbenoxolone was not significantly different from that
observed in control (94.2 ± 8.2% of control, P N 0.05, n = 7)
(Fig. 1A). At these different concentrations of carbenoxolone, there was no significant difference in the magnitude of
change observed in the noradrenaline-induced contractions.
For example, maximum force observed at 200 μM carbenoxolone was not significantly different from that observed
at 10 μM (P b 0.05, n = 7), indicating that 10 μM carbenoxolone also exerted the maximum effect on the contractions.
In previous studies, while exploring this range (10–200 μM)
of concentrations, we found that at concentration 10 μM and
below, carbenoxolone did not have any significant action on
the input resistance (Rin) of the cells in GPVD (Palani et al.,
2006). Our studies with carbenoxolone were therefore
conducted at concentrations between 10 and 200 μM.
The amplitude of the tonic component appeared to be
increased to 248.7 ± 40.3% of control (P b 0.01, n = 7, Fig. 1A).
However, as discussed earlier, this increase in the amplitude of
the tonic component was not significantly different from that
of the amplitude of the oscillatory component indicating that
this rise could be due to the smoothening and merger of the
oscillatory components with tonic component. Furthermore,
the development of tonic component, which reached the
peak along with the first group oscillatory component within
30–40 s in control contractions was drastically delayed and
was found to peak after 60–70 s (see 7, Fig. 1A).
in GPVD were investigated. We have restricted our study to
the initial components of the noradrenaline-induced contraction (over a time course b 2 min), which would facilitate
analysis of the dynamics of cell recruitment. Moreover, our
period of observation is adequate for the full development of
both tonic and oscillatory components of the contraction,
which can therefore be subjected to analysis in equal detail.
In all tissues examined (n = 7), the initial phase (0–2 min) of
the noradrenaline-induced contraction consisted of two
distinct components (Tsunobuchi and Gomi, 1990; Kato
et al., 1995), a slower tonic component and a component
consisting of rapid rhythmic contractions (the oscillatory
component) overlaid on the former (Fig. 1A, control). As
pharmacologically distinct mechanisms are involved in the
generation of tonic and oscillatory components of noradrenaline-induced contractions in GPVD (Kato et al., 1995), we
have analyzed the effects of heptanol and carbenoxolone on
these components separately along with their effect on the
maximum force developed (see Fig. 1B (control)).
Fig. 2. Effect of increasing concentrations of heptanol on maximum force,
tonic and oscillatory components of the noradrenaline-induced contractions.
Maximum force was monotonically reduced with increasing concentrations
of heptanol. Oscillatory component was abolished in presence of N900 μM
of heptanol.
59
3.2. Effect of heptanol on noradrenaline-induced contraction
Both the amplitude of spikes in the oscillatory component and their number were also reduced monotonically
with increasing concentrations of heptanol. Heptanol at
concentrations ≥ 900 μM completely abolished the oscillatory component. In contrast to carbenoxolone, lower
concentrations of heptanol (100 μM) significantly increased
oscillatory spike amplitude (Fig. 2) (P b 0.01, n = 7).
Heptanol reduced the maximum force in a concentration
dependent manner (Fig. 1B). Fig. 2 shows the concentration
dependent effect of heptanol on maximum force and
components of noradrenaline-induced contractions. With
2 min of noradrenaline incubation, 2 mM heptanol exerted
maximum reduction on both maximum force and tonic
component, the former (85.4 ± 18.2%) being much higher
than the later (28.8 ± 5.1%) (n = 7) (Fig. 2). Unlike in control
contraction, in the presence of heptanol (N300 μM) the tonic
component did not reach steady-state within the period of
observation, but continued to rise at a slower rate (Fig. 1B).
Since the effects of carbenoxolone and heptanol were
markedly different, it would appear that at least one of these
compounds may exert effects unrelated to its putative gap
junctional blocking actions. One of the principle non-selective
action reported for carbenoxolone and heptanol is their
modulatory action on VGCCs (in retina: Vessey et al., 2004;
in skeletal muscle: Squecco et al., 2004). To see if either of
these compound was affecting VGCCs in GPVD we have
compared their effect with those effects of nifedipine, a
specific blocker of VGCC involved in the generation of
noradrenaline-induced contraction i.e., L-type Ca2+ channels.
3.3. Effect of nifedipine (2 μM) on noradrenaline-induced
contraction in GPVD
Nifedipine reduced the maximum force elicited by
noradrenaline, by 82.4 ± 6.8% (Fig. 3) and the tonic component by 19.7 ± 6.4% (P b 0.01, n = 4), (Kato et al., 2000).
The reduction observed with nifedipine in maximum force
and the tonic component was not significantly different
from the reduction observed with 2 mM heptanol (P N 0.05,
n = 4). Similarly as observed in presence of heptanol
(N900 μM), nifedipine completely abolished the oscillatory
component. Also rate of rise of the tonic component was
slowed down in presence of nifedipine in a similar manner to
that of 2 mM heptanol. In summary the effect of nifedipine
was not significantly different to the effect of 2 mM heptanol
on noradrenaline-induced contractions (P N 0.05, n = 4) but
was very different to the effect of carbenoxolone on time
course and amplitude of the tonic and maximum force of
noradrenaline-induced contractions (P b 0.01, n = 4).
4. Discussion
The role of gap junctions in modulating physiological
responses of smooth muscles, has been addressed by the
use of chemicals purported to block gap junctions selectively (Nelson and Makielski, 1991). However, several of
the putative gap junction blockers used in the past have
been subsequently reported to possess non-gap junctional
actions as well (Rozental et al., 2001), necessitating caution in the interpretation of the results obtained with these
blockers (Squires et al., 2000; Keevil et al., 2000;
Matchkov et al., 2004). Two putative gap junction blockers
which have been used widely in the assessment of syncytial function are the alkanol, 1-heptanol, and the hydroxysteroid dehydrogenase inhibitor, carbenoxolone. Recent
reports have suggested that carbenoxolone over a concentration range of 10–200 μM blocks gap junctions more
selectively than does heptanol (Palani et al., 2006), which
has been suggested to have non-specific actions on ionic
channels (Margineanu and Klitgaard, 2001; Solomon et al.,
2003). In the present study we find, in the smooth muscle
of GPVD, that the effects of carbenoxolone are indeed
more consistent with gap junction block than are those of
heptanol. A comparison of the effects of these agents on
exogenous noradrenaline-induced contractions thus helps
in evolving a framework for the evaluation of the role of
gap junctions in shaping smooth muscle contractions, as
elaborated below.
4.1. Effects of heptanol
Fig. 3. Effect of nifedipine (2 μM) on noradrenaline-induced contractions in
guinea pig vas deferens. Duration of noradrenaline incubation is indicated at
the bottom by dashed horizontal line. Note similarity to the effect of heptanol
(N900 μM) on noradrenaline-induced contractions in Fig. 1.
We found that the noradrenaline-induced contraction in
GPVD was strikingly suppressed in the presence of heptanol.
At first sight this appears consistent with results of previous
studies which reported inhibition by heptanol of neurogenic
contractions of this organ (Palani and Manchanda, 2006;
Venkateswarlu et al., 1999), an effect interpreted as resulting
from disruption of co-ordinated contractions due to gap
junctional block. However, the findings of electrical studies
(Manchanda and Venkateswarlu, 1999) did not favor an
interpretation of the actions of heptanol in terms of cell-to-cell
uncoupling. In particular, heptanol failed to affect the salient
indices of syncytial coupling, both direct (input resistance and
60
cable potentials), as well as indirect (amplitude distributions
and time courses of spontaneous excitatory junction potentials
(SEJPs)). Indeed in other smooth muscles, heptanol has been
suggested to exert actions in addition to its gap junctional
blocking actions at the concentrations normally employed, eg.
activation of KCa channels and inhibition of Ca2+ channels
(Nelson and Makielski, 1991; Matchkov et al., 2004). The
proportion of these actions related to gap junctional block is
thought to depend on several factors, including the concentration of heptanol employed and the organ and species being
investigated.
In the present study, we found the effect of 2 mM heptanol,
a concentration at which heptanol has been widely reported to
exert selective gap junctional blocking actions, to be notably
similar to that of 2 μM nifedipine. This indicates that the
heptanol-induced changes in the contractions might be more
closely related to its action on L-type Ca2+ channels rather than
on gap junctions. Furthermore in rat vas deferens, where the
adrenergic mechanisms involved are thought to be similar to
those in GPVD, verapamil, another L-type Ca2+ channel
blocker, potentiated the oscillatory components at lower
concentrations than those required for its abolition (Boselli
et al., 1998), a pattern closely similar to that elicited by
heptanol in the present study in GPVD. In other tissues, too,
such as retina (Vessey et al., 2004) and skeletal muscle
(Squecco et al., 2004), there is growing evidence for the block
of L-type Ca2+ channels by heptanol. In conjunction with the
previously reported lack of heptanol's effects on passive
electrical properties of syncytium (Yamamoto et al., 1998;
Manchanda and Venkateswarlu, 1999), these observations
seem to indicate that the inhibitory effect of heptanol on
noradrenaline-induced contractions under the conditions of
our investigation might be primarily due to its inhibition of Ltype Ca2+ channels rather than of gap junctions, although
further investigation would be required to confirm this
possibility. It is conceivable, moreover, that in other smooth
muscle organs and under different conditions of investigation,
heptanol may exert a specific and selective block of gap
junctions, for which convincing evidence has been advanced,
e.g., in vascular smooth muscle (Christ, 1995; Christ et al.,
1999). Our results serve to issue a caveat that the primary effect
of this agent may vary between organs, leading to complications in interpretation, as also suggested by a number of other
studies (Manchanda and Venkateswarlu, 1999; Rozental et al.,
2001; Matchkov et al., 2004).
4.2. Effects of carbenoxolone
Our previous results show that in contrast to heptanol,
carbenoxolone, another putative gap junctional blocker,
increased the Rin of the cells and abolished cable potentials
in GPVD swiftly, markedly and reversibly. This increase in
input resistance and abolition of cable potentials constitutes
direct evidence for the suppression of cell-to-cell coupling
by carbenoxolone in the vas deferens. In view of this, the
effects of carbenoxolone on the profiles of noradrenaline-
induced contractions may more confidently be interpreted in
terms of disruption of cell-to-cell coupling.
4.2.1. Oscillatory component
Several lines of evidence across a range of smooth
muscles suggest that oscillatory contractions are mediated by
oscillatory Ca2+ waves. Propagation these Ca2+ waves are in
turn underpinned by gap junction mediated cell-to-cell
communication. For instance in GPVD, the noradrenalineinduced oscillatory contraction was completely abolished by
an L-type Ca2+ channel blocker (nifedipine) or block of
CICR by ryanodine (Lopez-Lopez et al., 1994; Sham et al.,
1995), indicating that extracellular Ca2+ entry and CICR are
important determinants of the oscillatory contraction (Imaizumi et al., 1999; Miriel et al., 1999; Tammaro et al., 2004).
In addition, imaging studies showed that the spread of CICRmediated Ca2+ oscillations across the tissue depended on gap
junctional communication (Lin et al., 2004). In rat tail artery,
presence of gap junction up-regulator or increased expression
of connexins was accompanied by an increase in the
amplitude of the oscillatory contraction (Tsai et al., 1995;
Slovut et al., 2004), while in murine intestinal smooth muscle,
inhibition of cell-to-cell coupling suppressed the spread of the
Ca2+ waves across the tissue, without affecting the generation
of the Ca2+ wave within individual cells (Sell et al., 2002;
Hennig, 2002). Bearing these findings in mind, we propose
that under the conditions of our study, carbenoxolone may
eliminate oscillatory contractions of the GPVD by blocking
the electrical coupling between the cells thereby eliminate
the Ca2+ oscillations at the cellular level within the tissue.
In this scenario even though CICR (brought about by the
action of noradrenaline) may result in [Ca2+] surge within
individual cells, block of gap junctions (due to the action of
carbenoxolone) would prevent both the electrical coupling
and the spread of Ca2+ within the tissue.
4.2.2. Maximum force
In previous studies, the total force developed due to
incubation of both vascular and non-vascular smooth muscle
with adrenergic agonists has been used as an index of the
ability of the agonist to recruit cells for contraction (Christ,
1995). In those studies, block of gap junctions using
heptanol (which in vascular smooth muscle appears to
have specific gap junction blocking activity: Christ et al.,
1999) reduced the agonist-induced contractions if the
duration of incubation with the agonist was brief
(b 40 min), but this reduction was overcome if agonist
exposure was prolonged (N 40 min). This indicated that with
increased duration of agonist incubation, the agonist in the
presence of gap junctional blocker might be able to act
directly on a volume of cells similar to those activated in the
control contraction, an effect which would overcome the
contraction-reducing effects of gap junctional block. In our
studies, noradrenaline when applied in the presence of
carbenoxolone was able to induce a maximum level of force
similar to control levels within our observation time period
61
of 2 min. Based on these findings, it can be tentatively
inferred that in GPVD (a) direct action of agonists can fully
compensate for the absence of gap junctional spread as in
vascular smooth muscle; (b) direct agonist action is achieved
relatively rapidly (b 2 min), perhaps implying a more rapid
diffusivity of agonist in the GPVD compared with vascular
smooth muscle.
Acknowledgements
We acknowledge the financial support provided by the
Department of Science & Technology (DST), India (SP/SO/
B-11/2000), and by the IIT-Bombay under the Cross
Disciplinary Research Grant.
References
4.2.3. Tonic component
In the presence of carbenoxolone, there is an apparent
increase in the tonic contraction. This is because, as mentioned
earlier, the amplitude of the tonic component was defined as
the amplitude of the “steady” or “baseline” force underlying the
oscillatory component. However, based on our results it is not
possible to assess the effect of carbenoxolone on the tonic
component unambiguously. This is because carbenoxolone also
smoothens out the oscillatory component, leaving steady force
which may reflect primarily the force generated by fusion of
fusion of asynchronous oscillatory contractions. Consequently
the net force in the presence of carbenoxolone consists of the
merged “tonic” and “fused oscillatory” contractions. Based on
present data it is not possible to discriminate the relative
contributions of “tonic” and “fused oscillatory” components
to the peak force generated in the presence of carbenoxolone,
hence the effect of carbenoxolone on the tonic component
alone cannot be stated with any certainty. This questions could
be resolved if a selective elimination could be achieved either of
the oscillatory component (e.g. with ryanodine, since the
oscillatory component is CICR-dependent) or of the tonic
component (e.g. with IP3 pathway blockers), allowing the effect
of carbenoxolone on each component alone to be studied.
However, it is not possible from these present results to
distinguish between the contribution of cell-cell communication between smooth muscle cells and between the smooth
muscle cells and the epithelial cells.
5. Conclusion
In conclusion, our results indicate that of the two
presumptive gap junctional blockers investigated, carbenoxolone appears to be a more specific gap junction blocker in
GPVD compared with heptanol under the conditions of our
experiments. These observations reinforce the concerns over
the non-gap junctional effects of heptanol and provide a
framework for the assessment of the effects of putative gap
junctional blockers. Our findings with carbenoxolone
indicate that in contractions induced by exogenous agonists,
block of cell-to-cell coupling may not affect the maximum
force generated, presumably due to recruitment of smooth
muscle cells direct action of agonist. However, gap junctional
coupling could play a significant modulatory role in shaping
the contractions, e.g. in the emergence of oscillatory
components. Similar modulatory mechanisms might assume
significance in smooth muscle systems where oscillatory
contractions play a dominant role, e.g. in the gastrointestinal
tract, portal vein and uterus.
Boselli, C., Bianchi, L., Barbieri, A., Grana, E., 1998. Effect of calcium
antagonists on the response to noradrenaline in the whole and bisected rat
vas deferens. Journal of Autonomic Pharmacology 18, 297–306.
Bültmann, R., Starke, K., 2001. Nucleotide-evoked relaxation of rat vas
deferens — a possible role for endogenous ATP released upon α1adrenoceptor stimulation. European Journal of Pharmacology 422,
197–202.
Chen, B.P., Mao, H.J., Fan, F.Y., Bruce, I.C., Xia, Q., 2005. Delayed
uncoupling contributes to the protective effect of heptanol against
ischaemia in the rat isolated heart. Clinical and Experimental
Pharmacology and Physiology 32, 655–662.
Christ, G.J., 1995. Modulation of a1-adrenergic contractility in isolated
vascular tissues by heptanol: a functional demonstration of the potential
importance of intercellular communication to vascular response
generation. Life Sciences 56, 709–721.
Christ, G.J., Spektor, M., Brink, P.R., Barr, L., 1999. Further evidence for
the selective disruption of intercellular communication by heptanol.
American Journal of Physiology 276, H1911–H1917.
Fanchaouy, M., Serir, K., Meister, J.J., Beny, J.L., Bychkov, R., 2005.
Intercellular communication: role of gap junctions in establishing the
pattern of ATP-elicited Ca2+ oscillations and Ca2+ -dependent currents in
freshly isolated aortic smooth muscle cells. Cell Calcium 37, 25–34.
Hennig, G.W., Smith, C.B., O'Shea, D.M., Smith, T.K., 2002. Patterns of
intracellular and intercellular Ca2+ waves in the longitudinal muscle layer
of the murine large intestine in vitro. Journal of Physiology 543, 233–253.
Haefliger, J.A., Nicod, P., Meda, P., 2004. Contribution of connexins to the
function of the vascular wall. Cardiovascular Research 1 62 (2), 345–356.
Imaizumi, Y., Ohi, Y., Yamamura, H., Ohya, S., Muraki, K., Watanabe, M.,
1999. Ca2+ spark as a regulator of ion channel activity. Japanese Journal
of Pharmacology 80, 1–8.
Kato, K., Tsutsui, I., Furuya, K., Ozaki, T., Yamagishi, S., 1995. Regional
differences in the contractile and intracellular Ca2+ responses of the
guinea-pig vas deferens to neurotransmitters and excess K+. Experimental Physiology 80, 721–733.
Kato, K., Furuya, K., Tsutsui, I., Ozaki, T., Yamagishi, S., 2000. Cyclic
AMP-mediated inhibition of noradrenaline-induced contraction and
Ca2+ influx in guinea-pig vas deferens. Experimental Physiology 85,
387–398.
Katsuragi, T., Tokunaga, T., Ogawa, S., Soejima, O., Sato, C., Furukawa, T.,
1991. Existence of ATP-evoked ATP release system in smooth muscles.
Journal of Pharmacology and Experimental Therapeutics 259, 513–518.
Keevil, V.L., Huang, C.L., Chau, P.L., Sayeed, R.A., Vandenberg, J.I., 2000.
The effect of heptanol on the electrical and contractile function of the
isolated, perfused rabbit heart. Pflugers Achieves 440, 275–282.
Lin, G.C., Rurangirwa, J.K., Koval, M., Steinberg, T.H., 2004. Gap
junctional communication modulates agonist-induced calcium oscillations in transfected HeLa cells. Journal of Cell Sciences 117, 881–887.
Lopez-Lopez, J.R., Shacklock, P.S., Balke, C.W., Wier, W.G., 1994. Local,
stochastic release of Ca2+ in voltage-clamped rat heart cells: visualization with confocal microscopy. Journal of Physiology 480, 21–29.
Manchanda, R., Venkateswarlu, K., 1999. Quantal evoked depolarizations
underlying the excitatory junction potential of the guinea-pig isolated
vas deferens. Journal of Physiology 520, 527–537.
Margineanu, D.G., Klitgaard, H., 2001. Can gap-junction blockade
preferentially inhibit neuronal hypersynchrony vs. excitability? Neuropharmacology 41, 377–383.
62
Matchkov, V.V., Rahman, A., Peng, H., Nilsson, H., Aalkjaer, C., 2004.
Junctional and nonjunctional effects of heptanol and glycyrrhetinic acid
derivates in rat mesenteric small arteries. British Journal of Pharmacology
142, 961–972.
Matsue, H., Yao, J., Matsue, K., Nagasaka, A., Sugiyama, H., Aoki, R.,
Kitamura, M., Shimada, S., 2006. Gap junction-mediated intercellular
communication between dendritic cells (DCs) is required for effective
activation of DCs. Journal of Immunology 176, 181–190.
Miriel, V.A., Mauban, J.R., Blaustein, M.P., Wier, W.G., 1999. Local and
cellular Ca2+ transients in smooth muscle of pressurized rat resistance
arteries during myogenic and agonist stimulation. Journal of Physiology
518, 815–824.
Nelson, W.L., Makielski, J.C., 1991. Block of sodium current by heptanol in
voltage-clamped canine cardiac Purkinje cells. Circulation Research 68,
977–983.
Nicholas, M., Roger, M., Andrew, S., Brian, S., 1992. Suramin: a selective
inhibitor of purinergic neurotransmission in the rat isolated vas deferens.
European Journal of Pharmacology 220, 1–10.
Palani, D., Manchanda, R., 2006. Effects of heptanol on neurogenic
contractions of vas deferens: a comparative study of stimulation frequency
in guinea pig and rat. Journal of Physiological Sciences 56 (1), 21–28.
Palani, D., Ghildyal, P., Manchanda, R., 2006. Effects of carbenoxolone on
syncytialelectrical properties and junction potentials of guinea-pig vas
deferens. Naunyn-Schmiedeberg's Archives of Pharmacology 374 (3),
207–214.
Perez Velazquez, J.L., Kokarovtseva, L., Sarbaziha, R., Jeyapalan, Z.,
Leshchenko, Y., 2006. Role of gap junctional coupling in astrocytic
networks in the determination of global ischaemia-induced oxidative stress
and hippocampal damage. European Journal of Neuroscience 23, 1–10.
Pietro, G., Daniele, C., Natale, B., Santo, G., Guido, F., Eugenio, D., Angela,
D., Giovambattista, D.S., 2004. Influence of carbenoxolone on the
anticonvulsant efficacy of conventional antiepileptic drugs against
audiogenic seizures in DBA/2 mice. European Journal of Pharmacology
484, 49–56.
Rozental, R., Srinivas, M., Spray, D.C., 2001. How to close a gap junction
channel: efficacies and potencies of uncoupling agents. Methods in
Molecular Biology 154, 447–476.
Sell, M., Boldt, W., Markwardt, F., 2002. Desynchronising effect of the
endothelium on intracellular Ca2+ concentration dynamics in vascular
smooth muscle cells of rat mesenteric arteries. Cell Calcium 32, 105–120.
Sham, J.S., Cleemann, L., Morad, M., 1995. Functional coupling of Ca2+
channels and ryanodine receptors in cardiac myocytes. Proceedings of
the National Academy of Sciences of the U. S. A. 92, 121–125.
Slovut, D.P., Mehta, S.H., Dorrance, A.M., Brosius, F.C., Watts, S.W., Webb,
R.C., 2004. Increased vascular sensitivity and connexin43 expression
after sympathetic denervation. Cardiovascular Research 62, 388–396.
Solomon, I.C., Chon, K.H., Rodriguez, M.N., 2003. Blockade of brain stem
gap junctions increases phrenic burst frequency and reduces phrenic burst
synchronization in adult rat. Journal of Neurophysiology 89, 135–149.
Squecco, R., Bencini, C., Piperio, C., Francini, F., 2004. L-type Ca2+ channel
and ryanodine receptor cross-talk in frog skeletal muscle. Journal of
Physiology 555, 137–152.
Squires, P.E., Hauge-Evans, A.C., Persaud, S.J., Jones, P.M., 2000.
Synchronization of Ca2+ — signals within insulin-secreting pseudoislets: effects of gap-junctional uncouplers. Cell Calcium 27, 287–296.
Tammaro, P., Smith, A.L., Hutchings, S.R., Smirnov, S.V., 2004.
Pharmacological evidence for a key role of voltage-gated K+ channels
in the function of rat aortic smooth muscle cells. British Journal of
Pharmacology 143, 303–317.
Tsai, M.L., Watts, S.W., Loch-Caruso, R., Webb, R.C., 1995. The role of gap
junctional communication in contractile oscillations in arteries from
normotensive and hypertensive rats. Journal of Hypertension 13,
1123–1133.
Tsunobuchi, H., Gomi, Y., 1990. Effects of ryanodine and nifedipine on
contractions induced by norepinephrine, acetylcholine, and potassium in
the isolated vas deferens of the guinea pig. Journal of Pharmacobiodynamics 13, 574–580.
Venkateswarlu, K., Dange, S.Y., Manchanda, R., 1999. Effect of heptanol on
the neurogenic and myogenic contractions of the guinea-pig vas
deferens. British Journal of Pharmacology 126, 227–234.
Verrecchia, F., Herve, J.C., 1997. Reversible inhibition of gap junctional
communication elicited by several classes of lipophilic compounds in
cultured rat cardiomyocytes. Canadian Journal of Cardiology 13,
1093–1100.
Vessey, J.P., Lalonde, M.R., Mizan, H.A., Welch, N.C., Kelly, M.E., Barnes, S.,
2004. Carbenoxolone inhibition of voltage-gated Ca channels and synaptic
transmission in the retina. Journal of Neurophysiology 92, 1252–1256.
Yamamoto, Y., Fukuta, H., Nakahira, Y., Suzuki, H., 1998. Blockade by
18beta-glycyrrhetinic acid of intercellular electrical coupling in guineapig arterioles. Journal of Physiology 1, 511, 501–508.