Department of Physiology, College of Medicine and Public Health, Ohio State University, Columbus, Ohio 43210-1218
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ABSTRACT |
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Starodub, Alexander M. and
Jackie D. Wood.
Histamine H2 Receptor Activated Chloride Conductance
in Myenteric Neurons From Guinea Pig Small Intestine.
J. Neurophysiol. 83: 1809-1816, 2000.
Whole cell
perforated patch-clamp methods were used to investigate ionic
mechanisms underlying histamine-evoked excitatory responses in small
intestinal AH-type myenteric neurons. When physiological concentrations
of Na+, Ca2+, and Cl were in the
bathing medium, application of histamine significantly increased total
conductance as determined by stepping to 50 mV from a holding potential
of
30 mV. The current reversed at a membrane potential of
30 ± 5 (SE) mV and current-voltage relations exhibited outward
rectification. The reversal potential for the histamine-activated
current was unchanged by removal of Na+ and
Ca2+ from the bathing medium. Reduction of Cl
from 155 mM to 55 mM suppressed the current when the neurons were in
solutions with depleted Na+ and Ca2+.
Current-voltage curves in solutions with reduced Cl
were
linear and the reversal potential was changed from
30 ± 5 mV to
7 ± 4 mV. Niflumic acid, but not anthracene-9-carboxylic acid
(9-AC) nor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS),
suppressed the histamine-activated current. A membrane permeable
analogue of cAMP evoked currents similar to those activated by
histamine. A selective histamine H2 receptor agonist
(dimaprit) mimicked the action of histamine and a selective histamine
H2 receptor antagonist (cimetidine) blocked the conductance
increase evoked by histamine. A selective adenosine A1
receptor agonist (CCPA) reduced the histamine-activated current and a
selective adenosine A1 receptor antagonist (CPT) reversed
the inhibitory action. The results suggest that histamine acts at
histamine H2 receptors to increase Cl
conductance in AH-type enteric neurons. Cyclic AMP appears to be a
second messenger in the signal transduction process. Results with a
selective adenosine A1 receptor agonist and antagonist add
to existing evidence for co-coupling of inhibitory adenosine A1 receptors and histamine H2 receptors to
adenylate cyclase in AH-type enteric neurons.
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INTRODUCTION |
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Slow synaptic excitation (sEPSP) is a
receptor-mediated, slowly activating depolarization of the membrane
potential found in enteric neurons in the guinea pig small and large
intestine and in the gastric antrum, but not the gastric corpus. The
depolarizing response is associated with increased input resistance and
enhanced excitability that is reflected by repetitive action potential discharge lasting for several seconds (Wood and Mayer
1978). Its occurrence in AH-type enteric neurons is associated
with suppression of the long-lasting hyperpolarizing afterpotentials
that characterize AH-type enteric neurons in their resting state
(Grafe et al. 1980
). Putative neurotransmitters and
paracrine/endocrine mediators evoke the response through activation of
a common ionic mechanism. Substances that mimic the slow excitatory
response include acetylcholine, biogenic amines, and peptides (reviewed
by Wood 1994
). Receptors for the mediators appear to be
linked by G-proteins to a common second messenger system. Classic
adenylate cyclase-cAMP signaling is implicated as the mechanism of
signal transduction (Palmer et al. 1986
). The underlying
ionic conductances responsible for generation of the response are not
well characterized. Available evidence suggests that conductance
changes include suppression of N-type Ca2+
channels, a Ca2+ leakage current,
Ca2+-activated K+ channels,
and an A-type K+ current (reviewed by Wood
1994
).
Results obtained from neurons in the guinea pig submucous plexus
suggest that in addition to suppression of K+
conductances, a cation conductance may be increased by the sEPSP mimetics substance P, muscarine, and 5-HT (Shen and Suprenant 1993). Shen and Suprenant (1993)
argued against
involvement of Cl
current whereas
Bertrand and Galligan (1994)
suggested that senktide, a
selective neurokinin-3 receptor agonist, activated
Cl
current coincident with suppression of
K+ conductance in guinea pig myenteric neurons.
In this study we used perforated patch-clamp recording methods to
investigate activation of Cl conductance as a
complementary mechanism in sEPSP-like responses in cultured AH-type
myenteric neurons from guinea pig small intestine. Histamine was
selected as the sEPSP mimetic for the study based on its importance in
enteric neuroimmune communication involving release from enteric mast
cells (Wood 1993
, 1998
). Release of histamine from mast
cells in the guinea pig intestine occurs during Type I hypersensitivity
reactions to
-lactoglobulin or Trichinella spiralis
antigen (Frieling et al. 1990a
,b
). The released
histamine acts as a paracrine mediator at histamine
H2 receptors to evoke sEPSP-like excitation in
the enteric neuronal cell soma. Aside from anaphylaxis, several lines
of evidence suggest that intestinal mast cells may be degranulated by
input from the CNS during psychogenic stress both in animal models and
humans (reviewed by Wood et al. 1999a
,b
).
A preliminary report of the work in the present paper has appeared in
abstract form (Starodub and Wood 1999a).
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METHODS |
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Tissue preparation
Methods used to prepare myenteric neuronal cultures in this
study were similar to those described in earlier reports (Xia et
al. 1991). The myenteric plexus was enzymatically dissociated from strips of longitudinal muscle by incubation in an enzyme solution
containing 20 mg collagenase type IA, 15 mg protease type IX, and 5 mg
deoxyribonuclease I in 15 ml Krebs solution for 15-25 min at 37°C in
a shaker bath. The digested tissue was washed several times with
ice-cold Krebs solution containing 5% antibiotic-antimycotic mixture
before collecting the dissociated ganglia. Suction pipettes and a
stereomicroscope were used to collect single ganglia. The ganglia, with
no visible smooth muscle present, were transferred into medium 199 supplemented with 15% L-glutamate, 10% heat-inactivated
fetal calf serum, 33 mM glucose, 1% Penn-Strep solution (10,000 units
penicillin and 10 mg/ml
1 streptomycin), and
0.5% gentamycin. They were transferred onto 22 × 22 mm cover
slips at the bottom of 33 mm plastic petri dishes and used for
patch-clamp studies the next day.
Solutions
The bathing solution was composed of (in mM) 120 NaCl, 6 KCl,
2.5 CaCl2, 1.2 MgCl2, 20 TEACl, 2 CsCl, 0.2 tetrodotoxin, and 10 HEPES (pH adjusted to 7.3 with
NaOH). -CgTx-MVIIC (300 µM) was used to suppress
Ca2+ currents. In cation substitution
experiments, Ca2+ and Na+
were replaced by equimolar Mg2+ and choline
cations respectively. In some experiments, the
Cl
concentration in the bathing solution was
reduced to 55 mM by substituting 100 mM sodium salt of
D-gluconic acid for 100 mM NaCl. Gluconate anion was used
because it does not permeate Cl
channels in
other preparations (Halm and Frizzel 1992
). The patch pipettes were filled with solution composed of (in mM) 50 CsCl, 50 Cs2SO4, 40 glucose, 10 HEPES (pH adjusted to 7.2 with CsOH), and amphotericin B 200 µg/ml.
Amphotericin B was initially prepared as a stock solution in DMSO and
dissolved in the pipette solution immediately before the experiments.
Agents used and sources were the following:
3-isobutyl-1-methylxanthine (IBMX), histamine, cimetidine,
anthracene-9-carboxylic acid (9-AC), niflumic acid,
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS),
8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate (ct-cAMP), N6,2'-O-dibutyryladenosine
3':5'-cyclic monophosphate (db-cAMP), -CgTx-MVIIC, and amphotericin
B all obtained from Sigma Biochemicals (St. Louis, MO); dimaprit,
2-chloro-N6-cyclopentyl-adenosine (CCPA), and
8-cyclopentyl-1,3-dimethylxanthine (CPT) were obtained from Research
Biochemicals International (Natick, MA).
Patch-clamp experiments
The cover slips with attached ganglia were washed free of
culture medium and placed into a custom made recording cell mounted on
the stage of an inverted microscope equipped with differential interference contrast optics, epi-fluorescence attachments, and 35-mm
camera (Nikon, Diaphot 300, Tokyo). The perforated patch configuration
(Horn and Marty 1988) was used to record whole cell currents. Preservation of the intraneuronal milieu was accomplished by
permeabilizing the membrane with amphotericin B after gigaseal formation. The intracellular ion concentrations were estimated as
described by Horn and Marty (1988)
. Serial resistances
<5 M
were achieved within 5-15 min after establishing stable
contact between the cell membrane and pipette tip. No swelling of the neurons was observed. The pipettes were fabricated from borosilicate glass capillary tubes (7052; World Precision Instruments, Sarasota, FL)
on a Flaming/Brown Model P-97 micropipette puller (Sutter Instruments,
San Francisco, CA). Tip resistances were ~1 M
. An Ag-AgCl
reference electrode was connected to the bath through an agar bridge
saturated with KCl solution. Ionic currents were recorded and voltage
clamp test pulses were applied with an Axopatch 200 amplifier and
Labmaster interfaced to an IBM computer with pClamp software (Axon
Instruments, Foster City, CA). The experiments were done at room
temperature (22-25°C). Averaged data are given as the mean ± SE. Student's t-test for paired data was used for statistical comparison and differences were accepted as significant for
P < 0.05.
Neuronal identification
At the end of each experiment the outline of the preparation and
locations of the electrode tip relative to identifiable landmarks were
sketched and a low-power photomicrograph with the electrode in place
was made for later identification of the recorded neurons in relation
to calbindin immunoreactivity. Lucifer yellow fluorescent marker was
injected and simultaneously viewed with ultraviolet illumination. The
presence of calbindin, as a marker for AH/Dogiel Type II neurons, was
determined with standard immuno-histochemical methods, which were
described in our earlier patch-clamp studies (Zholos et al.
1999). Retrospective localization of calbindin was the primary
criterion for identification as an AH-type neuron.
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RESULTS |
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Histamine-activated conductance
Both AH- and S-type myenteric neurons were studied [see
Bornstein et al. (1994) and Wood (1994)
for criteria used in classification of S- and AH-type enteric
neurons]. However, we restricted detailed analysis of the
histamine-activated ionic currents to calbindin-positive neurons
because the currents of interest were found mainly in this neuronal
population. Histamine-activated current was found in 81 of 183 calbindin-positive neurons and 3 of 47 calbindin-negative neurons.
Calbindin is a well recognized chemical code for Dogiel Type II neurons
with AH-type electrophysiologic behavior (Iyer et al.
1988
). Therefore calbindin-positive cells are referred to as
AH-type neurons in this paper.
Voltage-activated K+, Na+,
and Ca2+ currents were effectively suppressed by
the experimental conditions designed to do so (Zholos et al.
1999). The residual current after blockade of voltage-activated K+, Na+, and
Ca2+ currents remained stable throughout the
recording periods. The reversal potential for the control current with
Ca2+and Na+ present in the
bathing medium was
33.7 ± 2.5 mV for 56 neurons. The holding
potential was established at
30 mV to minimize the background current
and eliminate low voltage-activated Ca2+ current,
for which no specific blocker has been reported (Spedding and
Paoletti 1992
). High-voltage-activated (HVA)
Ca2+ currents were blocked by
-CgTx-MVIIC (300 µM) in the bathing solution. This toxin suppressed different HVA
Ca2+ currents (P, Q, and N) in our earlier work
on myenteric neurons (Starodub and Wood 1999b
). Bath
application of histamine (1 µM) evoked a current that was stable in
the presence of the agonist throughout the course of the experiments
(~30 min). Maximal increase in the total current evoked by stepping
the membrane potential from
30 to 50 mV amounted to a change from
13.2 ± 1.7 to 27.4 ± 5.3 pA pF
1 in
the 17 of 30 neurons tested that responded to histamine (Fig. 1, A and B). The
current activated by histamine was determined by digital subtraction of
the control current from the current recorded in the presence of the
agonist (Fig. 1C).
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Histamine-activated Cl current
The histamine-activated current reversed sign at 30 ± 5 mV
for the 17 neurons studied. The current and corresponding
current-voltage (I-V) relationship are shown in Fig.
2A. The I-V curve
exhibited outward rectification and reached saturation at potentials
more negative than
90 mV. With physiological concentrations of
Ca2+ and Na+ present, this
histamine-activated conductance could have been interpreted as either
nonspecific cation current (Shen and Suprenant 1993
), or
Cl
current (Bertrand and Galligan
1994
). To test the hypothesis that the histamine-activated
current was a nonspecific cation current, we removed
Ca2+ and Na+ from the
bathing solution by substituting Mg2+ and
choline, respectively. In Ca2+- and
Na+-free solution, the current at 50 mV was
12.5 ± 1.9 pA pF
1 in the 11 of 25 neurons
that responded to histamine. Figure 2B is an example of the
histamine-activated current and corresponding I-V
relationship. Elimination of cations from the bathing solution did not
change the reversal potential or form of the I-V curve but
did result in reduction in mean amplitude of the current. This
suggested that cations were not the charge carriers for the histamine-evoked current. Nevertheless, Na+ and
Ca2+ could have been indirectly affecting the
current because the observed reduction in amplitude was similar to that
reported for chloride current in cardiac myocytes (Bahinski et
al. 1989
; Harvey et al. 1990
). After finding
that the histamine response remained in Ca2+- and
Na+-free solution, we omitted these cations from
the bathing medium for the studies designed to test the second
hypothesis that the histamine-activated conductance was a
Cl
current. For this, we reduced the
concentration of Cl
in the bathing medium from
155 to 55 mM by substituting Cl
with gluconate,
an anion that is virtually impermeable for Cl
channels in other preparations (Halm and Frizzel 1992
).
Under our experimental conditions (i.e., perforated patches fully
permeable to Cl
) the concentration of
Cl
inside the cell was estimated to be ~50
mM. In Ca2+- and Na+-free
and reduced Cl
solution, the
histamine-activated current was lower in amplitude at 50 mV amounting
to 0.7 ± 0.3 pA pF
1 in 7 of 20 neurons
that responded to histamine (Fig. 3). The current activated at
100 mV remained unchanged at 1.9 ± 0.6 pA pF
1 in 7 of 20 neurons. The I-V
relationship became linear and intersected the abscissa at 7 ± 4 mV in the 7 responding neurons (Fig. 3). These results suggested that
Cl
was the charge carrier for the
histamine-activated current. The rectification observed with
physiological concentrations of extracellular Cl
was apparently caused by unequal
Cl
on opposite sides of the membrane, rather
than to intrinsic biophysical properties of the chloride channels.
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Niflumic acid inhibition
Another test of the hypothesis for Cl
involvement in histamine-mediated responses was whether the current
could be blocked by putative Cl
channel
blockers. There are no known specific, high-affinity blockers of
Cl
currents (Gogelein 1988
);
nevertheless, several substances are known to inhibit chloride
conductance in different systems including 9-AC in skeletal muscle
(Palade and Barchi 1977
) and epithelial preparations
(Welsh 1984
), DIDS in rabbit urinary bladder
(Hanrahan et al. 1985
), and niflumic acid in guinea pig
myenteric neurons (Bertrand and Galligan 1994
). In view
of the experience of others, we tested all three substances on the
histamine-activated current. Bath application of 0.4 mM niflumic acid
blocked 85 ± 8% of the histamine-activated Cl
conductance in four neurons, P < 0.05 (Fig.
4, A and
B). On the other hand, 9-AC and DIDS failed to have
consistent blocking effects. Because it is known that these inhibitors
of Cl
conductance can also affect other transport systems
(Gogelein 1988
), we preapplied each of the inhibitors
before histamine application. In the presence of 9-AC (4 mM), the
histamine-activated conductance was not changed significantly at
10.4 ± 2.1 pA pF
1 in 4 of 10 neurons
(P > 0.05). DIDS (0.5 mM) also did not suppress significantly the Cl
conductance. In DIDS, the
histamine-activated current was 11.5 ± 1.8 pA pF
1
in the 5 of 10 neurons that responded to histamine (P
>0.05). Preapplication of niflumic acid (0.4 mM) significantly
suppressed the action of histamine, with the histamine-activated
conductance averaging 3.3 ± 0.9 pA pF
1 in 2 of 22 neurons that responded to histamine (P < 0.05;
Fig. 4C).
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Membrane permeable cAMP analogues
Because, our laboratory has measured increased levels of cAMP
evoked by activation of histamine H2 receptors in
guinea pig small intestinal myenteric neurons (Xia et al.
1996), we tested the hypothesis that activation of
Cl
current by histamine involves elevation of
cAMP in the signal transduction cascade. Bath application of a membrane
permeable analogue of cAMP (0.5 mM ct-cAMP), alone or in combination
with the phosphodiesterase inhibitor IBMX (0.4 mM), evoked currents similar to those activated by histamine. In "sharp" microelectrode studies, ct-cAMP or IBMX mimicked the sEPSP-like actions of histamine (Palmer et al. 1986
). The amplitude of the current
evoked by ct-cAMP was 7.5 ± 1.4 pApF
1 in
11 neurons. The reversal potential and form of the cAMP-activated current were similar to that of the histamine-activated current under
similar ionic conditions (Figs. 2 and 5).
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Histamine receptor subtype
The excitatory actions of histamine and the associated elevation
of intraneuronal cAMP in guinea pig myenteric neurons are known to be
mediated by histamine H2 receptors (Wood
1992; Xia et al. 1996
). We used dimaprit, a
selective histamine H2 receptor agonist, to test
the suggestion that histamine-evoked changes in
Cl
conductance were mediated by histamine
H2 receptors. Bath application of dimaptit (1 µM) evoked a conductance change at 50 mV of 10.2 ± 1.7 pA
pF
1 in 11 of 17 neurons (Fig.
6A). Dimaprit (1 µM) also
activated the current in a bathing medium with
Cl
reduced to 55 mM. A decrease in outward
current without change in inward current amplitude was found when
Cl
concentration was reduced outside the
neurons. As a result, the current recorded with a ramp clamp protocol
was virtually linear and reversed sign at ~0 mV (Fig. 6B).
This supported the suggestion that Cl
is a
charge carrier for the dimaprit activated current. Moreover, bath
application of 100 µM cimetidine, a selective histamine
H2 receptor antagonist, suppressed the
histamine-activated Cl
current by 72 ± 11% in five neurons (P < 0.05; Fig.
7).
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Adenosine A1 agonist
Inhibition of adenylate cyclase by action of adenosine at
adenosine A1 receptors is known to have an
inhibitory action on histamine-evoked excitation of myenteric AH-type
neurons and elevation of intraneuronal cAMP (Tamura et al.
1995; Xia et al. 1997
). We tested the hypothesis
that activation of adenosine A1 receptors would
also suppress histamine-activated Cl
current.
Bath application of 1 µM CCPA, a selective A1
receptor agonist, reduced the histamine-activated current to 19 ± 6% (P < 0.05) of control values in seven neurons.
Bath application of 10 µM CPT, a selective A1
adenosine receptor antagonist, reversed the inhibitory effects from a
value of 19 ± 6 to 41 ± 7% of control current in the four
neurons tested (P < 0.05; Fig.
8).
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DISCUSSION |
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Histamine-activated current
The results suggest that activation of Cl
conductance is part of the excitatory action of histamine on AH-type
myenteric neurons. A histamine-evoked current was found in
approximately one-half of the calbindin-positive neurons and in only
5% of calbindin-negative neurons. This was consistent with earlier
findings in sharp electrode studies which suggested that ~50% of AH
neurons are depolarized by histamine (Nemeth et al.
1984
; Tamura and Wood 1992
). Whether a small
group of S-type neurons (i.e., calbindin-negative neurons) responded to
histamine was unclear. The proportion of calbindin-negative neurons in
our study was generally the same as reported by others. Iyer et
al. (1988)
reported that up to 20% of guinea pig enteric neurons with AH-type electrophysiologic behavior did not show calbindin
immunoreactivity. If it is assumed that many of the calbindin-negative
neurons with responses to histamine were AH neurons, then consideration
of the possibility that histamine-activated Cl
current is restricted to AH-Dogiel type II myenteric neurons is
justified. This would be consistent with observations that activation
of adenylate cyclase by forskolin and subsequent elevation of
intraneuronal cAMP mimic slow synaptic excitation in guinea pig
myenteric neurons with AH- but not S-type electrophysiologic behavior
(Palmer et al. 1986
).
The histamine-evoked current reversed at a holding potential of
approximately 30 mV. This is the theoretical reversal potential for
Cl
with our experimental protocol. Evidence of
outward rectification was apparent on the I-V curves. On the
basis of these results, the histamine-activated current could have been
interpreted as either a nonspecific cation current or a specific
Cl
current. Findings that removal of
Na+ and Ca2+ from the
bathing medium did not change either the reversal potential or shapes
of I-V curves suggested that neither
Na+ nor Ca2+ were charge
carriers for the current. On the other hand, reduction of the
Cl
concentration in the bathing solution to
match the intraneuronal Cl
concentration
significantly reduced the outward current without changing the inward
current. This procedure resulted in a linear I-V
relationship and shifted the reversal potential from ~0 to 7 ± 4 mV. These observations implicate Cl
as the carrier of
the histamine-activated conductance. The outwardly rectifying
properties of the current most likely reflected asymmetrical concentrations of Cl
across the cell membrane rather than
being an intrinsic property of the channel itself. These
characteristics of the histamine-activated current in enteric neurons
are reminiscent of the cAMP-activated Cl
conductance
found in cardiac cells (Harvey 1996
).
Removal of Ca2+ and Na+ from the bathing
medium resulted in decreased amplitude of the chloride current.
Although the cation dependence of the Cl current was not
studied in detail, we assumed that it was related to the same factors
reported for the cAMP-dependent chloride conductance in myocytes
(Hume and Harvey 1991
). Sodium ions may influence the
current by acting at a regulatory site instead of being a charge
carrier. Observations that the reversal potential for the current was
independent of Na+ concentration in the bathing medium is
consistent with this interpretation. An alternative possibility was
that Ca2+ might alter a Ca2+-dependent
conductance on entering the neuron through unblocked "leakage"
Ca2+ channels that have been postulated to influence
resting Ca2+- dependent K+ conductance in
AH-type enteric neurons (Wood 1994
). This was ruled out
by results showing no effects of removal of Ca2+ from the
bathing medium.
Pharmacology of the histamine-activated Cl current
Suppression of the histamine-activated current by niflumic acid
further supports Cl as the ionic carrier.
Niflumic acid is known to suppress Ca2+-,
volume-, and cAMP-activated chloride currents in other preparations (Currie et al. 1995
; Hughes and Segawa
1993
; Korn et al. 1991
; White and Aylwin
1990
). Neither DIDS nor 9-AC altered the histamine-activated current in the myenteric neurons. This was consistent with the observation of Shen and Suprenant (1993)
that currents
activated by substance P, muscarine, or serotonin in submucous neurons
were resistant to 9-AC.
The results with selective histamine H2 receptor
agonists and antagonists suggest that the histamine
H2 receptor mediates the action of histamine on
the Cl channels. Application of membrane
permeable cAMP analogues or dimaprit evoked currents that had similar
I-V relationships to the histamine-activated current. Like
histamine, the actions of both dimaprit and cAMP analogues were
independent of cation composition of the bathing medium and were
altered by reduction in the extracellular Cl
concentration. The results supplement existing evidence that histamine
acts at the histamine H2 receptor subtype on
enteric neurons of the guinea pig to elevate intracellular levels of
cAMP and trigger the cascade of events leading to sEPSP-like responses (Palmer et al. 1987a
; Xia et al. 1996
).
Our findings that a selective adenosine A1
receptor agonist blocked the stimulatory action of histamine on the
Cl current is consistent with earlier reports
that selective A1 receptor agonists suppress
histaminegic stimulation of cAMP formation in guinea pig myenteric
ganglia (Xia et al. 1997
). They conform also to an
earlier model for the presence of subtypes of inhibitory P1 purinoreceptors on myenteric neurons
(Christofi and Wood 1994
).
Our results are inconsistent with the conclusions of Bertrand
and Galligan (1995) that elevation of intraneuronal cAMP is not
associated with activation of Cl
channels in
guinea pig myenteric neurons. Bertrand and Galligan based their
conclusion on results obtained with single sharp electrode voltage-clamp methods which showed that the reversal potential for
forskolin-evoked depolarizing responses approximated the predicted K+ equilibrium potential. Current-voltage curves
for forskolin were reported to be best fit in a majority of their
neurons by a one-parameter model suggestive of a pure increase in
K+ conductance. Nevertheless, in 21% of the
neurons, analysis with a two-parameter model incorporating changes in
both K+ and Cl
conductance produced a significantly better fit for the I-V
curves than the one-parameter model.
Two sets of evidence support our conclusion that histamine-induced
stimulation of adenylate cyclase is involved in activation of the
histamine-evoked conductance we have interpreted as a
Cl current. First, histamine
H2 receptor activation both mimics sEPSP-like
responses and elevates cAMP in myenteric ganglia (Nemeth et al.
1986
; Xia et al. 1996
). Second, activation of
adenosine A1 receptors leads to inhibition of the
following: 1) forskolin-evoked sEPSP-like responses
(Palmer et al. 1987b
); 2) histamine-evoked sEPSP-like responses (Palmer et al. 1987a
,b
);
3) histamine-evoked elevation of cAMP in myenteric ganglia
(Xia et al. 1997
); and 4) the
histamine-evoked increase in conductance we interpret as Cl
current.
Significance of histamine-activated current
The slowly activating depolarizing response to histamine
H2 receptor activation in AH neurons is
associated with suppression of resting K+
conductance that is reflected by increased input resistance as determined in sharp microelectrode studies (Nemeth et al.
1984). However, decreased K+ conductance
cannot be the full explanation for the ionic mechanism underlying the
depolarization because changes in input resistance are often observed
to be biphasic with an increase in input resistance followed by a
decrease as membrane depolarization progresses to its peak during the
response to histamine. This change suggests that the opening of a
second set of conductance channels occurs in concert with closure of
K+ channels as the slowly activating depolarization
characteristic of sEPSP-like responses develops.
Our results suggest that the secondary decrease in input resistance,
observed with sharp microelectrodes as the membrane potential is
progressively depolarized from the resting potential by histaminergic action, could result from activation of Cl conductance.
The electrochemical gradient for Cl
in enteric neurons is
such that activation of Cl
conductance leads to
depolarization of the membrane potential. The reversal potential for
Cl
in guinea pig myenteric neurons was estimated by
Cherubini and North (1984)
to be
39 mV. This predicts
that the membrane potential of neurons responding to histamine by
opening Cl
conductance channels will become depolarized
relative to the resting potential. The histamine-activated current
could support the depolarized state of the membrane for as long as
histamine remained in the surrounding milieu because no inactivation
occurred over periods of ~30 min. This is reminiscent of findings
that the effects of activation of H2 histamine receptors on
AH-type myenteric neurons does not show tachyphylaxis during prolonged exposure to histamine in sharp microelectrode studies (Tamura and Wood 1992
). Inward Cl
current is much smaller
than the outward current in myenteric neurons; nevertheless, a new
equilibrium potential will be close to the reversal potential for
Cl
because background conductance is relatively small in
resting AH-type neurons (Baidan et al. 1992
).
Consequently, activation of the Cl
conductance could also
be a stabilizing factor that prevents depolarization to higher levels,
thereby protecting the neuron from detrimental depolarization.
The histamine-activated current may also contribute to spike
repolarization during the action potential in AH-type enteric neurons.
With the normal asymmetrical concentrations of Cl across
the cell membrane, the outwardly directed histamine-activated Cl
conductance is significantly larger than the inward
conductance. Therefore under normal physiological conditions,
activation of the Cl
conductance would be expected to
shorten the action potential. Considering that histamine also
suppresses A-type K+ current in myenteric neurons
(Starodub et al. 1998
), chloride conductance could
replace A-current as a significant factor in membrane repolarization
during spike discharge. This would be expected to support spike
discharge at higher frequencies as occurs during slow synaptic
excitation and sEPSP-like responses to paracrine mediators such as histamine.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-46941 to J. D. Wood.
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FOOTNOTES |
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Address for reprint requests: J. D. Wood, Dept. of Physiology, 300 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210-1218.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 August 1999; accepted in final form 23 November 1999.
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REFERENCES |
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