Actions of histamine on muscle and ganglia of the guinea pig gallbladder

Jason M. Hemming1, Fay A. Guarraci1, Tracy A. Firth1, Lee J. Jennings1, Mark T. Nelson2, and Gary M. Mawe1,2

Departments of 1 Anatomy and Neurobiology and 2 Pharmacology, College of Medicine, University of Vermont, Burlington, Vermont 05405


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Histamine is an inflammatory mediator present in mast cells, which are abundant in the wall of the gallbladder. We examined the electrical properties of gallbladder smooth muscle and nerve associated with histamine-induced changes in gallbladder tone. Recordings were made from gallbladder smooth muscle and neurons, and responses to histamine and receptor subtype-specific compounds were tested. Histamine application to intact smooth muscle produced a concentration-dependent membrane depolarization and increased excitability. In the presence of the H2 antagonist ranitidine, the response to histamine was potentiated. Activation of H2 receptors caused membrane hyperpolarization and elimination of spontaneous action potentials. The H2 response was attenuated by the ATP-sensitive K+ (KATP) channel blocker glibenclamide in intact and isolated smooth muscle. Histamine had no effect on the resting membrane potential or excitability of gallbladder neurons. Furthermore, neither histamine nor the H3 agonist R-alpha -methylhistamine altered the amplitude of the fast excitatory postsynaptic potential in gallbladder ganglia. The mast cell degranulator compound 48/80 caused a smooth muscle depolarization that was inhibited by the H1 antagonist mepyramine, indicating that histamine released from mast cells can activate gallbladder smooth muscle. In conclusion, histamine released from mast cells can act on gallbladder smooth muscle, but not in ganglia. The depolarization and associated contraction of gallbladder smooth muscle represent the net effect of activation of both H1 (excitatory) and H2 (inhibitory) receptors, with the H2 receptor-mediated response involving the activation of KATP channels.

motility; cholecystitis; ATP-sensitive K+ channel; innervation; mast cell; compound 48/80


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HISTAMINE IS A WELL-RECOGNIZED inflammatory mediator that, when released from mast cells, can cause vasodilation, increased vascular permeability, gastric secretion, and contraction of bronchiolar and gastrointestinal smooth muscle (1). Histamine is present in almost all mammalian tissue, and it has been found to be particularly abundant in tissues containing high numbers of mast cells such as skin, mesenteric endothelium, and intestinal mucosa (16).

The gallbladder wall is also rich in histamine-containing mast cells, which are distributed in the mucosa and muscularis/serosa layers (14). Studies (12, 14, 17, 19, 28) of gallbladder motility have demonstrated that histamine causes a contraction in human, pig, opossum, and guinea pig tissues. However, when histamine receptor subtype-specific compounds have been employed, it has been demonstrated that both H1 and H2 receptors are present, with H1 receptors causing a contraction and H2 receptors mediating a relaxation (6, 14, 28).

Although no studies of neuronal histamine responses have been conducted in the gallbladder, histamine responses have been detected in ganglia of the enteric nervous system. Electrophysiological studies (10, 25-27) have demonstrated the presence of H2 and H3 receptors in ganglia of the guinea pig small intestine and colon. These studies (10, 25-27) of enteric ganglia have demonstrated that activation of H2 receptors causes a membrane depolarization and a dramatic increase in neuronal excitability, whereas activation of H3 receptors causes a presynaptic inhibition of ACh release.

The aim of the present study was to examine the effects of endogenous and exogenous histamine on the electrical activity of individual gallbladder smooth muscle cells and on ganglionic neurotransmission. Recording techniques were used to evaluate responses to histamine and receptor subtype-specific analogs. In addition, compound 48/80, which degranulates mast cells, was employed to test the effects of endogenous histamine on gallbladder smooth muscle.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular Recording Studies

Guinea pigs of either sex, weighing between 250 and 350 g, were euthanized with deep halothane anesthesia and exsanguination. The abdominal cavity was then opened, and the gallbladder was removed and transferred to iced Krebs solution of the following composition (in mM): 121 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaH2PO4, and 8 glucose. The wall of the gallbladder was incised from the end of the cystic duct to the base and trimmed of any adherent liver tissue. Residual bile was rinsed away with Krebs solution, and the organ was stretched and pinned flat, mucosal side up, in recirculating iced Krebs solution. The mucosa and underlying connective tissue were gently removed with forceps under microscopic observation.

The gallbladder whole mount preparation was then transferred to a tissue chamber with a capacity of 3 ml and pinned serosal side up. The tissue chamber was placed onto the stage of the inverted microscope (Nikon, Diaphot), where the tissue was continuously perfused with Krebs solution (37°C), pH 7.4, that was aerated with 95% O2-5% CO2. Ganglia and bundles of smooth muscle were visualized at ×200 using Hoffman modulation contrast optics (Modulation Optics, Greenvale, NY). Wortmannin (100-400 nM), which inhibits myosin light chain kinase activity without altering electrical properties or Ca2+ transients in smooth muscle (2, 3), was added to the Krebs solution to inhibit tissue contractions and therefore increase the durations of cell impalements.

The electrophysiological methods that were used in this study to evaluate smooth muscle and neurons are similar to those previously described (15, 20, 30). Briefly, glass microelectrodes used for intracellular recording were filled with 2.0 M potassium chloride and had resistances in the range of 60-80 MOmega . Transmembrane potential was measured with an Axoclamp-2A amplifier (Axon Instruments), and outputs were displayed on an oscilloscope (Hitachi VC-6050). Electrical signals were recorded on VHS tapes via a pulse code modulator (model 525; A.R. Vetter, Rebersburg, PA), printed on thermal chart paper (Astro-Med, West Warwick, RI), and saved using the MacLab computer program (CB Sciences, Milford, MA). Synaptic inputs were elicited in gallbladder neurons using monopolar extracellular electrodes made from Teflon-coated platinum wire (25-µm diameter). Stimulation duration was 0.3-0.5 ms, and frequency was 0.5 Hz.

Patch-Clamp Studies

The whole-cell configuration of the patch-clamp technique was used to examine the actions of the H2 receptor agonist dimaprit (25 µM) on ATP-sensitive K+ (KATP) currents recorded from isolated gallbladder smooth muscle cells voltage clamped at -60 mV. Glibenclamide-sensitive currents were taken as a measure of KATP currents.

Gallbladder smooth muscle cells were isolated as described previously (9, 30). Briefly, guinea pig gallbladders, minus the mucosal layer, were cut into small strips and rinsed in Ca2+-free cell isolation solution (solution E) composed of (in mM) 55 NaCl, 80 monosodium glutamate, 2 MgCl2, 6 KCl, 10 glucose, and 10 HEPES (adjusted to pH 7.3 with NaOH). The pieces of gallbladder were transferred to an enzyme solution containing 1 mg/ml BSA, 1 mg/ml papain (23 U/mg; Worthington, Lakewood, NJ), and 1 mg/ml dithioerythreitol (Sigma Chemical, St. Louis, MO) and incubated at 37°C for 30-35 min. The tissue pieces were transferred to a tube containing solution E and 1 mg/ml BSA, 1 mg/ml collagenase (1.01 U/mg, Fluka, Milwaukee, WI), and 100 µM CaCl2 for an additional 8-12 min. The tissue was then rinsed in chilled enzyme solution and triturated with a flame-polished glass Pasteur pipette to yield single smooth muscle cells. Cells were stored in glass vials on ice until required.

Patch electrodes were filled with a solution containing (in mM) 102 KCl, 38 KOH, 10 NaCl, 1 MgCl2, 1 CaCl2, 10 EGTA, 0.1 Na2ATP, 0.1 ADP, 0.2 Na2GTP, 10 glucose, and 10 HEPES adjusted to pH 7.2 with KOH and bathed in a solution containing (in mM) 140 KCl, 1 MgCl2, 10 HEPES, 10 glucose, and 0.1 CaCl2 (pH 7.4).

Compounds were applied by either pressure microejection from glass micropipettes (0.01-1 mM in Krebs solution; 15- to 20-µM tip diameter) with pulses of nitrogen gas (300 kg/cm2, 10-500 ms in duration) or addition to the bathing solution. The distance between the tip of the microejection and the impaled smooth muscle cell was maintained between 50 and 100 µm. Histamine, wortmannin, compound 48/80, and dimaprit were purchased from Sigma Chemical. Mepyramine, ranitidine, and R-alpha -methylhistamine were purchased from Research Biochemicals International (Natick, MA). For stock solutions, all drugs were initially dissolved in distilled H2O, with the exception of wortmannin, which was dissolved in DMSO.

Toluidine Blue Staining

Gallbladder preparations were fixed in 0.6% formaldehyde and 0.5% acetic acid (pH 2.9) for 4-12 h at 4°C and then placed in 70% ethanol for 12 h. The tissues were subsequently dipped in 0.5% solution of toluidine blue in acetate buffer (pH 4.0) for 10 s, rinsed in a 0.05% acetate buffer (pH 4.0) for 10 s, and fully rinsed in distilled water for 5-10 s. The preparations were then mounted on glass slides and examined with standard bright-field optics.

Quantification and Statistics

Results are expressed as means ± SE of n cells. The Student's t-test was used to determine the significance of differences between sets of data. P <=  0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of Histamine Receptors in Gallbladder Smooth Muscle

Intracellular recordings were made from 154 guinea pig gallbladder smooth muscle cells in 84 preparations, and the mean membrane potential was -53.2 ± 0.6 mV. The properties of gallbladder smooth muscle cells impaled in the current study were comparable to those observed in previous studies (30). Typically, each action potential comprised four phases: a rapid depolarization upstroke, a transient repolarization downstroke, a plateau phase, and a complete repolarization back to the resting membrane potential. Spontaneous action potentials typically occurred at a frequency of 0.3-0.4 Hz. Occasionally, however, the normal low-frequency pattern of individual action potentials was disrupted by high-frequency bursts of action potentials.

Actions of histamine. Histamine (100 nM-1 mM) caused a concentration-dependent membrane depolarization that was associated with an increase in action potential frequency (Fig. 1). Membrane depolarizations (measured from the baseline membrane potential before the upstroke of an action potential) were detected at concentrations as low as 1 µM, and at the highest concentration tested (1 mM), histamine usually caused smooth muscle contractions resulting in the loss of the impalement. The amplitude of the maximal depolarization caused by 1 mM histamine was 27.2 ± 2.2 mV (range = 23.2-35.2 mV; n = 5).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1.   Histamine causes a concentration-dependent depolarization of gallbladder smooth muscle cells with an associated increase in action potential frequency. The graph demonstrates the concentration-effect relationship for the depolarization caused by histamine. Values are means ± SE of 5-24 cells. At concentrations of 1 mM or greater, the impalement was typically lost due to contraction of the tissue. The traces demonstrate representative responses to histamine at progressively increasing concentrations. The resting membrane potential of the cell represented in the top trace was -48 mV. The resting membrane potential of the cell represented in the other 3 traces was -50 mV.

Distinguishing H1 vs. H2 receptor-mediated responses. Gallbladder muscle strip tension studies have demonstrated that activation of H1 receptors causes an increase in tension, and activation of H2 receptors causes a decrease in muscle strip tension (6, 14, 28). To establish the relative contributions of H1 and H2 receptor activation to the membrane potential changes caused by histamine, histamine was superfused in the presence of either the H1 antagonist mepyramine or the H2 antagonist ranitidine. Application of histamine in the presence of the H2 antagonist ranitidine (10 µM; >= 15 min before histamine application) resulted in a shift to the left of the histamine concentration-effect curve (Fig. 2). In the presence of ranitidine, the amplitudes of the membrane depolarizations caused by histamine at concentrations of 1, 10, and 100 µM were significantly increased (Fig. 2; P <=  0.0005). Conversely, in the presence of mepyramine (1 µM; applied >= 15 min before histamine application), there was a transient elimination of action potentials associated with a concentration-dependent hyperpolarization (Fig. 3). The hyperpolarization was initially detected at a histamine concentration of 10 µM, and the maximum hyperpolarization (8.6 ± 1.3 mV, n = 5) was observed at 1 mM histamine.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2.   The H2 receptor antagonist ranitidine increased the potency of the excitatory action of histamine on gallbladder smooth muscle. The concentration-effect relationship of histamine alone (same data as in Fig. 1) is compared with that for histamine applied in the presence of 10 µM ranitidine. Values for ranitidine are means ± SE of 4-11 cells. * P <=  0.005, significant increase in histamine-stimulated depolarization. Traces demonstrate the potentiation of the histamine response when histamine was added to the bathing solution or added by pressure microejection in the presence of 10 µM ranitidine. The resting membrane potential of the cell shown in the top 2 traces was -52 mV. The resting membrane potential of the cell shown in the bottom 2 traces was -50 mV.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3.   Histamine causes a concentration-dependent hyperpolarization and a cessation of spontaneous action potentials in the presence of the H1 receptor antagonist mepyramine. The concentration-effect relationship of the hyperpolarization elicited by histamine in the presence of mepyramine (1 µM) is shown at left. Values are means ± SE of 5-9 cells. The representative traces at right were obtained from a single gallbladder smooth muscle cell that had a resting membrane potential of -57 mV.

To further examine the electrical response associated with activation of the H2 receptor, the H2 receptor agonist dimaprit was applied to the gallbladder preparations. Similar to the response to histamine in the presence of mepyramine, dimaprit caused a marked reduction in action potential generation and a concentration-dependent hyperpolarization. The threshold for the hyperpolarization was 1 µM dimaprit, and the peak response was detected at 1 mM (11.4 ± 0.7 mV, n = 4) (Fig. 4). At the highest concentrations of dimaprit (1 mM), some cells never fully recovered to the original resting membrane potential and action potential frequency.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   The H2 receptor agonist dimaprit caused a concentration-dependent hyperpolarization of gallbladder smooth muscle associated with a suppression of spontaneous action potentials. The concentration-effect relationship of the dimaprit-induced hyperpolarization is shown at left. Values are means ± SE of 4-7 cells. The representative traces at right were obtained from a single gallbladder smooth muscle cell that had a resting membrane potential of -48 mV.

H2 receptor activation leads to KATP channel opening. H2 receptor activation typically leads to the activation of the adenylate cyclase signal transduction pathway (8, 18). Because this pathway is tightly linked to the opening of KATP channels in gallbladder smooth muscle (29, 31), we tested whether KATP channels are involved in the H2 receptor-mediated responses in gallbladder smooth muscle. For these experiments, the KATP channel blocker glibenclamide (10 µM) was used, and as we have reported previously (31), glibenclamide caused a slight depolarization of the membrane as it entered the bathing solution. Glibenclaminde significantly reduced the amplitude of the hyperpolarization elicited by dimaprit (control, 11.5 ± 3.8 mV; glibenclamide, 0.4 ± 0.4 mV; P<= 0.025; n = 4; Fig. 5). Furthermore, in isolated gallbladder myocytes, dimaprit caused the activation of a glibenclamide-sensitive current (Fig. 5). The amplitude of the glibenclamide-sensitive component of the current activated by dimaprit was 29.9 ± 5.4 pA (n = 5).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5.   The hyperpolarization of gallbladder smooth muscle mediated by the activation of the H2 receptor is inhibited by the ATP-sensitive K+ (KATP) channel blocker glibenclamide. A: the traces are intracellular recordings from intact smooth muscle that demonstrate the inhibitory effect of glibenclamide on the hyperpolarization caused by dimaprit (resting membrane potential = -49 mV). B: the trace demonstrates a glibenclamide-sensitive current activated by dimaprit.

Actions of Histamine in Gallbladder Ganglia

Histamine, acting at H2 receptors, has been shown to have a direct excitatory effect on neurons in guinea pig small intestine and colon. Furthermore, histamine acting at H3 receptors causes presynaptic inhibition of ACh release in these ganglia (10, 26). Therefore, in the present study, intracellular recordings were made from individual guinea pig gallbladder neurons to examine the effects of histamine on neuronal excitability and synaptically evoked responses.

The H3 agonist R-alpha -methylhistamine was used to determine whether activation of presynaptic H3 receptors alters excitatory synaptic transmission in gallbladder neurons. In control conditions, the mean amplitude of the fast excitatory postsynaptic potential (fast EPSP) evoked by interganglionic fiber tract stimulation was 19.0 ± 3.5 mV. The amplitude of the fast EPSP was not significantly altered by the addition of the H3 agonist (100 µM) into the bath (amplitude = 20.1 ± 2.7 mV; n = 5; P > 0.05) (Fig. 6A).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 6.   Histamine does not have a presynaptic or direct effect on gallbladder ganglia. A: fast excitatory postsynaptic potentials were unaffected by superfusion of the H3 agonist R-alpha -methylhistamine (left) or histamine (right). Each trace represents an average of 10 consecutive events. B: neuronal responses to depolarizing current pulses (0.1 nA) were unaffected by superfusion of 100 µM histamine into the tissue bath. This cell generated a single action potential at the onset of a 0.3-nA depolarizing current pulse.

The amplitude of the fast EPSP was also measured in the presence of histamine. Histamine did not significantly alter the amplitude of the fast EPSP (control, 17.9 ± 4.7 mV; histamine, 20.4 ± 6.0 mV; n = 6; P > 0.05) (Fig. 6A), supporting the view that presynaptic histamine receptors are not present on cholinergic nerve terminals in gallbladder ganglia.

The direct effects of histamine and dimaprit on gallbladder neurons were also investigated. When histamine (10-100 µM) was applied to the bath, the mean membrane potential was not altered (control, -52.2 ± 2.0 mV; 10 µM histamine, -51.2 ± 2.0 mV; n = 4; P > 0.05; control, -51.8 ± 2.4 mV; 100 µM histamine, -52.1 ± 1.9 mV; n = 8) (Fig. 6B). Furthermore, the membrane potential of gallbladder neurons was not significantly affected by dimaprit (control, -49.6 ± 3.5 mV; 100 µM dimaprit, -48.1 ± 4.4 mV; n = 5) (data not shown). In addition, the responsiveness of the gallbladder neurons to depolarizing current pulses did not change in the presence of histamine (Fig. 6B) or the H2 agonist dimaprit. These findings suggest that histamine does not affect the excitability of gallbladder neurons.

Actions of Endogenous Histamine Released by Compound 48/80

Morphological studies involving toluidine blue histological staining, or immunostaining with antisera directed against histamine, have demonstrated an abundance of mast cells in the muscularis of the guinea pig gallbladder (14). Therefore, we tested whether release of endogenous histamine from mast cells could act on gallbladder smooth muscle. To accomplish this, we added the mast cell degranulator compound 48/80 (50 ng/ml) to the Krebs solution while recording from individual smooth muscle cells with intracellular recording electrodes.

Preliminarystudies were performed to confirm that mast cells in the gallbladder would degranulate in response to compound 48/80. These studies were conducted because only connective tissue mast cells, but not mucosal mast cells, respond to this compound and because the mast cells in the wall of the gallbladder have not been previously classified. Guinea pig gallbladders were divided into two whole mount preparations of equal size that were pinned flat to the bottom of a petri dish coated with Sylgard. One of the preparations was placed in a container of Krebs solution, and the other was placed in a container of Krebs solution with 50 ng/ml of compound 48/80. After 15 min, the preparations were fixed and histochemically stained with toluidine blue, which reveals mast cell granules. Mast cells were abundant in control preparations but were extremely sparse in preparations treated with compound 48/80 (Fig. 7; n = 4). These data indicate that compound 48/80 is an effective degranulator of guinea pig gallbladder mast cells.


View larger version (87K):
[in this window]
[in a new window]
 
Fig. 7.   Application of the mast cell degranulator compound 48/80 to gallbladder smooth muscle causes a mepyramine-sensitive depolarization. The photomicrographs depict gallbladder whole mount preparations stained with toluidine blue. The photomicrograph at left is from a preparation that was bathed in normal Krebs solution before fixation. The photomicrograph at right was bathed in compound 48/80 before fixation. Mast cells were abundant in the control preparation (arrows), but were not visible in the treated preparations. The traces demonstrate intracellular recordings from gallbladder smooth muscle. The trace at left shows a depolarization generated after the addition of compound 48/80 to the bath. When compound 48/80 was applied in the presence of the H1 receptor antagonist mepyramine, the cell did not depolarize and there was a reduction in spontaneous action potential frequency. Resting membrane potentials: left trace, -49 mV; right trace, -53 mV.

In the 12 cells tested with smooth muscle intracellular recording techniques, compound 48/80 elicited a depolarization of 7.9 ± 2.4 mV that was associated with a 46% increase in action potential frequency (control, 0.6 ± 0.06 Hz; compound 48/80, 0.8 ± 0.08 Hz; P = 0.001) (Fig. 7A). In the presence of compound 48/80 plus the H1 antagonist mepyramine, no change in resting membrane potential was detected, but there was a 43.17% decrease in action potential frequency (n = 5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Histamine is an inflammatory mediator that can alter gastrointestinal motility through direct actions on smooth muscle and by actions in local ganglia. The results of the current study indicate that histamine added exogenously or released from mast cells can greatly enhance gallbladder smooth muscle excitability through direct actions on the smooth muscle but does not directly affect ganglionic transmission.

Smooth Muscle

Previous studies (12, 14, 17, 19, 28) of gallbladder muscle strips and in vivo preparations have demonstrated that histamine causes a contraction of the gallbladder. Studies (6, 14, 28) involving receptor-specific analogs of histamine indicate that the contraction involves activation of gallbladder smooth muscle H1 receptors and that selective activation of H2 receptors can relax the gallbladder. The electrical properties of the effects of histamine on gallbladder smooth muscle are consistent with these findings. In the current study, histamine caused a concentration-dependent depolarization and increase in smooth muscle excitability. This response was difficult to study at high concentrations because histamine caused contractions, leading to a loss of the intracellular impalement. The net effect of histamine was apparently the result of activation of both H1 and H2 receptors, which had excitatory and inhibitory effects, respectively. Blockade of the H2 receptor increased the potency of histamine, shifting the concentration-effect curve for histamine to the left. Increases in both action potential frequency, which is Ca2+ dependent (30), and depolarization, which increases the open-state probability of the dihydropyridine-sensitive Ca2+ channels (30), result in increased intracellular Ca2+. This would in turn lead to enhanced excitation-contraction coupling. It is not yet clear how the activation of the H1 receptor causes the depolarization, but in other tissues, the H1 receptor is primarily coupled to Gq/11 proteins and stimulates phospholipase C (18). Whether this leads to activation of inward cationic currents, and/or suppression of K+ currents, is not clear at this time.

In our studies, application of the H2 agonist dimaprit or application of histamine in the presence the H1 receptor antagonist mepyramine caused a membrane hyperpolarization and a decline in the generation of spontaneous action potentials. The H2 receptor is a G protein-linked receptor coupled to the adenylate cyclase signal transduction pathway (18). We (29, 30) have previously demonstrated that activation of this pathway in gallbladder smooth muscle by calcitonin gene-related peptide or forskolin results in an increased KATP conductance, which leads to a membrane hyperpolarization. Data presented in the current study indicate that the H2 receptor-mediated membrane hyperpolarization also involves the activation of the KATP current. The KATP channel blocker glibenclamide suppressed hyperpolarizations that were activated by the H2 agonist dimaprit. Furthermore, dimaprit activated a glibenclamide-sensitive current in isolated smooth muscle cells. These data represent the first evidence that activation of the H2 receptor can activate the opening of KATP channels.

The role of the mast cell in gallbladder pathophysiology is not fully understood. Immunohistochemical studies (14) have demonstrated that histamine-immunoreactive mast cells are present within both the mucosa and muscularis/serosal layers of the guinea pig gallbladder. Because gallbladder nerves are not immunoreactive for histamine (Jennings and Mawe, unpublished observations), mast cells are the primary source of endogenous histamine within the gallbladder. It is quite possible that cholecystitis is associated with mast cell degranulation. Results from this study support the concept that endogenously released histamine can have a direct effect on gallbladder smooth muscle. Application of the mast cell degranulator compound 48/80 resulted in a depolarization of gallbladder smooth muscle that was associated with an increase in smooth muscle action potential frequency. Application of compound 48/80 in the presence of mepyramine led to a decrease in the frequency of spontaneous action potentials with little change in the membrane potential of gallbladder smooth muscle. Histological studies, using toluidine blue staining of mast cell whole mount preparations of the guinea pig gallbladder, demonstrated that compound 48/80 caused degranulation of the mast cells. Intracellular recordings demonstrated that the amplitude of the depolarization resulting from application of compound 48/80 correlates with a histamine concentration of ~10 µM, based on the concentration-effect curve for histamine. Furthermore, 10 µM histamine in the presence of mepyramine caused a decrease in action potential frequency with little change in membrane potential. Together, these data indicate that gallbladder smooth muscle can be exposed to histamine concentrations of at least 10 µM after mast cell degranulation; however, experimental conditions involving circulating solutions may result in an underestimate of the histamine concentrations that are involved. Local concentrations of histamine in tissues after mast cell degranulation can be as high as 0.1-1 mM (7).

Ganglia

Histamine has been shown to elicit direct and presynaptic actions in enteric and sympathetic ganglia (4, 5, 10, 25-27). In the gut, histamine has a direct, H2 receptor-mediated, excitatory effect on enteric neurons, involving a prolonged depolarization associated with the generation of action potentials. Histamine also acts presynaptically, on H3 receptors, in enteric ganglia to inhibit transmitter release. Data from the current study indicate that histamine receptors do not exist in gallbladder ganglia because histamine had no detectable effect, either pre- or postsynaptically, in these ganglia. This was an unexpected discovery considering the actions of histamine in other autonomic ganglia and the finding that gallbladder ganglia are an important target for various hormones, neuroactive compounds, and immune-mediating agents, such as cholecystokinin, norepinephrine, substance P, calcitonin gene-related peptide, and PGE2 (11, 13, 21-24). On the other hand, the lack of a neuronal response in gallbladder is consistent with the previously reported finding (14) that R-alpha -methylhistamine did not alter responses of guinea pig gallbladder muscle strips to electric field stimulation.

Concluding Remarks

In conclusion, histamine elicits its effects on gallbladder motility through direct actions on gallbladder smooth muscle. The smooth muscle response represents a balance between a H1 receptor-mediated excitatory response and a H2 receptor-mediated inhibitory response. Furthermore, we report here the novel finding that activation of the H2 receptor can lead to the activation of a KATP current and that this is the mechanism for the H2 receptor-mediated membrane hyperpolarization of gallbladder smooth muscle. Finally, histamine released from mast cells produces a depolarization of gallbladder smooth muscle, indicating that mast cell degranulation could contribute to pathophysiological changes in gallbladder tone that are associated with cholecystitis.


    ACKNOWLEDGEMENTS

We thank Michelle Anderson for excellent technical assistance with the toluidine blue staining experiments.


    FOOTNOTES

This work was supported by National Institutes of Health Grants NS-26995, DK-45410, and HL-44455.

Address for reprint requests and other correspondence: G. M. Mawe, Given C 423, Dept. of Anatomy and Neurobiology, Univ. of Vermont, Burlington, VT 05405 (E-mail: gmawe{at}zoo.uvm.edu).

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. §1734 solely to indicate this fact.

Received 26 January 2000; accepted in final form 31 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Babe, KS, and Serafin WE. Histamine, bradykinin and their antagonists. In: Goodman and Gilman's The Pharmacological Basis of Therapeutics, edited by Hardman J, and Limbird L.. New York: McGraw-Hill, 1996, p. 581-600.

2.   Burdyga, TV, and Wray S. The effect of inhibition of myosin light chain kinase by wortmannin on intracellular [Ca2+], electrical activity and force in phasic smooth muscle. Pflügers Arch 436: 801-803, 1998[ISI][Medline].

3.   Burke, EP, Gerthoffer WT, Sanders KM, and Publicover NG. Wortmannin inhibits contraction without altering electrical activity in canine gastric smooth muscle. Am J Physiol Cell Physiol 270: C1405-C1412, 1996[Abstract/Free Full Text].

4.   Christian, EP, Undem BJ, and Weinreich D. Endogenous histamine excites neurones in the guinea-pig superior cervical ganglion in vitro. J Physiol (Lond) 409: 297-312, 1989[Abstract].

5.   Christian, EP, and Weinreich D. Presynaptic histamine H1 and H3 receptors modulate sympathetic ganglionic synaptic transmission in the guinea-pig. J Physiol (Lond) 457: 407-430, 1992[Abstract].

6.   Coruzzi, G, Pozzoli C, Poli E, Coppelli G, and Bertaccini G. Effects of histamine H2 receptor agonists and antagonists on the isolated guinea pig gallbladder. Fundam Clin Pharmacol 13: 84-90, 1999[ISI][Medline].

7.   Dale, MM, and Foreman JC. Histamine as a mediator of allergic and inflammatory reactions. In: Textbook of Immunopharmacology, edited by Dale MM, Foreman JC, and Fan T-PD.. London: Blackwell Scientific Publications, 1994, p. 123-130.

8.   Del Valle, J, and Gantz I. Novel insights into histamine H2 receptor biology. Am J Physiol Gastrointest Liver Physiol 273: G987-G996, 1997[Abstract/Free Full Text].

9.   Firth, TA, Mawe GM, and Neslon MT. Pharmacology and modulation of KATP channels by protein kinase C and phosphatases in gallbladder smooth muscle. Am J Physiol Cell Physiol 278: C1031-C1037, 2000[Abstract/Free Full Text].

10.   Frieling, T, Cooke HJ, and Wood JD. Histamine receptors on submucous neurons in guinea pig colon. Am J Physiol Gastrointest Liver Physiol 264: G74-G80, 1993[Abstract/Free Full Text].

11.   Gokin, AP, Jennings LJ, and Mawe GM. Actions of calcitonin gene-related peptide (CGRP) in guinea pig gallbladder ganglia. Am J Physiol Gastrointest Liver Physiol 271: G876-G883, 1996[Abstract/Free Full Text].

12.   Hanyu, N, Dodds WJ, Layman RD, Hogan WJ, and Colton DG. Effect of two new cholecystokinin antagonists on gallbladder emptying in opossums. Am J Physiol Gastrointest Liver Physiol 260: G258-G264, 1991[Abstract/Free Full Text].

13.   Jennings, LJ, and Mawe GM. PGE2 hyperpolarizes gallbladder neurons and inhibits synaptic potentials in gallbladder ganglia. Am J Physiol Gastrointest Liver Physiol 274: G493-G502, 1998[Abstract/Free Full Text].

14.   Jennings, LJ, Salido GM, Pozo MJ, Davison JS, Sharkey KA, Lea RW, and Singh J. The source and action of histamine in the isolated guinea-pig gallbladder. Inflamm Res 44: 447-453, 1995[ISI][Medline].

15.   Jennings, LJ, Xu QW, Firth TA, Nelson MT, and Mawe GM. Cholesterol inhibits spontaneous action potentials and calcium currents in guinea pig gallbladder smooth muscle. Am J Physiol Gastrointest Liver Physiol 277: G1017-G1026, 1999[Abstract/Free Full Text].

16.   Kaliner, M, and Metcalfe D. The Mast Cell in Health and Disease. New York: Dekker, 1993.

17.   Kaplan, GS, Bhutani VK, Shaffer TH, Tran N, Koslo RJ, and Wolfson MR. Gallbladder mechanics in newborn piglets. Pediatr Res 18: 1181-1184, 1984[Abstract].

18.   Leurs, R, Smit MJ, and Timmerman H. Molecular pharmacological aspects of histamine receptors. Pharmacol Ther 66: 413-463, 1995[ISI][Medline].

19.   Mack, AJ, and Todd JK. A study of human gall bladder muscle in vitro. Gut 9: 546-549, 1968[ISI][Medline].

20.   Mawe, GM. Intracellular recording from neurones of the guinea-pig gall-bladder. J Physiol (Lond) 429: 323-338, 1990[Abstract].

21.   Mawe, GM. The role of cholecystokinin in ganglionic transmission in the guinea-pig gall-bladder. J Physiol (Lond) 439: 89-102, 1991[Abstract].

22.   Mawe, GM. Noradrenaline acts as a presynaptic inhibitory neurotransmitter in ganglia of the guinea-pig gall-bladder. J Physiol (Lond) 461: 378-402, 1993.

23.   Mawe, GM. Tachykinins as mediators of slow EPSPs in guinea-pig gall-bladder ganglia. Involvement of neurokinin-3 receptors. J Physiol (Lond) 485: 513-524, 1995[Abstract].

24.   Mawe, GM, Gokin AP, and Wells DG. Actions of cholecystokinin and norepinephrine on vagal inputs to ganglionic cells in guinea pig gallbladder. Am J Physiol Gastrointest Liver Physiol 267: G1146-G1151, 1994[Abstract/Free Full Text].

25.   Nemeth, PR, Ort CA, and Wood JD. Intracellular study of effects of histamine on electrical behaviour of myenteric neurones in guinea-pig small intestine. J Physiol (Lond) 355: 411-425, 1984[Abstract].

26.   Tamura, K, Palmer JM, and Wood JD. Presynaptic inhibition produced by histamine at nicotinic synapses in enteric ganglia. Neuroscience 25: 171-179, 1988[ISI][Medline].

27.   Tokimasa, T, and Akasu T. Histamine H2 receptor mediates postsynaptic excitation and presynaptic inhibition in submucous plexus neurons of the guinea-pig. Neuroscience 28: 735-744, 1989[ISI][Medline].

28.   Wise, WE, Jr, LaMorte WW, Gaca JM, Schoetz DJ, Jr, Birkett DH, and Williams LF, Jr. Reciprocal H1- and H2-histamine receptors in guinea pig gallbladder. J Surg Res 33: 146-150, 1982[ISI][Medline].

29.   Zhang, L, Bonev AD, Mawe GM, and Nelson MT. Protein kinase A mediates activation of ATP-sensitive K+ currents by CGRP in gallbladder smooth muscle. Am J Physiol Gastrointest Liver Physiol 267: G494-G499, 1994[Abstract/Free Full Text].

30.   Zhang, L, Bonev AD, Nelson MT, and Mawe GM. Ionic basis of the action potential of guinea pig gallbladder smooth muscle cells. Am J Physiol Cell Physiol 265: C1552-C1561, 1993[Abstract/Free Full Text].

31.   Zhang, L, Bonev AD, Nelson MT, and Mawe GM. Activation of ATP-sensitive potassium currents in guinea-pig gall-bladder smooth muscle by the neuropeptide CGRP. J Physiol (Lond) 478: 483-491, 1994[Abstract].


Am J Physiol Gastrointest Liver Physiol 279(3):G622-G630
0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (5)
Google Scholar
Articles by Hemming, J. M.
Articles by Mawe, G. M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Hemming, J. M.
Articles by Mawe, G. M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online