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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 H(2) antagonist ranitidine, the response to histamine was potentiated. Activation of H(2) receptors caused membrane hyperpolarization and elimination of spontaneous action potentials. The H(2) response was attenuated by the ATP-sensitive K(+) (K(ATP)) 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 H(3) 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 H(1) 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 H(1) (excitatory) and H(2) (inhibitory) receptors, with the H(2) receptor-mediated response involving the activation of K(ATP) channels.

Elevated cholesterol decreases agonist-induced contractility and enhances stone formation in the gallbladder. The current study was conducted to determine if and how the electrical properties and ionic conductances of gallbladder smooth muscle are altered by elevated cholesterol. Cholesterol was delivered as a complex with cyclodextrin, and effects were evaluated with intracellular recordings from intact gallbladder and whole cell patch-clamp recordings from isolated cells. Cholesterol significantly attenuated the spontaneous action potentials of intact tissue. Furthermore, calcium-dependent action potentials and calcium currents were reduced in the intact tissue and in isolated cells, respectively. However, neither membrane potential hyperpolarizations induced by the ATP-sensitive potassium channel opener, pinacidil, nor voltage-activated outward potassium currents were affected by cholesterol. Hyperpolarizations elicited by calcitonin gene-related peptide were reduced by cholesterol enrichment, indicating potential changes in receptor ligand binding and/or second messenger interactions. These data indicate that excess cholesterol can contribute to gallbladder stasis by affecting calcium channel activity, whereas potassium channels remained unaffected. In addition, cholesterol enrichment may also modulate receptor ligand behavior and/or second messenger interactions.

Gallbladder prostaglandin E2 (PGE2) levels are significantly elevated in pathophysiological conditions, resulting in changes in gallbladder motility or secretion that may involve actions of the prostanoid in intramural ganglia. This study was undertaken to examine the effects of PGE2 on neurons of the intramural ganglia of the guinea pig gallbladder. Application of PGE2 by microejection or superfusion elicited a complex triphasic change in the resting membrane potential (RMP). For example, application of PGE2 by microejection (100 microM) resulted in a brief hyperpolarization (mean duration 11.1 +/- 1.3 s), followed by a mid-phase repolarization toward or above RMP (mean duration 50.7 +/- 8.1 s), and finally a long-lasting hyperpolarization (mean duration 157.3 +/- 36.7 s). Associated with these PGE2-evoked alterations in RMP were changes in input resistance measured via injection of hyperpolarizing current pulses. An examination of the action potential afterhyperpolarization (AHP) during the PGE2-evoked response revealed an attenuation of both the amplitude and duration of the AHP. However, only a slight increase in excitability of gallbladder neurons in the presence of PGE2 was evident in response to depolarizing current pulses, and PGE2 did not cause the cells to fire spontaneous action potentials. Application of PGE2 reduced the amplitudes of both fast and slow excitatory synaptic potentials. These results suggest that increased prostaglandin production may decrease ganglionic output and therefore contribute to gallbladder stasis.

Background/aims: Gallbladder neurons are important governors of gallbladder function. In animal models, gallbladder ganglia can be regulated both by neural and hormonal inputs. The purpose of this study was to demonstrate the feasibility of obtaining recordings from human gallbladder neurons., Methods: Human gallbladders (n = 33) were bathed in oxygenated Krebs solution (37 degrees C) containing the vital fluorescent stain 4-Di-2-ASP to localize the ganglia. Cells were characterized using conventional intracellular recording techniques., Results: The mean resting membrane potential of human gallbladder neurons was -51.2 +/- 1.8 mV (n = 11). Depolarizing current pulses elicited only 1-4 spikes regardless of the amplitude or duration of the stimulus. Afterspike hyperpolarizations had a mean duration of 144.5 +/- 19.2 ms (n = 10). Anodal break excitation was not recorded with hyperpolarizing current pulses. Fiber tract stimulation elicited fast excitatory postsynaptic potentials in all neurons tested., Conclusion: Intracellular recordings of human gallbladder neurons utilizing 4-Di-2-ASP are thus feasible, but are very problematic due to the density of connective tissue overlying the ganglia. As human and guinea pig gallbladder neurons have similar basic electrical properties, the guinea pig may be an appropriate model for further electrophysiological studies into gallbladder disease.

The muscle and epithelial tissues of the gallbladder are regulated by a ganglionated plexus that lies within the wall of the organ. Although these ganglia are derived from the same set of precursor neural crest cells that colonize the gut, they exhibit structural, neurochemical and physiological characteristics that are distinct from the myenteric and submucous plexuses of the enteric nervous system. Structurally, the ganglionated plexus of the guinea pig gallbladder is comprised of small clusters of neurons that are located in the outer wall of the organ, between the serosa and underlying smooth muscle. The ganglia are encapsulated by a shell of fibroblasts and a basal lamina, and are devoid of collagen. Gallbladder neurons are rather simple in structure, consisting of a soma, a few short dendritic processes and one or two long axons. Results reported here indicate that all gallbladder neurons are probably cholinergic since they all express immunoreactivity for choline acetyltransferase. The majority of these neurons also express substance P, neuropeptide Y, and somatostatin, and a small remaining population of neurons express vasoactive intestinal peptide (VIP) immunoreactivity and NADPH-diaphorase enzymatic activity. We report here that NADPH-diaphorase activity, nitric oxide synthase immunoreactivity, and VIP immunoreactivity are expressed by the same neurons in the gallbladder. Physiological studies indicate that the ganglia of the gallbladder are the site of action of the following neurohumoral inputs: 1) all neurons receive nicotinic input from vagal preganglionic fibers; 2) norepinephrine released from sympathetic postganglionic fibers acts presynaptically on vagal terminals within gallbladder ganglia to decrease the release of acetylcholine from vagal terminals; 3) substance P and calcitonin gene-related peptide, which are co-expressed in sensory fibers, cause prolonged depolarizations of gallbladder neurons that resemble slow EPSPs; and 4) cholecystokinin (CCK) acts presynaptically within gallbladder ganglia to increase the release of acetylcholine from vagal terminals. Results reported here indicate that hormonal CCK can readily access gallbladder ganglia, since there is no evidence for a blood-ganglionic barrier in the gallbladder. Taken together, these results indicate that gallbladder ganglia are not simple relay stations, but rather sites of complex modulatory interactions that ultimately influence the functions of muscle and epithelial cells in the organ.

The actions of calcitonin gene-related peptide (CGRP) have been determined from intracellular recordings obtained from gallbladder neurons in intact whole mount preparations. In most cells, pressure microejection of CGRP elicited a slow, monophasic depolarization, 4 mV in amplitude, that was associated with a decrease in input resistance and increased excitability. The CGRP-induced depolarization was attenuated in a low-Na+ solution and had a reversal potential of -8 mV. In 10% of the cells, microejection of CGRP elicited a biphasic response that was composed of a rapid transient depolarization followed by a slow depolarization that was similar to the monophasic response. Addition of CGRP (1-10 nM) to the bathing solution elicited a monophasic depolarization and desensitized the cells to applications of CGRP by microejection. Forskolin, applied either by microejection or bath application, also depolarized gallbladder neurons and produced cross-desensitization to CGRP. Responses to substance P were not enhanced by CGRP, and CGRP did not affect fast synaptic responses. It is concluded that CGRP may contribute to a local axon reflex response in gallbladder ganglia.

We have investigated the effects of histamine on motility of the gallbladder and characterized the receptor types involved. Histamine and the histamine H1-receptor agonist, 2-thiazolylethylamine (2-TEA) contracted the isolated guinea-pig gallbladder strip in a dose dependent manner. The contractile response to histamine was shifted to the right by the H1-receptor antagonist, mepyramine. In pre-contracted gallbladder strips, the H2-receptor agonist dimaprit reduced the tension generated in a dose dependent fashion. The histamine H2-receptor antagonist, ranitidine shifted the histamine concentration effect curve to the left and attenuated the dose dependent relaxations elicited at high concentrations. The histamine H3-receptor agonist, (R)-alpha-methylhistamine (RMHA) elicited dose dependent contraction of the tissue which was significantly inhibited in the presence of mepyramine. The effects of electrical field stimulation (EFS) on the strips were not significantly altered by the presence of RMHA (10(-10) - 10(-7) M) indicating little pre-synaptic H3 activity in this tissue. Histamine immunoreactivity (IR) was detected in gallbladder whole mount preparations of the mucosa and the muscularis/serosa. The histamine IR appeared cell bound in cells of varying morphological characteristics but no IR was detected in nerve fibres or cell bodies (ganglia). Alcian blue staining was consistent with the distribution of histamine IR cells as mast cells. The results indicate that histamine is distributed in the guinea-pig gallbladder and it can regulate contractile activity via activation of H1 and H2 but not H3 receptors.