Anthony P. Pagano

Ranitidine and Nizatidine Stimulate Antral Smooth Muscle Contractility via Excitatory Cholinergic Mechanisms

Histamine type 2 receptor antagonists (H2RAs)have been found to alter gastric motility. The aims ofthis study were to determine if H2RAs affect antralcontractility in vitro and the mechanism of this effect. Guinea pig antral muscle strips werepinned in an organ bath after removing the mucosa, andcircular muscle tension was measured using an isometricforce transducer. Gastric myocytes were isolated from guinea pig stomach using collagenasedigestion, and cell lengths were measured using an imageanalysis system. In muscle strips, ranitidine andnizatidine increased the amplitude of spontaneous phasic antral contractions in aconcentration-dependent fashion with thresholdconcentrations of 5 μM. The order of potency for theH2RAs was ranitidine = nizatidine ≫ cimetidine >famotidine. The contractile effects of ranitidine and nizatidine werereduced, but not abolished, by tetrodo- toxin andomega-conotoxin GVIA and nearly abolished by atropine.In isolated cells, ranitidine and nizatidine, but notfamotidine or cimetidine, induced concentration-dependentcell shortening, with maximal shortening at 10 μM.These contractile effects of ranitidine and nizatidinein isolated cells were inhibited by atropine. Ranitidine and nizatidine increase antral contractility;this effect appears to be mediated by an interactionbetween ranitidine and nizatidine on cholinergicpathways with both direct effects on smooth musclecholinergic receptors and indirect effects by increasingcholinergic neurotransmission.

PACAP and VIP inhibit pyloric muscle through VIP/PACAP-preferring receptors

UNLABELLED Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with structural homology to vasoactive intestinal polypeptide (VIP). Two receptor types for PACAP have been described: PACAP preferring receptors are selective for PACAP; whereas VIP/PACAP preferring receptors have similar affinity for both PACAP and VIP. Both VIP and PACAP are present in enteric nerves at the pylorus. VIP is known to exert inhibitory effects on pyloric muscle; the effect of PACAP is unknown. The aims of this study were to determine the effect of PACAP on pyloric muscle and to characterize the PACAP receptor. METHODS Rabbit pyloric muscle strips were cut parallel to circular muscle fibres and placed in muscle baths. The effect of PACAP and VIP were quantitated as percent of basal motility index (MI). RESULTS PACAP-27, PACAP-38, and VIP had dose dependent inhibitory effects on the spontaneous phasic contractions of the pylorus. The PACAP-27- induced relaxation was inhibited by the PACAP receptor antagonist PACAP6-27, but was not affected by tetrodotoxin. VIP also had dose dependent inhibitory effects on pyloric muscle. The VIP relaxation was inhibited by PACAP6-27, but not affected by tetrodotoxin. CONCLUSIONS These studies indicate that, similar to VIP, PACAP inhibits pyloric muscle. The inhibitory effect of the PACAP receptor antagonist on both PACAP and VIP-induced relaxation suggest that PACAP and VIP act at the same receptor, a VIP/PACAP preferring receptor.

Effect of modulating voltage-dependent calcium channels on cholecystokinin and acetylcholine-induced contractions of the guinea pig gallbladder.

UNLABELLED The aim of this study was to investigate the role of extracellular Ca2+ utilization in cholecystokinin (CCK) and acetylcholine-induced guinea pig gallbladder contractions by using agents that modulate influx of extracellular Ca2+ through voltage-dependent calcium channels. METHODS Guinea pig gallbladder muscle strips were studied isometrically at Lmax in vitro. RESULTS (1) Acetylcholine and CCK caused dose-dependent contractions, with EDmax of 10(-4) and 10(-6) M, respectively. (2) Preventing influx of extracellular Ca2+ by incubation in Ca(2+)-free/0.1 mM EGTA solution inhibited the acetylcholine (10(-4) M)-induced contraction by 60 +/- 3% compared to only 46 +/- 5% (P < 0.05) for CCK (10(-6) M)-induced contraction. (3) Nifedipine (3 microM) inhibited the response to acetylcholine (10(-4) M) by 54 +/- 3%, compared to only 34 +/- 3% (P < 0.01) for CCK (10(-6) M). (4) Bay K 8644 (10(-7) M) significantly increased (P < 0.05) the contractile responses to low doses of each agonist: acetylcholine (10(-6) M) by 121 +/- 44% and CCK (10(-9) M) by 94 +/- 31%, but had no effect on the contraction to the EDmax of each agonist. CONCLUSIONS These studies demonstrate: (1) acetylcholine and CCK cause guinea pig gallbladder contraction by both intracellular Ca2+ release and influx of extracellular Ca2+ through voltage-dependent calcium channels; (2) the CCK-induced contraction is more dependent on intracellular Ca2+ than is acetylcholine; and (3) acetylcholine and CCK-induced contractions can by modulated by manipulating influx of extracellular Ca2+ through voltage-dependent calcium channels.

Electric Field Stimulation-Induced Guinea Pig Gallbladder Contractions (Role of Calcium Channels in Acetylcholine Release)

Gallbladder motility is modulated by intrinsiccholinergic neurons. The aims of this study were todetermine: (1) the effect of electric field stimulation(EFS) on guinea pig gallbladder smooth muscle, and (2) the role of calcium channels inmediating neurotransmitter release. Gallbladder musclestrips were studied isometrically in vitro. EFS (1-16Hz, 100 V, 0.5-msec pulse width, 30-sec train duration) was used to activate the intrinsic nerves.Exogenous acetylcholine was also used to directlystimulate the smooth muscle. EFS produced afrequency-dependent contractile response that wascompletely abolished by tetrodotoxin. EFS-induced contractions at 16Hz were suppressed by 84 ± 4% with atropine,whereas hexamethonium had no effect. The L-type calciumchannel blocker, nifedipine, reduced EFS contractions by 51 ± 4%, whereas it reduced contractionsto acetylcholine by only 11 ± 5%. The N-typecalcium channel blocker, omega-conotoxin GVIA, reducedEFS-induced contractions by 22 ± 9%, but did notaffect acetylcholine-induced contractions. EFS-induced contractions of theguinea pig gallbladder are primarily mediated byactivation of postganglionic cholinergic neurons. Theacetylcholine release from these cholinergic neurons is regulated by L- and N-type calcium channels.The inhibitory effect of calcium channel blockers on thegallbladder seen in vivo may be in part related toinhibition of acetylcholine release from the intrinsic cholinergic nerves of thegallbladder.

Pharmacological analysis of receptors for bombesin-related peptides on guinea pig gallbladder smooth muscle

The aim of this study was to characterize the receptor(s) for bombesin (BN) and its homologues (gastrin releasing peptide, GRP; neuromedin B, NMB; neuromedin C, NMC) in guinea pig gallbladder muscle strips. Dose-dependent contractions were induced by all peptides tested (potency: BN = GRP > NMC > NMB, but with similar efficacy: BN = GRP = NMC = NMB). The contractions were resistant to tetrodotoxin, atropine, phentolamine, and propranolol. BN tachyphylaxis (1 microM) abolished subsequent contractile responses to BN, GRP and NMC; and partially antagonized the response to NMB (66 +/- 7% inhibition). NMB tachyphylaxis (10 microM) markedly inhibited subsequent contractile responses to NMB (78 +/- 5%); and partially antagonized the contractile response to BN (36 +/- 4%), GRP (31 +/- 12%) and NMC (22 +/- 2%). At 1 microM, both [D-Phe6, Des-Met14]-BN(6-14) ethylamide and ICI 216, 140, two BN receptor antagonists, reduced the contractile actions of BN (82 +/- 4% and 59 +/-8% inhibition, respectively), GRP (75 +/- 11% and 45 +/- 5%), and NMC (73 +/- 9% and 51 +/- 6%) while having no marked effect on NMB contractions. Our pharmacological approaches (receptor tachyphylaxis and differential antagonism) provide support for two types of receptors for BN-like peptides on guinea pig gallbladder smooth muscle: a GRP-preferring receptor and a NMB-preferring receptor.

Investigation of Endogenous of Guinea Pig Gallbladder Using Nicotinic Agonist Stimulation

Gallbladder motility is modulated by intrinsicnerves, the identities of which are not wellestablished. The aim of this study was to determine theeffect of nicotinic receptor stimulation of intrinsic nerves on gallbladder muscle contractility.Guinea pig gallbladder muscle strips were studied invitro . Histamine 1 μM was used to increase baselinetone. The nicotinic receptor agonist,1,1-dimethyl-4-phenylpiperazinium (DMPP), produced a biphasic responsecharacterized by an initial transient contractionfollowed by a sustained relaxation. The initialcontraction was inhibited by the neural blockertetrodotoxin, the nicotinic antagonist hexamethonium, and the muscarinicantagonist atropine, but not by a substance P receptorantagonist or a bombesin receptor antagonist. Therelaxation response to DMPP was not affected bytetrodotoxin, but was reduced by hexamethonium andomega-conotoxin GVIA, an inhibitor of neurotransmitterrelease. The relaxation response was reduced by thenitric oxide synthase inhibitor L-NAME, but not by avasoactive intestinal peptide antagonist or propranolol.DMPP produces a biphasic response in the guinea piggallbladder. The initial contractile response ismediated by nicotinic receptors on the cell body or axon of cholinergic nerves. The relaxation responseappears to result, in part, from activation of nicotinicreceptors on nerve terminals of nitric oxide-releasingnerves. These results suggest nicotinic receptors have heterogeneity in location depending onexcitatory or inhibitory neuronal function.

Subtypes of muscarinic receptors regulating gallbladder cholinergic contractions

Parkman, Henry P., Anthony P. Pagano, and James P. Ryan. Subtypes of muscarinic receptors regulating gallbladder cholinergic contractions. Am. J. Physiol. 276 (Gastrointest. Liver Physiol. 39): G1243–G1250, 1999.—The aim of this study was to determine the functional role of muscarinic receptor subtypes regulating gallbladder cholinergic contractions. Electrical field stimulation (EFS; 16 Hz) produced contractile responses of guinea pig gallbladder muscle strips in vitro that were inhibited by 1 μM tetrodotoxin (2 6 2% of control) and 1 μM atropine (1 6 1% of control), indicating activation of intrinsic cholinergic nerves. Exogenous ACh (5 μM)-induced contractions were inhibited by atropine (1 6 1% of control) but not tetrodotoxin (102 6 1% of control), indicating a direct effect on smooth muscle. The M1 receptor antagonist pirenzepine (10 nM) had no effect on ACh-induced contractions but inhibited EFS-induced contractions by 11 6 3%. The M2 antagonist methoctramine (10 nM) had no effect on ACh-induced contractions but augmented EFS-induced contractions by 5 6 2%. The M3 antagonist 4-DAMP (10 nM) inhibited ACh-induced contractions by 14 6 4% and EFSinduced contractions by 22 6 5%. In conclusion, specific M1, M2, and M3 receptors modulate gallbladder muscle contractions by regulating ACh release from cholinergic nerves and mediating the contraction. Cholinergic contractions are mediated by M3 receptors directly on the smooth muscle. M2 receptors are on cholinergic nerves and function as prejunctional inhibitory autoreceptors. M1 receptors are on cholinergic nerves and function as prejunctional facilitatory autoreceptors.