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1.
Gut ; 57(12): 1654-64, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18682519

ABSTRACT

BACKGROUND: Acid in the oesophageal lumen is often sensed as heartburn. It was hypothesised that luminal CO(2), a permeant gas, rather than H(+), permeates through the epithelium, and is converted to H(+), producing an afferent neural signal by activating chemosensors. METHODS: The rat lower oesophageal mucosa was superfused with pH 7.0 buffer, and pH 1.0 or pH 6.4 high CO(2) (P(CO2) = 260 Torr) solutions with or without the cell-permeant carbonic anhydrase (CA) inhibitor methazolamide (MTZ, 1 mM), the cell-impermeant CA inhibitor benzolamide (BNZ, 0.1 mM), the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (CPZ, 0.5 mM) or the acid-sensing ion channel (ASIC) inhibitor amiloride (0.1 mM). Interstitial pH (pH(int)) was measured with 5',6'-carboxyfluorescein (5 mg/kg intravenously) loaded into the interstitial space, and blood flow was measured with laser-Doppler. RESULTS: Perfusion of a high CO(2) solution induced hyperaemia without changing pH(int), mimicking the effect of pH 1.0 perfusion. Perfused MTZ, BNZ, CPZ and amiloride all inhibited CO(2)-induced hyperaemia. CA XIV was expressed in the prickle cells, with CA XII in the basal cells. TRPV1 was expressed in the stratum granulosum and in the muscularis mucosa, whereas all ASICs were expressed in the prickle cells, with ASIC3 additionally in the muscularis mucosa. CONCLUSIONS: The response to CO(2) perfusion suggests that CO(2) diffuses through the stratum epithelium, interacting with TRPV1 and ASICs in the epithelium or in the submucosa. Inhibition of the hyperaemic response to luminal CO(2) by CA, TRPV1 and ASIC inhibitors implicates CA and these chemosensors in transduction of the luminal acid signal. Transepithelial CO(2) permeation may explain how luminal H(+) equivalents can rapidly be transduced into hyperaemia, and the sensation of heartburn.


Subject(s)
Carbon Dioxide/metabolism , Esophagus/metabolism , Gastroesophageal Reflux/metabolism , Hyperemia/metabolism , TRPV Cation Channels/metabolism , Acid Sensing Ion Channels , Amiloride/pharmacology , Animals , Benzolamide/pharmacology , Capsaicin/analogs & derivatives , Capsaicin/antagonists & inhibitors , Carbon Dioxide/pharmacokinetics , Carbonic Anhydrase Inhibitors/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Esophageal pH Monitoring , Esophagus/blood supply , Gastroesophageal Reflux/complications , Hyperemia/chemically induced , Male , Methazolamide/pharmacology , Mucous Membrane/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Rats , Rats, Sprague-Dawley , Sodium Channels/metabolism , TRPV Cation Channels/antagonists & inhibitors
2.
J Physiol Pharmacol ; 58(4): 767-91, 2007 Dec.
Article in English | MEDLINE | ID: mdl-18195487

ABSTRACT

We measured villous cell intracellular pH (pH(i)) and solute diffusion between the bathing media and the epithelial cells in stripped, chambered mouse duodenum. Apical perfusion of a high CO2 solution rapidly acidified the upper villous cells with recovery after its removal. Apical zoniporide (ZP) enhanced CO(2)-induced acidification. Serosal ZP, dimethylamiloride (DMA) or stilbene anion transport inhibitors failed to alter CO(2)-induced acidification, whereas serosal high CO(2) buffer acidified the upper villous cells. Serosal 5-hydroxytryptamine rapidly acidified the upper villous cells. All serosally-perfused fluorescent compounds stained the crypt area, but not the villi or villous cells. In contrast, intravenous carboxyfluorescein quickly diffused into the interstitial space of the entire mucosa, and mucosally perfused fluorescent compound rapidly penetrated the epithelial cell layer. In muscle-stripped duodenum mounted in a small-aperture perfusion chamber, serosal solutes can readily diffuse only to the crypt cell region, whereas access to the villous epithelial cells is diffusion-limited. In contrast, rapid villous cell responses to serosally applied solutes are best explained by neural reflexes. Limited viability of the villous cells and impaired structural stability of the villi further limit long-term, villous cell functional studies of mucosal preparations mounted in small aperture diffusion chambers.


Subject(s)
Duodenum/metabolism , Intestinal Mucosa/metabolism , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Anions , Biological Transport/drug effects , Carbon Dioxide/metabolism , Diffusion , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Fluoresceins/metabolism , Fluorescent Dyes/metabolism , Guanidines/pharmacology , Hydrogen-Ion Concentration , Male , Mice , Mice, Inbred C57BL , Pyrazoles/pharmacology , Serotonin/pharmacology , Sodium-Bicarbonate Symporters/antagonists & inhibitors , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Solutions , Stilbenes/metabolism
3.
Mol Cell Neurosci ; 20(4): 695-711, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12213449

ABSTRACT

UNLABELLED: Opioid peptides have been detected in the auditory and vestibular efferent neurons where they colocalize with the major neurotransmitter, acetylcholine. We investigated the function of opioids to modulate neurotransmission mediated by hair cell's alpha9/alpha10-containing nicotinic acetylcholine receptors (alpha9/alpha10nAChRs). The endogenous opioid peptides, endomorphin-1 (mu agonist) and dynorphin B (kappa agonist), but not a delta agonist [D-Pen2,D-Pen-5]enkephalin, inhibited the acetylcholine-evoked currents in frog saccular hair cells and rat inner hair cells. This inhibition was noncompetitive, voltage-independent, and was accompanied by an acceleration of the rate of current decay. Selective mu- and kappa-opioid receptor antagonists did not block the inhibition, although partial reduction by naloxone was observed. All opioid antagonists tested also reduced the acetylcholine response. Endomorphin-1 and dynorphin B inhibited the acetylcholine-evoked currents in alpha9/alpha10-expressing Xenopus oocytes. Because oocytes lack opioid receptors, it provides strong evidence for the direct interaction of opioid peptides with alpha9/alpha10nAChR. CONCLUSION: alpha9/alpha10nAChR is a target for modulation by endomorphin-1 and dynorphin B, efferent cotransmitters in the inner ear.


Subject(s)
Dynorphins/physiology , Ear, Inner/physiology , Endorphins/physiology , Neurotransmitter Agents/physiology , Oligopeptides/physiology , Receptors, Nicotinic/metabolism , Acetylcholine/pharmacology , Animals , Anura , Cochlea/drug effects , Cochlea/physiology , Dynorphins/pharmacology , Electric Conductivity , Endorphins/pharmacology , Enkephalin, D-Penicillamine (2,5)-/pharmacology , Hair Cells, Auditory/drug effects , Hair Cells, Auditory/physiology , In Vitro Techniques , Narcotic Antagonists , Oligopeptides/pharmacology , Oocytes/drug effects , Oocytes/metabolism , Protein Isoforms/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/physiology , Saccule and Utricle/cytology , Saccule and Utricle/drug effects , Saccule and Utricle/physiology , Synapses/drug effects , Synapses/physiology , Xenopus laevis
4.
Dig Dis Sci ; 47(5): 967-73, 2002 May.
Article in English | MEDLINE | ID: mdl-12018922

ABSTRACT

Gastroduodenal mucus may play a critical role in defending the epithelium from luminal acid and in the creation of a microenvironment suitable for H. pylori. We measured transmucus permeation of H+, HCO3-, and CO2 with an in vitro perfusion chamber through freshly harvested or partially purified porcine gastric mucin. pH and CO2 concentrations were measured with selective ion electrodes; HCO3- and CO2 concentrations were derived. Viscosity was measured by rotational microviscometry. Mucin viscosity was directly related to concentration. There was a large variation in viscosity among native mucus from antrum, corpus, and duodenum. The highest viscosity was found in the antral mucus; duodenal mucus had the lowest. Diffusion coefficients of duodenal mucus for H+ and HCO3- were significantly lower than those from corpus and antrum. CO2 diffusion coefficients were invariant. In conclusion, despite large variations in viscosity, antral and corpus gastric mucus were similar in terms of ion diffusion. Surprisingly, the low viscosity duodenal mucus was a more potent barrier to ion diffusion than was gastric mucus. Consequently, duodenal mucus may play a more important role in inhibiting ion diffusion than its gastric counterpart.


Subject(s)
Bicarbonates/metabolism , Carbon Dioxide/metabolism , Duodenum/metabolism , Gastric Mucosa/metabolism , Hydrogen/metabolism , Intestinal Mucosa/metabolism , Animals , Diffusion , Hydrogen-Ion Concentration , In Vitro Techniques , Mucins/metabolism , Pyloric Antrum/metabolism , Swine , Viscosity
5.
J Clin Invest ; 108(12): 1807-16, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11748264

ABSTRACT

Secretion of bicarbonate from epithelial cells is considered to be the primary mechanism by which the duodenal mucosa is protected from acid-related injury. Against this view is the finding that patients with cystic fibrosis, who have impaired duodenal bicarbonate secretion, are paradoxically protected from developing duodenal ulcers. Therefore, we hypothesized that epithelial cell intracellular pH regulation, rather than secreted extracellular bicarbonate, was the principal means by which duodenal epithelial cells are protected from acidification and injury. Using a novel in vivo microscopic method, we have measured bicarbonate secretion and epithelial cell intracellular pH (pH(i)), and we have followed cell injury in the presence of the anion transport inhibitor DIDS and the Cl(-) channel inhibitor, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). DIDS and NPPB abolished the increase of duodenal bicarbonate secretion following luminal acid perfusion. DIDS decreased basal pH(i), whereas NPPB increased pH(i); DIDS further decreased pH(i) during acid challenge and abolished the pH(i) overshoot over baseline observed after acid challenge, whereas NPPB attenuated the fall of pH(i) and exaggerated the overshoot. Finally, acid-induced epithelial injury was enhanced by DIDS and decreased by NPPB. The results support the role of intracellular bicarbonate in the protection of duodenal epithelial cells from luminal gastric acid.


Subject(s)
Bicarbonates/metabolism , Cytoprotection , Duodenum/metabolism , Gastric Acid/metabolism , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Hydrogen-Ion Concentration , Intestinal Mucosa/metabolism , Nitrobenzoates/pharmacology , Rats , Rats, Sprague-Dawley , Sodium-Bicarbonate Symporters/analysis , Staining and Labeling
6.
Acta Otorhinolaryngol Ital ; 21(3 Suppl 66): 16-23, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11677835

ABSTRACT

The problem of deciding, which, of several drug actions is the 'true' mechanism of action is an ancient and difficult one in pharmacology. Sometimes the problem is that each investigator may see his described action as through a tunnel, his vision not encompassing other possibilities. To help with the process of deciding the 'true' mechanism of action, the pharmacologist Philip Seeman has offered some guidelines. A few of his guidelines apply in the case of betahistine (BH). One is--does the drug have access to the proposed site of action? A second is--are the concentrations at which the drug acts at the candidate mechanism achievable in the patient? The three candidate sites of BH action are vascular, central nervous system and inner ear. There is obvious evidence that a vascular site as well as a vestibular end organs site are possible. There is also evidence that BH gains access to the central nervous system albeit achieving lower concentrations there than in plasma. Whether BH crosses the blood-labyrinthine barrier is not known. Then there is the guideline of similarity of clinically-achievable and experimental concentrations. Implicit in this guideline, without data to the contrary, is the assumption that the plasma concentration of a drug is roughly the concentration at the active site. This may or may not be true.


Subject(s)
Betahistine/pharmacology , Vasodilator Agents/pharmacology , Vestibule, Labyrinth/drug effects , Histamine/physiology , Humans , Vestibule, Labyrinth/physiology
7.
Acta Otorhinolaryngol Ital ; 21(3 Suppl 66): 8-15, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11677837

ABSTRACT

Betahistine has been used to treat several vestibular disorders of both central and peripheral origin. The objective of this work was to study the betahistine action mechanism at the vestibular end organs. Experiments were carried out in wild larval axolotl (Ambystoma tigrinum). Multiunit extracellular recordings were obtained from the semicircular canal nerve using a suction electrode. Betahistine (10 microM to 10 mM, n = 32) inhibited the basal spike discharge of the vestibular afferent neurons with an IC50 of 600 microM. To define the site of action of betahistine, its interactions with antagonists of nitric oxide sintethizing enzyme, cholinergic drugs, and excitatory amino acids were studied. Betahistine 1 mM (n = 5) was coadministered with NG-nitro-L-arginine 3 microM. The action of betahistine remained as in control experiments. Betahistine 1 mM reduced the excitatory action of carbachol (200 microM, n = 5) in a 30 +/- 3.4%. Cholinergic antagonists atropine (10 microM, n = 3) and d-tubocurarine (10 microM, n = 3) did not modify betahistine actions. Betahistine 1 mM also reduced kainic acid (10 microM, n = 4) excitatory action in 45.5 +/- 9.8%. These results corroborate that betahistine has a peripheral inhibitory action in the spike discharge of the afferent neurons in the vestibule. This action seems to involve neither NO production nor modifications in the release of acetylcholine from the efferent fibers. The inhibitory action of betahistine seems to be due to a postsynaptic binding site on the afferent neurons.


Subject(s)
Betahistine/pharmacology , Vasodilator Agents/pharmacology , Vestibule, Labyrinth/drug effects , Ambystoma , Animals , Vestibule, Labyrinth/physiology
8.
Acta Otorhinolaryngol Ital ; 21(3 Suppl 66): 24-30, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11677836

ABSTRACT

Betahistine is widely used in the treatment of peripheral and central vestibular disorders. Till now the anti-vertigo effect of the drug was though to be mainly due to an action of betahistine on inner ear or cerebral microcirculation or on some structures of the CNS, chiefly the vestibular nuclei. Vertigo, however is, in most cases, of peripheral origin but it remains unknown whether betahistine, or some of its metabolities, may directly affect the vestibular system at peripheral level. Pharmacokinetic studies have in fact demonstrated that betahistine is transformed, mainly at the hepatic level, in aminoethylpyridine (M1), hydroxyethylpyridine (M2) and, finally, in pyridylacetic acid (M3) which is excreted with the urine. All these substances are therefore present in the body fluids of subjects treated with betahistine, and thus might have pharmacological effects. The goal of the present study was to investigate whether betahistine or some of its metabolites could exert any effect on vestibular receptors. To this end, the effects of the drugs (10(-7)-10(-2) M) have been examined on frog semicircular canals, an animal model well suited for this purpose. The effects of betahistine and of its metabolites have been evaluated by recording ampullar receptor activity both at rest and during mechanical stimulation of the sensory organ. The results demonstrated that both betahistine and one of its metabolites, the aminoethylpyridine (M1), exert effects quite similar on ampullar receptors; both these substances in fact could reduce greatly ampullar receptor resting discharge but had scanty effects on mechanically-evoked responses. This observation might justify betahistine and possibly M1 anti-vertigo effects. In fact vertigo is normally due to uncontrolled changes in vestibular receptor resting discharge. It is therefore probable that any factor able to reduce vestibular receptor resting firing rate and, in consequence, its variations, may have, as final effect, an anti-vertigo action. The observation that betahistine and M1 have similar effects might be of some clinical interest. In fact, on the basis of our data, the hypothesis may be put forward that the anti-vertigo action of betahistine is at first achieved by betahistine itself and then sustained and prolonged in time by M1.


Subject(s)
Betahistine/metabolism , Betahistine/pharmacology , Vasodilator Agents/metabolism , Vasodilator Agents/pharmacology , Vestibule, Labyrinth/drug effects , Vestibule, Labyrinth/metabolism , Animals , Female , Male , Rana esculenta
9.
Am J Physiol Gastrointest Liver Physiol ; 280(6): G1083-92, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11352800

ABSTRACT

We studied the role of duodenal cellular ion transport in epithelial defense mechanisms in response to rapid shifts of luminal pH. We used in vivo microscopy to measure duodenal epithelial cell intracellular pH (pH(i)), mucus gel thickness, blood flow, and HCO secretion in anesthetized rats with or without the Na(+)/H(+) exchange inhibitor 5-(N,N-dimethyl)-amiloride (DMA) or the anion transport inhibitor DIDS. During acid perfusion pH(i) decreased, whereas mucus gel thickness and blood flow increased, with pH(i) increasing to over baseline (overshoot) and blood flow and gel thickness returning to basal levels during subsequent neutral solution perfusion. During a second brief acid challenge, pH(i) decrease was lessened (adaptation). These are best explained by augmented cellular HCO uptake in response to perfused acid. DIDS, but not DMA, abolished the overshoot and pH(i) adaptation and decreased acid-enhanced HCO secretion. In perfused duodenum, effluent total CO(2) output was not increased by acid perfusion, despite a massive increase of titratable alkalinity, consistent with substantial acid back diffusion and modest CO(2) back diffusion during acid perfusions. Rapid shifts of luminal pH increased duodenal epithelial buffering power, which protected the cells from perfused acid, presumably by activation of Na(+)-HCO cotransport. This adaptation may be a novel, important, and early duodenal protective mechanism against rapid physiological shifts of luminal acidity.


Subject(s)
Bicarbonates/metabolism , Duodenum/metabolism , Intestinal Mucosa/metabolism , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Acids/metabolism , Acids/pharmacology , Adaptation, Physiological , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Carbon Dioxide/metabolism , Diffusion , Duodenum/drug effects , Hydrogen-Ion Concentration , In Vitro Techniques , Intestinal Mucosa/blood supply , Intestinal Mucosa/drug effects , Rats , Regional Blood Flow/drug effects
10.
Hear Res ; 152(1-2): 25-42, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11223279

ABSTRACT

In frog vestibular organs, efferent neurons exclusively innervate type II hair cells. Acetylcholine, the predominant efferent transmitter, acting on acetylcholine receptors of these hair cells ultimately inhibits and/or facilitates vestibular afferent firing. A coupling between alpha9-nicotinic acetylcholine receptors (alpha9nAChR) and apamin-sensitive, small-conductance, calcium-dependent potassium channels (SK) is thought to drive the inhibition by hyperpolarizing hair cells thereby decreasing their release of transmitter onto afferents. The presence of alpha9nAChR in these cells was demonstrated using pharmacological, immunocytochemical, and molecular biological techniques. However, fewer than 10% of saccular hair cells dissociated using protease VIII, protease XXIV, or papain responded to acetylcholine during perforated-patch clamp recordings. When present, these responses were invariably transient, small in amplitude, and difficult to characterize. In contrast, the majority of saccular hair cells ( approximately 90%) dissociated using trypsin consistently responded to acetylcholine with an increase in outward current and concomitant hyperpolarization. In agreement with alpha9nAChR pharmacology obtained in other hair cells, the acetylcholine response in saccular hair cells was reversibly antagonized by strychnine, curare, tetraethylammonium, and apamin. Brief perfusions with either protease or papain permanently abolished the alpha9-nicotinic response in isolated saccular hair cells. These enzymes when inactivated became completely ineffective at abolishing the alpha9-nicotinic response, suggesting an enzymatic interaction with the alpha9nAChR and/or downstream effector. The mechanism by which these enzymes render saccular hair cells unresponsive to acetylcholine remains unknown, but it most likely involves proteolysis of alpha9nAChR, SK, or both.


Subject(s)
Hair Cells, Vestibular/drug effects , Hair Cells, Vestibular/physiology , Peptide Hydrolases/pharmacology , Receptors, Nicotinic/physiology , Acetylcholine/pharmacology , Animals , Cell Separation , Immunohistochemistry , Neural Inhibition/physiology , Neurons, Afferent/drug effects , Neurons, Afferent/physiology , Patch-Clamp Techniques , RNA, Messenger/metabolism , Rana pipiens , Receptors, Nicotinic/genetics , Saccule and Utricle/innervation
11.
Hear Res ; 152(1-2): 67-76, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11223282

ABSTRACT

In the present work we studied the regional expression of voltage-dependent Ca channels in hair cells from the frog semicircular canals, employing whole-cell patch-clamp on isolated and in situ hair cells. Although Ca channels are thought to play a major role in afferent transmission, up to now no data were available regarding their distribution in vestibular organs. The problem appears of interest, especially in the light of recent results showing the presence of multiple Ca current components in semicircular canal hair cells. Our data suggest the presence, in all regions of the crista ampullaris, of two classes of cells, one displaying an inactivating Ca current (R1) and one lacking it. In the former cells, Ca current amplitude decreased from the central to the peripheral zone (the maximal currents being observed in the intermediate zone). Only L-type and R2 current components displayed regional differences in expression, whereas the size and properties of R1, although variable among cells, were not regionalized. However, in cells lacking R1, Ca current amplitudes were similar regardless of cell shape and location. The possible contributions of this Ca current distribution to afferent discharge properties are discussed.


Subject(s)
Calcium/physiology , Hair Cells, Auditory/physiology , Semicircular Canals/innervation , Animals , Barium/physiology , Calcium Channel Blockers/pharmacology , Electric Conductivity , Hair Cells, Auditory/cytology , Hair Cells, Auditory/drug effects , Kinetics , Nimodipine/pharmacology , Rana esculenta , Rana pipiens
12.
Am J Physiol Gastrointest Liver Physiol ; 280(3): G470-4, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11171630

ABSTRACT

We previously showed that the duodenal hyperemic response to acid occurs through activation of capsaicin-sensitive afferent nerves with subsequent release of vasodilatory substances such as calcitonin gene-related peptide (CGRP) and nitric oxide. We then tested the hypothesis that similar factors regulate duodenal mucus gel thickness. Gel thickness was optically measured using in vivo microscopy in anesthetized rats. Duodenal mucosae were superfused with pH 7.0 buffer with vanilloid receptor agonist capsaicin, bradykinin, or PGE(2) injection or were challenged with pH 2.2 solution, with or without the vanilloid antagonist capsazepine, human CGRP-(8-37), N(G)-nitro-L-arginine methyl ester, and indomethacin. Other rats underwent sensory ablation with high-dose capsaicin pretreatment. Acid, bradykinin, capsaicin, and PGE(2) all quickly thickened the gel. Antagonism of vanilloid and CGRP receptors, inhibition of nitric oxide synthase, and sensory deafferentation delayed gel thickening, suggesting that the capsaicin pathway mediated the initial burst of mucus secretion that thickened the gel. Indomethacin abolished gel thickening due to acid, bradykinin, and capsaicin. Inhibition of gel thickening by indomethacin in response to multiple agonists suggests that cyclooxygenase activity is essential for duodenal gel thickness regulation. Duodenal afferent neural pathways play an important role in the modulation of cyclooxygenase-mediated physiological control of gel thickness.


Subject(s)
Capsaicin/analogs & derivatives , Duodenum/enzymology , Intestinal Mucosa/enzymology , Mucus/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Signal Transduction/physiology , Animals , Bradykinin/administration & dosage , Capsaicin/administration & dosage , Cyclooxygenase Inhibitors/administration & dosage , Dinoprostone/administration & dosage , Dose-Response Relationship, Drug , Duodenum/drug effects , Enzyme Inhibitors/administration & dosage , Injections, Intravenous , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Mucus/drug effects , Perfusion , Rats , Signal Transduction/drug effects
13.
Hear Res ; 149(1-2): 167-77, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11033256

ABSTRACT

Nicotinic acetylcholine (nACh) receptors are known to be targets for modulation by a number of substances, including the opiates. It is known that acetylcholine (ACh) coexists with opioid peptides in cochlear efferent neurons, and such a colocalization has been proposed for the vestibular system. In the present study we test the hypothesis that morphine, an opioid receptor agonist with a broad spectrum of selectivity, modulates alpha9nACh receptor-mediated responses in frog vestibular hair cells. Morphine dose-dependently and reversibly inhibited ACh-induced currents as recorded by the perforated patch-clamp method. In the presence of morphine the ACh dose-response curve was shifted to the right in a parallel fashion, suggesting a competitive interaction. However, naloxone did not antagonize the inhibition produced by morphine. To test the hypothesis that morphine could interact with the alpha9nACh receptor without the involvement of opioid receptors, experiments were performed using Xenopus laevis oocytes injected with the alpha9nACh receptor cRNA. The currents activated by ACh in Xenopus oocytes, a system that lacks opioid receptors, were also dose-dependently inhibited by morphine. We conclude that morphine inhibits the alpha9nACh receptor-mediated response in hair cells and Xenopus oocytes through a mechanism which does not involve opioid receptors but may be a direct block of the alpha9nACh receptor.


Subject(s)
Morphine/pharmacology , Narcotics/pharmacology , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/physiology , Acetylcholine/pharmacology , Animals , Electric Conductivity , Hair Cells, Auditory/drug effects , Hair Cells, Auditory/physiology , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Oocytes/metabolism , Rana pipiens , Xenopus laevis
14.
J Infect Dis ; 182(3): 799-807, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10950774

ABSTRACT

Mechanisms responsible for the rapid tissue destruction in gas gangrene are not well understood. To examine the early effects of Clostridium perfringens exotoxins on tissue perfusion, a rat model of muscle blood flow was developed. Intramuscular injection of a clostridial toxin preparation containing both phospholipase C (PLC) and theta-toxin caused a rapid (1-2 min) and irreversible decrease in blood flow that paralleled formation of activated platelet aggregates in venules and arterioles. Later (20-40 min), aggregates contained fibrin and leukocytes, and neutrophils accumulated along vascular walls. Flow cytometry confirmed that these clostridial toxins or recombinant PLC induced formation of P-selectin-positive platelet aggregates. Neutralization of PLC activity in the clostridial toxin preparation completely abrogated human platelet responses and reduced perfusion deficits. It is concluded that tissue destruction in gas gangrene is related to profound attenuation of blood flow initiated by activation of platelet responses by PLC.


Subject(s)
Clostridium perfringens , Exotoxins/metabolism , Gas Gangrene/physiopathology , Muscles/blood supply , Animals , Flow Cytometry , Humans , Microcirculation , P-Selectin/metabolism , Platelet Aggregation , Rats , Regional Blood Flow , Type C Phospholipases/metabolism
15.
J Infect Dis ; 182(3): 808-15, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10950775

ABSTRACT

Clostridium perfringens gas gangrene is a fulminant infection, and radical amputation remains the single best treatment. It has been hypothesized that rapid tissue destruction is related to tissue hypoxia secondary to toxin-induced vascular obstruction, and previous studies demonstrated that phospholipase C (PLC) caused a rapid and irreversible decrease in skeletal muscle blood flow that paralleled the formation of intravascular aggregates of activated platelets, fibrin, and leukocytes. In this study, flow cytometry demonstrated that PLC stimulated platelet/neutrophil aggregation in a gpIIbIIIa-dependent fashion. Pretreatment of animals with heparin or depletion of leukocytes reduced blood-flow deficits, and aggregate formation caused by PLC. It is concluded that fulminant tissue destruction in gas gangrene results from profound attenuation of blood flow caused by PLC-induced, gpIIbIIIa-mediated formation of heterotypic platelet/polymorphonuclear leukocyte aggregates. Therapeutic strategies that target gpIIbIIIa may prevent vascular occlusion, maintain tissue viability, and provide an alternative to radical amputation for patients with this infection.


Subject(s)
Gas Gangrene/pathology , Muscle, Skeletal/pathology , Platelet Activation , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Type C Phospholipases/metabolism , Animals , Clostridium perfringens , Flow Cytometry , Gas Gangrene/metabolism , Granulocytes/metabolism , Heparin/pharmacology , Microscopy, Video , Muscle, Skeletal/blood supply , Rabbits , Rats , Regional Blood Flow , Sheep
16.
Am J Physiol Gastrointest Liver Physiol ; 279(2): G437-47, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10915654

ABSTRACT

We examined the dynamic regulation of mucus gel thickness (MGT) in vivo in rat duodenum in response to luminal acid, cyclooxygenase (COX) inhibition, and exogenous PGE(2). An in vivo microscopic technique was used to measure MGT with fluorescent microspheres in urethan-anesthetized rats. Duodenal mucosa was topically superfused with pH 7.0 or pH 2.2 solutions with or without PGE(2) and indomethacin treatments. Glycoprotein concentration of duodenal loop perfusates was measured with periodic acid/Schiff (PAS) or Alcian blue (AB) staining. MGT and perfusate glycoprotein concentration were stable during a 35-min perfusion with pH 7.0 solution. Acid exposure increased MGT and PAS- and AB-positive perfusate glycoprotein concentrations. Indomethacin pretreatment increased both PAS- and AB-positive perfusate glycoprotein at baseline; subsequent acid superfusion decreased perfusate glycoproteins and gel thickness. PGE(2) (1 mg/kg iv) simultaneously increased MGT and PAS-positive perfusate glycoprotein concentrations followed by a transient increase in AB-positive glycoprotein concentration, suggesting contributions from goblet cells and Brunner's glands. Parallel changes in MGT and perfusate glycoprotein concentration in response to luminal acid and PGE(2) suggest that rapid MGT variations reflect alterations in the balance between mucus secretion and exudation, which in turn are regulated by a COX-related pathway. Luminal acid and PGE(2) augment mucus secretion from goblet cells and Brunner's glands.


Subject(s)
Brunner Glands/enzymology , Duodenum/enzymology , Mucus/metabolism , Acids/pharmacology , Alcian Blue , Animals , Brunner Glands/chemistry , Brunner Glands/cytology , Coloring Agents , Cyclooxygenase Inhibitors/pharmacology , Dinoprostone/pharmacology , Duodenum/chemistry , Duodenum/cytology , Fluoresceins , Fluorescent Dyes , Frozen Sections , Glycoproteins/analysis , Glycoproteins/metabolism , Goblet Cells/enzymology , Hydrogen-Ion Concentration , Immunoblotting , Indomethacin/pharmacology , Male , Microspheres , Mucous Membrane/chemistry , Mucous Membrane/cytology , Mucous Membrane/enzymology , Mucus/drug effects , Periodic Acid-Schiff Reaction , Polyvinyls , Prostaglandin-Endoperoxide Synthases/metabolism , Rats , Rats, Sprague-Dawley
17.
Hear Res ; 146(1-2): 17-27, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10913880

ABSTRACT

Acetylcholine (ACh) is the dominant transmitter released from inner ear efferent neurons. In frog vestibular organs, these efferent neurons synapse exclusively with type II hair cells. Hair cells isolated from the frog saccule hyperpolarize following the application of 50 microM ACh, thereby demonstrating the presence of an ACh receptor. A role for Cl(-) in the response of hair cell-bearing organs to efferent nerve activation or ACh application was suggested some years ago. Perfusion with solutions in which most of the Cl(-) was replaced by large impermeant anions decreased the cholinergic inhibition of afferent firing in the cat and turtle cochleas, and frog semicircular canal. Our previous work in the intact organ demonstrated that substitution of large impermeant anions for Cl(-) or use of Cl(-) channel blockers reduced the effect of ACh on saccular afferent firing. Using the perforated-patch clamping technique, replacement of Cl(-) by methanesulfonate, iodide, nitrate, or thiocyanate attenuated the hyperpolarizing response to ACh in hair cells isolated from the frog saccule. The chloride channel blockers picrotoxin and 4,4'-dinitrostilbene-2,2'-disulfonic acid were also tested and found to inhibit the ACh response. Thus, the present work demonstrates that the effects of Cl(-) substitutions or Cl(-) channel blockers on the ACh response in the intact saccule can be explained completely by effects on the hair cell. Evidence is also presented for the presence of the messenger RNA for a calcium-dependent chloride channel in all hair cells but especially saccular hair cells. This channel may be involved in the response to ACh. The precise role for chloride in this response, whether as a distinct ion current, as a transported ion, or as a permissive ion for other components, is discussed.


Subject(s)
Acetylcholine/pharmacology , Chlorides/metabolism , Hair Cells, Auditory/drug effects , Hair Cells, Auditory/metabolism , Animals , Cats , Chloride Channels/antagonists & inhibitors , Chloride Channels/genetics , Chloride Channels/metabolism , In Vitro Techniques , Membrane Potentials/drug effects , Patch-Clamp Techniques , Picrotoxin/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rana pipiens , Stilbenes/pharmacology
18.
Am J Physiol ; 277(2): G268-74, 1999 08.
Article in English | MEDLINE | ID: mdl-10444439

ABSTRACT

We tested the hypothesis that the duodenal hyperemic response to acid occurs through activation of capsaicin-sensitive afferent nerves with subsequent release of vasodilatory substances such as calcitonin gene-related peptide (CGRP) and nitric oxide (NO). Laser-Doppler flowmetry was used to measure duodenal blood flow in urethan-anesthetized rats. Duodenal mucosa was superfused with pH 7. 0 buffer with capsaicin or bradykinin or was acid challenged with pH 2.2 solution, with or without vanilloid receptor antagonists, a CGRP receptor antagonist, an NO synthase (NOS) inhibitor, or a cyclooxygenase inhibitor. The selective vanilloid receptor antagonist capsazepine (CPZ) dose dependently inhibited the hyperemic response to acid and capsaicin but did not affect bradykinin-induced hyperemia. Ruthenium red was less inhibitory than capsazepine. Selective ablation of capsaicin-sensitive nerves, CGRP-(8-37), and N(G)-nitro-L-arginine methyl ester inhibited acid-induced hyperemia, but indomethacin did not. We conclude that luminal acid, but not bradykinin, stimulates CPZ-sensitive receptors on capsaicin-sensitive afferent nerves of rat duodenum. Activation of these receptors produces vasodilation via the CGRP-NO pathway but not via the cyclooxygenase pathway. Acid appears to be the endogenous ligand for duodenal vanilloid receptors.


Subject(s)
Acids/metabolism , Chemoreceptor Cells/physiology , Duodenum/innervation , Duodenum/metabolism , Animals , Bradykinin , Capsaicin/analogs & derivatives , Capsaicin/antagonists & inhibitors , Capsaicin/pharmacology , Duodenum/blood supply , Hyperemia/chemically induced , Hyperemia/physiopathology , Hyperemia/prevention & control , Male , Rats , Rats, Sprague-Dawley , Receptors, Drug/antagonists & inhibitors , Regional Blood Flow/drug effects , Time Factors
19.
Hear Res ; 125(1-2): 154-62, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9833969

ABSTRACT

This research sought to test the presence and function of metabotropic excitatory amino acid receptors (mGluR) in the frog semicircular canal (SCC). The mGluR agonist +/- 1-aminocyclopentane-trans-1,3-dicarboxylate (ACPD) produced an increase in afferent firing rates of the ampullar nerve of the intact posterior canal. This increase was not due to a stimulation of cholinergic efferent terminals or the acetylcholine (ACh) receptor, since atropine, in concentrations which blocked the response to exogenous acetylcholine, did not affect the response to ACPD. Likewise, ACPD effects were not due to stimulation of postsynaptic NMDA receptors, since the NMDA antagonist D(-)-2-amino-5-phosphonopentanoate (AP-5) did not affect the response to ACPD, reinforcing the reported selectivity of ACPD for mGluRs. When the SCC was superfused with artificial perilymph known to inhibit hair cell transmitter release (i.e. low Ca-high Mg), ACPD failed to increase afferent firing. This suggests that the receptor activated by ACPD is located on the hair cell. Pharmacological evidence suggested that the mGluRs involved in afferent facilitation belong to Group I (i.e. subtypes 1 and 5). In fact, the Group III agonist AP-4 had no effect, and the ACPD facilitatory effect was blocked by the Group I mGluR antagonists (S)-4-carboxyphenylglycine (CPG) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Additional pharmacological evidence supported the presence of Group I mGluRs. Interestingly, the mGluR antagonists, AIDA and 4CPG, by themselves did not affect the resting firing rates of ampullar afferents. This may suggest that the mGluRs are not involved in resting activity but perhaps only in evoked activity (as suggested in Guth et al. (1991) Hear. Res. 56, 69-78). In addition, the mRNA for the mGluR1 has been detected in hair cells of both SCC, utricle, and saccule. In summary, the evidence points to an mGluR localized to the hair cell (i.e. an autoreceptor) which may be activated to produce a positive feedback augmentation of evoked but not resting transmitter release and thus affect afferent activity.


Subject(s)
Receptors, Metabotropic Glutamate/physiology , Vestibule, Labyrinth/physiology , Afferent Pathways/drug effects , Afferent Pathways/physiology , Animals , Auditory Pathways/drug effects , Auditory Pathways/physiology , Cycloleucine/analogs & derivatives , Cycloleucine/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Feedback , Hair Cells, Auditory/drug effects , Hair Cells, Auditory/physiology , In Vitro Techniques , Models, Biological , RNA/genetics , Rana pipiens , Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/genetics , Semicircular Canals/drug effects , Semicircular Canals/innervation , Semicircular Canals/physiology , Vestibule, Labyrinth/drug effects , Vestibule, Labyrinth/innervation
20.
Hear Res ; 123(1-2): 31-40, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9745953

ABSTRACT

Previous studies have shown that galvanic stimulation of semicircular canal organs can modulate their afferent discharge. However, it has not been resolved whether this modulation derived from direct stimulation of hair cells, afferent nerve fibers, some combination of the two, or some as yet unknown path. This problem is addressed in the present study. Experiments were designed first to determine the gross current path necessary for the DC current to modulate afferent firing. These led to the conclusion that the current path had to flow between endolymph and perilymph across the neuroepithelium. Next, the various components in this established path were considered: the afferents, the hair cells, between the hair cells, or some combination of the three. These experiments led to the conclusion that the current pathway was across the hair cells causing transmitter release and thus affecting afferent activity.


Subject(s)
Benzodiazepines , Hair Cells, Auditory/physiology , Semicircular Canals/physiology , Synaptic Transmission/physiology , Amiloride/pharmacology , Animals , Anti-Anxiety Agents/pharmacology , Electric Stimulation , Epithelium/physiology , Excitatory Amino Acid Antagonists/pharmacology , Hair Cells, Auditory/drug effects , In Vitro Techniques , Nerve Fibers/drug effects , Nerve Fibers/physiology , Neurotransmitter Agents/antagonists & inhibitors , Potassium Chloride , Rana pipiens , Receptors, AMPA/antagonists & inhibitors , Semicircular Canals/drug effects , Synaptic Transmission/drug effects
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