Discovery of a series of 2-(1H-pyrazol-1-yl)pyridines as ALK5 inhibitors with potential utility in the prevention of dermal scarring.

A series of 2-(1H-pyrazol-1-yl)pyridines are described as inhibitors of ALK5 (TGFß receptor I kinase). Modeling compounds in the ALK5 kinase domain enabled some optimization of potency via substitutions on the pyrazole core. One of these compounds PF-03671148 gave a dose dependent reduction in TGFß induced fibrotic gene expression in human fibroblasts. A similar reduction in fibrotic gene expression was observed when PF-03671148 was applied topically in a rat wound repair model. Thus these compounds have potential utility for the prevention of dermal scarring.

Interaction of delavirdine with human liver microsomal cytochrome P450: inhibition of CYP2C9, CYP2C19, and CYP2D6.

Delavirdine, a non-nucleoside inhibitor of HIV-1 reverse transcriptase, is metabolized primarily through desalkylation catalyzed by CYP3A4 and CYP2D6 and by pyridine hydroxylation catalyzed by CYP3A4. It is also an irreversible inhibitor of CYP3A4. The interaction of delavirdine with CYP2C9 was examined with pooled human liver microsomes using diclofenac 4'-hydroxylation as a reporter of CYP2C9 catalytic activity. As delavirdine concentration was increased from 0 to 100 microM, the K(M) for diclofenac metabolism rose from 4.5+/-0.5 to 21+/-6 microM, and V(max) declined from 4.2+/-0.1 to 0.54+/-0.08 nmol/min/mg of protein, characteristic of mixed-type inhibition. Nonlinear regression analysis revealed an apparent K(i) of 2.6+/-0.4 microM. There was no evidence for bioactivation as prerequisite to inhibition of CYP2C9. Desalkyl delavirdine, the major circulating metabolite of delavirdine, had no apparent effect on microsomal CYP2C9 activity at concentrations up to 20 microM. Several analogs of delavirdine showed similar inhibition of CYP2C9. Delavirdine significantly inhibited cDNA-expressed CYP2C19-catalyzed (S)-mephenytoin 4'-hydroxylation in a noncompetitive manner, with an apparent K(i) of 24+/-3 microM. Delavirdine at concentrations up to 100 microM did not inhibit the activity of CYP1A2 or -2E1. Delavirdine competitively inhibited recombinant CYP2D6 activity with a K(i) of 12.8+/-1.8 microM, similar to the observed K(M) for delavirdine desalkylation. These results, along with previously reported experiments, indicate that delavirdine can partially inhibit CYP2C9, -2C19, -2D6, and -3A4, although the degree of inhibition in vivo would be subject to a variety of additional factors.

Microsomal metabolism of delavirdine: evidence for mechanism-based inactivation of human cytochrome P450 3A.

Administration of delavirdine, an HIV-1 reverse transcriptase inhibitor, to rats or monkeys resulted in apparent loss of hepatic microsomal CYP3A and delavirdine desalkylation activity. Human CYP3A catalyzes the formation of desalkyl delavirdine and 6'-hydroxy delavirdine, an unstable metabolite, while CYP2D6 catalyzes only desalkyl delavirdine. CYP2D6 catalyzed desalkyl delavirdine formation was linear with time (up to 30 min) but when catalyzed by cDNA expressed CYP3A4 or human liver microsomes the reaction rate declined progressively with time. Coincubation with triazolam showed that delavirdine caused a time- and NADPH-dependent loss of CYP3A4 activity in human liver microsomes as measured by triazolam 1'-hydroxylation. The catalytic activity loss was saturable and was characterized by a Ki of 21.6 +/- 8.9 microM and a kinact of 0.59 +/- 0.08 min-1. An apparent partition ratio of 41 was determined with cDNA expressed CYP3A4, based on the substrate depletion method. Incubation of [14C]delavirdine with microsomes from several species resulted in irreversible association with an approximately 50 kDa protein, as demonstrated by SDS-PAGE/autoradiography. Binding to the protein was NADPH dependent, glutathione insensitive, proportional to the level of CYP3A expression and was inhibited by ketoconazole, a specific CYP3A inhibitor. NADPH-dependent irreversible binding to human and rat total microsomal protein was demonstrated following exhaustive extraction of microsomal protein. Binding was decreased in the presence of glutathione and appeared to be related to expression level of CYP3A. These results suggest that delavirdine can inactivate CYP3A and has the potential to slow the metabolism of coadministered CYP3A substrates.

Metabolism of delavirdine, a human immunodeficiency virus type-1 reverse transcriptase inhibitor, by microsomal cytochrome P450 in humans, rats, and other species: probable involvement of CYP2D6 and CYP3A.

The metabolism of delavirdine was examined using liver microsomes from several species with the aim of comparing metabolite formation among species and characterizing the enzymes responsible for delavirdine metabolism. Incubation of 10 microM [14C]delavirdine with either an S9 fraction from human jejunum or liver microsomes from rat, human, dog, or monkey followed by high pressure liquid chromatography analysis showed qualitatively similar metabolite profiles among species with the formation of three significant metabolites. The major metabolite was desalkyl delavirdine; however, the identity of MET-7 and MET-7a (defined by high pressure liquid chromatography elution) could not be unambiguously established, but they seem to be related pyridine hydroxy metabolites, most likely derived from 6'-hydroxylation of the pyridine ring. The apparent KM for delavirdine desalkylation activity ranged from 4.4 to 12.6 microM for human, rat, monkey, and dog microsomes, whereas Vmax ranged from 0.07 to 0.60 nmol/min/mg protein, resulting in a wide range of intrinsic clearance (6-135 microL/min/mg protein). Delavirdine desalkylation by microsomes pooled from several human livers was characterized by a KM of 6.8 +/- 0.8 microM and Vmax of 0. 44 +/- 0.01 nmol/min/mg. Delavirdine desalkylation among 23 human liver microsomal samples showed a meaningful correlation (r = 0.96) only with testosterone 6beta-hydroxylation, an indicator of CYP3A activity. Among ten human microsomal samples selected for uniform distribution of CYP3A activity, formation of MET-7 was strongly correlated with CYP3A activity (r = 0.95) and with delavirdine desalkylation (r = 0.98). Delavirdine desalkylation was catalyzed by cDNA-expressed CYP2D6 (KM 10.9 +/- 0.8 microM) and CYP3A4 (KM 5.4 +/- 1.4 microM); however, only CYP3A4 catalyzed formation of MET-7 and MET-7a. Quinidine inhibited human liver microsomal delavirdine desalkylation by about 20%, indicating a minor role of CYP2D6. These findings suggest the potential for clinical interaction with coadministered drugs that are metabolized by or influence the activity of CYP3A or CYP2D6.

Effect of adenosine and histamine receptor stimulation on canine histamine release to pentagastrin.

The effects of adenosine and histamine 2 and histamine 3 receptor agonists on the regulation of gastric histamine release were examined in anesthetized mixed-breed dogs. All compounds were infused directly into the gastrosplenic artery to avoid perturbations in systemic hemodynamics, and the gastric histamine release was stimulated with pentagastrin. The histamine concentration in plasma samples was measured utilizing gas chromatography-negative-ion chemical ionization mass spectroscopy. Pentagastrin consistently stimulated gastric histamine release with the peak stimulation occurring at 5 min, while neither 30 nor 100 microM of adenosine altered the effect of pentagastrin on histamine release. In addition, theophylline at 20 microg/ml exhibited no effect on stimulated histamine release. The histamine 2 receptor agonist dimaprit, at 1 and 3 microM, attenuated pentagastrin-stimulated histamine release at the 5-min time period, but the difference was not sustained at later time points (histamine release from 1.4 +/- 0.6 to 92 +/- 18 ng/min at 5 min with pentagastrin alone; from 1.2 +/- 0.5 to 32 +/- 11 ng/min with pentagastrin plus 1 microM dimaprit, and from 2.0 +/- 1.1 to 32 +/- 9 ng/min with pentagastrin plus 3 microM dimaprit), while the H2 receptor antagonist cimetidine exhibited no effect on pentagastrin-stimulated histamine release. The histamine 3 receptor agonist (R)-alpha-methylhistamine attenuated the pentagastrin-stimulated histamine release at the 5- and 10-min time periods only at 1 microM without showing any effect at the higher (3 microM) concentration. Thioperamide, a H3 receptor antagonist, did not modify pentagastrin-stimulated histamine release. These data demonstrate that adenosine has no modulatory role on gastric histamine release, but histamine via H2 and H3 histamine receptors could modulate its own release but only to a modest degree as compared with the potent effect of the paracrine hormone somatostatin.

Secretin inhibits canine gastric acid secretion in response to pentagastrin by modulating gastric histamine release.

The effect of secretin on pentagastrin- and gastrin-stimulated gastric histamine release and acid secretion was examined in the anesthetized dog model, where all compounds were infused directly into the artery supplying the gastric corpus. Secretin at an infusion rate of 10 ng/kg/min resulted in approximately 90% inhibition of gastric secretion in response to pentagastrin (20 ng/kg/min), whereas at the physiological postprandial concentration of 40 pg/ml it inhibited gastric secretion by approximately 55%. Gastric acid stimulated by gastrin I at the physiological post-prandial concentration of 150 pg/ml was inhibited by secretin at 40 pg/ml by approximately 80%. Pentagastrin stimulated histamine release to a peak of 168 +/- 34 ng/min, which was inhibited to 14 +/- 8 ng/min with the high concentration of secretin and to 85 +/- 21 ng/min at 40 pg/ml secretin. Gastrin I (150 pg/ml) stimulated histamine release to a peak of 10.6 +/- 4.6 ng/min, which was inhibited to 2.1 +/- 0.5 ng/min by secretin (40 pg/ml). Because secretin has been reported to stimulate gastric somatostatin release, we examined the somatostatin secretory rate concomitant with histamine release. Both doses of secretin stimulated gastric somatostatin release, compared with pentagastrin alone. The present data demonstrate that secretin, even at physiological concentrations, can inhibit gastric acid secretion in response to gastrin/pentagastrin, and one of the mechanisms of inhibition involves modulation of gastric histamine release. This effect of secretin on histamine release may be either direct, at the histamine-containing endocrine cells, or indirect, through somatostatin release.

The effect of beta andrenoceptor stimulation of canine stomach on pentagastrin-stimulated histamine release.

The effect of beta adrenergic agonist isoproterenol, infused directly into the gastrosplenic artery in anesthetized mixed-breed dogs, on pentagastrin-stimulated gastric histamine release was examined to clarify the potential mechanisms by which beta adrenoceptor stimulation results in gastric acid inhibition. Two doses of isoproterenol (3 and 10 ng/kg/min) were infused with pentagastrin; histamine and n(tau)-methyl-histamine concentrations were measured in arterial and gastric venous samples, and their gastric secretory rates were calculated. Both doses of isoproterenol decreased histamine-secretory rate to pentagastrin from a peak of 234 +/- 51.5 ng/min with vehicle to 17.7 +/- 4.2 ng/min with the 3 ng/kg/min dose of isoproterenol and to 8.6 +/- 2.9 ng/min with the 10 nk/kg/min dose of isoproterenol. The change in N(tau)-methyl-histamine-secretory rate paralleled the histamine-secretory rate. Concomitantly with the histamine-secretory rate, the effect of beta adrenoceptor agonist on gastric somatostatin secretion was also examined. The lower dose of isoproterenol stimulated somatostatin-secretory rate from 4.0 +/- 1.8 to 31.8 +/- 10.3 ng/min, and the higher dose of isoproterenol increased somatostatin-secretory rate from 6.0 +/- 3.1 to 61.5 +/- 21.5 ng/min, whereas isoproterenol potentiated pentagastrin-stimulated gastric somatostatin release. These data demonstrate that isoproterenol is a potent inhibitor of pentagastrin-stimulated gastric histamine release, and the mechanism may be related to the concomitant somatostatin release. Thus, the most likely mechanism by which beta adrenoceptor stimulation results in inhibition of gastric acid secretion is through down-regulation of gastric histamine release.

The role of gastric histamine release in the acid secretory response to pentagastrin and methacholine in the dog.

We have previously demonstrated that both pentagastrin and methacholine can stimulate histamine release from the canine stomach during short term administration of the secretagogues into the gastrosplenic artery. In this study we tested the hypothesis that gastric histamine release determines the acid secretory response to acid secretagogues. Increasing doses of pentagastrin (2, 6, and 20 ng/kg/min) and methacholine (0.1, 0.3, and 1 micrograms/min) were infused into the gastrosplenic artery in dogs, while gastric acid output, histamine and N tau-methyl histamine secretory rates were monitored. Histamine and N tau-methyl histamine concentrations in plasma were measured using GC/NICI-MS. Increasing doses of pentagastrin resulted in increasing gastric output. Total histamine secretory rate expressed as the sum of histamine and N tau-methyl histamine secretory rate showed a significant increase above basal with the two highest doses of pentagastrin. Regression analysis correlating the dose of pentagastrin to gastric acid output gave a correlation coefficient of 0.586 which was very significant. Regression analysis correlating the total histamine secretory rate to acid output gave a correlation coefficient of 0.498 which was also very significant. Increasing doses of methacholine also resulted in a dose-dependent increase in acid output. Histamine secretory rates showed a statistically significant increase above basal only at the 1 microgram/min infusion rate, however, the total histamine secretory rates (histamine + N tau-methyl histamine) were no longer significant at any of the doses of methacholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of somatostatin and prostaglandins on gastric histamine release to pentagastrin.

The effects of gastric acid antisecretory agents prostaglandins E2, I2 (PGE2, PGI2) and somatostatin on pentagastrin-stimulated gastric histamine and N tau-methyl histamine secretory rates were examined in anesthetized mixed breed dogs. We infused two gastric acid antisecretory doses of PGE2 and PGI2 to test the effect of prostaglandins on pentagastrin-stimulated gastric histamine release. Neither dose of PGE2 and PGI2 had an effect on pentagastrin-stimulated histamine and N tau-methyl histamine release, even though the prostaglandins caused marked gastric vasodilation. In addition, the infusion of the higher dose of PGE2 and PGI2 alone had no effect on histamine secretory rates. In contrast, somatostatin inhibited both pentagastrin-stimulated gastric histamine release by approximately 95% as well as basal histamine release by approximately 60%. Somatostatin also inhibited the pentagastrin-stimulated N tau-methyl histamine secretory rates. The results indicate that neither PGE2 nor PGI2 at antisecretory doses affect pentagastrin-stimulated gastric histamine release, but somatostatin has a very potent inhibitory effect in that regard. Our data suggest that the mechanisms by which prostaglandins and somatostatin affect gastric acid secretion may be diverse.

The role of gastric secretagogues in regulating gastric histamine release in vivo.

The effect of short-term intragastric arterial infusion of pentagastrin and methacholine on histamine and N tau-methyl histamine secretory rates was evaluated in mongrel dogs in vivo. Doses of pentagastrin and methacholine were chosen that stimulate gastric acid secretion equivalently. Histamine and N tau-methyl histamine secretory rates were evaluated by measuring the arterial and gastric venous plasma histamine and N tau-methyl histamine concentrations at several time points during the secretagogue infusion, and gastric blood flow was continuously monitored. Histamine and N tau-methyl histamine plasma concentrations were analyzed by stable isotope dilution technique using gas chromatography/negative ion-chemical ionization mass spectrometry. Histamine and N tau-methyl histamine secretory rates were calculated by subtracting the arterial from the venous plasma concentrations and multiplying the difference by gastric plasma flow. Infusion of pentagastrin resulted in large pulsed increase of histamine release from 1.5 +/- 0.7 ng/min at time 0 to 72 +/- 20 ng/min at 5 minutes, which decreased to a plateau of 20 +/- 8 ng/min at 20 minutes. N tau-Methyl histamine secretory rate increased from 6.7 +/- 1.9 ng/min at baseline to a maximum of 42.5 +/- 13.1 ng/min at 10 minutes, and the increase was maintained for the duration of the pentagastrin infusion. Methacholine infusion was associated with a small but sustained increase in histamine release, from a baseline of 1.6 +/- 0.6 ng/min to 5.9 +/- 1.7 ng/min at 5 minutes. N tau-Methyl histamine secretory rate was unchanged by methacholine. Gastric blood flow changes to pentagastrin roughly paralleled the extent of histamine release, but methacholine is a gastric vasodilator in its own right. Our data indicate that pentagastrin is a much more effective stimulator of gastric histamine release than methacholine and that the overall role of histamine in gastrin-stimulated acid output is likely to be different from the role of histamine in cholinergic-mediated gastric acid output.

The effect of age on the sensitivity of the alpha 1-adrenoceptor to phenylephrine and prazosin.

Certain beta-adrenoceptor-mediated functions seem to diminish with age; however, information on alpha-adrenoceptor-mediated function is sparse and often conflicting but overall suggests little age-related change. To assess an age-related alteration in the responsiveness to an alpha 1-adrenergic agonist and to estimate changes in the apparent affinity of the alpha 1-adrenoceptor for the antagonist prazosin, we infused phenylephrine into 12 healthy elderly subjects and 12 healthy young subjects before and after an oral dose of prazosin, and we compared the shift in the dose-response curves for the two groups. With this protocol we were unable to detect any age-related decline in sensitivity of the alpha-adrenoceptor to either agonist or antagonist. However, oral prazosin resulted in higher plasma concentrations and a consistently greater hypotensive effect in the elderly subjects than in the young subjects. We concluded that there was no difference in alpha 1-adrenoceptor sensitivity in the elderly persons, but that the kinetics of prazosin may be altered and that the response of the blood pressure to prazosin was increased because of the kinetic changes and possibly the physiologic changes associated with aging.

Analysis of histamine and N tau-methylhistamine in plasma by gas chromatography-negative ion-chemical ionization mass spectrometry.

Current methods of quantitation of histamine and its major metabolite N tau-methylhistamine are inaccurate and insensitive to the very low concentrations that exist in plasma samples. Therefore, an accurate and sensitive method for quantification in plasma has been developed using the stable isotope dilution assay with negative ion-chemical ionization mass spectrometry. For histamine, after the addition of [2H4]histamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH2C6F5)3-histamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m/z 430/434. For N tau-methylhistamine, after the addition of N tau-[2H3]methylhistamine to 2 ml of plasma, the plasma sample is deproteinized, extracted into butanol, back extracted into HCl, derivatized to the heptafluorobutyryl derivative (C3F7CO2)2-N tau-methylhistamine, purified on silica gel columns, and then quantified with negative ion-chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions m/z 497/500. The precision of the histamine assay is 3.1% and the accuracy is 95.5 +/- 2.5% while the precision of the N tau-methylhistamine assay is 1.9% and the accuracy is 106.8 +/- 1.9%. The lower limits of sensitivity are 1 pg for histamine and 6 pg for N tau-methylhistamine injected on column. Using the assay in three normal human volunteers, plasma concentrations of histamine were 130, 92, and 85 pg/ml, and of N tau-methylhistamine were 229, 228, and 216 pg/ml. This assay provides a very sensitive and accurate method of quantitation of histamine and N tau-methylhistamine in plasma samples.

Endogenous adenosine modulates gastric acid secretion to histamine in canine parietal cells.

We have recently demonstrated the presence of A1 adenosine receptors on canine parietal cells which are involved in the inhibition of histamine-stimulated acid secretion. In order to demonstrate the importance of endogenously generated adenosine on acid secretion we examined the effect of compounds that either increase or decrease the availability of adenosine to the A1 receptor on histamine-stimulated parietal cell aminopyrine (AP) accumulation. Inclusion of 10 microM 8-phenyltheophylline, an adenosine receptor antagonist, with the cells resulted in a 35 +/- 12% and 31 +/- 9% increase in parietal cell AP accumulation at histamine concentrations of 1 microM and 10 microM, respectively. The effect of 8-phenyltheophylline was specific to histamine in that it did not affect carbachol-stimulated AP accumulation or dibutyryl cyclic AMP-stimulated AP accumulation. Inclusion of 1 microM dipyridamole, an inhibitor of adenosine transport, resulted in a 34 +/- 6% and 31 +/- 5% decrease in parietal cell AP accumulation at histamine concentrations of 1 microM and 10 microM, respectively. Again the effect of dipyridamole was specific to histamine in that it did not affect either carbachol- or dibutyryl cyclic AMP-stimulated AP accumulation. The addition of adenosine deaminase, 500 mU/ml, resulted in an enhanced histamine-stimulated AP accumulation at all the histamine concentrations. The effect was specific to histamine because the enzyme had no effect on either carbachol- or dibutyryl cyclic AMP-stimulated AP uptake. Our present data suggest that endogenous adenosine generated by the gastric cells can interact with parietal cell adenosine receptors to modulate acid secretion to histamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin E2 and [14C]arachidonic acid release by carbachol in the isolated canine parietal cell.

Parietal cells have the capacity to synthesize prostaglandins (PGs) and these PGs have a significant effect on parietal cell acid secretion. We examined the stimuli responsible for the control of PG production by parietal cells. Two approaches were utilized. One consisted of measuring PGE2 concentration in the incubation media containing dispersed canine parietal cells stimulated with increasing concentrations of carbachol, histamine plus a phosphodiesterase inhibitor and pentagastrin. PGE2 was measured by radioimmunoassay. The other consisted of measuring the release of radioactive arachidonic acid by parietal cells prelabeled with [4C]arachidonic acid to increasing concentrations of the secretagogues. Both techniques gave very similar results. Only carbachol stimulated the release of PGE2 as well as [14C]arachidonic acid into the incubation media. The increase in PGE2 release was from a base line of 44.3 +/- 10.8 pg/ml to 51.0 +/- 11.7, 55.2 +/- 11.1 and 69.3 +/- 14.3 pg/ml at carbachol concentrations of 10(-6), 10(-5) and 10(-4) M, respectively. In prelabeled cells, carbachol stimulated 1072 +/- 141 and 1264 +/- 155 cpm/ml above basal at concentrations of 10(-5) and 10(-4) M, respectively. Neither histamine nor pentagastrin stimulated PGE2 or [14C]arachidonic acid release significantly at any concentration. The effect of carbachol on the release of [14C]arachidonic acid was blocked by atropine and the exclusion of calcium from the incubation media. Our data suggest that the cholinergic tone to the stomach with subsequent interaction of acetylcholine with muscarinic receptors determines the amount of PG production by the parietal cells. Prostaglandins may then modulate acid secretion by limiting the combined, potentiated effects of the secretagogues.

Somatostatin's ability to inhibit gastric acid is not prostaglandin-mediated in the dog.

The role of prostaglandins in the effect of somatostatin to inhibit cholinergically stimulated gastric acid output was examined in gastric fistula dogs. Methacholine stimulation of gastric acid output to a maximum of 810 +/- 423 mu eq of [H+] per 15 min was inhibited by 10 nM somatostatin to the same extent either in the presence and/or the absence of indomethacin. In contrast, histamine-induced acid secretion was stimulated weakly by 10 nM somatostatin. Our data do not support a role for prostaglandins in somatostatin's action to inhibit cholinergically stimulated acid secretion.

Adenosine receptors on canine parietal cells modulate gastric acid secretion to histamine.

Administration of theophylline has been shown to enhance gastric acid secretion in humans. Because theophylline has been reported to be a poor inhibitor of phosphodiesterase but a better adenosine receptor antagonist, we tested the hypothesis that there are inhibitory "R site" adenosine receptors on parietal cells. Utilizing isolated dispersed canine parietal cells, we measured acid secretion by the [14C]aminopyrine accumulation technique. We tested the effect of increasing concentrations of 2-chloroadenosine (10(-7), 10(-6), 10(-5) M) and L-N6-phenylisopropyl adenosine (L-PIA) (10(-7), 10(-6), and 10(-5) M), stable analogs of adenosine with specificity for the R sites, on aminopyrine uptake produced by submaximal stimulating concentrations of histamine (1 microM) plus isobutyl methylxanthine (3 microM) or carbachol (1 microM). Histamine-stimulated parietal cell aminopyrine uptake was 4.3- +/- 0.4-fold above basal; 2-chloroadenosine inhibited this response in a dose-dependent fashion with a 57 +/- 6% inhibition at 10(-5) M.L-PIA also inhibited histamine-stimulated aminopyrine uptake with a 67 +/- 11% inhibition at 10(-5) M. Carbachol-stimulated aminopyrine uptake was 5.8- +/- 1.6-fold above basal, but 2-chloroadenosine had no significant effect on this response. Theophylline, 300 microM, and 8-phenyltheophylline, 10 microM, reduced the inhibitory effect of 2-chloroadenosine. 8-Phenyltheophylline was inactive in inhibiting the parietal cell phosphodiesterase activity and the IC50 of theophylline for phosphodiesterase was 1 mM. Because prostaglandins inhibit parietal cell uptake of aminopyrine in a pattern similar to 2-chloroadenosine, we also tested the possibility that prostaglandins are involved in the 2-chloroadenosine response.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholinergic mechanism of acid secretion in the dog: an in vivo and in vitro comparison.

The effect of cholinergic stimulation on gastric acid secretion was examined in mongrel dogs using an in vivo and an in vitro preparation for comparison. The gastric fistula dog was used for the in vivo studies, and methacholine was infused directly into the artery supplying the fundus of the stomach to avoid systemic hemodynamic changes. Isolated parietal cells were used for the in vitro studies. In vivo, methacholine infused at 1 microgram/min was found to stimulate gastric acid secretion that was inhibited 98.5 +/- 1.4% by intravenously administered metiamide (H2 blocker) and 72 +/- 11% by intra-arterially administered glucagon. Glucagon had no effect on histamine stimulated acid secretion. In vitro, increasing concentrations of methacholine from 10(-7), 10(-6) to 10(-5) M stimulated [14C]aminopyrine uptake into parietal cells in a concentration dependent manner. This effect of methacholine was unaltered by 10(-4) M metiamide or 10(-6) M glucagon. However, atropine 10(-5) M totally blocked the stimulatory effect of methacholine. Our data suggest that the cholinergic mechanism of acid secretion is different when examined in vivo vs. in vitro even in the same species. In vivo histamine dependency has a major contribution to the cholinergic mechanism of acid secretion, whereas in vitro the interaction of the cholinergic agonist at the muscarinic receptor results in acid stimulation that does not require the presence of histamine.

Adenosine: a modulator of gastric acid secretion in vivo.

We tested the hypothesis that there are adenosine receptors in the gastric fundus responsible for the modulation of acid secretion to secretagogues. We utilized anesthetized gastric fistula dogs and infused both the secretagogues (histamine, 0.5 microgram/min and methacholine, 1 microgram/min) and adenosine through the gastric artery supplying the fundus of the stomach to avoid systemic effects of the drug. Adenosine infused to a concentration of 30 microM inhibited histamine-stimulated acid secretion from 197 +/- 25 to 68 +/- 19 microEq/15 min and inhibited methacholine-stimulated acid secretion from 280 +/- 48 to 81 +/- 24 microEq/15 min. Intravenously infused theophylline to a plasma concentration of 17 micrograms/ml (94 microM) blocked the inhibitory effect of adenosine on gastric acid secretion but did not affect the inhibitory effect of prostaglandin E2 on gastric acid secretion. In contrast, the specific phosphodiesterase inhibitor RO 20 1724 did not alter the inhibitory effect of adenosine on gastric acid secretion. In addition to its effect on gastric acid secretion, adenosine was found to be a potent vasodilator of gastric blood vessels. Our data suggest that there are inhibitory adenosine "R" receptors in the gastric fundus that modulate acid secretion to both histamine and methacholine.