Isolated rat gastric mucosal cells: optimal conditions for cell harvesting, measures of viability and direct cytoprotection.

Controversial data have been obtained with direct cellular protection by prostaglandins and sulfhydryls. In the present studies, we compared the merits and liabilities of currently available cell viability assays, some of which have not been previously employed in studies of the gastric mucosa. We also tested the hypothesis that length of incubation of isolated cells with protective agents might influence the degree of cellular damage. Gastric mucosal cells were isolated from nonfasted rats and digested with various concentrations of pronase and/or EGTA. Cell viability was assessed by trypan blue and fast green exclusion, fluorescein diacetate hydrolysis, chromium release, LDH release, mitochondrial succinate dehydrogenase activity and nuclear fluorescence induced by ethidium bromide. The experiments revealed that both pronase and EGTA are needed to obtain mucosal cells with optimal yield and initial viability and that sequential additions of pronase (30 min) and EGTA (30 min), rather than their combination (60 min), increased viability without decreasing yield. Cell viability and yield were better when pronase was used in the first incubation. For LD50 determinations, a cell suspension was incubated with ethanol (0%-15%) for 5 min. For studying the effects of protective agents, the cells were pretreated with 16,16-dmPGE2 or cysteamine HCl at 37 degrees C for 30 min before a 5-min exposure to 7.5% or 15% ethanol. The LD50 value for ethanol injury was approximately 15% for all assays except LDH, where the LD50 value was 8.5%. Preincubating gastric mucosal cells for 30 min with 16,16-dmPGE2 or cysteamine resulted in no preservation of cell viability. However, when cells were preincubated with one of the protective agents for 60 min and then exposed to 8% or 10% ethanol for 5 min, partial protection was observed when assessed by succinate dehydrogenase activity and, in certain cases, by LDH release. We conclude that all seven cell viability assays yield a measurable LD50 value for ethanol-induced cell injury, but the results may vary by as much as 82%. Low concentrations of both pronase and EGTA are needed to obtain isolated mucosal cells with both high yield and initial viability. Biochemical measures of mitochondrial activity and nuclear damage provide reliable evidence of cell viability and should be used to complement membrane permeability assays. Long preincubation of cells (60 min) with protective agents resulted in only slight protection of mitochondrial function, in contrast to the rapid induction of gastroprotection seen with these compounds in vivo. We therefore surmise that processes that contribute to organ protection occur faster and more efficiently than those that control direct cell injury and protection.

Effects of a novel 5-HT1A receptor agonist, E4424, on gastric adherent mucus levels following restraint stress in rats.

Several novel arylpiperazine serotonin 1A receptor agonists, developed as anxiolytics, have antisecretory and gastroprotective effects in rats. E4424 (2-¿4-[4-(4-chloropyrazol-1-yl)butyl]-1-piperazinyl ¿pyrimidine; Lesopitron dihydrochloride), has potent anti-gastric secretory and antiulcer effects. Preliminary data indicated an enhancing effect of E4424 on gastric mucus that may underlie its gastroprotective actions. We therefore tested the effects of acute and chronic administration of E4424 and of a reference 5-HT1A receptor agonist, 8-OHDPAT [8-hydroxy-2-(di-n-propylamino)tetralin], on gastric mucus levels in rats subjected to cold-restraint stress, a procedure associated with depletion of gastric mucus and the development of mucosal injury. Acute oral administration of E4424 increased adherent mucus levels by 12%, 11%, and 13%, relative to controls. Chronic E4424 significantly increased gastric mucus relative to controls (69% increase). Acute oral treatment with 8-OHDPAT did not affect gastric mucus level. Acute intraperitoneal 8-OHDPAT slightly increased mucus levels. Chronic twice per day 8-OHDPAT did not affect mucus levels; however, chronic once per day treatment with 8-OHDPAT significantly elevated gastric mucus levels at the highest doses used. For E4424, there is a strong correlation between reduction of gastric mucosal injury and increase in gastric mucus level, suggesting that the action of E4424 on glandular mucus levels is an important mechanism underlying its gastroprotective effects.

Central and peripheral dopamine D1/DA1 receptor modulation of gastric secretion and experimental gastric mucosal injury.

1. Dopamine D1 (central)/DA1 (peripheral) receptors are believed to influence gastrointestinal function and pathology. 2. When given i.c.v. or i.p., an agonist (SKF38393) and an antagonist (SCH23390) of this DA receptor subtype inhibit and enhance, respectively, gastric secretion and gastric mucosal injury. 3. When given both i.c.v. and i.p., their respective effects in the gut were amplified. 4. Antagonist or agonist given i.p., blocked the corresponding protective and worsening effect of the agonist or antagonist given i.c.v. 5. Both central and peripheral D1/DA1 receptors modulate gastric function and response to injury.

Efaroxan acts peripherally to block the antisecretory and gastroprotective effects of moxonidine in rats.

I1-imidazoline receptor activation by moxonidine has potent antigastric secretory and gastroprotective effects in rats. We therefore tested whether an imidazoline receptor antagonist, efaroxan, would influence gastric secretion and block the antisecretory and antiulcer effects of moxonidine. When given intracerebroventricularly (i.c.v.), moxonidine inhibited basal acid output in conscious rats to a maximum of 38%. Moxonidine given i.p. also significantly increased gastric adherent mucus levels in rats subjected to cold-restraint stress. Efaroxan alone given i.c.v., did not influence gastric secretion nor did it affect moxonidine's ability to decrease gastric secretion. Similarly, peripherally administered efaroxan did not block the antisecretory effect of moxonidine given i.c.v. However, when both compounds were given i.p., efaroxan pretreatment at all but the lowest doses significantly blocked the antigastric secretory effect of moxonidine. Efaroxan alone (i.p.) did not influence stress-induced gastric mucosal injury or adherent mucus levels. However, pretreatment of rats with efaroxan i.p. significantly blocked the mucus-preserving effect of i.p. moxonidine. These results demonstrate that central (i.c.v.) or peripheral (i.p.) administration of the I1-imidazoline receptor agonist moxonidine is associated with gastroprotection. The ability of i.p. efaroxan to block the effects of i.p. moxonidine but not i.c.v. moxonidine indicates that imidazoline receptors located centrally and peripherally may represent two unique sites associated with gastroprotection.

Agmatine, an endogenous imidazoline receptor agonist, increases gastric secretion and worsens experimental gastric mucosal injury in rats.

The present experiments tested the actions of a putative endogenous imidazoline receptor agonist, agmatine, on gastric secretion and on experimental gastric mucosal injury in rats. Agmatine, given i.p. (0.5-20.0 mg/kg) or i.c.v. (0.5-2.5 micrograms), augmented basal gastric acid secretion in conscious rats to a maximum of 40% when given i.p. and 44% when given i.c.v. Agmatine also potentiated total secretory volume as well as gastric acid and pepsin output in pylorus-ligated rats. When administered before exposure to stress, agmatine significantly decreased gastric glandular mucus levels and exacerbated stress-induced gastric mucosal injury. These results are in contrast to our data showing that an exogenous agonist of I1-imidazoline receptors, moxonidine, is a potent antisecretory and gastroprotective agent. A precise physiological role for agmatine in blood pressure regulation and in gastrointestinal function awaits clarification. However, it is possible that agmatine functions as an "inverse agonist" at central imidazoline receptors, resulting in hypertension, augmented gastric secretion and exacerbated gastric mucosal injury.

Some novel 5-hydroxytryptamine1A (5-HT1A) receptor agonists reduce gastric acid and pepsin secretion, reduce experimental gastric mucosal injury and enhance gastric mucus in rats.

The present studies examined the actions of a series of novel arylpiperazine 5-hydroxytryptamine1A (5-HT1A) agonists, developed originally for anxiolytic efficacy, in several models of gastric secretion and experimental gastric mucosal injury. These models included conscious gastric acid secretion, pylorus ligation (gastric acid and pepsin secretion), stress-induced gastric mucosal injury, ethanol-induced gastric mucosal damage and gastric adherent mucus levels. 2-(4-[4-(4-Nitropyrazol-1- yl)butyl]-1-piperazinyl)pyrimidine (E4414) and 2-(4-[4-(4-chloropyrazol-1-yl)butyl]-1-piperazinyl)pyrimidine dihydrochloride (E4424) significantly inhibited gastric acid secretion in conscious as well as in pylorus-ligated rats. Both compounds also significantly reduced pepsin secretion in pylorus-ligated animals. E4414 and E4424 significantly reduced both stress-induced and ethanol-induced gastric mucosal injury, and both compounds significantly maintained gastric adherent mucus levels in rats subjected to stress. The antisecretory and gastroprotective actions of E4414 and E4424 were of significantly greater magnitude than those of the reference 5-HT1A agonists, buspirone and 8-hydroxy-2-(di-n- propylamino)tetralin. These results suggest that some novel 5-HT1A agonists exert gastroprotection not only through reduction of aggressive elements in the gut (acid and pepsin secretion) but also through enhancement of defensive gastrointestinal factors such as adherent mucus.

Effects of the selective I1 imidazoline receptor agonist, moxonidine, on gastric secretion and gastric mucosal injury in rats.

1. Previous reports of the effects of alpha 2-adrenoceptor stimulation on gastric secretion are inconsistent because it was not clear whether the compounds were activating alpha 2-adrenoceptors and/or newly described imidazoline receptors. In the present experiments, the effects of moxonidine, an I1-imidazoline receptor agonist and antihypertensive agent, on gastric secretion and on experimental gastric mucosal injury were examined. 2. Moxonidine (0.01, 0.1 and 1.0 mg kg-1, i.p.) potently inhibited basal (non-stimulated) gastric acid secretion in conscious rats with an ED50 of 0.04 mg kg-1. Two hours following administration of the highest dose of moxonidine (1.0 mg kg-1), gastric acid output was completely suppressed. Moxonidine also significantly increased intragastric pH, at the two highest doses. 3. The alpha 2-adrenoceptor agonist, clonidine (0.01, 0.1 and 1.0 mg kg-1, i.p.) decreased basal acid secretion at the lowest dose (37%) and at the highest dose (46%), while the intermediate dose did not affect gastric acid output. 4. In an ethanol-induced model of gastric mucosal injury, moxonidine decreased the length of lesions at the lowest and highest doses (0.01 and 1.0 mg kg-1) as well as the number of the lesions, at the highest dose (1.0 mg kg-1). 5. In pylorus-ligated rats, moxonidine significantly decreased acid secretion (all doses), total secretory volume (1.0 mg kg-1) as well as pepsin output (1.0 mg kg-1). 6. In comparison to clonidine, moxonidine appears to be a more potent anti-secretory and gastric-protective compound. These data indicate a potential role for imidazoline receptor agonists in the management of gastroduodenal diseases associated with hypertension. The relative contribution of the central and peripheral effects of moxonidine to these gastrointestinal actions remains to be determined.

A dopamine D3 receptor agonist, 7-hydroxy-N,N-di-n-propyl-2-aminotetralin, reduces gastric acid and pepsin secretion and experimental gastric mucosal injury in rats.

Dopamine D1/DA1 agonists are associated with significant gastroprotective and antisecretory effects. The new dopamine DA3 agonist, 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OHDPAT), possesses neural and behavioral properties that are similar to those of DA1 agonists. In the present experiments, the effects of 7-OHDPAT on gastric acid secretion in conscious and anaesthetized rats, on restraint stress-induced gastric mucosal injury and on gastric adherent mucus levels were examined. 7-OHDPAT (2.5, 5.0 and 10.0 mg/kg) reduced significantly basal gastric acid secretion in conscious rats (66%, 92% and 78% inhibition, respectively). 7-OHDPAT also reduced significantly gastric acid and pepsin secretion in pylorus-ligated rats. 7-OHDPAT reduced stress-induced gastric mucosal injury at doses of 1.0, 5.0 and 10.0 mg/kg. Gastric adherent mucus was preserved only at the 5.0 mg/kg dose. Neither pretreatment with the DA1 antagonist, SCH23390, nor with the DA2 antagonist, eticlopride, affected 7-OHDPAT-induced reductions in gastric secretion or gastric mucosal injury. Although dopamine DA3 receptors exhibit greater amino acid sequence homology with DA2 receptors than with DA1 receptors, the possibility nevertheless exists that some of the gastrointestinal actions of dopamine and its agonists--both anti-secretory and gastroprotective--may be exerted through activation of DA3 receptors.

The antisecretory effects of clozapine, a dopamine D4 receptor antagonist, are blocked by the dopamine D1 receptor antagonist, SCH23390.

Dopaminergic compounds affect gastric secretion and response to experimental gastric mucosal injury. We showed previously that the novel dopamine D4 receptor antagonist, clozapine, significantly reduces gastric acid secretion and restraint stress-induced gastric lesions. Because the selectivity of clozapine for D4 receptors has recently been questioned, we tested the ability of a known D1 receptor blocker, SCH23390, to affect clozapine-induced reduction in gastric acid secretion. SCH23390 given i.p. or i.c.v., at doses that did not affect gastric acid secretion, significantly blocked the anti-secretory effect of clozapine, administered either peripherally or centrally. These data suggest that neither clozapine nor SCH23390 exhibit as high a degree of selectivity for the dopamine D4 and D1 receptor, respectively, as previously believed.

Chlorotyrosine exerts renal effects and antagonizes renal and gastric responses to atrial natriuretic peptide.

3-Chloro-L-tyrosine (3CT) is an inhibitor of tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis. In vivo inhibition of tyrosine hydroxylase results in lower catecholamine levels. 3CT (0.5 mg/kg), administered as a bolus i.v. to anesthetized uninephrectomized rats, elicited increases of 72% and 44% in urinary sodium concentration and volume, respectively, whereas a dose of 1 mg/kg caused increases of 27% and 29%. 3CT, 1 mg/kg, resulted in a 2-fold increase in plasma aldosterone (ALD); 0.5 mg/kg was without significant effect. At a dose of 1 mg/kg 3CT significantly antagonized the renal effects of atrial natriuretic peptide (ANP) (1.5 micrograms kg-1 min-1 by intrarenal infusion), expressed as an enhanced excretion of urine volume (102 +/- 14 vs. 70 +/- 11 microliters/min) and sodium (16.1 +/- 1.8 vs. 11.5 +/- 1.7 microEq/min) and increased osmolar clearance (171 +/- 12 vs. 144 +/- 13 microliters/min). A dose of 0.5 mg/kg of 3CT did not produce these same responses to ANP. The increased urine flow caused by 3CT may reflect reduced norepinephrine synthesis. The inverse dose-effect relationship of 3CT on urine flow rate may result from concomitant depletion of dopamine (DA) and elevated circulating ALD. The antagonism of 3CT on responses to ANP is not at the receptor level, because 3CT did not compete for [125I] ANP binding or inhibit ANP-stimulated guanylate cyclase in kidney cell membranes. It was proposed that the reduced basal sympathetic and renal DA tone, together with the elevated ALD level, account for this antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)

Restraint stress in biomedical research: an update.

Since the publication of our initial review of restraint stress in 1986, much work has continued with this technique, either as a tool for the investigation of other pharmacological, physiological, or pathologic phenomena or with restraint stress itself serving as the object of the study. As we noted in 1986, the major use of restraint has been for the induction of stress responses in animals and, more specifically, for the investigation of drug effects, particularly as they affect typical stress-related pathology--gastrointestinal, neuroendocrine, and immunological agents have been extensively studied. In compiling this update on restraint stress and its effects, we noted an increasing emphasis on central nervous system mechanisms in peripheral disease, especially gastrointestinal disease. In particular, many CNS-active agents have been tested for their effects on gastric and duodenal lesion formation and gastric secretion, including antidepressants, antipsychotics, anxiolytics, noradrenergic, serotonergic, dopaminergic, and peptidergic compounds. Some of these agents are especially active in the gastrointestinal tract even when administered centrally, further solidifying the concept of a brain-gut axis. The present update includes studies of: methods and procedures, pre-restraint manipulations, post-restraint/healing effects, and drug effects. In addition, a current bibliography of reports that have employed restraint is included.

Central antisecretory and peripheral gastroprotective effects of GBR 12909, a selective dopamine uptake inhibitor.

A variety of dopaminergic compounds influence gastric secretion and response to injury. In particular, agonists of the D1 receptor are gastroprotective when given either centrally of peripherally. In the present studies, we show that an indirect dopamine (DA) promoter, GBR 12909, a selective DA uptake inhibitor given ip but not icv, protects against restraint-cold stress-induced gastric mucosal injury. This protection likely occurred through preservation of gastric adherent mucus, since all doses of GBR 12909 resulted in gastric mucus levels at or near control (non-stressed) values. When given, ip, GBR 12909 did not influence basal gastric acid secretion in conscious rats, however, when given icv, GBR 12909 inhibited gastric acid secretion with an ED50 of about 0.5 microgram (1.13 umoles). We conclude that both central and peripheral DA contributes to gastrointestinal integrity through reduction of aggressive elements in the gut as well as by enhancing gastric mucosal defence.

Clozapine, a dopamine DA4 receptor antagonist, reduces gastric acid secretion and stress-induced gastric mucosal injury.

Dopamine and its agonists modulate a variety of gastrointestinal functions. In light of the increasing attention directed toward novel dopamine receptors and compounds that are active at these sites, we examined the effects of a dopamine D4 antagonist and putative antipsychotic, clozapine, in a model of conscious basal gastric acid secretion and in a model of stress-induced gastric mucosal injury. At a dose of 10.0 mg/kg i.p., clozapine significantly inhibited basal gastric acid secretion by 84% relative to vehicle. Lower doses (2.5 and 5.0 mg/kg) were inactive. Doses of 2.5, 5.0 and 7.5 mg/kg i.p. all significantly reduced restraint stress-induced gastric mucosal injury in rats. The highest dose inhibited gastric lesions by 70% relative to vehicle. We conclude that dopamine D4 receptors, present in high concentrations in mesolimbic brain regions, modulate gastric function and pathology in addition to mesolimbic D1 receptors, whose role in gastrointestinal function is already established.

Central nervous system and gut interactions: dopamine and experimental gastroduodenal lesions.

There is increasing evidence for brain regulation of gastroduodenal function and pathological responses. This laboratory has demonstrated a significant role for dopamine (DA) as a modulator of gastrointestinal function and disease. Using models of both acute (ethanol restraint stress; cysteamine) and chronic (iodoacetamide-induced gastritis) gastroduodenal mucosal injury, as well as tests of gastric secretory function (conscious basal gastric acid secretion; pylorus ligation; ex vivo gastric chamber), we have shown that DA, particularly DA1/D1 receptor agonists are powerful gastroprotective agents. This action is demonstrable upon peripheral administration as well as central (particularly intramesolimbic) administration. DA1/D1 agonists such as SKF38393 and SKF75670C reduce experimental gastric mucosal injury and secretion while antagonists of these receptors, including SCH23390, worsen experimental gastroduodenal lesions and augment secretion. That there exists a significant central component to DA-induced gastroprotection is demonstrated by data showing that rats assessed as anxiety prone, develop a greater degree of experimentally induced gastric damage, require greater amounts of DA agonists for 50% gastroprotection and respond to exogenous stress challenge with greater central DA turnover and loss, relative to rats assessed as low in anxiety. Very recently, we showed that dopamine D4 receptor blockade by clozapine and activation of dopamine D3 receptors by 7-hydroxy-N, N-di-n-propyl-2-aminotetralin (7-OHDPAT) are also associated with antiscretory and gastroprotective effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Stress-induced reactivation of latent herpes simplex virus infection in rat lumbar dorsal root ganglia.

Clinical reports suggest that stress precipitates recurrent cutaneous Herpes simplex virus (HSV) infection, presumably by reactivating latent infection in sensory ganglia with subsequent centrifugal axonal spread to the skin. As an initial test of this hypothesis, rats with latent HSV, type-1, (HSV-1) infection in lumbar dorsal root ganglia (DRG) were exposed to a well-characterized acute stressor that produced gastric ulcers (U) and elevated plasma corticosterone (CS) concentrations. Stress-induced reactivation of latent HSV infection was suggested by the earlier appearance of cytopathic effect (CPE) in human foreskin fibroblast monolayers co-cultivated with ganglia from stressed rats than from nonstressed ones (4.5 +/- 0.2 and 6.4 +/- 0.4 [mean +/- SEM] days respectively; p < 0.001). No CPE was detected in monolayers co-cultivated with ganglia from non-infected rats. These initial results suggest that acute stress reactivates latent HSV-1 ganglionic infection.

Dopamine DA1 receptor activation reduces experimentally-induced gastritis in rats.

1. A selective dopamine DA1 receptor agonist, SKF38393, reduced the severity of experimentally-induced gastritis in rats. 2. This is the first such demonstration with a dopamine DA1 receptor agonist. 3. When considered together with other reported actions of D1/DA1 receptor agonists, these data confirm a significant gastroprotective role for dopamine.

Effects of neuropeptide Y and [Leu31,Pro34] neuropeptide Y on experimental gastric lesion formation and gastric secretion in the rat.

The present study examined the effects of neuropeptide Y (NPY) and a selective NPY1 receptor agonist, leucine31 proline34 neuropeptide Y ([leu31,pro34]NPY) on gastric lesion formation and gastric secretion in three preparations: Basal gastric acid secretion in conscious rats, restraint-induced gastric lesion formation and acid and pepsin output and gastric mucosal damage in pylorus-ligated rats. The hypothesis that benextramine, a non-selective NPY receptor antagonist, could attenuate responses to NPY or [leu31,pro34]NPY was also tested. Both NPY and [leu31,pro34]NPY (i.p. and i.c.v.) decreased basal gastric acid output, restraint-induced gastric lesion formation, and acid and pepsin secretion and gastric mucosal damage in pylorus-ligated rats. The magnitude of inhibition of secretion and of ulcer reduction was significantly greater for [leu31,pro34]NPY than for NPY at comparable doses. Benextramine blocked the protective effect of NPY and [leu31,pro34]NPY against restraint-induced gastric mucosal injury. Both central and peripheral treatment with benextramine blocked the antisecretory effects of centrally administered NPY and [leu31,pro34]NPY. These data were consistent with both a central and a peripheral action of NPY on the gut, possibly through Y1 receptors.

Use of a polypropylene mesh in fixing a chronic intracerebroventricular cannula.

Surgical implantation of an intracerebroventricular cannula allows investigators to determine the effects of central administration of compounds on physiological/pathological events taking place in the periphery. Attempts to examine the influence of repeated, chronic drug administration on gastrointestinal function have been hampered by the tendency of cannulae to become dislodged and lost during the course of such studies. We describe herein the insertion of surgical polypropylene mesh between the skull and the fixative during surgical implantation of an intracerebroventricular (i.c.v.) cannula. This inexpensive addition to a standard surgical procedure extends the period of time during which the cannula remains viable for i.c.v. drug administration. Extending the active life of the cannula, in turn, allows the investigator to reduce the number of animals used.

Effects of the Ca2+ chelators EGTA and EDTA on ethanol- or stress-induced gastric mucosal lesions and gastric secretion.

Ca2+ modulates gastric function and dysfunction as well as the release of cysteine proteases and metalloproteinases which have been implicated in the pathogenesis of gastric mucosal lesions. We thus tested the hypothesis that pretreatment with the Ca2+ chelators, ethylene diamine tetraacetic acid (EDTA) and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) might reduce the experimental gastric mucosal damage induced by restraint cold stress or 1.0 ml of 75% ethanol. Other rats were prepared with chronic indwelling gastric cannulas and the effects of EDTA and EGTA on conscious basal gastric acid output were assessed. In addition, rats were pretreated with EGTA or EDTA prior to pylorus ligation and their effects on acid and pepsin output assessed. Both EDTA and EGTA reduced significantly the extent of ethanol-induced gastric mucosal damage as well as the degree of stress-induced gastric lesions. To further characterize the mechanism of Ca2+ chelator protection against ethanol-induced gastric lesions, some rats were vagotomized or adrenalectomized prior to treatment with EGTA or EDTA, followed by ethanol or stress. Both adrenalectomy and vagotomy abolished gastroprotection by EGTA and slightly reduced that induced by EDTA in both models of experimental gastric mucosal injury. Both EDTA and EGTA reduced significantly basal gastric acid output, an effect which persisted for at least 2 h following their administration. Both compounds also decreased significantly acid and pepsin output in pylorus-ligated rats. We conclude that Ca2+ chelators attenuate both acid-dependent and acid-independent gastric lesions.(ABSTRACT TRUNCATED AT 250 WORDS)