Resistance mutations selected in vivo under therapy with anti-HIV drug HBY 097 differ from resistance pattern selected in vitro.

The quinoxaline derivative HBY 097, an orally active nonnucleoside inhibitor of HIV-1 reverse transcriptase (NNRTI), showed an efficient suppression of viral load in a dose-escalating phase I study with mean trough concentrations increasing from 137-1299 ug/l [Rübsamen-Waigmann et al., Lancet 349:1517]. Half-maximal inhibitory concentrations (IC50) for viruses grown from the patients at entry of the study were 0.1-3 nM, except for one patient who had a virus with reduced susceptibility to HBY 097 at entry (IC50: 160 nM). During therapy, only two patients developed a virus with a moderately increased IC50 (2.2 and 15 nM). This reduced susceptibility was associated with the known NNRTI-resistance mutation K ==> N at position 103, in contrast to resistance selection in vitro, which had yielded predominant mutations at positions 179 and 190. The Tyr mutation at position 181, inducing high resistance for other NNRTIs, was never observed. The resistant virus at study entry (IC50 = 160 nM) had a mutation at position 103 as well, combined with an AZT resistance mutation (K ==> R) at position 70, suggesting that nucleoside-resistance mutations may help increasing resistance to HBY 097. This is in line with our in vitro selection studies, where resistance mutations at the 'nucleoside sites' 74 and 75 increased the resistance phenotype of NNRTI mutations. Our findings highlight the crucial importance of IC50 determinations from cultured virus for determination of phenotypic resistance development during therapy and demonstrate that in vivo resistance development cannot be predicted from in vitro selection.

Gastrointestinal receptors and drugs in motility disorders.

The review describes gastrointestinal receptors which are of therapeutic interest for the treatment of motility disorders. It updates the present knowledge on muscarinic, adrenergic, dopamine, opioid and dihydropyridine receptors, their subtypes, cellular sites and functional role as drug targets. On the basis of this pharmacological receptor concept, drugs like bethanechol, clonidine, lidamidine, metoclopramide, domperidone, cisapride, loperamide and nifedipine are evaluated in proven and potential indications. In view of the very complex regulation of motility, our understanding of receptors is still fragmentary and our tools to treat motility disorders do not fulfil all therapeutic requirements. This review tries to point out the areas of particular need for further basic research and the prospects of further drug development.

Rational pharmacotherapy of gastrointestinal motility disorders.

Nervous control of gastrointestinal motility is extremely complex, is regulated by the enteric system, the "brain of the gut", and modulated by extrinsic nerves. This system with its multiplicity of transmitters and receptors does not always allow a clear interpretation of experimental data, especially with compounds lacking specificity. In this review the complex situation is described particularly in relation to receptor populations (cholinergic, adrenergic, dopamine, histamine, 5-hydroxytryptamine, opioid, gamma-aminobutyric acid (GABA), prostanoid and dihydropyridine receptors), therapeutic aspects of drugs and their usefulness in children. Newer principles with known drugs and promising new compounds with a more appropriate kinetic or fewer side-effects, deriving from distinct pharmacological groups, as candidates for the treatment of gastrointestinal disorders are considered e.g. anticholinergics (prifinium or actilonium bromide), adrenergic alpha 2-agonists (clonidine, lidamidine) for diarrhoea in diabetic neuropathy, adrenergic beta-blockers for shortening postoperative ileus (propranolol), dopamine receptor antagonists (metoclopramide, domperidone, alizapride) and another prokinetic substance (cisapride) which may be useful for a number of applications as gastro-oesophageal reflux, gastro-paresis, intestinal pseudo-obstruction, cystic fibrosis and constipation, morphine derivatives (e.g. loperamide) for intractable diarrhoea and calcium antagonists (e.g. nifedipine) for achalasia. Increasing experience in digestive tract pharmacology and reliable clinical studies will furthermore be the basis for a more specific and better tolerated therapy of gastrointestinal motility disorders in adults and children.

Modulation of rat gastric mucosal prostaglandin E2 release by dietary linoleic acid: effects on gastric acid secretion and stress-induced mucosal damage.

We studied chronic intake of diets deficient in or supplemented with linoleic acid to determine whether it affects gastric acid secretion, release of prostaglandin E2, and stress-induced lesions. For 8-10 wk rats were fed three dietary regimens supplying 3.5% (control group), 0.3%, and 10% of total calories as linoleic acid. We found that diets deficient in linoleic acid (0.3%) reduced release of prostaglandin E2 into the gastric lumen (-77%) and increased basal (+133%) and pentagastrin-stimulated acid secretion (+93%) and the area of cold restraint-induced gastric mucosal lesions (+280%), when compared with the control group. Diets supplemented with linoleic acid (10%) increased prostaglandin E2 release into the gastric lumen (+106%) and reduced basal (-44%) and pentagastrin-stimulated acid secretion (-78%) and the area of cold restraint-induced mucosal.lesions (-80%). Prevention of these lesions by the 10% linoleic acid diet was confirmed by quantitative histology. Pretreatment with indomethacin (8 mg/kg intraperitoneally) abolished the effects of the 10% linoleic acid diet on prostaglandin formation, acid secretion, and mucosal injury. We conclude that in rats chronic intake of dietary linoleic acid reduces acid secretion and prevents cold restraint-induced mucosal lesions, possibly because of augmented synthesis of endogenous prostaglandins in the gastric mucosa.

Effect of rioprostil, an oral prostaglandin E1 (PGE1) analogue, on lower esophageal sphincter pressure and on the motility of the distal esophagus in healthy volunteers.

The influence of rioprostil on the resting pressure of the lower esophageal sphincter (LESP) and on the bolus-stimulated contraction wave amplitude of primary peristalsis was investigated in 9 healthy male volunteers receiving placebo or 300 and 600 micrograms of rioprostil orally in a randomised, double-blind, threefold cross over study. Manometry was performed using the low-compliance pneumohydraulic infusion system. Rioprostil in a dose of 600 micrograms slightly increased LESP and contraction wave amplitudes measured 5 cm and 10 cm above LES. The duration of the peristaltic contractions was not altered. We conclude that rioprostil in doses which inhibit effectively gastric acid and pepsin secretion and heal peptic ulcers has no inhibitory effects on esophageal motility. Thus rioprostil may be a candidate to treat reflux esophagitis and studies are warranted to establish its efficacy.

A calmodulin antagonist inhibits histamine-stimulated acid production by isolated rat parietal cells.

The role of calmodulin in the regulation of histamine-stimulated parietal cell function was studied in isolated rat parietal cells using [14C]aminopyrine uptake as a quantitative index of acid production. In enriched (77-87%) intact parietal cells the calmodulin antagonist naphthalene sulfonamide W 7 dose-dependently inhibited the response to 10(-4) M histamine (IC50: 2 X 10(-6) M). The mechanism of this inhibition was examined further with two other stimuli of H+-production: forskolin which directly activates the parietal cell adenylate cyclase without interacting at the histamine H2-receptor and dbcAMP which mimics the biological action of cAMP without preceding activation of adenylate cyclase. W 7 effectively inhibited the responses to 10(-4) M forskolin (IC50: 6 X 10(-7) M), 10(-3) M dbcAMP (IC50: 10(-6) M) and to 10(-2) M K+ (IC50: 3 X 10(-6) M). The action of W 7 followed non-competitive kinetics since the antagonist reduced the entire range of the concentration-response curves without shifting them rightwards towards higher concentrations of the respective stimulants. The effect of W 7 was reversed by washing the cells. ATP-induced [14C]aminopyrine uptake into digitonin-permeabilized oligomycin-inhibited parietal cells reflects H+-production independent of oxidative phosphorylation and was also inhibited by W 7 (IC50: 10(-5) M). Inhibition of K+-stimulated H+/K+-ATPase activity required even higher W 7-concentrations (IC50: 1.4 X 10(-4) M). Our data suggest that calmodulin might be involved in the intracellular mediation of the response to histamine. Between histamine-induced cAMP-generation and the H+-secreting tubulovesicular system W 7 seems to inhibit an intracellular step that finally activates the H+/K+-ATPase. Yet, direct inhibition of the ATPase requires W 7 concentrations of questionable specificity and is unlikely to be the mechanism behind the action of W 7 on the parietal cell response to histamine.

Dose response relationship of relaxin on the pubic ligament of the mouse.

In the present work, the dose-response relationship of highly purified porcine relaxin has been examined on broadening of the pubic ligament in mice. Using the method of Steinetz et al. with 7 days of oestriol priming, higher sensitivity of the pubic ligament was attained in mice with an original weight of 10 g than in those with a weight of 20 g. S-shaped curves were obtained; by increasing the relaxin doses after maximal effect had been reached a decreased action was observed. With presomen priming, a relaxin effect was established after only 2 days instead of the usual 8 days but the action was markedly less than in the Steinetz test.

Effect of glucagon on adenylate cyclase activity and acid production of isolated human parietal cells.

The direct effect of glucagon on human parietal cell function in vitro was tested by measuring adenylate cyclase (AC) activity and H+ production in homogenates of human gastric mucosa obtained during surgery or at biopsy. Cells isolated from mucosa obtained during surgery showed an increase in AC with histamine and glucagon. In parietal cell enriched fractions (75%) glucagon and histamine stimulated AC much more effectively than in parietal cell depleted fractions (15% and 7%). In contrast, glucagon did not affect basal or histamine stimulated 14C amino pyrine uptake. In homogenates of mucosal biopsy specimens 2 X 10(-7) mol/l glucagon enhanced AC activity by 76% (corpus) and 20% (antrum). In the same homogenates 10(-4) mol/l histamine caused a stimulation by 161% (corpus) and 38% (antrum). In fundic biopsy specimens glucagon displayed a biphasic concentration response curve with an increase at 10(-10) mol/l (46% above basal AC activity) and a maximum at 2 X 10(-7) mol/l (97%). Histamine elicited the maximal response (192%) at 10(-3) mol/l. Increasing histamine and glucagon concentrations caused additive stimulation of AC. Ranitidine did not change AC in response to glucagon but abolished the effect of histamine. Data suggest that the glucagon action is mediated by separate (glucagon?) receptors. As H+ production was not affected by glucagon, the coexistence of two AC systems in the human parietal cell is postulated: One that is activated via histamine H2-receptors and which stimulated H+ production; another that is activated by glucagon and is directed towards other, possibly metabolic effects.

Effects of glucagon and histamine on human parietal cells.

Since in vivo pancreatic glucagon inhibits gastric acid secretion it was of interest to test its direct effect on human parietal cell function in vitro by measuring adenylate cyclase (AC) activity and H+ production. Cells were isolated from human gastric mucosa obtained at surgery for peptic ulcer. In enriched (75%) parietal cells glucagon and histamine stimulated AC much more effectively than in the parietal cell depleted (15%, 7%) fractions. In contrast basal and histamine-stimulated [14C] aminopyrine uptake, an indirect measure of parietal cell H+ production, was not affected by glucagon. In homogenates of mucosal biopsy specimens 2 X 10(-7) mol/l glucagon enhanced AC activity by 76% (corpus) and 20% (antrum), respectively; in the same homogenates 10(-4) mol/l histamine caused a stimulation by 161% (corpus) and 38% (antrum). In fundic biopsy specimens glucagon displayed a biphasic concentration response curve with an increase at 10(-10) mol/l (46% above basal AC activity) and a maximum at 2 X 10(-7) mol/l (97%); histamine elicited the maximal response (192%) at 10(-3) mol/l. Histamine (10(-5), 10(-4), 10(-3) mol/l) and glucagon (10(-10) to 10(-6) mol/l) caused additive stimulation of AC. Ranitidine did not change AC in response to glucagon but abolished the effect of histamine. Our data demonstrate that glucagon stimulates an AC bound to the parietal cells. This response is not blocked by ranitidine suggesting that the glucagon action is mediated by a separate receptor, possibly by a glucagon-receptor. Furthermore we have shown that glucagon in contrast to its effects on AC does not affect H+ production.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphologically different biopsy specimens of the human gastric mucosa. II. Adenylate cyclase activity in response to prostaglandin E2 and histamine.

Adenylate cyclase (AC) in response to prostaglandin E2 (PGE2) and histamine was studied in morphologically different biopsy specimens from human gastric mucosa. The activities of the enzyme were log-normally distributed and did not differ between males and females. PGE2 activated AC in a concentration-dependent manner in normal gastric mucosa (n = 57), chronic superficial gastritis (GI, 18), chronic gastritis with beginning atrophy (GII, 10), chronic atrophic gastritis (GIII, 24), gastric ulcer (GU, 39), duodenal ulcer (DU, 32), and biopsies of patients operated according to Billroth II (BII, 20) and was most efficacious in GIII and BII. Histamine, which was studied in normal gastric mucosa (n = 27), DU (n = 20), GU (n = 13), and BII (n = 18), stimulated AC most efficaciously and potently in DU, was less effective in normal gastric mucosa and GU, and had no effect at all in BII. Cimetidine treatment of DU patients did not change the PGE2 action, while the degree of stimulation by histamine was reduced. The data indicate characteristic differences of the PGE2- and histamine-sensitive AC in the mucosal samples of these patients.

[Isolated human gastric mucosa cells--studies on physiologic and pharmacologic regulatory mechanisms]. / Isolierte menschliche Magenschleimhautzellen--Untersuchungen physiologischer und pharmakologischer Regulationsmechanismen.

Cells were isolated by use of collagenase, EDTA and pronase form human gastric mucosa obtained at peptic ulcer surgery (n = 61) or at Whipple's operations (n = 6). Enriched parietal cell fractions were prepared by isopycnic centrifugation with Percoll. H+ production, intracellular instrinsic factor and histamine content were maximal in the low density fraction containing 75% parietal cells and--among other nonparietal cell types--mast cells. H+ production, intrinsic factor secretion and adenylate cyclase-activity responded to histamine stimulation in a concentration dependent manner. Response was blocked by histamine H2 receptor antagonists (rantidine, famotidine). Dibutyryl cAMP and the phosphodiesterase inhibitor IMX were the most powerful stimuli whereas carbachol, hexoprenaline and pentagastrin were less effective. Prostaglandin E2 and 6-keto-PGF2 alpha occurred in the highest concentrations in the low density cell fraction. PG production increased linearly for 15 min and seemed to be influenced by the intracellular calcium level.

[14C]-aminopyrine accumulation by isolated human parietal cells.

Gastric mucosal cells were isolated from resected fundic mucosa of peptic ulcer patients and used to determine H+ production by [14C]-aminopyrine (AP) uptake. H+ production was stimulated by histamine linearly for 40 min and in a concentration-dependent manner reaching a maximum of 228% of basal at 10(-4) mol/l histamine. This response was reduced by the histamine H2-receptor antagonist ranitidine (IC50 = 3 X 10(-6) mol/l). Pentagastrin and carbachol induced a small but significant increase at 10(-7) and 10(-4) mol/l, respectively. At low histamine concentrations the response to carbachol plus histamine was nearly additive. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was a powerful stimulant of basal AP uptake but failed to augment the histamine effect. IBMX-induced AP uptake was reduced by ranitidine suggesting that, at least in part, the response to IBMX is mediated by release of endogenous histamine. Isopycnic centrifugation with Percoll resulted in fractions with 7 and 72% parietal cells, respectively. Basal AP uptake amounted to 203 cpm/10(6) cells in the parietal cell depleted fraction whereas in the enriched preparation the basal rate (4,787 cpm/10(6) cells) exceeded the uptake which was to be expected due to 10-fold parietal cell enrichment. When calculated as percentage of the basal H+ production the histamine-stimulated AP uptake was less in the enriched fraction than in the parietal cell depleted or in the nonfractionated preparation. Basal AP uptake was reduced by ranitidine more effectively in the enriched fraction suggesting background stimulation by endogenous histamine which might impair the response to exogenous stimulants. This functional evidence was paralleled by electron microscopical identification of mast cells copurified in the enriched parietal cell fraction.

Morphologically different biopsy specimens of the human gastric mucosa. I. The use of enzymatic cell isolation for quantitative determination of parietal cells.

Single biopsies of human gastric mucosa from controls and different groups of patients were used for enzymatic cell isolation by pronase and collagenase and subsequent count of parietal and nonparietal cells. This procedure was tested in regard to its validity and delivered the following cell numbers. Total gastric cells/mg wet weight gastric mucosa: normal gastric mucosa [controls (C), n = 95] 31,500 +/- (SEM) 1,490, chronic superficial gastritis (GI; n = 49) 36,300 +/- 2,770, chronic gastritis with beginning atrophy (GII; n = 36) 44,100 +/- 3,050 (p less than 0.025), chronic atrophic gastritis (GIII; n = 12) 40,100 +/- 5,760, duodenal ulcer (DU; n = 26) 29,340 +/- 2,280, gastric ulcer (GU; n = 23) 37,090 +/- 3,000, gastric resection according to Billroth I (BI; n = 7) 57,480 +/- 12,360 (p less than 0.005) and Billroth II (BII; n = 12) 52,560 +/- 6,730 (p less than 0.005). Parietal cells/mg wet weight gastric mucosa: 1,910 +/- 490 (C), 1,980 +/- 140 (GI), 1,700 +/- 200 (GII), 1,170 +/- 220 (GIII, p less than 0.025), 2,580 +/- 240 (DU, p less than 0.05), 1,690 +/- 150 (GU), 1,500 +/- 250 (BI), 1,360 +/- 320 (BII). Parietal cell concentration (density) did not differ in males and females and did not change with age. The method delivers relevant cell numbers, is suitable to detect qualitative differences and can be used for the interpretation of biochemical studies.

Effects of hormones (calcitonin, GIP) and pharmacological antagonists (ranitidine and famotidine) on isolated rat parietal cells.

The rationale for the present study was to compare calcitonin and gastric inhibitory polypeptide (GIP) versus two histamine H2 receptor antagonists with respect to their potency of inhibiting parietal cell functions. Adenylate cyclase activity and acid production ([14C]aminopyrine uptake) of isolated rat parietal cells were stimulated by histamine. At 10(-7) and 10(-6) mol/l, calcitonin and GIP reduced the response to histamine by 10-20% following noncompetitive kinetics. Ranitidine and famotidine (MK 208) inhibited the response to histamine by about 50% at 10(-7)-10(-6) mol/l, and at 10(-5) mol/l abolished the histamine effect. On a molar basis famotidine turned out to be 6 times more potent than ranitidine. Both antagonists revealed competitive kinetics. Our data suggest direct inhibition of the parietal cells by the tested compounds which were shown to interfere at the adenylate cyclase cAMP system or at the histamine H2 receptor. However, compared to the histamine H2 receptor antagonists, hormonal inhibition is less pronounced and mediated by a different mechanism.

Cyclic GMP and acid production in isolated gastric cells of the rat.

Just as cAMP is regarded as an intracellular mediator of histamine, so has cGMP been connected with cholinergic stimulation of gastric acid secretion. The object of the present investigation was to study the possible role of cellular cGMP on 14C-aminopyrine uptake, an indirect measure of parietal cell H+-production, by using mixtures of isolated rat gastric cells and fractions with different parietal cell content. Cellular cAMP and cGMP. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) enhanced the cAMP and cGMP of gastric cells in a time- and concentration-dependent manner, by 98 and 124% (1 mmol/l) and was included in all further studies. In parietal cell enriched fractions, histamine elevated cAMP by 109% (100 mumol/l) without changing cGMP while carbachol did not influence either nucleotide. Various thiols and nitrogen compounds strongly enhanced cellular cGMP, e.g. hydroxylamine and L-cysteine (1 mmol/l) by 527 and 656%, whereas changes in cAMP were minimal. The hydroxylamine response occurred in parietal cell depleted and enriched fractions. 14C-aminopyrine (AP) uptake. IBMX alone reduced the basal AP uptake, potentiated the effect of histamine, and inhibited the effect of carbachol, which alone stimulated basal accumulation by 302%. The most efficacious stimulant of parietal cell H+-production was dibutyryl cAMP (582%, 100 mumol/l), whereas dibutyryl cGMP was without effect. However, this latter compound (1 mmol/l) reduced AP accumulation due to dibutyryl cAMP almost completely. Thiols and nitrogen compounds all more or less reduced AP uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine content and release of isolated rat gastric mucosal cells.

Enzymatically dispersed rat gastric cells were subdivided (Percoll) in fractions (F1, F2, F3) with different number of parietal cells (PC) and the intracellular histamine content was estimated (ng/10(6) cells): F1 (9% PC): 17 +/- 4 (SEM), F2 (26% PC): 80 +/- 9 and F3 (77% PC): 134 +/- 15. Histidinedecarboxylase showed the same pattern of distribution, F1 low, F2 medium, F3 high activity. Incubated F3 cells constantly released histamine (19 +/- 3 ng/10(6) cells/h), a process which could be stimulated by carbachol, forskolin and hexoprenaline. The data suggest: rat gastric histaminocytes copurify with PC, background stimulation by histamine should be considered in isolated cell systems, vagal stimulation may release histamine within the gastric mucosa.

H+ production by isolated cells from human gastric mucosa.

Gastric mucosal cells were isolated from human mucosa obtained at surgery. H+ production was indirectly estimated by 14C aminopyrine (AP) uptake. The maximal response to histamine occurred after 30 min of incubation whereas intrinsic factor (IF) secretion was maximal after only 7.5 to 15 min. According to the concentration response curve 10(-4) mol/1 histamine proved to be the most effective concentration, the response to which was completely inhibited by ranitidine. Carbachol, dibutyryl cAMP and IMX also enhanced AP uptake, IMX being even more powerful than histamine. Carbachol and IMX failed to potentiate the response to histamine. Parietal cell fractions enriched by a Percoll density gradient revealed a pronounced background stimulation so that additional stimulation by test agents was less effective than in non-fractionized cells.

Prostaglandin formation by isolated gastric parietal and nonparietal cells of the rat.

Rat gastric cells isolated by pronase and subdivided by Percoll into 3 fractions (F1, F2, F3) were used to study prostaglandin E2 (PGE2) formation and, as an indirect measure of parietal cell H+ production, [14C]-aminopyrine uptake. Cells that had not been fractionated, with 20 to 25% parietal cells, contained at 0 degree C 1.7 +/- 0.35 (s.e.mean) ng PGE2 10(8) cells-1. During incubation at 37 degrees C these cells steadily synthesized up to 4.36 +/- 0.73 ng PGE2 10(8) cells-1 from endogenous substrate. Indomethacin in concentrations higher than 10(-6) mol 1(-1) inhibited this basal formation completely, but 10(-4) mol 1(-1) did not reduce the cellular PGE2 level below 1.4 +/- 0.2 ng 10(8) cells-1. Arachidonic acid in concentrations higher than 10(-5) mol 1(-1) evoked an abundant formation of PGE2, and 10(-4) mol 1(-1) built up a plateau of over 7.5 +/- 1.65 ng PGE2 10(8) cells-1 within 15 min. PGE2 formation in cell fractions increased significantly with the number of parietal cells per assay tube. Indomethacin (10(-8) to 10(-4) mol 1(-1] did not influence the histamine-stimulated uptake of [14C]-aminopyrine, while arachidonic acid (10(-5) to 10(-4) mol 1(-1] inhibited this process. PGE2 formation in response to arachidonic acid was prevented by indomethacin, but the inhibition of aminopyrine uptake by arachidonic acid could not be prevented by indomethacin. The data suggest that isolated gastric cells of the rat sustain constant PGE2 synthesis in vitro, which is more pronounced in parietal than in mucosal and chief cells. PGE2 may exert different effects within distinct gastric cell types.

Intrinsic factor secretion from isolated human gastric mucosal cells.

Human gastric mucosal cells were isolated from the resected fundic mucosa of peptic ulcer patients. The intracellular content and secretion of intrinsic factor were estimated by binding to cyano[57Co]cobalamin. The content was maximal in the enriched parietal cell fraction which also displayed the highest H+ production as measured by amino[14C] pyrine uptake. Secretagogues evoked full response after 15 min of incubation: pentagastrin (181% of basal secretion), carbachol (208%), histamine (250%) and dibutyryl cyclic adenosine monophosphate (304%). The phosphodiesterase inhibitor isobutylmethylxanthine was slightly more effective even than dibutyryl cAMP. The response to histamine was abolished by ranitidine, indicating activation of adenylate cyclase via histamine H2 receptors, but remained unaffected by atropine, which in turn blocked the carbachol effect, whereas ranitidine was ineffective. The mean formation rate was 8.4 fmol intrinsic factor/10(6) cells per h under basal conditions and 14.3 fmol in response to histamine.