WHO informal consultation on revision of guidelines on evaluation of similar biotherapeutic products, virtual meeting, 30 June - 2 July 2021.

The WHO informal consultation was held to promote the revision of WHO guidelines on evaluation of similar biotherapeutic products (SBPs) adopted by the Expert Committee on Biological Standardization (ECBS) in 2009. It was agreed in the past consultations that the evaluation principles in the guidelines are still valid, but a review was recommended to provide more clarity and case-by-case flexibility. The opportunity was therefore taken to review the experience and identify areas where the current guidance could be more permissive without compromising its basic principles, and where additional explanation could be provided regarding the possibility of reducing the amount of data needed for regulatory approval. The meeting participants applauded the leading role taken by the WHO in providing a much-needed streamlined approach for development and evaluation of SBPs which will provide efficient and cost-effective product development and increase patient access to treatments. It was recognized that the principles as currently described in the draft WHO guidelines are based on sound science and experience gained over the last fifteen years of biosimilar approvals. However, since these guidelines when finalised will constitute the global standard for biosimilar evaluation and assist national regulatory authorities in establishing revised guidance and regulatory practice in this complex area, it was felt that further revision and clarity on certain perspectives in specific areas was necessary to dispel uncertainties arising in the current revised version. This report describes the principles in the draft guidelines, including topics discussed and consensus reached.

Synthetic gestagens exert differential effects on arterial thrombosis and aortic gene expression in ovariectomized apolipoprotein E-deficient mice.

BACKGROUND AND PURPOSE: Combined hormone replacement therapy with oestrogens plus the synthetic progestin medroxyprogesterone acetate (MPA) is associated with an increased risk of thrombosis. However, the mechanisms of this pro-thrombotic effect are largely unknown. The purpose of this study was to: (i) compare the pro-thrombotic effect of MPA with another synthetic progestin, norethisterone acetate (NET-A), (ii) determine if MPA's pro-thrombotic effect can be antagonized by the progesterone and glucocorticoid receptor antagonist mifepristone and (iii) elucidate underlying mechanisms by comparing aortic gene expression after chronic MPA with that after NET-A treatment. EXPERIMENTAL APPROACH: Female apolipoprotein E-deficient mice were ovariectomized and treated with placebo, MPA, a combination of MPA + mifepristone or NET-A for 90 days on a Western-type diet. Arterial thrombosis was measured in vivo in a photothrombosis model. Aortic gene expression was analysed using microarrays; GeneOntology and KEGG pathway analyses were conducted. KEY RESULTS: MPA's pro-thrombotic effects were prevented by mifepristone, while NET-A did not affect arterial thrombosis. Aortic gene expression analysis showed, for the first time, that gestagens induce similar effects on a set of genes potentially promoting thrombosis. However, in NET-A-treated mice other genes with potentially anti-thrombotic effects were also affected, which might counterbalance the effects of the pro-thrombotic genes. CONCLUSIONS AND IMPLICATIONS: The pro-thrombotic effects of synthetic progestins appear to be compound-specific, rather than representing a class effect of gestagens. Furthermore, the different thrombotic responses elicited by MPA and NET-A might be attributed to a more balanced, 'homeostatic' gene expression induced in NET-A- as compared with MPA-treated mice.

SNARE proteins and rab3A contribute to canalicular formation in parietal cells.

SNARE proteins - rab3A - parietal cells - H+/K+-ATPase When stimulated by histamine, acetylcholine, or gastrin the luminal compartments of oxyntic parietal cells display conspicuous morphological changes. The luminal plasma membrane surface becomes greatly expanded, while the cytoplasmic tubulovesicles are decreased in parallel. Due to these membrane rearrangements the H+/K(+)-ATPase obtains access to the luminal surface, where proton secretion occurs. The stimulation-induced translocation of H+/K(+)-ATPase involves a fusion process. Exocytotic membrane fusion in neurons is achieved by the highly regulated interaction of mainly three proteins, the vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 (synaptosomal-associated protein of 25 kDa), also referred to as SNARE proteins. Using immunofluorescence microscopy we analysed the subcellular distribution of neuronal synaptic proteins and rab3A in resting and stimulated parietal cells from pig and rat. In resting cells all synaptic proteins colocalized with the H+/ K(+)-ATPase trapped in the tubulovesicular compartment. After stimulation, translocated H+/K(+)-ATPase showed a typical canalicular distribution. Syntaxin, synaptobrevin, SNAP25 and rab3A underwent a similar redistribution in stimulated cells and consequently localized to the canalicular compartment. Using immunoprecipitation we found that the SNARE complex consisting of synaptobrevin, syntaxin and SNAP25, which is a prerequisite for membrane fusion in neurons, is also assembled in parietal cells. In addition the parietal cell-derived synaptobrevin could be proteolytically cleaved by tetanus toxin light chain. These data may provide evidence that SNARE proteins and rab3A are functionally involved in the stimulation-induced translocation of the H+/K(+)-ATPase.

SAP 97 is a potential candidate for basolateral fixation of ezrin in parietal cells.

Acid secretion in gastric parietal cells is preceded by a dramatic increase in surface area of the apical membrane compartment, due to fusion of the H+/K(+)-ATPase-containing tubulovesicles. The resulting canaliculi must be fixed for a period of minutes by cytoskeletal elements to sustain acid secretion. Using immunofluorescence microscopy, the cytoskeletal linker molecule, ezrin, localizes to the apical canalicular membrane of parietal cells. Antibodies against ezrin precipitate H+/K(+)-ATPase and beta-actin. In addition to its apical localization, ezrin is found to be colocalized at the basolateral compartment with synapse-associated protein (SAP) 97. Immunoprecipitation confirms a direct binding of SAP 97 and ezrin. We conclude that ezrin is fixed to the basolateral compartment by SAP 97. Upon stimulation of acid secretion, ezrin moves to the apical surface where it might stabilize the canalicular microvilli by connecting to beta-actin and H+/K(+)-ATPase, thereby sustaining acid secretion.

Stimulation of pepsinogen release from chief cells by Helicobacter pylori: evidence for a role of calcium and calmodulin.

To define the mechanisms by which Helicobacter pylori stimulates pepsinogen secretion, the in vitro release of pepsinogen was studied using a preparation of pig chief cell monolayers. Helicobacter pylori induced a time- and concentration-dependent release of pepsinogen into the medium, with about a three-fold increase in pepsinogen secretion over controls found after 45 min of incubation. 3x10(7) H. pylori produced 50% of the maximal response found at a H. pylori count of 2x10(8). The action of H. pylori did not depend on the presence of the vacuolating toxin (vacA) and the cytotoxin-associated protein (cagA). Dibutyryl-cAMP and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also markedly stimulated pepsinogen secretion and enhanced the stimulatory effect of H. pylori. Helicobacter pylori-stimulated pepsinogen release was inhibited by lanthanum and the calmodulin antagonist W-7, but not by the L-type Ca2+ channel blocker nifedipine, TMB-8, an agent that blocks the release of Ca2+ from intracellular stores, the protein kinase C inhibitor staurosporine and the protein kinase A inhibitor H-8. It is suggested that H. pylori directly stimulates pepsinogen release from gastric chief cells and that this effect is mediated via the calcium/calmodulin messenger branch.

Effects of PGE2 and of different synthetic PGE derivatives on the glycosylation of pig gastric mucins.

The glycosylation of pig gastric mucins, discharged in response to prostaglandin (PG) E2 and to three synthetic PGE-derivatives (misoprostol, nocloprost, rioprostil) was compared. After a 20 h culture period in the absence or presence of 1 micromol/l of one of the PGs, mucins were isolated by gel chromatography and their glycosylation characterized by their linkage to a panel of lectins. For all tested PGs, a significantly increased lectin linkage to mucin glycoproteins of high molecular weight was detected; no significant effects were observed for low molecular weight glycoproteins. Within the stimulatory pattern, major effects were found for the linkage of peanut agglutinin and soybean agglutinin, suggesting predominant effects on the expression of galactose and N-acetyl-galactosamine. Only minor effects were found for sialic acid, mannose, N-acetyl-glucosamine and fucose expression, as evidenced by the linkage of Sambucus nigra agglutinin, Concanavalin A, Datura stramonium agglutinin and Ulex europaeus I agglutinin. All PGs exerted a similar stimulatory pattern. However, at the indicated concentration, misoprostol (281 +/- 36% of control) rendered a significantly higher overall effect than PGE2 (208 +/- 31%), whereas the increases induced by nocloprost (237 +/- 35%) and rioprostil (202 +/- 35%) were not significantly different from the PGE2 effects. These results, suggesting similar stimulatory effects of PGE2 and of the tested synthetic PGs on glycosylation of mucin oligosaccharides, discharged from mucous cells during an in vitro culture, may, at least in part, explain clinical findings that during an impairment of the endogenous PG synthesis, the tested synthetic PGs are effective exogenous substitutes for endogenous E-type prostaglandins and act as anti-ulcer drugs.

Pepsinogen synthesis during long-term culture of porcine chief cells.

The purpose of this study was to characterize time-dependent changes in pepsinogen (PG) synthesis of porcine gastric chief cells during long-term monolayer culture. Porcine chief cells were isolated by pronase/collagenase treatment of fundic mucosa and enriched by density gradient and counterflow centrifugation. PG isoenzymes were identified in [L-35S]methionine-labelled cultured chief cells by native polyacrylamide gel electrophoresis followed by phosphor imager analysis, protease detection and immunoblots with specific PG A and C antibodies. The obtained results suggest that porcine chief cell cultures, after an initial settling period, reached an approximate steady state in total protein content and synthesis as well as in PG content and isoenzyme pattern from days 3 to 9 of culture. The latter was characterized by the presence of at least two PG A and two PG C isoenzymes. During the supposed steady-state total PG synthesis averaged out at 34 +/- 2% of total protein synthesis, as detected by [L-35S]methionine incorporation, due to the synthesis of, mainly, PG A2 and, to a much lesser extent, PG C and A1. In line with an active secretion, PG A2 proportion was on average significantly higher in released (44 +/- 3%) than in intracellular labelled proteins (19 +/- 2%). In addition, PG release from chief cells cultured for 6 and 9 days could be stimulated by cholecystokinin-octapeptide. These data suggest that porcine chief cells in monolayer culture are a model well suited for the quantitative and qualitative characterization of PG isoenzyme synthesis and release during long-term investigations, for which an establishment of a culture steady state appears to be a useful prerequisite.

Effects of PGE2 on amount and composition of high molecular weight glycoproteins released by human gastric mucous cells in primary culture.

The aim of this study was to determine effects of prostaglandin E2 (PGE2) on amount and composition of high molecular weight glycoproteins (HMG), released by human gastric mucous cells in primary culture. PGE2 stimulated the release of HMG, as evidenced by measurement of total carbohydrate and protein content, in a concentration-dependent manner. At the maximally tested concentration of 10(-5) mol/l, the increase amounted to 53% and 85%, over controls, for carbohydrate and protein, respectively. The stimulated release was accompanied by alterations of HMG glycosylation. As detected by lectin-ELISA, there was a relative decrease in N-acetyl glucosamine and an increase in mannose and galactose content. The sialic acid content increased in parallel to the total carbohydrate content. These results suggest that PGE2 plays a regulatory role in the synthesis and secretion of HMG by human gastric mucous cells.

Recovery of cholecystokinin response of porcine gastric chief cells during monolayer culture.

Cell isolation may impair secretory chief cell functions. To evaluate whether a monolayer culture results in a recovery, we compared the effects of cholecystokinin (CCK) octapeptide (CCK-8) on pepsinogen release from freshly isolated and from cultured porcine chief cells. CCK-8 had no significant effect on freshly isolated porcine chief cells but stimulated pepsinogen release from 36- and 60-hour cultured cells with EC50 values of 180 and 130 nmol/l, respectively. Maximal stimulation, achieved at a concentration of 1 micromol/l, amounted to 289 +/- 63 (p <0.01) and 401 +/- 64% (p <0.01) of the respective control value. In addition, the CCK-8 concentration-response curve for 60-hour, but not for 36-hour cultured chief cells displayed a second stimulatory peak at a CCK-8 concentration of 100 pmol/l (266 +/- 55% of control value, p < 0.05) with an EC50 value of 16 pmol/l. The CCKA-receptor antagonist devazepide (10 nmol/l) prevented the stimulatory effect of 1 micromol/l CCK-8 on pepsinogen release of 60-hour cultured cells. The adenylate cyclase activator forskolin (10 micromol/l) potentiated the low concentration CCK-8 effect, shifting the peak stimulation to a CCK-8 concentration of 10 pmol/l, and inhibited the high concentration CCK-8 effect on 60-hour cultured cells. These results indicate a time-dependent recovery of the CCK response of porcine gastric chief cells in monolayer culture and suggest that this model has an advantage over freshly isolated chief cells with regard to the pharmacological characterization of CCK effects.

Characterization of CCK receptor-mediated effects on intracellular calcium of porcine chief cells.

Effects of cholecystokinin (CCK) receptor agonists and antagonists on intracellular calcium ¿[Ca2+]i¿ of isolated porcine chief cells were determined by FURA2 fluorometry. CCK-8 increased [Ca2+]i with an EC50 of 6 nmol/l. The CCKB receptor preferring agonists gastrin-17-I, desulfated CCK-8 and CCK-4 had only small stimulatory effects (< 12% of maximal CCK-8 effect, EC50's in the low nanomolar range) and did not inhibit the CCK-8 response, suggesting that they were acting at CCKB but not, as partial agonists, at CCKA receptors. A71378 had its main stimulatory effect in low and a slight additional effect in high concentrations (EC50 80 pmol/l and > 1 mumol/l), respectively, while A72962 had its main stimulatory effect in high concentrations (EC50 > 1 mumol/l). The CCK receptor antagonists L364.718, L365.260, CBZ-CCK27-32 and dibutyryl cGMP inhibited CCK-8 (100 nmol/l) response concentration-dependently with IC50's of 540 pmol/l, 2mumol/l, 3 mumol/l and 250 mumol/l, respectively. These results suggest that CCK effects on [Ca2+]i of porcine chief cells are mainly (> 80%) mediated via CCKA receptors, which differ from guinea-pig and rabbit chief cell receptors by a higher distinction capacity between selective CCKA and CCKB, receptor agonists (A71378 versus A72962) and antagonists (L364.718 versus L365.260) and by the apparent lack of activation by desulfated CCK-8 and gastrin-17-I. Isolated porcine chief cells therefore appear to be a favourable "in vitro" system to characterize CCKA receptor specific compounds.

Pharmacological regulation of gastric mucous glycoprotein secretion.

Gastric mucous glycoproteins (GMGs) are an important protective component of the gastric 'mucus bicarbonate barrier'. The characterization of drug effects on GMG metabolism is difficult because the quantification of GMGs poses analytical problems and because indirect drug effects (e.g. on other gastric secretory functions) can influence GMG metabolism or quantification and thereby complicate the interpretation of in-vivo experiments. The use of suitable in-vitro systems, in particular of isolated gastric mucous cells, helped to resolve the latter problem and enabled the characterization of direct effects of prostaglandins, gastric acid secretagogues, peptide hormones, growth factors, adrenoceptor agonists, and synthetic compounds (e.g. teprenone) on GMG metabolism. Furthermore, from these experiments, evidence has accumulated that the cyclic AMP system, the inositol trisphosphate/calcium/protein kinase C system and the cyclic GMP system are involved in the intracellular transmission of drug effects on GMG metabolism.

Prostaglandin E2 alters terminal glycosylation of high molecular weight glycoproteins, released by pig gastric mucous cells in vitro.

The gastric mucus layer consists of high molecular weight glycoproteins (HMG). E-Type prostaglandins (PGs) stimulate total HMG release from isolated gastric mucous cells. We determined the effects of PGE2 on HMG glycosylation. Pig gastric mucous cells were cultured for 20 h with 1 mumol/l PGE2. Released HMG were isolated by gel chromatography and periodic acid-Schiff (PAS)-positive sugars and protein-bound [14C]GlcNAc were determined. Monosaccharides terminally linked to HMG oligosaccharide chains were monitored by lectin enzyme linked immunosorbent assay (ELISA): N-acetylglucosamine (GlcNAc) with Datura stramonium agglutinin, N-acetylgalactosamine (GalNAc) with soy bean agglutinin, fucose (Fuc) with Ulex europaeus I agglutinin and sialic acids (Sial) with Sambucus nigra agglutinin. PGE2 stimulated total HMG release, indicated by an increase of PAS-positive sugars to 170% and [14C]GlcNAc to 220% of controls. Terminal GlcNAc increased to 128%, GalNAc to 133%, Fuc to 165% and Sial to 182%. In addition to stimulation of total HMG release, PGE2 caused alterations of HMG glycosylation, which may modulate HMG viscosity and microbiological barrier function.

Effects of prostaglandins on [Ca2+]i and adenylate cyclase activity in isolated porcine gastric mucous cells.

In porcine gastric mucous cells, isolated enzymatically from the fundic mucosa and enriched by counterflow centrifugation, PGE2 (1 microM) increased adenylate cyclase activity to 225% and, distinct from that documented for other species, also [Ca2+]i, measured fluorimetrically with Fura2/AM, in Ca(2+)-containing and Ca(2+)-free incubation medium to 182% and 165% of control values, respectively. PGF2 alpha, PGD2, the stable prostacyclin analogue iloprost and the thromboxane-mimetic U46619 had no significant effects on adenylate cyclase activity and [Ca2+]i. Histamine (10 microM) stimulated adenylate cyclase activity to 236% of control value, an effect which could be blocked by the H2-receptor antagonist ranitidine. However, histamine and the activators of the cAMP system forskolin and dibutyryl cAMP had no significant effect on [Ca2+]i, indicating that an activation of the adenylate cyclase/cAMP system per se does not result in an increase in [Ca2+]i. These data suggest that prostanoids stimulate adenylate cyclase activity and [Ca2+]i in gastric mucous cells via activation of EP-receptors linked to both second messenger systems.

Effects of EP-receptor subtype specific agonists and other prostanoids on adenylate cyclase activity of duodenal epithelial cells.

Rank order of agonist potency for activation of adenylate cyclase by the naturally occurring prostanoids PGE2, PGF2 alpha, PGD2, the stable PGI2 analogue iloprost, and the TXA2 mimetic U 46619, provides evidence for the existence of a distinct PGE-receptor on guinea-pig duodenal enterocytes. The PGE-receptor is likely to be of the EP2-subtype since the specific EP2-agonist 11-deoxy-PGE1 stimulated adenylate cyclase activity with a 20-fold higher potency than the EP1-agonist 17-phenyltrinor-PGE2 and the EP3-agonists MB 28767 and GR 63799. In addition, sulprostone (acting on both EP1- and EP3-receptors) was ineffective. Since the specific EP1-antagonist SC 19220 did not inhibit PGE2-stimulated adenylate cyclase activity, the involvement of EP1-receptors could be further excluded. The synthetic prostaglandin E-analogues misoprostol and nocloprost stimulated adenylate cyclase almost identically, though they were about 10-fold less potent than the natural PGE2.

Effects of histamine and activators of the cyclic AMP system on protein synthesis in and release of high molecular weight glycoproteins from isolated gastric non-parietal cells.

1. Glycoprotein and protein synthesis in and release from pig isolated, enriched gastric mucous cells were measured by the incorporation of N-acetyl-[14C]-D-glucosamine and [3H]-L-leucine, respectively, into cellular and released acid precipitable material. 2. Histamine and activators of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) system maximally stimulated total protein and glycoprotein synthesis in and release from the cells at concentrations of histamine (10 microM), forskolin (10-100 microM), 3-isobutyl-1-methylxanthine (100 microM), and dibutyryl cyclic AMP (1-3 mM), respectively. In the presence of 3-isobutyl-1-methylxanthine (30 microM) histamine stimulation was enhanced. 3. As shown by gel chromatography, stimulation by histamine (100 microM), forskolin (10 microM), 3-isobutyl-1-methylxanthine (100 microM) and dibutyryl cyclic AMP (1 mM) resulted in a release of high molecular weight (approximately 2 x 10(6) daltons) glycoproteins from the cells. The histamine H2-receptor antagonist, ranitidine (100 microM), blocked the effect of histamine. 4. We conclude that cyclic AMP-dependent processes are involved in the regulation of protein and glycoprotein synthesis in and the release of high molecular weight (mucous) glycoproteins from pig gastric non-parietal cells and that histamine may be a physiological activator of this system.

Effects of PGE2, carbenoxolone, cholecystokinin, and the thromboxane-mimetic U46619 on protein and glycoprotein production by isolated pig gastric mucosal cells.

We studied the effects of prostaglandin (PG) E2, carbenoxolone, cholecystokinin-octapeptide (CCK-OP), and the thromboxane-mimetic U46619, all known to stimulate gastric mucus secretion in vivo, on protein and glycoprotein synthesis in and release from isolated and enriched pig gastric mucous cells, as measured by the incorporation of [3H]L-leucine and N-acetyl-[14C]D-glucosamine respectively into cellular and released acid insoluble material. PGE2 stimulated glycoprotein and protein synthesis (EC50 7 and 30 nmol/L, respectively) and release (EC50 50 and 140 nmol/L, respectively) in a concentration-dependent manner, whereas carbenoxolone, CCK-OP and U46619 failed to enhance the incorporation of the tracers. We conclude that stimulation of mucus secretion by PGE2 is related to direct effects on protein and glycoprotein production of gastric non-parietal cells, whereas indirect effects may be involved in the stimulation by carbenoxolone, CCK-OP, and U46619.

Stimulation of glycoprotein and protein synthesis in isolated pig gastric mucosal cells by prostaglandins.

The purpose of this study is to evaluate the effects of different prostaglandin derivatives on protein and glycoprotein synthesis and secretion in isolated and enriched pig gastric mucous cells, as measured by the incorporation of [3H]L-leucine and N-acetyl-[14C]D-glucosamine respectively into acid insoluble macromolecules (AIM). PGE2 and 16,16-dimethyl-PGE2 enhanced the incorporation of the amino sugar into cellular (EC50 8 and 75 nmol/l) and secreted (EC50 30 and 270 nmol/l) AIM in a concentration dependent manner during a 20 hours incubation. After incubation for eight hours or more they also stimulated the incorporation of [3H]L-leucine into cellular AIM. PGF2 alpha was considerably less potent (EC50 greater than 1 mumol/l) than the E-type prostaglandins. Iloprost, a stable prostacyclin analogue, was ineffective.

Effects of histamine on protein and glycoprotein production of isolated pig gastric mucosal cells.

The production of glycoprotein and protein by isolated pig gastric non-parietal cells was measured by incorporation of N-acetyl-[14C]-D-glucosamine [( 14C]GlcNAc) and [3H]-L-leucine [( 3H]Leu), respectively, into acid insoluble material (AIM). Histamine enhanced incorporation of the tracers into cellular and released AIM in a concentration-dependent manner. The H2 receptor antagonist ranitidine completely blocked the effects of histamine (100 mumol/l) on [3H]Leu incorporation into cellular and released AIM (IC50 37 and 32 mumol/l, respectively) but had no inhibitory effect on the 16,16-dimethylprostaglandin-E2 - and forskolin-stimulated tracer incorporation. The H1 receptor antagonist mepyramine did not inhibit the histamine effect. We conclude that histamine is a stimulant of protein, via H2 receptors, and glycoprotein production of isolated pig gastric non-parietal cells.

Incorporation of N-acetyl-[14C]D-glucosamine and [3H]L-leucine by isolated pig gastric mucosal cells.

Glycoprotein and protein production of isolated pig gastric mucosal cells were determined by the incorporation of N-acetyl-[14C]D-glucosamine ([14C]GlcNAc) and [3H]L-leucine ([3H]Leu) into acid-insoluble macromolecules (AIM). In four cell fractions (F1-F4), obtained by counterflow centrifugation, specific [14C]GlcNAc incorporation was greatest in the mucous cell-enriched F2. Tracer incorporation by F2 cells, proceeded linearly up to 20 h, was inhibited by cycloheximide or incubation at 0 degree C, and enhanced by PGE2 1 mumol/l. Gel chromatography of released AIM revealed that PGE2-stimulated [14C]GlcNAc incorporation was predominantly directed into high molecular weight (2 X 10(6) daltons) glycoproteins, whereas [3H]Leu incorporation was mainly related to proteins of albumin-like molecular weight. We conclude that incorporation of [14C]GlcNAc by enriched pig gastric mucous cells (F2), further analyzed by gel chromatography, is a suitable probe to study the production of high molecular weight gastric mucous glycoproteins in vitro.

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.