Richard Magous

Acid secretion and membrane reorganization in single gastric parietal cell in primary culture

Summary— A digitally‐enhanced videomicroscopy study of rabbit gastric parietal cells in primary culture was performed using alternate observations with differential interference contrast and fluorescence optics of cells mounted and perfused on a temperature‐controlled microscope stage. The effect of histamine, a physiological effector of acid secretion, was followed. Isolated parietal cells possess an internal apical vacuole, which kept the cell in a pseudopolarized state. This apical vacuole is a site of acid secretion. This was demonstrated by the direct visualization of the uptake of the fluorescent weak base 9‐amino acridine and of the concomitant enormous swelling of the acid vacuole which reached an estimated size of 3–7 times the normal cell volume. This morphological change of shape and acidification of apical vacuoles was fully reversible and cells could respond to successive stimulations. A quantitative study of these events provided a value of the acid accumulation index for each single cell in response to histamine. Individual cell response varied within a factor of 7. The cellular localization of the proton pump complex responsible for acid secretion and of the major components of the secretory microvilli, actin and ezrin, a histamine‐dependent phosphorylation target of protein kinase A, were detected by indirect immunofluorescence microscopy in resting and stimulated cells. Both actin and ezrin colocalized at the apical vacuole membrane in resting and stimulated cells, whereas the proton pump shifted from an intracytoplasmic pool to the apical vacuole membrane upon stimulation.

Gastrointestinal hormone receptors on isolated smooth muscle cells from gastric antrum of the rabbit

The regulation by gastrointestinal polypeptide hormones of contraction and relaxation of functionally isolated smooth muscle cells from gastric antrum of the rabbit has been investigated. Gastrin, cholecystokinin (CCK-8) and motilin induced a rapid contraction of isolated cells: significant response occurred within a 5-sec incubation with these peptides and maximal response (40% decrease in cell length) after 30 sec. A higher sensitivity of smooth muscle cells to gastrin and CCK-8 than to motilin stimulations was demonstrated (EC50 = 10 pM for both gastrin and CCK-8 and EC50 = 1 nM for motilin). The minimal gastrin fragment required to get full contraction was the C-terminal pentapeptide amide common to gastrin and CCK. Proglumide inhibited gastrin- or CCK-8- but not motilin-induced contractions with an IC50 of 50 microM. contraction induced by gastrin and motilin required normal levels of extracellular calcium, whereas that due to CCK-8 seemed to be independent of extracellular calcium. Vasoactive intestinal polypeptide (VIP) caused a relaxation of smooth muscle cells maximally contracted by carbachol or CCK-8 or gastrin (EC50 = 2.2 nM) with a parallel increase in intracellular cAMP content.

Common or distinct receptors for gastrin and cholecystokinin in gastric mucosa

The differentiation between gastrin (HG) and cholecystokinin (CCK) receptors in gastric mucosa was examined on isolated parietal (F3) and non-parietal (F1) cells from rabbit fundic mucosa separated by elutriation. Direct binding assays on enriched cell populations were performed using 125I-labeled HG-17, 125I-labeled CCK-8 and 125I-labeled CCK-39 as probes. (1) On F1 cells, the dissociation constants (Kd) for the two labeled CCKs were nearly the same (62 pM for CCK-8 and 74 pM for CCK-39) but the binding capacity for CCK-8 was 2-times higher than for CCK-39. HG-17 also bound to this cell population, but its Kd value as about 2-times higher (110 pM) than that of CCK. The presence of two distinct classes of sites on F1 cells can be suggested from competition studies: one more specific for CCK, which bound CCK-8 and CCK-39 with the same affinity, and another class more specific for gastrin, which bound CCK-8 and HG-17 with the same affinity and CCK-39 with a low affinity. (2) On F3 cells, CCK-8 and HG-17 bound with similar affinities (Kd values 81 pM for CCK-8 and 87 pM for HG-17), but CCK-39 did not specifically bind to this cell population. The presence of a binding site more specific for HG than for CCK on F3 cells was confirmed by competition studies in which CCK-33 competed for binding with labeled HG-17 and labeled CCK-8 with a 50-times lower affinity than the other peptides.

M3-subtype muscarinic receptor that controls intracellular calcium release and inositol phosphate accumulation in gastric parietal cells

The muscarinic receptor subtype which triggers acid secretion was investigated in isolated rabbit gastric parietal cells. Cytosolic free Ca2+ concentration ([Ca2+]i), measured with the fluorescent indicator FURA-2, increased rapidly after full agonist (carbachol) stimulation (6-8 sec), then returned to an intermediate sustained value. Other M2-agonists, oxotremorine and arecoline, produced a partial [Ca2+]i increase, whereas M1-agonists, pilocarpine and [4-m-chlorophenylcarbamoyloxyl]-2-butynyl-trimethylammonium, were without any significant effect. [Ca2+]i rise was inhibited by selective muscarinic antagonists: atropine greater than 4-diphenylacetoxy-N-methyl-piperidine methbromide greater than quinuclidinylbenzilate (QNB) greater than pirenzepine greater than 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H- pyrido[2,3-b][1,4]benzodiazepine-6-one, this sequence being characteristic of the involvement of an M3-subtype. This inhibition was shown to be stereoselective; dexetimide and (-)QNB were more potent than levetimide and (+)QNB. The IC50 values for inhibition of [Ca2+]i increase by muscarinic antagonists were in good agreement with those obtained for inhibition of phospholipase C activation. In conclusion, the muscarinic receptor that controls acid secretion appears to be of the M3-subtype and the biochemical events coupled to the activation of this receptor system are also controlled through the same subtype.

Gastrin and CCK-8 induce inositol 1,4,5-trisphosphate formation in rabbit gastric parietal cells

The role of phosphoinositide turnover in the mediation of acid secretion was examined in an enriched preparation of isolated rabbit parietal cells (75%). Both gastrin and CCK-8 (octapeptide of cholecystokinin) stimulated [14C]aminopyrine (AP) uptake by cells (EC50 0.07 +/- 0.03 nM (gastrin) and 0.093 +/- 0.065 nM (CCK-8] and increased [3H]inositol phosphates cellular contents (EC50 0.142 +/- 0.016 nM (gastrin) and 0.116 +/- 0.027 nM (CCK-8] in a parallel fashion. In addition, the EC50 values for both phenomenon were quite similar to the Kd values obtained from binding experiments. HPLC analysis of the different [3H]inositol phosphates produced under gastrin or CCK-8 stimulation showed a 2-fold increase in [3H]Ins(1,4,5)P3 levels within 5 s with a concomitant increase in [3H]Ins(1,4)P2 content within 15 s. A low but significant rise in [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,3,4)P3 cellular contents was also observed. No difference between gastrin- and CCK-8-induced inositol phosphates production could be shown. We can conclude that gastrin and CCK-8 display an identical profile of action, suggesting that they stimulate the acid secretory function of parietal cells through the same receptor site coupled to the Ins(1,4,5)P3 production.

Biphasic kinetics of inositol 1,4,5-trisphosphate accumulation, in gastrin-stimulated parietal cells Effects of pertussis toxin and extracellular calcium

The effect of Pertussis toxin (PTx) and extracellular Ca2+ were investigated on gastrin‐induced Ins(1,4,5)P3 mass level in isolated gastric parietal cells. Basal Ins(1,4,5)P3 content was 5.48±0.49 pmol/500 000 cells. Gastrin (10 nM) induced a rapid increase in Ins(1,4,5)P3 content which was maximal after 15 s and corresponded to 2–2.5‐fold basal level; this Ins(1,4,5)P3 content then decreased within 30 s. After a longer time of gastrin exposure (> 1 min), a sustained and unexpected increased in Ins(1,4,5)P3 accumulation was observed which was maximal at 7.5 min (corresponding to 2.3–2.8‐fold basal value) and slightly decreased thereafter. PTx treatment of cells (200 ng/ml) for 3 h or removal extracellular Ca2+ did not affect the rapid rise, but drastically reduced the sustained increase in Ins(1,4,5)P3 content (60–100% inhibition); this inhibition was not evident after 10 min of hormone stimulation. Furthermore, diltiazem, a Ca2+ channel blocker, led to a similar inhibition of the sustained increase. We concluded that: (i) gastrin induced rapid increase in Ins(1,4,5)P3 content via a mechanism insensitive to PTx and to extracellular Ca2+, and (ii) gastrin induced a sustained increase in Ins(1,4,5)P3 level via a mechanism sensitive to PTx and to extracellular Ca2+. Even though the rapid rise in Ins(1,4,5)P3 content may be involved in the intracellular Ca2+ mobilization occurring after the first seconds of hormone stimulation, the physiological role of the sustained Ins(1,4,5)P3 increased level remains to be elucidated.

Intracellular coupling of prostaglandin inhibition of acid secretion in isolated rabbit gastric parietal cells.

Acid secretion from isolated rabbit gastric parietal cells can be stimulated by gastric secretagogues, histamine (cyclic-AMP pathway) and carbachol (inositol phosphate pathway). Prostaglandins (PG) from E series are potent inhibitors of acid secretion. The intracellular mechanism of this inhibition was examined by using a stable PGE1-analogue, misoprostol. Aminopyrine (AP) accumulations due to histamine, IBMX and forskolin were dose-dependently inhibited by misoprostol, whereas a weak but significant biphasic effect on carbachol-induced AP accumulation was observed. The cyclic-AMP formation induced by histamine and IBMX were also inhibited by misoprostol in a non-competitive way. The potent effect of forskolin on cyclic-AMP levels was not modified by misoprostol in parietal cells, whereas it was potentiated in non-parietal cells. The inhibitory effect of misoprostol on AP accumulation was reduced by incubation of parietal cells with Bordetella pertussis toxin (IAP) but not with Cholera toxin (CT). Pretreatment of the cells with IAP did not alter cyclic-AMP levels of resting and histamine-stimulated parietal cells but abolished the inhibitory effect of misoprostol. Treatment with CT increased basal and histamine-stimulated cyclic-AMP levels and masked the inhibitory effect of misoprostol. The biphasic effect of misoprostol on carbachol-stimulated AP accumulation in parietal cells was confirmed on carbachol-stimulated phospholipase C activity and on [Ca2+]i stimulated by carbachol. These data confirm a direct and specific effect of the prostanoid on the Gi-subunit of the adenylate cyclase coupled to the histamine H2-receptor, and a biphasic effect on the phospholipase C pathway of the parietal cells.

Relationship between inositol 1,4,5-trisphosphate mass level and [14C]aminopyrine uptake in gastrin-stimulated parietal cells

The relationship between gastrin-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content and [14C]aminopyrine ([14C]AP) uptake (an index of in vitro acid secretion) was investigated in a population of highly enriched rabbit parietal cells (90 +/- 5%). Gastrin induced a rapid rise in Ins(1,4,5)P3 content which was maximal within 15 s of stimulation (2- to 2.5-fold basal level) followed by a rapid decrease within 30 s; a high Ins(1,4,5)P3 level could also be observed after a longer time of hormone stimulation (180 s). Gastrin dose-dependently induced Ins(1,4,5)P3 accumulation and [14C]AP uptake; both dose-response curves were similar (EC50 approximately 0.1 nM). Furthermore, L-365,260 (3-(acylamino)benzodiazepine), a selective gastrin/CCK-B receptor antagonist, dose-dependently inhibited Ins(1,4,5)P3 production and [14C]AP accumulation induced by 10 nM gastrin with a similar potency (IC50 approximately 1-2 nM). These results led us to conclude that Ins(1,4,5)P3 is involved in gastrin-stimulated acid secretory activity of gastric parietal cells.

Signal transduction pathways of muscarinic receptors in circular smooth muscle from the rabbit caecum

The effects of muscarinic acetylcholine receptor stimulation on phosphoinositides breakdown and adenylate cyclase activity were examined in the circular smooth muscle of the rabbit caecum. InMyo-[3H]inositol-labeled circular smooth muscle cells, carbachol caused a concentration-dependent increase in [3H]inositol phosphates ([3H]IPs) accumulation (EC50 of 3±1 μM). The M1-selective antagonist pirenzepine (PRZ), the M2-selective AF-DX 116 (11-2[[2-[(diethyl-amino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepin-6-one) and the M3-selective para-fluoro-hexahydrosiladifenidol (p-F-HHSiD) inhibited the carbachol-induced [3]inositol phosphates accumulation with the following order of potency: p-F-HHSiD>PRZ>AF-DX 116. In saponin-permeabilized circular smooth muscle cells, carbachol and GTPγ[S] elicited a concentration-dependent increase in [3H]inositol phosphates accumulation. The concentration-response curve for GTPγ[S] was shifted to the left when cells were incubated with 1 μM carbachol. The [3H]inositol phosphates accumulation elicited by simultaneous addition of 0.1 μM GTPγ[S] and 1 μM carbachol to permeabilized cells was significantly decreased (78.28±18.23% inhibition) when cells were preincubated for 5 min with 0.1 mM GDPβ[S]. In nonpermeabilized cells, pertussis toxin did not alter the carbachol-induced increase in [3H]inositol phosphates accumulation. On the other hand, the 0.1 mM carbachol-induced inhibition of forskolin-stimulated adenylate cyclase activity in circular smooth muscle homogenates was significantly reversed by atropine and AF-DX 116, whereas PRZ and p-F-HHSiD were ineffective (muscarinic antagonists were used at 1 μM final concentration). Moreover, the carbachol-induced inhibition of the cyclic AMP accumulation elicited by 10 μM isoproterenol was abolished by pertussis toxin pretreatment of isolated circular smooth muscle cells. In conclusion, our data suggest that in circular smooth muscle of rabbit caecum, the muscarinic receptor stimulation of [3H]inositol phsophates accumulation is mediated by M3 subtype receptors coupled to a pertussis toxin-insensitive G protein, whereas inhibition of adenylate cyclase activity is mediated by M2 subtype receptors coupled to a pertussis toxin-sensitive GTP-binding protein Gi.