Pertussis toxin-sensitive and pertussis toxin-insensitive inhibition of parietal cell response to GLP-I and histamine WOLFGANG SCHEPP, JOHANNA SCHMIDTLER, KERSTIN VOLKER SCHUSDZIARRA, AND MEINHARD CLASSEN Department of Medicine II, Technical University of Munich, 8000 Munich 80, Federal Republic of Germany Johanna Schmidtler, Kerstin Schepp, Wolfgang, Dehne, Volker Schusdziarra, and Meinhard Classen. Pertussis toxin-sensitive and pertussis toxin-insensitive inhibition of parietal cell response to GLP-1 and histamine. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G660-G668, 1992.-We have recently shown that in rat parietal cells the glucagon-like peptide 1 (GLP-1) variants 7-36 amide, l-37, and l-36 amide stimulate H+ production as indirectly measured by [‘*Cl aminopyrine (AP) accumulation. This response to the GLP-1 peptides was intracellularly mediated by activation of adenylate cyclase and by adenosine 3’,5’-cyclic monophosphate (CAMP) as second messenger. In the present study, we compared prostaglandin (PG)E2, somatostatin, and the protein kinase A antagonist Rp-adenosine-3’,5’-monophosphorothioate (Rp-CAMPS) with respect to their inhibitory effects on parietal cell function induced by GLP-1 or histamine. PGE2 and somatostatin noncompetitively inhibited AP accumulation and CAMP production in response to the GLP-1 variants and histamine (I&J: [mean inhibitory concn 5 x lo-’ M PGE,; 3 x low7 M somatostatin]; at their maximal concentrations PGE2 (10m7 M) and somatostatin (low6 M) caused 85 and 65% inhibition, respectively. Treatment with pertussis toxin (PT; 250 rig/ml; 4 h) reversed the inhibitory effect of PGE2 and somatostatin on AP accumulation and CAMP production. At 2 x 10s3 M (I&: 3 x low4 M) Rp-CAMPS completely inhibited AP accumulation induced by the GLP-1 variants or histamine; this effect was insensitive to PT. Specificity of Rp-CAMPS as protein kinase A inhibitor is suggested by inhibition of AP accumulation in response to Sp-CAMPS and N6,02-dibutyryl adenosine 3’,5’-cyclic phosphate sodium, and forskolin, activators of protein kinase A and adenylate cyclase, respectively. We conclude that the parietal cell responses to GLP-1 and histamine are inhibited by identical mechanisms. Effects of PGE2 and somatostatin are mediated by the PTsensitive subunit of adenylate cyclase Gi, whereas Rp-CAMPS interferes with CAMP-dependent mechanisms that are insensitive to PT. glucagon-like peptide 1; prostaglandin E2; somatostatin; Rpadenosine-3’,5’-monophosphorothioate; Sp-adenosine-3’,5’monophosphorothioate; rat parietal cell; hydrogen ion production; adenosine 3’,5’-cyclic monophosphate PEPTIDE 1 (GLP-1) is derived from the glucagon precursor by specific posttranslational processing in the intestine. In addition to the complete amino acid sequence, GLP-l-( l-37), three molecular variants have been identified, namely the nonamidated peptide GLP-l-(7-37), the COOH-terminal amidated GLP-l(l-36), and the NH2-terminal truncated GLP-l-(7-36) amide, The latter two peptides are released into the circulation on stimulation by nutrients (for review, see Ref. 12). The physiological relevance of the GLP-1 variants for the regulation of gastric acid secretion remains to be clarified. GLP-l-(7-36) amide and GLP-l-(1-36) amide have been shown to inhibit pentagastrin-stimuGLUCAGON-LIKE

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lated gastric acid secretion in humans (26); however, in a recent human study GLP-l-(7-36) failed to do so (19). In accordance with the latter report is our observation that GLP-l-( 7-36) fails to affect gastric acid secretion in anesthetized rats (27). Thus available in vivo data do not unequivocally support a physiological role of GLPl-(7-36) amide as an enterogastrone. On the other hand we have recently shown that in isolated rat parietal cells GLP-l-(7-36) amide, l-37, and l-36 amide stimulate [14C]aminopyrine accumulation, an indirect measure of H+ production (27). This response is not blocked by the histamine Hz-receptor antagonist ranitidine and involves activation of adenylate cyclase (27). Thus it is conceivable that stimulation by GLP-1 of parietal cells is mediated by the same cascade of transduction mechanisms that is activated by histamine, i.e., adenylate cyclase, adenosine 3’,5’-cyclic monophosphate (CAMP)-dependent protein kinase A, and consecutive protein phosphorylation steps (7, 8, 18, 31, 32, 34). If so, GLP-l-induced H+ production should be sensitive to the same inhibitors that are known to reduce the response to histamine. The focus of the present study in rat parietal cells is on inhibition by prostaglandin E, (PGE,) and somatostatin at the level of adenylate cyclase, and on inhibition by a CAMP analogue devoid of stimulatory properties at the level of protein kinase A. In parietal cells, histamine-induced production of H+ and CAMP is potently inhibited by PGE, (30, 38) and somatostatin (30) via specific receptors (20, 28). In canine (5, 20), rabbit (9), and rat parietal cells (1, Schmidtler and Schepp, unpublished observation), inhibition by PGE, or somatostatin was completely reversed by pertussis toxin (PT). This pharmacological tool causes irreversible ADP ribosylation of the a-subunit of Gi, the guanosine 5’-triphosphate-binding inhibitory protein of adenylate cyclase (14,33). As a consequence of PT treatment, the inhibitory pathway of this enzyme is blocked, rendering PGE, and somatostatin incapable of inhibiting parietal cell function. Thus both inhibitors apparently interact with specific receptors on the parietal cell surface, which are coupled to Gi. On the other hand, Rp- and Sp-adenosine-cyclic-3’,5’monophosphothioate (Rp- and Sp-CAMPS, respectively) act at an intracellular site distal to CAMP production. Rp- and Sp-CAMPS are diastereomers of an analogue of natural CAMP in which one of the two exocyclic oxygen atoms in the cyclic phosphate moiety is replaced by sulfur. Equatorial thio substitution leads to the R-isomer, whereas axial modification yields the S-compound. The suffix “p” indicates that R/S nomenclature refers to

0 1992 the American

Physiological

Society

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sac technique as previously described (15). The resulting crude cell preparation (20 t 4% parietal cells) was suspended in a modified Krebs-Henseleit buffer of the following composition (in mM): 0.5 NaH2P04, 1.0 Na2HP04, 20 NaHC03, 70 NaCl, 5 KCl, 1.0 CaC12, 1.5 MgC12, 11 glucose, 50 HEPES (as acid), 1,4dithiothreitol (15.5 mg/lOO ml), and bovine serum albumin (1 mg/ml). The medium was adjusted to pH 7.4 by tris(hydroxymethyl)aminomethane-HCl (1.0 mM) and was used for fractionation of the crude cell suspension by counterflow elutriation (rotor JE 6B, run in a 52-21 M/E centrifuge, Beckman Instruments, Glenrothes, UK). According to our recently described elutriation protocol (24), crude cells were loaded into the rotor at a flow rate of 18 ml/min and a rotor velocity of 2,500 revolutions/min (rpm). Nonparietal cells were washed out at 40 ml/min and 2,000 rpm, and a fraction enriched in parietal cells (76 t 4%) was collected at 58 ml/min and 2,000 rpm, respectively. Cell viability (trypan blue exclusion) exceeded 95%. As described previously (27), enriched parietal cells (5 X 106/ ml) were incubated in DMEM at 37°C for 4 h in the absence of test agents. In experiments studying the effect of PT on PGE2-, somatostatin-, or Rp-CAMPS-induced inhibition, identical cell preparations were incubated in parallel in the presence or absence of PT (250 rig/ml DMEM) (27). After this preinMATERIALS AND METHODS cubation, the cell suspensions were immediately used for studies on parietal cell function. Materials Measurement of H+ production and CAMP production. AcAll reagents were of analytical grade and were purchased cumulation of the weak base [14C]aminopyrine served as an from the indicated sources. Dimethyl- [‘4C]aminopyrine (sp act indirect quantitative index of H+-production within the parietal 114 mCi/mol) was from Amersham Buchler (Braunschweig, cells (29). Our modification of the original procedure has been FRG). Quickszint 2000 scintillation cocktail and Biolute S described before (27). Briefly, 400 ~1 of the cell suspension was tissue solubilizer were from Zinsser Analytic (Frankfurt, FRG). incubated in flat-bottomed plastic vials in a shaking bath at Bovine serum albumin, N-2-hydroxyethylpiperazine-N-237°C. At the beginning of the incubation, [14C]aminopyrine ethanesulfonic acid (HEPES), N6,3-isobutylmethylxanthine (0.04 &i/tube) was added together with all test agents (i.e., (IMX), histamine, N6,02-dibutyryl adenosine 3’,5’-cyclic phosstimuli and inhibitors). After 30 min, 200 ~1 of the suspension phate sodium salt (DBcAMP), and Dulbecco’s modified Eagle’s were layered over 1,000 ~1 of medium C and spun down in an medium (DMEM) containing HEPES and glutamine were from Eppendorf table centrifuge at 15,000 rpm for 10 s. The pellet Serva (Heidelberg, FRG). PT, PGE2, and carbamylcholine (carwas dissolved with 300 ~1 Biolute S. Thereafter, 4 ml scintillabachol) were from Sigma (Munich, FRG). Synthetic GLP-Ition cocktail was added, and radioactivity was determined in a (7-36) amide, GLP-l-(1-37), GLP-l-(1-36) amide, and so- liquid scintillation counter (LSD 1801, Beckman). According matostatin-( l-14) were from Peninsula Europe (St. Helens, to our previous data (27), the following were used as maximally UK). Rp-CAMPS and Sp-CAMPS were from BioLog (Bremen, and equally effective concentrations: lo-’ M GLP- l- (7-36) FRG). Forskolin was from Calbiochem (La Jolla, CA). Na2amide, 10s6 M GLP-l-(1-37), and low6 M GLP-l-(1-36) EDTA, pronase E, trypan blue, 1,4-dithiothreitol, dimethyl amide. sulfoxide (DMSO), and all buffer constituents were from Merck To amplify the responses to histamine or GLP-1, the incu(Darmstadt, FRG). bation medium (DMEM) contained 10V4 M IMX (27), an Stock solutions (10V4 M) of PGE2 were prepared in ethanol. inhibitor of phosphodiesterase. In rat parietal cells, concentraAliquots were stored at -70°C for up to 8 wk and diluted with tion response curves have shown that in the presence of 10s4 saline immediately before being added to the cell incubations. M IMX maximal stimulation of [14C]aminopyrine accumuThus when cells were incubated in the presence of the maximal lation is achieved with lOa M histamine and lo-’ M GLP-lconcentration of PGE2 ( 10M7 M) the final ethanol concentration (7-36) amide, respectively (24, 25, 27). At the concentration was 0.001% and affected neither the response to histamine nor studied, IMX does not exert an effect of its own on [‘“Clthat to GLP-1. DMSO at final concentrations ~0.02% was used aminopyrine accumulation in rat parietal cells (25). Cells stimas vehicle for forskolin; at these concentrations, DMSO did not ulated with Sp-CAMPS, DBcAMP, and carbachol were incuaffect parietal cell function. Histamine, Sp-CAMPS, Rpbated in the absence of IMX. Experiments with carbachol were CAMPS, DBcAMP, and carbachol were dissolved in 0.9% NaCl. performed in the presence of 3 x 10B3 M calcium Stock solutions of GLP-1, somatostatin, and PT were prepared The ratio of [14C]aminopyrine taken up in the parietal cells in saline containing 0.1% bovine serum albumin. Aliquots of to that in the undiluted incubation medium was calculated as all test agents were stored at -70°C for up to 8 wk and diluted described previously (29). Thus the ratio of [ 14C]aminopyrine with DMEM immediately before being added to the cell incuaccumulation was determined under basal conditions and in bations. the presence of the test agents. [14C]aminopyrine accumulation in the presence of 0.1 mM dinitrophenol represents nonspecific incorporation and was subtracted from the test values. Data Methods were normalized as the percentage of the maximal effect of the respective stimulus (GLP-I, histamine, Sp-CAMPS, DBcAMP, Cell isolation, parietal cell enrichment, and culture. Isolation forskolin, or carbachol). of mucosal cells from the stomachs of six female Wistar rats Parietal cell CAMP production was measured as previously (130-150 g, Charles River, Sulzfeld, FRG) per experiment was performed by pronase E digestion with the use of the everted described (27). After 30 min of incubation in DMEM containing

phosphorus. Both diastereomers are characterized by remarkable stability and membrane permeability (4). RpCAMPS binds to the regulatory subunit of the CAMPdependent protein kinase but does not dissociate the catalytic subunit (21). Thus Rp-CAMPS is a competitive inhibitor for activators of CAMP-mediated signal response transduction (22). In contrast, Sp-CAMPS mimics the effects of natural CAMP by dissociating the catalytic subunit, thereby activating the CAMP-dependent protein kinase (21, 22). The effects of Rp- and SpCAMPS have not yet been studied in parietal cells. It was the rationale of our present work to investigate whether PGEz and somatostatin inhibit GLP-l-induced rat parietal cell function via PT-sensitive mechanisms. Furthermore, we studied whether Rp-CAMPS, modulating the target of CAMP rather than the site of its production, inhibits the parietal cell response to GLP-1 and whether this action is sensitive to PT. The effects of these inhibitors on histamine-stimulated parietal cells were determined for comparison.

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10m4 M IMX and the test agents, 400 ~1 absolute ethanol was added to 400-~1 cell suspension (5 x lo6 cells/ml). After centrifugation at 8,000 g for 5 min, CAMP was determined in the upper aqueous phase by a commercial kit (RIANEN CAMP[ 1251],Du Pont, Dreieich, FRG). Cross-reactivity of the antibody was ~0.02% for other nucleotides [guanosine 3’,5’-cyclic monophosphate (cGMP), GMP, ATP, ADP, AMP]. Results (fmole. lo6 cells-l.30 min) were normalized as the percentage of the maximal response to the respective stimulus. Statistical analysis. Data were expressed as means t SE from 5-8 independent experiments in each of which all combinations of test agents were studied in quadruplicate. On the basis of the absolute values, analysis of variance for multiple determinations was performed followed by Student’s t test for unpaired data as post hoc test for calculation of statistically significant differences between individual treatments.Values of P < 0.05 were considered significant. RESULTS

Effects of PGE2 on Parietal Cell Function Inhibition by PGE2 of [ W]aminopyrine accumulation. [ 14C]aminopyrine accumulation in response to maximally effective concentrations of GLP-1-( 7-36) amide, GLP-l-(1-37), or GLP-l-(7-36) amide was inhibited by PGE, in a concentration-dependent manner. The lowest PGE, concentration to be effective was lo-’ M. Mean inhibitory concentration (I&) values were 2.5 t 0.8,4.2 t 1.9 and 2.8 t 1.0 X lo-’ M PGEB for inhibition of the responses to the respective GLP-1 variants. At 10B7M, PGEz reduced the response to the GLP-1 variants by 8595% (P < O.Ol), respectively (Fig. 1). The concentration-response curve of [14C]aminopyrine

PARIETAL

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accumulation in response to each of the three GLP-1 variants was studied in the presence and absence of low8 M PGE2. The prostaglandin reduced the maxima of the curves by 66-81% (P < 0.01) without causing a rightward shift toward higher GLP-1 concentrations (n = 5 independent cell preparations; not shown). These data are consistent with the assumption that inhibition by PGE2 follows noncompetitive kinetics. Reversal by PT of PGE2-induced inhibition. At 10v7 M, PGE2 inhibited [14C]aminopyrine accumulation in response to low4 M histamine or to maximally effective concentrations of the three GLP-1 variants (P < 0.01). After pretreatment of the identical cell preparations with PT, PGEz failed to exert statistically significant inhibition (histamine: P = 0.63; GLP-I-( 7-36) amide: P = 0.30; GLP-l-(1-37): P = 0.64; GLP-l-(1-36) amide: P = 0.37 vs. stimulus plus PGE2). Thus PT treatment of the cells before commencement of the [14C]aminopyrine accumulation study almost completely ‘reversed inhibition by PGE2 (Fig. 2). Effects of Somatostatin on Parietal Cell Function Inhibition by somatostatin of [ 14C]aminopyrine accumulation. Somatostatin was an effective inhibitor of GLP-l-induced [ 14C]aminopyrine accumulation. Higher concentrations were needed when compared with PGE2: the lowest concentration to inhibit the responses to the three GLP-1 variants was 10m8M somatostatin. I& values were 7.1 t 2.4 X 10m8M, 2.8 t 1.0 X 10s7M, and 1.8 & 0.6 X 10v7 M somatostatin, for inhibition of the - PT

t PT

I GLP-1 (7-36)

GLP-1 (l-37)

GLP-1 (l-36)

1O-8

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M

0 ALONE 0 t PGE2

Cl ALONE n + PGE2

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9

8

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9

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Fig. 1. Effect of graded concentrations of PGE, (solid symbols) on [ 14C] aminopyrine accumulation in response to lo-” M glucagon-like peptide 1 (GLP-l)-(7-36) amide (0; Left), 10s6 M GLP-l-(1-37) (0; middle), and 10V6 M GLP-l-(1-36) amide (A; right). In presence of (IMX) [ 14C]aminopyrine acculow4 M N6,3-i so b u t y 1me th y 1xanthine mulation ratio was 2.7 +: 0.6 under basal conditions, and 47.1 1: 5.3, to GLP-l-(7-36), GLP-l43.8 Ifr 4.9, and 44.2 k 5.5 in response (l-37), or GLP-l-( l-36) amide, respectively. Results were normalized to response (=lOO%) elicited by respective stimulant above basal ratio and are given as means -t- SE from n = 6 independent experiments comparing all 3 GLP-1 peptides in identical cell preparations. * P < 0.05; ** P < 0.01 vs. GLP-1 alone. ’

Fig. 2. Effect of 10m7 M PGE, (hatched bar) on [14C]aminopyrine accumulation in response to 10e4 M histamine (open bar), lo-” M GLPl-(7-36) amide, 10m6 M GLP-l-(1-37), and 10m6 M GLP-l-(1-36) amide, respectively (solid bar). Identical cell preparations were studied without (left) or with pertussis toxin (PT) pretreatment (250 rig/ml; 4 h; right). In control cells (Left), [ 14C] aminopyrine accumulation ratio in presence of 10m4 M IMX was 2.3 t 0.8 under basal conditions and 50.9 & 7.4, 47.6 t 6.1, 45.9 t 5.8, and 47.2 2 6.1 in response to histamine, GLP-l-(7-36) amide, GLP-I-(1-37), and GLP-l-(1-36) amide, respectively. In PT-treated cells in presence of 10m4 M IMX (right), corresponding ratios were 3.0 ? 0.5, 52.2 -I- 7.4, 49.3 t 6.8, 48.5 t 7.4, and 48.1 t 6.8, respectively. Results were normalized to response (=lOO%) elicited by respective stimulant above basal ratio and are given as means t SE from n = 8 independent experiments. ** P < 0.01 vs. stimulus alone.

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responses to GLP-l-(7-36) amide, GLP-l-(1-37), and GLP-l-(1-36) amide, respectively. As no higher somatostatin concentrations were tested, the apparently maximal inhibition was achieved with low6 M somatostatin, a concentration that reduced the responses to the three GLP-1 variants by 60-75%, respectively (P c 0.01; Fig. 3). Similar to PGE2, 10D7 M somatostatin lowered the entire range of the concentration-response curves of the three GLP-1 peptides by 4153% without shifting the maxima rightward toward higher GLP- 1 concentrations (n = 5 independent cell preparations; not shown). Thus somatostatin likewise appears to act as a noncompetitive inhibitor of GLP-l-induced [14C]aminopyrine accumulation. Reversal by PT of somatostatin-induced inhibition. Histamine-stimulated [ 14C]aminopyrine accumulation was reduced by 34.0 t 2.3% by 10e6 M somatostatin (P < 0.05). On the other hand, inhibition by somatostatin of the response to the three GLP-1 variants was more effective: only 30-40% of the response to the respective GLP-1 peptide persisted in the presence of somatostatin (P < 0.01). After pretreatment of the identical cell preparations with PT, somatostatin failed to exert statistically significant inhibition (histamine: P = 0.88; GLP-l-(7-36) amide: P = 0.73; GLP-l-(1-37): P = 0.78; GLP-l-(1-36) amide: P = 0.50 vs. stimulus plus somatostatin). Thus the inhibitory effect of somatostatin on histamine-induced as well as on GLP-l-induced [‘“Claminopyrine accumulation was completely reversed (Fig. 4) . GLP-1 (7-36)

GLP-1 (l-37)

1O-8 M

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0 ALONE n t SOMATOSTATIN

0 ALONE . t SOMATOSTATIN

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Fig. 3. Effect of graded concentrations of somatostatin (solid symbols) on [14C]aminopyrine accumulation in response to lo-” M GLP-l(7-36) amide (0; Left), 10V6 M GLP-l-(1--37) (0; middle), and 10e6 M GLP-l-(1-36) amide (A; right). In presence of lo-* M IMX [‘“Claminopyrine accumulation ratio was 2.5 ~fr 0.6 under basal conditions, and 46.4 t 4.7, 46.0 t 4.9, and 46.2 t 5.0 in response to GLP-l(7-36) amide, GLP-l-(1-37), or GLP-l-(1-36) amide, respectively. Results were normalized to response (=lOO%) elicited by respective stimulant above basal ratio and are given as means t SE from n = 8 independent experiments comparing all 3 GLP-1 peptides in identical cell preparations. * P < 0.05; ** P < 0.01 vs. GLP-1 alone.

t PT

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z 0

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% z 2 c u tf

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Fig. 4. Effect of 10s6 M somatostatin (hatched bar) on [14C]aminopyrine accumulation in response to 10m4 histamine (open bar), lo-’ M GLP-l-(7-36) amide, 10F6 M GLP-l-(1-37), and 10m6 M GLP-l(l-36) amide, respectively (solid bar). Identical cell preparations were studied without (left) or with pretreatment with PT (250 rig/ml; 4 h; right). In controls (Left) and in PT-treated cells (right) [ 14C]aminopyrine accumulation ratios in presence of 10T4 M IMX were identical with those indicated in Fig. 3 legend. Results were normalized to response (=lOO%) elicited by respective stimulant above basal ratio and are given as means t SE from n = 8 independent experiments. * P < 0.05; ** P < 0.01 vs. stimulus alone.

Inhibition by PGEz and somatostatin of CAMP production Compared with [ 14C]aminopyrine accumulation, maximal stimulation of CAMP production requires slightly higher histamine concentrations in parietal cells of guinea pig (3), dog (29, 31), and rat (27); the same holds for GLP-1-( 7-36) amide in rat parietal cells (27). Accordingly, we studied the effects of PGEx and somatostatin on CAMP production in response to 10V3 M histamine and 10B7 M GLP-l-(7-36) amide, respectively. In the absence of PT, PGEZ at lOa M inhibited CAMP production in response to low3 M histamine or 10D7 M GLP-l-(7-36) amide by 75.1 t 5.3 and by 45.9 t 6.6% of the response to the respective stimulus (P < 0.01). In the same cell preparations, 10B6 M somatostatin inhibited the response to histamine and GLP-l-(7-36) amide by 52.3 t 6.1 and 48.4 t 6.6%, respectively. Pretreatment of the cells with PT resulted in complete reversal of these inhibitory effects of PGE2 and somatostatin (Fig. 5). Effects of Rp-CAMPS on [ 14C]aminopyrine

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Parietal cell response to Sp-CAMPS and effect of RpCAMPS on receptor and postreceptor stimuli. In n = 3 separate cell preparations, Sp-CAMPS caused concentration-dependent stimulation of [ 14C]aminopyrine accumulation. Stimulation was initiated at 3 x 10V5 M and was maximal at 3 x low4 M. [14C]aminopyrine accumulation ratio was 2.1 t 0.5 under basal conditions, and 53.9 k 11.2, 61.0 & 12.4, and 59.8 t 12.1 in response to 3 x lOa M Sp-CAMPS, 3 X 10B4 M DBcAMP, and 3 x 10D5 M forskolin, respectively. In the identical cell preparations these concentrations of DBcAMP and forskolin were found to be maximally effective. Thus Sp-CAMPS

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HIST

E ii

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HIST

GLP-1 (7-36) lo-’ M

GLP-1

(7-36)

(7-36)

s2 120=g

00 Lk

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2% gg 60na. g 40nu 20 20=LL ,\” OPGE2 SOMATOSTATIN

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7 6

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Fig. 5. Effect of PGE2 or somatostatin (hatched bar) on CAMP production in response to 10d3 M histamine (open bar) or 10m7 M GLP-l(7-36) amide (solid bar). Identical cell preparations were tested without (left) or with PT pretreatment (250 rig/ml; 4 h; right). In control cells (left), CAMP production (fmol lo6 cells-’ 30 min-‘) was 1,262.7 t 178.5 under basal conditions and 5349.8 & 911.1, and 4,569.7 2 686.2 in response to histamine and GLP-l-(7-36) amide, respectively. In PT-treated cells (right) corresponding data were 1,313.l k 173.6, 6,219.7 t 587.8, and 5,411.6 & 951.6, respectively. Results were normalized to response (=lOO%) elicited by respective stimulant above basal and are given as means k SE from n = 8 independent experiments. ** P C 0.01 vs. stimulus alone. l

-

l-t7 5

4

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a -log M

5

4

3

2

Fig. 6. Effect of graded concentrations of Rp-CAMPS (solid symbols) on [ 14C]aminopyrine accumulation in response to 10V4 M histamine (0; left) or 10m8 M GLP-l-(7-36) amide (0; right). In presence of 10m4 M IMX [‘4C]aminopyrine accumulation ratio was 2.7 t 0.7 under basal conditions, and 51.6 t 7.3 and 48.2 t 6.1 in response to histamine and GLP-l-(7-36) amide, respectively. Results were normalized to response (=lOO%) elicited by respective stimulant above basal ratio and are given as means t SE from n = 5 independent experiments comparing histamine and GLP-l-(7-36) amide in identical cell preparations. *P < 0.05; ** P < 0.01 vs. stimulus alone.

l

reached 87-90% of the maximal efficacy of DBcAMP or forskolin (not shown). Rp-CAMPS (10m5to 2 x 10B3M) had no effect on basal [ 14C]aminopyrine accumulation (not shown). However, the responses to 10B4 M histamine or 10v8 M GLP-l(7-36) amide were inhibited in a concentration-dependent manner: ICsOwas 3.9 t 1.7 x 10D4M Rp-CAMPS in the presence of histamine and 1.3 t 0.5 X 10s4 M RpCAMPS in GLP-l-(7-36) amide-stimulated cells. At 2 x 10B3M Rp-CAMPS completely abolished the response to either stimulus (Fig. 6). To determine the specificity of Rp-CAMPS the antagonist was tested versus three postreceptor stimuli of CAMP-mediated [14C]aminopyrine accumulation: forskolin (low5 M), which activates the catalytic subunit of adenylate cyclase, as well as DBcAMP (10m4M) and SpCAMPS (3 x 10m4M), activators of the CAMP-dependent protein kinase A. Rp-CAMPS caused concentration-dependent inhibition (Table 1). With the respective stimuli, I& values were 1.1 t 0.4, 5.6 t 2.1, and 0.8 t 0.3 X 10m4 M for Rp-CAMPS, which at 2 X 10B3 M completely abolished stimulation. In an additional attempt to further determine the specificity of Rp-CAMPS, we studied the effect of the CAMP antagonist on carbachol-induced parietal cell function, which is thought to be mediated by Ca’+/phosphoinositol-dependent mechanisms (9). [14C]Aminopyrine accumulation in response to 10B4 M carbachol was inhibited by Rp-CAMPS, albeit with lower potency (Table 1). Up to 6 X 10m4M Rp-CAMPS failed to significantly inhibit the response to carbachol, although these concentrations reduce stimulation by histamine and GLP-l-(7-36) amide by 60-70% (Fig. 6);

thus at least the effect of up to 6 X 10B4M Rp-CAMPS can be considered specific. At l-2 X low3 M Rp-CAMPS significantly inhibited carbachol-induced [ 14C]aminopyrine accumulation, although not completely and less effectively than the responses to histamine and GLPl-( 7-36) amide (Table 1). Inhibition by 10s3 M RpCAMPS of carbachol-stimulated [14C]aminopyrine accumulation was gradually overcome by addition of 10V4to 10B3M DBcAMP (Table 2) suggesting specificity of RpCAMPS even at these concentrations. Failure of PT to reverse Rp-CAMPS-induced inhibition. At 3 x 10V4 M, a concentration close to the I&,, RpCAMPS inhibited histamine-induced and GLP-l-( 7-36) amide-induced [14C]aminopyrine accumulation in the absence as well as in the presence of PT (Fig. 7). Thus the inhibitory effect of Rp-CAMPS was completely resistant to PT and does not appear to involve Gi or other PT sensitive cellular transduction mechanisms. DISCUSSION

The present study demonstrates that in rat parietal cells stimulation by GLP-1 peptides, similar to that by histamine, is inhibited by PGE, and somatostatin and that the action of these inhibitors involves a PT-sensitive substrate. On the other hand, inhibition by Rp-CAMPS of the response to the GLP-1 variants and histamine is independent of PT-sensitive mechanisms. These data indicate that there are striking similarities between the GLP-1 peptides and histamine with respect not only to stimulatory but also to inhibitory cellular transduction mechanisms that control the response to both secretagogues. Production of CAMP by parietal cell adenylate cyclase mediates the stimulatory response to both GLP-1 peptides (27) and histamine (31) and appears to be the effector at which PGE, and somatostatin interfere. In the present study, during stimulation with either the

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Table 1. Inhibition by Rp-CAMPS of [14C] aminopyrine accumulation in response to CAMP-dependent postreceptor stimuli and to carbachol Rp-CAMPS, 1o-5

Forskolin DBcAMP Sp-CAMPS Carbachol

(10e5 M) (lo-* M) (3 x lo-* (lo-” M)

1o-4

93.2rt4.2 89.926.8 95.5t5.7 94.928.7

M)

3 x 1o-4

49.8t6.7* 70.3*5.3* 54.8&6.7* 87.8k6.1

M 6 x lO-4

40.4+5.3-t 64.7t7.2*

32.9*6.0-f 40.3+6.8t

87.6tll.l

76.6t9.4

1o-3

25.7+7.8? 28.5+lO.O”f 20.3+6.3-t 37.8+11.7”f

2 x 1o-3

0-t ot ot 20.5+6.3t

Results were normalized to response (=lOO%) elicited by the respective stimulant above basal ratio and are given as means & SE. Effect of graded concentrations of Rp-CAMPS on [14C]aminopyrine accumulation in response to forskolin, DBcAMP, Sp-CAMPS and carbachol. [‘“Claminopyrine accumulation ratio was 2.3 t 0.4 under basal conditions and 52.1 t 7.4, 54.5 * 8.3, and 47.9 t 5.3 in response to Sp-CAMPS, DBcAMP, and forskolin, respectively (n = 6 independent experiments comparing all 3 stimuli in identical cell preparations). In additional n = 6 experiments where carbachol was used as stimulus, [14C]aminopyrine accumulation ratio was 2.6 zt 0.5 under basal conditions and 23.4 & 3.5 in response to low5 M carbachol. * P < 0.05; t P < 0.01 vs. stimulus alone.

Table 2. Reversal by DBcAMP of Rp-CAMPS-induced inhibition of [ 14C]aminopyrine accumulation in response to carbachol

Carbachol

Carbachol Rp-CAMPS

100

38.4t4.7*

Carbachol + Rp-CAMPS + DBcAMP (1O-4 M)

+

Carbachol + Rp-CAMPS + DBcAMP (3 x 1O-4 M)

76.9210.8

114.9kl4.4

Effect of DBcAMP on Rp-CAMPS (10V3 [ ‘*C]aminopyrine accumulation in response [14C]aminopyrine accumulation ratio was 2.6 tions and 23.4 t 3.5 in response to 10V5 normalized to response to carbachol (=lOO%) t SE from n = 6 independent experiments. alone.

174.3t23.8*

M)-induced inhibition of to carbachol ( 10B5 M). & 0.5 under basal condicarbachol. Results were and are given as means * P c 0.01 vs. carbachol

-PT

t PT

I

'

HIST

Rp-CAMPS

Carbachol + Rp-CAMPS + DBcAMP (1O-3 M)

(3x)

GLP-1

4

4

HIST

44

GLP-1

4

4

4

4

- log M

Fig. 7. Effect of Rp-CAMPS (hatched bar) on [14C]aminopyrine accumulation in response to lo-* M histamine (open bar), 10s8 M GLP-l(7-36) amide, low6 M GLP-l-(1-37), and low6 M GLP-l-(1-36) amide, respectively (solid bar). Identical cell preparations were studied without (left) or with pretreatment with PT (250 rig/ml; 4 h; right). In control cells (left), [‘*Cl aminopyrine accumulation ratio in presence of lo-* M IMX was 2.4 k 0.8 under basal conditions and 50.5 t 7.1, 48.1 t 6.4, 46.0 of: 5.6, and 45.7 t 6.8 in response to histamine, GLP-l(7-36), GLP-l-(1-37), and GLP-l-(1-36) amide, respectively. In PT-treated cells (right), the corresponding ratios were 2.8 & 0.5, 51.7 t 7.3, 49.6 t 7.1, 48.1 f: 7.4, and 47.3 t 6.0, respectively. Results were normalized to response (=lOO%) elicited by respective stimulant above basal ratio and are given as means t SE from n = 6 independent experiments. ** P < 0.01 vs. stimulus alone.

GLP-1 peptides or histamine, PGE2 caused parallel inhibition of [14C]aminopyrine accumulation as well as of CAMP production indicating that PGE2 acts by reducing adenylate cyclase activity. The potency by which PGE2 inhibits the response to the GLP-1 peptides is similar to that by which the prostanoid reduces histamine-stimulated [14C]aminopyrine accumulation in isolated parietal cells from various species (I&o = 5 X 10-l’ to 1 X 10D8 M) (30, 38). Likewise, we found that PGE, is equally effective in inhibiting GLP-l- and histamine-stimulated parietal cell H+ production. PGE, has been reported to reduce the response to histamine by up to 7O%, a range that is confirmed by our present findings with the GLP1 peptides and histamine. Similar conclusions can be drawn from our results with somatostatin, which during stimulation by either the GLP-1 peptides or histamine caused parallel inhibition of CAMP production and [14C]aminopyrine accumulation. Reduction by somatostatin of GLP-l-induced CAMP production confirms and extends recent data (11). This study had shown that the analogue sandostatin (SMS 201-955) reduces GLP-l-(7-36) amide-stimulated CAMP production in rat gastric glands. Our data localize this effect to enriched parietal cells and correlate it to a biological response, i.e., inhibition of H+ production. In the present study, nanomolar to micromolar somatostatin concentrations inhibited the response to the GLP-1 peptides; this potency was similar to that previously reported in histamine-stimulated parietal cells (30). However, the present data demonstrate that the inhibitory efficacy of somatostatin was lower in histamine-stimulated than in GLP-l-stimulated [ 14C]aminopyrine accumulation. This discrepancy cannot be explained by more effective inhibition of GLP-l-induced than of histamineinduced adenylate cyclase activity, because we observed that CAMP production in response to both stimuli was equally well reduced by somatostatin. Furthermore, ranitidine failed to alter the response to GLP-1-( 7-36) amide in our enriched parietal cell fraction (27); this result does not support the hypothesis that GLP-l(7-36) amide might release endogenous histamine in the cell fraction studied thus allowing somatostatin to reduce the response to GLP-1 by inhibiting both histamine release and parietal cell function. It might, however, be hypothesized that, in addition to adenylate cyclase, the GLP-1 peptides activate a CAMP-independent trans-

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duction mechanism that is not induced by histamine and which is inhibited by somatostatin in a PT-sensitive manner. The latter postulate is based on our observation that PT completely reverses inhibition by somatostatin of the response to the GLP-1 variants. This hypothesis would fit the general concept that in various cell types somatostatin interacts with multiple mechanisms beyond adenylate cyclase, e.g., protein kinase C-mediated G-cell function (23), ion antiporters (2), and calcium channels (16, 17), the latter being sensitive to PT (16). Inhibition by somatostatin of such CAMP-independent mechanisms would explain the higher efficacy of somatostatin in reducing GLP- 1- compared with histamine-stimulated [‘“Cl aminopyrine accumulation in the present study. However, a CAMP-independent GLP-l-sensitive mechanism remains elusive as yet: the GLP-1 variants were not more effective than histamine in stimulating [‘“Cl aminopyrine accumulation; furthermore, in rat parietal cells the GLP-1 peptides do not induce the breakdown of membrane inositol phosphates (27). Thus we are presently unable to interpret the higher efficacy of somatostatin versus GLP-l-induced parietal cell function. Inhibition by PGEZ and somatostatin of the response to GLP-1 peptides as well as to histamine involves PTsensitive mechanisms. Because pretreatment of the parietal cells with PT completely reversed inhibition by PGE2 and somatostatin, the effect of both appears to be entirely mediated via a PT substrate. Thus PT-insensitive GTP-binding proteins or mechanisms independent of GTP-binding proteins do not mediate inhibition by PGE2 and somatostatin in rat parietal cells. Our data are consistent with the view that Gi, the inhibitory subunit of adenylate cyclase, is the transduction mechanism that entirely mediates inhibition by PGE, and somatostatin. This hypothesis is supported by our observation that reversal by PT of the inhibition of [ 14C]aminopyrine accumulation mirrors that of CAMP production. Although confirming in the rat previous observations in histamine-stimulated canine parietal cells (5, 20), the present study for the first time yields evidence that Gi mediates inhibition by PGEZ and somatostatin of the response to GLP-1 peptides. The present data suggest that Rp- and Sp-CAMPS are useful pharmacological tools for studying parietal cell function at the postreceptor level. Sp-CAMPS is thought to offer certain advantages over forskolin and DBcAMP, which activate parietal cell function at different intracellular levels. Forskolin triggers CAMP production by interacting with the catalytic subunit of adenylate cyclase. However, in various cell systems forskolin exerts additional effects that cannot be attributed to stimulation of adenylate cyclase (13, 35-37, 40). DBcAMP is thought to activate the CAMP-dependent protein kinase A. However, intracellular hydrolysis of DBcAMP yields butyrate, the effects of which potentially superimpose CAMP-mediated results (39). In contrast, Sp-CAMPS is more resistant to degradation with virtually no appearance of metabolites producing undesired effects (4). Thus Sp-CAMPS can be used as a postreceptor stimulus avoiding potential disadvantages of forskolin and DBcAMP. In the present study, Sp-CAMPS reached 80-90% of the efficacy of forskolin and DBcAMP in stimulating

PARIETAL

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[‘4C]aminopyrine accumulation. These data indirectly indicate that in rat parietal cells not X0-20% of the response to DBcAMP or forskolin might be due to CAMP-independent mechanisms. However, our main reason in using Sp-CAMPS was to test the specificity of the inhibitory versus the stimulatory analogue. By blocking Sp-CAMPS-induced H+ production, the inhibitory diastereomer, Rp-CAMPS, appears to exert a specific CAMP-related effect on parietal cell function. The lower potency of the inhibitory compared with the stimulatory analogue could be explained by the lo-fold lower binding affinity of Rp-CAMPS for protein kinase A (2l), although this notion requires definitive confirmation in parietal cells. Potency and efficacy of RpCAMPS against Sp-CAMPS were comparable to those required for inhibition of the response to forskolin and DBcAMP. These data are compatible with the view that Rp-CAMPS blocks the cascade of cellular transduction mechanisms distally to CAMP production and at a step on which all three postreceptor activators converge, i.e., most probably the CAMP-dependent protein kinase A. Moreover, with potency and efficacy similar to those revealed versus the postreceptor stimuli, Rp-CAMPS inhibited [ 14C] aminopyrine accumulation in response to the GLP-1 peptides and histamine. These data indirectly confirm that the GLP-1 peptides induce parietal cell function via the same transduction mechanisms that are activated by histamine (6, 7, 18, 31, 32, 34) and which are adequately probed at the postreceptor level by forskolin, DBcAMP, and Sp-CAMPS, respectively. Finally, because blockade by Rp-CAMPS of the CAMPdependent transduction mechanisms completely prevented [ 14C]aminopyrine accumulation in response to GLP-l-(7-36) amide, our data do not support the assumption of an additional CAMP-independent mechanism mediating GLP-l-stimulated parietal cell function. With respect to the sensitivity to PT, inhibition by Rp-CAMPS was completely different from that brought about by PGEz or somatostatin. Rp-CAMPS interferes with protein kinase A, the target mediating the biological effect of CAMP, rather than with adenylate cyclase, the site of CAMP production that is inhibited by PGEz and somatostatin. Thus Rp-CAMPS bypasses Gi thus causing PT-resistant inhibition of the parietal cell response to the GLP-1 peptides and to histamine. Inhibition, although incomplete, by Rp-CAMPS of carbachol-stimulated [“Cl aminopyrine accumulation was somewhat surprising because cholinergic stimulation of parietal cell function is thought to be mediated primarily by Ca2+/phosphoinositol-dependent mechanisms (8). We found that Rp-CAMPS-induced inhibition of the response to carbachol is reversed by DBcAMP. This suggests that the effect on cholinergically induced parietal cell function is due to specific interaction of Rp-CAMPS with protein kinase A or some other CAMP-binding site. Thus we speculate that in rat parietal cells maximal cholinergic stimulation might require a functionally active protein kinase A in addition to the well-recognized Ca”+/phosphoinositol-mediated signaling mechanisms. In the parietal cell, potentiating interactions between CAMP and Ca2+/phosphoinositide-dependent mechanisms are known to occur (30), and it is conceivable that

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Rp-CAMPS, by inhibiting protein kinase A, might prevent this potentiation. However, the specific interaction mechanisms impaired by Rp-CAMPS-induced blockade of protein kinase A were not a subject of the present study and certainly have to await identification by further investigations. In summary, the present study has identified a pattern of PT-sensitive and PT-insensitive mechanisms that inhibit rat parietal cell H+ production in response to the GLP-1 variants. These mechanisms are located at different levels of the cascade of CAMP-mediated signal response transduction, and they appear to be the same mechanisms that control the parietal cell response to histamine. Thus our data substantiate the hypothesis that at the cellular level the GLP-1 peptides modulate rat parietal cell function via the same pathways by which histamine controls H+ production. This study was supported by a grant to W. Schepp from the Deutsche Forschungsgemeinschaft (Sche 229/2-4). Address for reprint requests: W. Schepp, Dept. of Internal Medicine II, Technical Univ. of Munich, Ismaninger Strasse 22, 8000 Munich 80, FRG. Received

14 February

1991; accepted

in final

form

26 November

1991.

REFERENCES 1. Atwell, M. M., and P. J. Hanson. Effect of pertussis toxin on the inhibition of secretory activity by prostaglandin EZ, somatostatin, epidermal growth factor and 12-O-tetradecanoylphorboll3-acetate in parietal cells from rat stomach. Biochim. Biophys. Acta 971: 282-288,1988. 2. Barber, D. L., M. E. McGuire, and M. B. Ganz. ,&Adrenergic and somatostatin receptors regulate Na-H exchange independent of CAMP. J. BioZ. Chem. 264: 21038-21042,1989. 3. Batzri, S., and J. Dyer. Aminopyrine uptake by guinea pig gastric mucosal cells. Mediation by cyclic AMP and interaction among secretagogues. Biochim. Biophys. Acta 675: 416-426, 1981. 4. Braumann, T., C. Erneux, G. Petridis, W.-D. Stohrer, and B. Jastorff. Hydrolysis of cyclic nucleotides by a purified cGMPstimulated phosphodiesterase: structural requirements for hydrolysis. Biochim. Biophys. Acta 871: 199-206, 1986. 5. Chen, M. C. Y., D. A. Amirian, M. Toomey, M. J. Sanders, and A. H. Soll. Prostanoid inhibition of canine parietal cells: mediation by the inhibitory guanosine triphosphate-binding protein of adenylate cyclase. Gustroenterology 94: 1121-1129, 1988. 6. Chew, C. S. Parietal cell protein kinases: selective activation of type I cyclic AMP-dependent protein kinase by histamine. J. Biol. Chem. 260: 7540-7550,1985. 7. Chew, C. S., and M. R. Brown. Histamine increases phosphorylation of 27- and 40-kDa parietal cell proteins. Am. J. Physiol. 253 (Gastrointest. Liver Physiol. 16): G823-G829, 1987. 8. Chiba, T., K. Fisher, J. Park, E. B. Segun, B. W. Agranoff, and T. Yamada. Carbamylcholine and gastrin induce inositol lipid turnover in canine gastric parietal cells. Am. J. Physiol. 255 (Gustrointest. Liver Physiol. 18): G99-G105, 1988. 9. Choquet, A., A. Leonard, R. Magous, and J. P. Bali. Intracellular coupling of porstaglandin inhibition of acid secretion in isolated rabbit gastric parietal cells. Biochem. Phurmucol. 39: 19051911,199o. 10. Eissele, R., H. Koop, and R. Arnold. Effect of glucagon-like peptide-l on gastric somatostatin and gastrin secretion in the rat. Scund. J. GustroenteroL 25: 449-454, 1990. 11. Gespach, C., A. Hansen, and J. Holst. Differential regulation of membrane receptors sensitive to histamine (H&pe), isoproterenol (&type) and glucagon-like peptides by the somatostatin analogue Sandostatin in rat gastric glands. Agents Actions 27: 169172,1989. 12. Goke, R., H.-C. Fehmann, and B. Goke. Glucagon-like peptide1 (7-36) amide is a new incretin/enterogastrone candidate. Eur. J. CZin. Inuest. 21: 135-144, 1991.

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33. Ui, M. Islet-activating protein, pertussis toxin: a probe for functions of the inhibitory guanosine nucleotide regulatory component of adenylate cyclase. Trends PharmacoZ. Sci. 5: 227-229, 1984. 34. Urushidani, T., D. K. Hanzel, and J. G. Forte. Characterization of an 80-kDa phosphoprotein involved in parietal cell stimulation. Am. J. Physiol. 256 (Gustrointest. Liver Physiol. 19): Gl070G1080,1989. 35. Van Halen, F., and E. Keck. Forskolin inhibition of glucose transport in bone cell cultures through a CAMP-independent mechanism. Bone 9: 89-92,1988. 36. Wagoner, P. K., and B. S. Palotta. Modulation of acetylcholine receptor desensitization by forskolin is independent of CAMP. Science Wash. DC 240: 1655-1657,1988. 37. White, M. M. Forskolin alters acetylcholine receptor gating by a

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mechanism independent of adenylate cyclase activation. Mol. PharmacoZ. 34: 427-430, 1988. 38. Whittle, B. J. R., and J. R. Vane. Prostanoids as regulators of gastrointestinal function. In: Physiology of the Gastrointestinal Tract, edited by L. R. Johnson. New York: Raven, 1987, vol. 1, p. 143-180. 39. Yusta, B., J. Ortiz-Caro, A. Pascual, and A. Aranda. Comparison of the effects of forskolin dibutyryl CAMP in neuroblastoma cells: evidence that some of the actions of dibutyryl CAMP are mediated by butyrate. J. Neurochem. 51: 1808-1818, 1988. 40. Zuenkler, B. J., G. Trube, and T. Ohno-Shosaku. Forskolininduced block of delayed rectifying potassium channels in pancreatic ,&cells is not mediated by CAMP. Pfluegers Arch. 411: 613619,1988.

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