Stimulation of rat parietal cell function by histamine and GLP-1-(7-36) amide is mediated by Gs alpha

Abstract

It was the aim of the present study to determine in rat parietal cells whether Gs alpha, the stimulatory subunit of adenylate cyclase, mediates adenosine 3',5'-cyclic monophosphate (cAMP)-dependent parietal cell function in response to histamine and glucagon-like peptide 1 (GLP-1)-(7-36) amide. Cytoplasmic membrane from enriched (83 +/- 5%) rat parietal cells were incubated for 30 min with 30 microCi/ml [32P]NAD+ and 40 micrograms/ml preactivated cholera toxin (CT), a pharmacological tool for activation of Gs alpha. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography revealed [32P]ADP ribosylation of Gs alpha represented by three proteins with molecular masses ranging from 42 to 45 kDa. In intact parietal cells, CT (10(-12)-10(-8) M) caused marked stimulation of [14C]aminopyrine accumulation and cAMP production confirming the functional importance of Gs alpha in regulation of H+ production. Identical membrane preparations were preincubated (2 h, 4 degrees C) in parallel with and without RM/1, a rabbit polyclonal anti-Gs alpha-antibody. Subsequently, adenylate cyclase was stimulated by histamine, GLP-1-(7-36) amide, CT, or forskolin. At a 1:10 dilution, the antiserum completely abolished adenylate cyclase activity in response to maximal concentrations of histamine, GLP-1-(7-36) amide, and CT while reducing forskolin stimulation by only 22.0 +/- 4.9%. At 1:50, RM/1 reduced responses to histamine, GLP-1-(7-36) amide and CT by 20-30% but failed to inhibit forskolin-stimulated enzyme activity. At 1:100, the antiserum was ineffective versus all stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual bradykinin B2 receptor signalling in A431 human epidermoid carcinoma cells: activation of protein kinase C is counteracted by a GS-mediated stimulation of the cyclic AMP pathway

“…Pretreatment of A431 membranes with AS 348, which is selective for the C-terminal region of α s [32], did in fact significantly decrease the stimulation of adenylate cyclase by CTX and isoprenaline, which are known to act via G s . Obviously antibodies directed against the C-terminus of G s , which is responsible for the interaction with the receptor, also affect the CTX target site [28]. The stimulatory effect of forskolin, which directly activates the catalytic subunit of adenylate cyclase, was only slightly affected by AS 348 in the concentration used.…”

“…This prompted us to study the effect of an anti-α s antibody on BK-induced adenylate cyclase activation. Since the C-terminus of G protein α-subunits is important for their activation by receptors, antibodies directed against this region suppress G α function and were recently successfully used as tools for studying the functional importance of G s [28,36] or G i [37,38] proteins in agonist-regulated adenylate cyclase activity. The specificity of the anti-α s antiserum AS 348 was shown previously [31,32].…”

“…For each column cAMP recovery was separately estimated by using [$H]cAMP. Alternatively, some experiments were performed without labelled ATP by using a modified procedure in accordance with Schmidtler et al [28] (method B). The assay medium contained all reagents as in method A, except [α-$#P]ATP and cAMP.…”

Glucagon‐like peptide‐1 receptor expression in Xenopus oocytes stimulates inositol trisphosphate‐dependent intracellular Ca2+ mobilization

“…It is generally accepted that the effect of GLP‐1(7–36)amide is mediated by the G‐protein subunit G sα [9]. However, this is most likely not the case in Xenopus oocytes since pretreatment for >20 h with 100 ng/ml cholera toxin or pertussis toxin did not affect the amplitude of the GLP‐1(7–36)amide‐evoked Cl − current (Fig.…”

Helicobacter pyloriinduces apoptosis of rat gastric parietal cells

“…Highly enriched rat gastric PC were prepared as previously described (27). A total of 80 preparations was used (5 rats/preparation).…”