Pertussis Toxin-sensitive Guanine Nucleotide Binding Research Articles

Phorbol ester dissociates endothelin-stimulated phosphoinositide hydrolysis and arachidonic acid release in vascular smooth muscle cells

Endothelin-stimulated { 3H}-inositol phosphate formation and { 3H}-arachidonic acid release were measured in cultured vascular smooth muscle cells from rabbit renal artery. Both responses were partially inhibited by pretreatment with pertussis toxin, indicating the involvement of pertussis toxin-sensitive guanine nucleotide binding regulatory proteins in the coupling processes. Pretreatment with the phorbol ester PMA inhibited endothelin-stimulated { 3H}-inositol phosphate formation, but potentiated endothelin-stimulated { 3H}-arachidonic acid release, suggesting that these two coupling processes occur in a parallel and independent manner in vascular smooth muscle cells.

Techniques used in the identification and analysis of function of pertussis toxin-sensitive guanine nucleotide binding proteins.

Techniques used in the identification and analysis of function of pertussis toxin-sensitive guanine nucleotide binding proteins.

Differential effect of pertussis toxin on pre- and postjunctional α2-adrenoceptors in the cardiovascular system of the pithed rat

The role of the pertussis toxin-sensitive guanine nucleotide binding regulatory protein in pre- and postjunctional α 2-adrenoceptor-mediated responses has been investigated in the pithed rat. Pertussis toxin (50 μg/kg i.v., administered 3 days prior to experimentation) markedly inhibited the α 2-adrenoceptor-mediated vasopressor response to B-HT 933, but had no effect on the α 2-adrenoceptor-mediated cardiac neuroinhibitory effect of B-HT 933. Thus, postjunctional α 2-adrenoceptor activation in vascular smooth muscle, but not prejunctional α 2-adrenoceptor activation on sympathetic neurons, utilizes a pertussis toxin-sensitive guanine nucleotide binding regulatory protein.

The D2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein.

Dopaminergic inhibition of prolactin release from the anterior pituitary may be mediated through both the adenylate cyclase and Ca2+ mobilization/phosphoinositide pathways. The D2-dopamine receptor of the bovine anterior pituitary has been partially purified by affinity chromatography on CMOS-Sepharose (immobilized carboxymethyleneoximinospiperone). Reinsertion of these partially purified receptor preparations into phospholipid vesicles reconstituted guanine nucleotide-sensitive high affinity agonist binding, agonist-promoted GTPase and 35S-labeled guanosine 5'-O-(thiotriphosphate) [( 35S]GTP gamma S) binding activity in these preparations. Pertussis toxin treatment of the purified receptor preparation abolished agonist-stimulated GTPase and guanine nucleotide-sensitive high affinity agonist binding. These observations suggest that the receptor copurifies with an endogenous, pertussis toxin-sensitive guanine nucleotide binding protein (N). [32P]ADP-ribosylation of affinity-purified D2 receptor preparations by pertussis toxin revealed the presence of a substrate of Mr 39,000-40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps generated using elastase of the [32P]ADP-ribosylated endogenous N protein, transducin, and Ni and No from brain revealed similarities but not identity between the endogenous pituitary N protein and brain Ni and No. Immunoblotting of the partially purified D2 receptor preparations showed an Mr 39,000-40,000 band with an Ni-specific antiserum raised against a synthetic peptide, and with RV3, an No-specific anti-serum, but not with CW6, an antiserum strongly reactive with brain Ni. Several lines of evidence indicate that endogenous pituitary N protein is functionally coupled to the D2 receptor. As measured by [35S]GTP gamma S binding, ratios of 0.2-0.6 mol N protein/mol receptor were observed. Association of N protein with the D2 receptor was increased by agonist pretreatment and decreased by guanine nucleotides. These results suggest that No and/or a form of Ni distinct from the Mr 41,000 pertussis toxin substrate (Ni) is the predominant N protein functionally coupled with the D2-dopamine receptor of anterior pituitary.

Inositol 1,4,5-triphosphate-induced granule secretion in platelets. Evidence that the activation of phospholipase C mediated by platelet thromboxane receptors involves a guanine nucleotide binding protein-dependent mechanism distinct from that of thrombin.

Phosphoinositide hydrolysis in platelets stimulated by thrombin is thought to be regulated by a pertussis toxin-sensitive guanine nucleotide binding protein (G protein) referred to as Gp. The present studies examine the role of Gp in platelet responses to the thromboxane A2 analogue U46619 and in the pathway by which the phosphoinositide hydrolysis product inositol 1,4,5-triphosphate (IP3) causes secretion. In permeabilized platelets, U46619 caused phosphatidic acid formation and secretion, which were abolished by the G protein inhibitor, guanosine 5'-O-(2-thiophosphate) (GDP beta S). Unlike thrombin, however, U46619-induced phosphoinositide hydrolysis was unaffected by pertussis toxin, and U46619 was unable to inhibit the [32P]ADP ribosylation of the 42-kD pertussis toxin substrate in platelets. IP3-induced secretion, which is known to depend upon intracellular Ca release and subsequent arachidonic acid metabolism, was also inhibited by GDP beta S, as was Ca-induced secretion. These observations suggest that platelet thromboxane A2 (TxA2) receptors are coupled to a toxin-resistant form of Gp distinct from the one that is coupled to thrombin receptors, and that TxA2-stimulated phosphoinositide hydrolysis may serve as a feedback mechanism by which stimuli for arachidonic acid release, such as IP3 and Ca, amplify responses to agonists.