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Regulation of guanylate cyclase in guinea-pig islets of Langerhans.

ABSTRACT: 1. Guanylate cyclase activity was determined in homogenates of guinea-pig islets of Langerhans by measurement of the conversion of [alpha-(32)P]GTP into cyclic [(32)P]GMP, the reaction products being separated on columns of neutral alumina. 2. The pH optimum of the enzyme was 7.3; it showed a requirement for bivalent cations, the effectiveness of the cations tested being Mn(2+)>>Ca(2+)>Mg(2+). 3. About 70% of enzyme activity was sedimented by centrifugation at 105000g for 60min; activity was increased 2.3-fold by treatment of homogenates with 0.1% Triton X-100. 4. Guanylate cyclase activity of homogenates was increased by acetylcholine, secretin or pancreozymin, but was inhibited by adrenaline, noradrenaline or ATP. Insulin, glucagon, prostaglandins E(1) or E(2), glucose, F(-), diazoxide or glibenclamide were ineffective. 5. Determination of cyclic GMP amounts in islets by radioimmunoassay showed a basal concentration of 2.0pmol/mg of protein, which was increased by incubation of the islets in the presence of acetylcholine or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, but was unaffected by glucose. 6. Dibutyryl cyclic GMP had significant stimulatory effects on rates of insulin biosynthesis in isolated rat islets of Langerhans. 7. These results suggest a possible role for cyclic GMP in the regulation of insulin biosynthesis and secretion.

SUBMITTER: Howell SL

PROVIDER: S-EPMC1168289 | biostudies-other | 1974 Aug

REPOSITORIES: biostudies-other

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Project description:Heme oxygenase-1 knockout, H(mox)1(-/-), mice exhibit exacerbated vascular lesions after ischemia-reperfusion and mechanical injury. Surprisingly, we found no studies that reported contractile responses and sensitivity to vasorelaxants in H(mox)1(-/-) mice. The contractile responses [superior mesenteric arteries (SMA), from female H(mox)1(-/-) mice] exhibited increased sensitivity to phenylephrine (p < 0.001). Cumulative addition of acetylcholine relaxed SMA, with the residual contraction remaining 2 times higher in H(mox)1(-/-) mice (p < 0.001). Sodium nitroprusside (SNP, an NO donor) and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole [YC-1; acts directly on soluble guanylate cyclase (sGC)] led to further relaxation, yet the residual contraction remained 2 to 3 times higher in H(mox)1(-/-) than H(mox)1(+/+) mice (p < 0.001). Branches from H(mox)1(-/-) mesenteric and renal arteries also showed reduced relaxation (p < 0.025). Relaxation of SMA was measured to 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl] methoxy}phenyl)ethyl]amino}benzoic acid (BAY 60-2770), which is a more effective activator of oxidized/heme-free sGC; and to 5-cyclopropyl-2-{1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}-pyrimidin-4-ylamine (BAY 41-2272), a more effective stimulator of reduced sGC. H(mox)1(-/-) arteries were 15 times more sensitive to BAY 60-2770 (p < 0.025) than were H(mox)1(+/+) arteries. Pretreatment with 1H-[1,2,4]oxadiazolo[3,4-a]quinoxalin-1-one (ODQ), an oxidizer of sGC, predictably shifted the BAY 60-2770 response of H(mox)1(+/+) to the left (p < 0.01) and BAY 41-2272 response to the right (p < 0.01). ODQ had little effect on the responses of H(mox)1(-/-) arteries, indicating that much of sGC was oxidized/heme-free. Western analyses of sGC in SMA indicated that both ?1 and ?1 subunit levels were reduced to <50% of H(mox)1(+/+) level (p < 0.025). These findings support the hypothesis that the antioxidant function of H(mox)1 plays a significant role in the maintenance of sGC in a reduced state, which is resistant to degradation and is sensitive to NO. This function may be especially important in reducing vascular damage during ischemia-reperfusion injury.

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Regulation of guanylate cyclase in guinea-pig islets of Langerhans.

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