Lamm M L, Ekstrom R C, Maizels E T, Rajagopalan R M, Hunzicker-Dunn M
Department of Cell, Molecular, and Structural Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Endocrinology. 1994 Apr;134(4):1745-54. doi: 10.1210/endo.134.4.8137739.
Because the acute homologous phase of desensitization of the LH/CG-sensitive adenylyl cyclase in porcine follicles is readily demonstrated in a cell-free membrane preparation, it follows that any enzyme(s) required to achieve desensitization must be present in the membranes and must be activated upon LH/CG receptor activation. The purpose of the following studies was to determine whether modulation of endogenous membrane protein kinases, with activators or inhibitors, or addition of exogenous protein kinases affected desensitization of the LH/CG-sensitive adenylyl cyclase. The effects of these potential modulators were evaluated in both the presence and absence of ligand (hCG)-stimulated receptor activation. To this end, membranes were incubated in the presence or absence of hCG (stage 1) and then assayed for adenylyl cyclase activity in the presence or absence of hCG (stage 2). The results showed that although porcine follicular membranes rich in LH/CG-sensitive adenylyl cyclase activity also exhibited cAMP-dependent [protein kinase-A (PKA)], cGMP-dependent (PKG), lipid-dependent (PKC), Ca2+/calmodulin, and casein kinase-I and -II activities, only full hCG-stimulated adenylyl cyclase activity (measured with BSA in stage 1 and hCG in stage 2) was reduced upon addition of exogenous PKC (to the stage 1 incubation). hCG-dependent desensitization of cAMP synthesis (measured with hCG in stages 1 and 2) was unaffected by activators or inhibitors of endogenous PKA, PKC, or PKG, by an inhibitor of casein kinases and kinases in the beta-adrenergic receptor kinase family, or by the addition of exogenous active PKA, PKC, or rhodopsin kinase to the stage 1 incubation. These results suggest that the acute homologous phase of hCG-dependent desensitization of adenylyl cyclase activity in follicular membranes is not regulated by PKA, PKC, PKG, or messenger-independent heparin-sensitive protein kinases.
由于在无细胞的膜制剂中很容易证明猪卵泡中促黄体生成素/绒毛膜促性腺激素（LH/CG）敏感的腺苷酸环化酶脱敏的急性同源阶段，因此可以推断，实现脱敏所需的任何酶都必须存在于膜中，并且必须在LH/CG受体激活时被激活。以下研究的目的是确定用激活剂或抑制剂调节内源性膜蛋白激酶，或添加外源性蛋白激酶是否会影响LH/CG敏感的腺苷酸环化酶的脱敏。在有或没有配体（hCG）刺激的受体激活的情况下，评估了这些潜在调节剂的作用。为此，将膜在有或没有hCG的情况下孵育（阶段1），然后在有或没有hCG的情况下测定腺苷酸环化酶活性（阶段2）。结果表明，尽管富含LH/CG敏感的腺苷酸环化酶活性的猪卵泡膜也表现出cAMP依赖性[蛋白激酶A（PKA）]、cGMP依赖性（PKG）、脂质依赖性（PKC）、Ca2+/钙调蛋白以及酪蛋白激酶-I和-II活性，但仅在添加外源性PKC（到阶段1孵育中）后，完全hCG刺激的腺苷酸环化酶活性（在阶段1用牛血清白蛋白测量，在阶段2用hCG测量）才降低。cAMP合成的hCG依赖性脱敏（在阶段1和2用hCG测量）不受内源性PKA、PKC或PKG的激活剂或抑制剂、酪蛋白激酶和β-肾上腺素能受体激酶家族中的激酶的抑制剂的影响，也不受在阶段1孵育中添加外源性活性PKA、PKC或视紫红质激酶的影响。这些结果表明，卵泡膜中腺苷酸环化酶活性的hCG依赖性脱敏的急性同源阶段不受PKA、PKC、PKG或不依赖信使的肝素敏感蛋白激酶的调节。
