Niroomand F, Bangert M, Philipps C, Rauch B
University of Heidelberg, Innere Medizin III, Germany.
Mol Pharmacol. 1993 Jan;43(1):90-5.
To differentiate the effects of GDP and GTP on adenylyl cyclase regulation, adenylyl cyclase in canine sarcolemmal membranes was studied under conditions where only 3-12% of added GDP was converted to GTP by membrane-associated nucleoside diphosphate kinase. Adenylyl cyclase was stimulated up to 180% by GDP at 7-fold lower concentrations than required for stimulation by GTP (GDP half-maximal activation, 120 nM; GTP half-maximal activation, 830 nM). Transphosphorylation of GDP to GTP was blocked completely by the addition of 3 mM UDP. However, UDP did not affect GDP-mediated adenylyl cyclase activation, and guanosine 5'-O-(2-thiodiphosphate) had the same effect on adenylyl cyclase activity as did GDP, indicating that GDP-mediated stimulation of adenylyl cyclase was not due to transphosphorylation of GDP to GTP. Carbachol inhibited GDP-stimulated adenylyl cyclase activity even without addition of GTP; however, this inhibition was clearly dependent upon the endogenous formation of GTP. Half-maximal adenylyl cyclase inhibition by carbachol required the addition of either 330 nM GDP or 25 nM GTP. Taking into account a 3-12% conversion of GDP to GTP by membrane-associated nucleoside diphosphate kinase, sufficient GTP was generated from GDP to support receptor-mediated inhibition of adenylyl cyclase. In addition carbachol-mediated adenylyl cyclase inhibition in the presence of GDP, but not GTP, was blocked completely by 3 mM UDP. In conclusion, GDP-activated adenylyl cyclase could be inhibited by carbachol in the presence of GTP concentrations that were 34-fold below the concentrations needed for GTP-mediated activation of stimulatory guanine nucleotide-binding proteins. In addition, at low GTP concentrations carbachol reduced adenylyl cyclase to levels below "basal" activity (activity in the absence of guanine nucleotides). Although indirectly, these results suggest that carbachol-mediated inhibition of adenylyl cyclase may be independent of Gs activity and possibly due to direct interaction of inhibitory guanine nucleotide-binding proteins and adenylyl cyclase.
为了区分GDP和GTP对腺苷酸环化酶调节的影响，我们在膜相关核苷二磷酸激酶仅将3%-12%添加的GDP转化为GTP的条件下，研究了犬肌膜中的腺苷酸环化酶。与GTP刺激所需浓度相比，GDP在低7倍的浓度下就能将腺苷酸环化酶的活性刺激高达180%（GDP的半最大激活浓度为120 nM；GTP的半最大激活浓度为830 nM）。添加3 mM UDP可完全阻断GDP向GTP的转磷酸化。然而，UDP并不影响GDP介导的腺苷酸环化酶激活，5'-O-(2-硫代二磷酸)鸟苷对腺苷酸环化酶活性的影响与GDP相同，这表明GDP介导的腺苷酸环化酶刺激并非由于GDP向GTP的转磷酸化。即使不添加GTP，卡巴胆碱也会抑制GDP刺激的腺苷酸环化酶活性；然而，这种抑制明显依赖于GTP的内源性形成。卡巴胆碱对腺苷酸环化酶的半最大抑制需要添加330 nM GDP或25 nM GTP。考虑到膜相关核苷二磷酸激酶将GDP转化为GTP的转化率为3%-12%，从GDP中产生的GTP足以支持受体介导的腺苷酸环化酶抑制。此外，在存在GDP而非GTP的情况下，3 mM UDP可完全阻断卡巴胆碱介导的腺苷酸环化酶抑制。总之，在GTP浓度比GTP介导的刺激性鸟嘌呤核苷酸结合蛋白激活所需浓度低34倍的情况下，卡巴胆碱可抑制GDP激活的腺苷酸环化酶。此外，在低GTP浓度下，卡巴胆碱可将腺苷酸环化酶活性降低至“基础”活性（无鸟嘌呤核苷酸时的活性）以下。尽管是间接的，但这些结果表明，卡巴胆碱介导的腺苷酸环化酶抑制可能独立于Gs活性，可能是由于抑制性鸟嘌呤核苷酸结合蛋白与腺苷酸环化酶的直接相互作用。
