Stable cell lines, derived from NG108-15 cells and transfected to express both the β2-adrenoceptor and adenylate cyclase type II, were produced and examined. The absence of adenylate cyclase type II in the parental cells and its presence in these clones was demonstrated by reverse transcriptase-PCR. Total cellular levels of adenylate cyclase were increased in a number of clones between 3- and 8-fold, as assessed by guanine nucleotide-stimulated specific high-affinity binding of [3H]forskolin to cellular membranes. Basal adenylate cyclase activity was markedly elevated compared with a clone expressing similar levels of the β2-adrenoceptor in the absence of adenylate cyclase type II. Each of NaF, forskolin and guanosine 5´-[β,γ-imido]triphosphate (a poorly hydrolysed analogue of GTP) produced substantially higher levels of adenylate cyclase activity in membranes of the clones positive for expression of adenylate cyclase type II than was achieved with the parental cells. Both isoprenaline, acting at the introduced β2-adrenoceptor, and iloprost, acting at the endogenously expressed IP prostanoid receptor, stimulated adenylate cyclase activity to much higher levels in the clones expressing adenylate cyclase type II compared with the clone lacking this adenylate cyclase; however, the concentration–effect curves for adenylate cyclase stimulation by these two agonists were not different between parental cells and clones overexpressing adenylate cyclase type II. A maximally effective concentration of the β-adrenoceptor partial agonist ephedrine displayed similar intrinsic activity and potency to stimulate adenylate cyclase in membranes of clones both with and without adenylate cyclase type II. Both secretin and 5´-N-ethylcarboxamidoadenosine (acting at an endogenous A2 adenosine receptor) were also able to produce substantially greater maximal activations of adenylate cyclase in the clones expressing excess adenylate cyclase type II, without alterations in agonist intrinsic activity or potency. These results demonstrate that the maximal output of the stimulatory arm of the adenylate cyclase cascade can be increased by increasing total levels of adenylate cyclase in the genetic background of NG108-15 cells.
Agonist regulation of adenylate cyclase activity in neuroblastoma × glioma hybrid NG108-15 cells transfected to co-express adenylate cyclase type II and the β2-adrenoceptor. Evidence that adenylate cyclase is the limiting component for receptor-mediated stimulation of adenylate cyclase activity
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David J. MACEWAN, Gun-Do KIM, Graeme MILLIGAN; Agonist regulation of adenylate cyclase activity in neuroblastoma × glioma hybrid NG108-15 cells transfected to co-express adenylate cyclase type II and the β2-adrenoceptor. Evidence that adenylate cyclase is the limiting component for receptor-mediated stimulation of adenylate cyclase activity. Biochem J 15 September 1996; 318 (3): 1033–1039. doi: https://doi.org/10.1042/bj3181033
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