Positively cooperative cAMP phosphodiesterase attenuates cellular cAMP responses.

We have shown that growth of S49 WT mouse lymphoma cells for 24 hr in 3 nM epinephrine produced very significant desensitization of subsequent cellular cAMP responses to challenges with higher concentrations of epinephrine. The effects of this long-term treatment (LTT) were obvious in intact cells and also when adenylate cyclase activity was measured in semi-purified membranes. Beta 2-adrenergic receptors (beta 2AR) were decreased by LTT, and the desensitization of adenylate cyclase was due at least in part to this down-regulation. When mouse L cells transfected with WT beta 2AR from hamster lung (L-WT beta 2AR cells) were subjected to LTT, the attenuation of adenylate cyclase in membranes was obvious, but the consequences of LTT on intact L-WT beta 2AR cells were highly equivocal. That is, when the effects of epinephrine on cellular cAMP levels were measured in LTT or control L-WT beta 2AR cells little desensitization was apparent. Further, cellular cAMP excursions in response to even very high concentrations of epinephrine were very small in control L-WT beta 2AR cells as compared to control S49 WT cells. Subsequent experiments have shown that L-WT beta 2AR cells possess a phosphodiesterase (PDE) which demonstrates marked positive cooperativity with cAMP with a Hill coefficient of 2. The EC50 for cAMP hydrolysis was approximately 30 nM in cell free preparations. cGMP was a positive allosteric effector of the L-WT beta 2AR cell PDE. Further, when cellular cAMP levels in intact L-WT beta 2AR cells were raised above a threshold by treatment with 0.5 microM forskolin and 2 mM IBMX with the epinephrine challenge, the effect of LLT became obvious in the intact cell system. These data demonstrate that cAMP responses to hormones are greatly decreased in systems where the predominant PDE demonstrates positive cooperativity for cAMP.