The amount and activity of nucleoside diphosphate kinase are increased in sarcolemmal membranes of failing human myocardium, resulting in a substantial receptor-independent inhibition of adenylyl cyclase activity.

1 A receptor-independent activation of heterotrimeric G proteins by plasma membrane-associated nucleoside diphosphate kinase (NDPK) has been demonstrated in vivo, and elevated levels of NDPK were found in purified sarcolemmal membranes of patients with end-stage heart failure. 2 Among 22 consecutive patients with chronic heart failure who underwent cardiac transplantation, those treated with a b-blocker (n ¼ 8) had a 65% lower NDPK content and activity in the cardiac sarcolemma, compared to patients with similar base line characteristics who had no b-blocker therapy (n ¼ 14). 3 The lower NDPK was associated with a reduced NDPK-dependent, G i -mediated inhibition of adenylyl cyclase activity, as assessed by in vitro measurement of adenylyl cyclase activity in the presence of GDP or its kinase-resistant analog guanosine 5 0 -O-(2-thio)diphosphate (GDPbS). 4 We further tested whether treatment with a b-adrenergic agonist would induce an increase in sarcolemmal NDPK. Rats treated with isoproterenol developed myocardial hypertrophy, and NDPK in the sarcolemma rose by 60% during 14 days of treatment. The b-blocker propranolol prevented both effects. When hypertrophy was induced with thyroid hormone, NDPK did not increase. 5 In conclusion, chronic activation of b-adrenergic receptors increases the binding of NDPK to cardiac sarcolemma, where it may activate heterotrimeric G proteins.

Although both opioid receptors and endogenous opioids are abundant in cardiac tissues, the signal transduction pathways of opioids in cardiac sarcolemmal membranes have yet to be identified. In highly purified canine cardiac sarcolemmal membranes, binding of the opioid receptor antagonist [3H]diprenorphine and effects of mu, delta and kappa agonists on low Km GTPase and adenylyl cyclase were measured. Equilibrium binding of [3H]diprenorphine revealed a maximal binding capacity of 7.2 pmol/mg protein and a Kd of 1.3 nmol/l. In the presence of GTP, (D-Pen2,5, p-Cl-Phe4) enkephalin and (D-Arg6) dynorphin A 1-13 fragment both inhibited adenylyl cyclase by 20-25% (from 206 +/- 30 to 164 +/- 28 pmol.min-1.mg protein-1, EC50 6 mumol/L, and from 254 +/- 109 to 204 +/- 90 pmol.min-1.mg protein-1, EC50 8 mumol/L, respectively; P < 0.001). Both substances stimulated low Km GTPase by 20% and 13%, respectively (from 12.7 +/- 3.0 to 15.2 +/- 3.7 pmol.min-1.mg protein-1, EC50 12 mumol/L, P < 0.01, and from 9.1 +/- 2.8 to 10.4 +/- 3.2 pmol.min-1.mg protein-1, EC50 6 mumol/L, P < 0.05, respectively). These effects were blocked by the opioid receptor antagonist naltrexone and by pretreatment of sarcolemmal membranes with pertussis toxin. The mu opioid receptor agonists (D-Ala2, Me Phe4, Gly-[ol]5)enkephalin and morphiceptin had no effect on either adenylyl cyclase or low Km GTPase activities. These data suggest that in cardiac sarcolemma, opioid receptors are coupled to pertussis toxin sensitive G proteins and mediate inhibition of adenylyl cyclase activity.

Low concentrations of GDP and its stable analog guanosine 5'-O-(2-thio)diphosphate (GDP beta S) have been shown to stimulate adenylyl cyclase activity in canine cardiac sarcolemmal membranes independent from a phosphate transfer reaction. The mechanism of this stimulation was further examined. The stable GTP analog guanosine 5'-(beta gamma-imino)triphosphate (Gpp(NH)p) increased basal adenylyl cyclase activity and inhibited forskolin-stimulated activity with EC50 (half-maximal effective concentration) values of 0.7 mumol/l and 10 nmol/l, respectively. In the presence of GDP beta S (5 mumol/l), which increased basal activity by about 150%, addition of Gpp(NH)p inhibited adenylyl cyclase activity by up to 50% with an EC50 value of 40 nmol/l. Activation of cardiac muscarinic acetylcholine receptors by carbachol amplified this Gpp(NH)p-induced inhibition of GDP beta S-stimulated adenylyl cyclase activity. The stimulatory effect of GDP beta S and the inhibitory effect of Gpp(NH)p on GDP beta S-stimulated adenylyl cyclase activity were both attenuated by increasing the Mg2+ concentration or substituting Mn2+ for Mg2+ in the assay. Furthermore, both effects were strongly reduced or abolished upon pretreatment of the sarcolemmal membranes with a low concentration of the SH reagent N-ethylmaleimide (10 mumol/l). These results suggest that the stimulatory effect of GDP beta S on basal adenylyl cyclase activity in canine cardiac sarcolemmal membranes is caused by inactivation of G(i) proteins, which are then rendered susceptible to activation by Gpp(NH)p and inhibitory receptors.

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