Opioid receptor agonists activate pertussis toxin-sensitive G proteins and inhibit adenylyl cyclase in canine cardiac sarcolemma

Abstract

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.

Diminution of contractile response by κ-opioid receptor agonists in isolated rat ventricular cardiomyocytes is mediated via a pertussis toxin-sensitive G protein

“…The present study demonstrates, that the direct cardiodepressive effects of U-50,488 and dynorphin A (1-8) in isolated adult rat ventricular cardiomyocytes are abolished after pertussis toxin pretreatment, thus clearly indicating the involvement of a pertussis toxin-sensitive G i/o protein. These findings support and extend previous biochemical studies (Ela et al 1993;Niroomand et al 1996;Zimlichman et al 1996). In crude membrane preparations from neonatal cardiomyocytes pertussis toxin concentration dependently reduces affinity of the selective κ-opioid receptor agonist U69593, indicating an interaction between κ-opioid receptors and G i/o proteins (Zimlichman et al 1996).…”

“…In cardiac tissue or cell preparations opioids cause alterations in cyclic AMP levels (Lee and Wong 1987;Yamada et al 1991;Zhang and Wong 1998), inhibition of adenylyl cyclase activity (Niroomand et al 1996), activation of phospholipase C (Sheng et al 1997;Zhang and Wong 1998), stimulation of phosphoinositol turnover (Ventura et al 1991b,c), reduction of calcium currents (Xiao et al 1993), stimulation of protein kinase C (Ventura et al 1991a;Ela et al 1993) and elevation of intracellular cyclic GMP levels (Muraki et al 1989). Taken together, the shallow concentration/response curves for U-50,488 and dynorphin A (1-8) observed in the present study may reflect concentration-dependent activation of different second messengers, rather than interaction of different types of myocardial opioid receptors.…”

“…Therefore, it is questionable whether acidification is the mechanism underlying the decrease in contractile response exerted by opioids in adult ventricular myocytes. Additionally, pertussis toxin blocks the inhibition of adenylyl cyclase activity induced by the κ-opioid receptor agonist (D-Arg 6 ) dynorphin A (1-13) or the δ-opioid receptor agonist (D-Pen 2,5 ,p-Cl-Phe 4 ) enkephalin in sarcolemmal membranes from whole canine hearts (Niroomand et al 1996). Thus, it is possible that the blockade of the inhibitory effects on the adenylyl cyclase by opioid receptor agonists is one mechanism underlying the herein described pertussis toxin-sensitive contraction decreasing effects of opioid receptor agonists in adult cardiomyocytes.…”

β 2 -Adrenergic and Several Other G Protein–Coupled Receptors in Human Atrial Membranes Activate Both G s and G i

“…PTX (50 ng/L) was activated by incubation with 100 mmol/L DTT and 0.25% SDS for 30 minutes at 30°C as described previously, 13 with minor modifications, and mixed with atrial membranes. A second tube containing the same volumes of all constituents, with the exception of H 2 O in place of PTX, was treated in the same fashion and used as a control.…”

Age Increases Cardiac Gαi2 Expression, Resulting in Enhanced Coupling to G Protein-coupled Receptors

“…Pertussin Toxin Treatment of Ventricular Membranes for AC Assays-PTX (50 ng/ l) was activated by incubation with 100 mM DTT and 0.25% SDS for 30 min at 30°C as described (34). The activation reaction was stopped by the addition of four volumes of 1 mg/ml ice-cold bovine serum albumin (BSA).…”