Regulation of adenylate cyclase by adenosine and other agonists in rat myocardial sarcolemma

Abstract

The presence of adenosine receptors coupled to adenylate cyclase in rat heart sarcolemma is demonstrated in these studies. Heart sarcolemma was isolated by the hypotonic shock-Lithium bromide treatment method. This preparation contained negligible amounts (2–4%) of contamination by other subcellular organelles such as mitochondria, sarcoplasmic reticulum, and myofibrils as verified by electron microscopic examination. In addition this preparation was also devoid of endothelial cells, since angiotensinconverting enzyme activity was not detected in this preparation. N-Ethylcarboxamide adenosine (NECA), l- N 6-phenylisopropyladenosine (PIA), and adenosine N′-oxide (Ado N′-oxide) were all able to stimulate adenylate cyclase in heart sarcolemma, but not in crude homogenate, with an apparent K a of 3–7 μM. The activation of adenylate cyclase by NECA was dependent on the concentrations of metal ions such as Mg 2+ or Mn 2+. The maximal stimulation was observed at lower concentrations of the metal ions (0.2–0.5 mM). At 5 m m Mg 2+ or Mn 2+, the stimulation by NECA was completely abolished. The stimulatory effect of NECA on adenylate cyclase was also dependent on guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine. In addition, 2′-deoxyadenosine showed an inhibitory effect on adenylate cyclase. The myocardial adenylate cyclase was also stimulated by β-adrenergic agonists, dopamine and glucagon, and inhibited by cholinergic agonists such as carbachol and oxotremorine. The stimulation of adenylate cyclase by NECA was found to be additive with maximal stimulation obtained by epinephrine. These data suggest that rat heart sarcolemma contains adenosine (Ra), β-adrenergic, dopaminergic, glucagon, and cholinergic receptors, and the stimulation of adenylate cyclase by epinephrine and adenosine occurs by distinctly different mechanism or adenosine and epinephrine stimulate different cyclase populations.