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1 Cyclic GMP (cGMP) has been shown to be an important modulator of cardiac contractile function. A major component of cGMP regulation of contractility is cGMP-mediated inhibition of the cardiac calcium current (I Ca ). An under-appreciated aspect of cyclic nucleotide signalling is hydrolysis of the cyclic nucleotide (i.e., breakdown by phosphodiesterases (PDEs)). The role of cGMP hydrolysis in regulating I Ca has not been studied. Thus the purpose of this study was to investigate if inhibition of cGMP hydrolysis can modulate I Ca in isolated guinea-pig ventricular myocytes. 2 Zaprinast, a selective inhibitor of cGMP-speci®c PDE (PDE5), caused a signi®cant increase in cGMP levels in myocytes, but was without a ect on basal or b-adrenergic stimulated cAMP levels (consistent with its actions as a speci®c inhibitor of PDE5). 3 Zaprinast inhibited I Ca that was pre-stimulated with cAMP elevating agents (isoproterenol, a badrenergic agonist; or forskolin, a direct activator of adenylate cyclase). The e ect of zaprinast was greatly reduced by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). 4 Zaprinast also signi®cantly inhibited basal I Ca when perforated-patch or whole-cell recording with physiological pipette calcium concentration (10 77 M) was used. However, this e ect was not observed when using standard calcium-free whole-cell recording conditions. 5 These results indicate that inhibition of cGMP hydrolysis can decrease both basal and cAMPstimulated I Ca . Thus, cGMP hydrolysis may likely be an important step for physiological modulation of I Ca . This regulation may also be important in disease states in which cGMP production is increased and PDE5 expression is altered, such as heart failure.

The rat ventricular action potential shortens after birth. The contribution of increases in the transient outward current (Ito) to postnatal action potential shortening was assessed by measuring Ito in isolated cells and by determining the effect of 2 mM 4-aminopyridine (4-AP) on the action potentials of papillary muscles. 4-AP had no effect on 1-day action potential duration at 25% repolarization (APD25), and 1-day cells had little Ito. In 8- to 10-day muscles, 4-AP caused a small, but significant, increase in APD25. Ito increased slightly between day 1 and days 8-10, but this increase was not significant. Most of the increase in Ito (79%) and in the response to 4-AP (64%) occurred between days 8-10 and adult; however, approximately 75% of the APD25 shortening took place by days 8-10. Thus, while Ito may contribute to repolarization in late neonatal and adult cells, the different time courses of action potential shortening and increases in Ito suggest that changes in Ito are unlikely to be responsible for most of the postnatal action potential shortening.

This study was designed to determine whether alpha-receptor-stimulated monovalent ionic fluxes in rat aorta required calcium, and, if so, whether both extracellular calcium and cellularly stored calcium are active. Calcium removal in the presence of 10 mM magnesium (to maintain membrane stability) inhibited the norepinephrine-stimulated increase in potassium-42 and chloride-36 efflux. However, the norepinephrine-stimulated increase in sodium-24 influx was relatively resistant to calcium depletion. Protocols were designed to measure the time course for the changes in potassium-42 efflux and contraction when calcium was removed or replaced in the presence of norepinephrine. The dose-dependent effect of a calcium antagonist (diltiazem) was also measured. A close correlation (r = 0.94) was found between inhibition of contraction and potassium-42 effluxes which followed the regression: % potassium-42 response = 1.0 X (% contraction) + 1.8%). The slope of 1.0 and intercept near zero suggests the hypothesis that norepinephrine-stimulated potassium-42 efflux and contraction are codependent on cellular calcium concentration. This co-dependence held for short phasic responses (approximately 1 minute), as well as longer tonic responses (greater than or equal to 5 minutes). It appears that calcium-dependent potassium-42 effluxes can be supported by both the influx of extracellular calcium and release of cellular stores. It is concluded that calcium-dependent potassium channels (and possibly chloride channels) are operative in rat aorta and are an important component of the graded membrane response to norepinephrine. The sodium channels, however, do not appear to share this same calcium dependency.

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The purpose of this study was to evaluate the effect of calcium antagonists on basal tension and the elevated

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SUMMARY Factors that lead to supersensitivity of vascular smooth muscle to norepinephrine during aldosterone-salt-induced hypertension in rats appear to reside beyond ligand-a-adrenergic receptor binding, which we have shown previously to be normal. The objective of this study was to determine whether significant shifts occur in the coupling between receptors and the production of putative second messengers. Measures of [ 3 H]mjo-inositol phosphates in aorta (endothelium removed) exhibited a concentration-dependent increase to norepinephrine, with the 50% response shifted significantly to the left in the hypertensive group (7.0 ± 0.9 x 10~7 M in 8 control rats vs 1.1 ± 0.2 x 10" 7 M in 8 hypertensive rats; p<0.001). The production of [ 32 P]phosphatidic acid was also shifted (6.5 ± 2.5 x 10" 7 M in 16 control vs 1.9 ± 0.8 x 10" 7 M in 12 hypertensive rats; p<0.05). The functional responses of 42 K efflux and contraction to norepinephrine were also significantly shifted threefold to 15-fold in the hypertensive group (p<0.001), but the 50% response typically occurred at a 10 to 100 times lower concentration than that for the production of mvo-inositol phosphates and phosphatidic acid. The amplification between receptor occupancy and functional responses apparently occurs beyond the production of phosphoinositide metabolites. The fivefold shift in the 50% response of biochemical end points for the hypertensive group accounted for most of the shift (sixfold) in the functional end points. It is concluded that the increased efficacy in the hypertensive group resulted more from shifts in the relation between receptor occupancy and production of phosphoinositide metabolites than from shifts in the action of these metabolites on functions that control to catecholamines has been associated with several models of hypertension. 1 * 3 Moreover, it precedes the elevation of blood pressure in the mineralocorticoid-salt hypertensive rat, thus implicating supersensitivity as a pathogenic factor. '• 4 -5 A systematic characterization of a-adrenergic receptors revealed no significant alteration in receptor type (a,), equilibrium dissociation constants, or maximum binding (receptor concentration) of aortic smooth muscle from the aldosterone-salt hypertensive rat (AHR).6 7 Analyses of the ligand binding and dose-response curves revealed that the agonist dissociation constant (A" A ) for norepinephrine (NE) was not changed in AHR, while the efficacy was 4.4 times higher. These findings support the conclusion that postreceptor events underlie the supersensitivity in AHR.There has been an explosion of information relating phosphoinositide metabolites to cellular regulation in many tissues, including smooth muscle.8 "' 2 The regulation of phospholipase C (PLC) by a-adrenergic receptor occupancy is a potential site for the development of supersensitivity. PLC acts on phosphatidylinositol 4,5-bisphosphate to form mjo-inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG), which are thought to be important regulators of calcium rele...