We examined the action of levosimendan, a new Ca2+-sensitizing inodilator, on isolated porcine coronary arteries. Vessel rings were studied in isometric myographs. Arterial cyclic adenosine monophosphate (cAMP) levels were determined by radioimmunoassay. Levosimendan (10(-7)-10(-3) M) completely relaxed arteries preconstricted by prostaglandin F2alpha (PGF2alpha) with a pD2 (-logEC50) value of 3.99 +/- 0.05 (n = 6-9 in all experiments). Pretreatment with levosimendan also prevented contraction induced by PGF2alpha. The vasorelaxation produced by levosimendan (10(-7)-10(-3) M) was not attenuated by removal of the endothelium. Levosimendan (10(-7)-10(-3) M) relaxed contractions induced by 30 mM K+ as well as 80 mM K+, whereas the K+ channel opener levcromakalim selectively relaxed contraction induced by 30 mM K+. Neither the cyclooxygenase inhibitor indomethacin nor the beta-adrenoceptor blocker propranolol influenced levosimendan-induced vasorelaxation. The Ca2+-entry blocker isradipine failed to relax arteries precontracted by endothelin-1 in Ca2+-free/EGTA medium. However, levosimendan (10(-7)-3 x 10(-3) M) completely relaxed endothelin-1-induced contractions in this medium. Levosimendan potentiated the relaxant effect of a cAMP-stimulating drug, isoprenaline, but also that of nitroglycerin and isradipine. At a maximal effective concentration, it increased arterial tissue contents of cAMP twofold. In conclusion, levosimendan produces coronary vasorelaxation by a mechanism that seems to be endothelium independent and not mediated by K+ channel opening, Ca2+-entry blockade, release of cyclooxygenase products, or beta-adrenoceptor stimulation. Accumulation of cAMP may possibly participate in vasorelaxation at high concentrations of levosimendan, but a cAMP-independent mechanism seems to be involved at lower concentrations.

1 The haeme-containing soluble guanylyl cyclase (a 1 b 1 -heterodimer) is a major intracellular receptor and e ector for nitric oxide (NO) and carbon monoxide (CO) and mediates many of their biological actions by increasing cyclic GMP. We have synthesized new oxadiazolo-benz-oxazins and have assessed their inhibitory actions on guanylyl cyclase activity in vitro, on the formation of cyclic GMP in cultured cells and on the NO-dependent relaxation of vascular and non-vascular smooth muscle. 2 Soluble guanylyl cyclase, puri®ed to homogeneity from bovine lung, was inhibited by 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS 2028) in a concentration-dependent and irreversible manner (IC 50 30 nM for basal and 200 nM for NO-stimulated enzyme activity). Evaluation of the inhibition kinetics according to Kitz & Wilson yielded a value of 8 nM for K i , the equilibrium constant describing the initial reversible reaction between inhibitor and enzyme, and 0.2 min 71 for the rate constant k3 of the subsequent irreversible inhibition. Inhibition was accompanied by a shift in the soret absorption maximum of the enzyme's haem cofactor from 430 to 390 nm. 3 S-nitroso-glutathione-enhanced soluble guanylyl cyclase activity in homogenates of mouse cerebellum was inhibited by NS 2028 (IC 50 17 nM) and by 17 structural analogues in a similar manner, albeit with di erent potency, depending on the type of substitution at positions 1, 7 and 8 of the benzoxazin structure. Small electronegative ligands such as Br and Cl at position 7 or 8 increased and substitution of the oxygen at position 1 by -S-,-NH-or -CH 2 -decreased the inhibition. 4 In tissue slices prepared from mouse cerebellum, neuronal NO synthase-dependent activation of soluble guanylyl cyclase by the glutamate receptor agonist N-methyl-D-aspartate was inhibited by NS 2028 (IC 50 20 nM) and by two of its analogues. Similarly, 3-morpholino-sydnonimine (SIN-1)-elicited formation of cyclic GMP in human cultured umbilical vein endothelial cells was inhibited by NS 2028 (IC 50 30 nM). 5 In prostaglandin F 2a -constricted, endothelium-intact porcine coronary arteries NS 2028 elicited a concentration-dependent increase (65%) in contractile tone (EC 50 170 nM), which was abolished by removal of the endothelium. NS 2028 (1 mM) suppressed the relaxant response to nitroglycerin from 88.3+2.1 to 26.8+6.4% and induced a 9 fold rightward shift (EC 50 15 mM) of the concentrationrelaxation response curve to nitroglycerin. It abolished the relaxation to sodium nitroprusside (1 mM), but did not a ect the vasorelaxation to the K ATP channel opener cromakalim. Approximately 50% of the relaxant response to sodium nitroprusside was recovered after 2 h washout of NS 2028. 6 In phenylephrine-preconstricted, endothelium-denuded aorta of the rabbit NS 2028 (1 mM) did not a ect relaxant responses to atrial natriuretic factor, an activator of particulate guanylyl cyclase, or forskolin, an activator of adenylyl cyclase. 7 NO-dependent relaxant responses in non-vascular smooth muscle were a...