calcimycin and diacetylmonoxime

In chronic liver injury, hepatic stellate cells (HSCs) have been implicated as regulators of sinusoidal vascular tone. We studied the relative role of Ca(2+)-dependent and Ca(2+)-independent contraction pathways in rat HSCs and correlated these findings to in situ perfused cirrhotic rat livers. Contraction of primary rat HSCs was studied by a stress-relaxed collagen lattice model. Dose-response curves to the Ca(2+) ionophore A-23187 and to the calmodulin/myosin light chain kinase inhibitor W-7 served to study Ca(2+)-dependent pathways. Y-27632, staurosporin, and calyculin (inhibitors of Rho kinase, protein kinase C, and myosin light chain phosphatase, respectively) were used to investigate Ca(2+)-independent pathways. The actomyosin interaction, the common end target, was inhibited by 2,3-butanedione monoxime. Additionally, the effects of W-7, Y-27632, and staurosporin on intrahepatic vascular resistance were evaluated by in situ perfusion of normal and thioacetamide-treated cirrhotic rat livers stimulated with methoxamine (n = 25 each). In vitro, HSC contraction was shown to be actomyosin based with a regulating role for both Ca(2+)-dependent and -independent pathways. Although the former seem important, an important auxiliary role for the latter was illustrated through their involvement in the phenomenon of "Ca(2+) sensitization." In vivo, preincubation of cirrhotic livers with Y-27632 (10(-4) M) and staurosporin (25 nM), more than with W-7 (10(-4) M), significantly reduced the hyperresponsiveness to methoxamine (10(-4) M) by -66.8 +/- 1.3%, -52.4 +/- 2.7%, and -28.7 +/- 2.8%, respectively, whereas in normal livers this was significantly less: -43.1 +/- 4.2%, -40.2 +/- 4.2%, and -3.8 +/- 6.3%, respectively. Taken together, these results suggest that HSC contraction is based on both Ca(2+)-dependent and -independent pathways, which were shown to be upregulated in the perfused cirrhotic liver, with a predominance of Ca(2+)-independent pathways.

Topics: Actomyosin; Amides; Animals; Calcimycin; Calcium; Cell Shape; Cells, Cultured; Diacetyl; Enzyme Inhibitors; Liver; Male; Marine Toxins; Methoxamine; Myocytes, Cardiac; Myocytes, Smooth Muscle; Myosin-Light-Chain Kinase; Oxazoles; Protein Kinase Inhibitors; Pyridines; Rats; Rats, Wistar; Signal Transduction; Staurosporine; Sulfonamides; Vasoconstrictor Agents

The contribution of chloride and potassium to the 4-aminopyridine (4-AP)-resistant transient outward current was investigated in dog cardiac myocytes. Whole cell currents were recorded at 37 degrees C in single cells dissociated from epicardial and midmyocardial regions of the canine ventricle. Sodium-calcium exchange current and voltage-dependent transient outward potassium current (IA) were blocked in sodium-free solutions containing 2 mM 4-AP; sodium channels were inactivated by the -50-mV holding potential. When patch pipettes contained 0.4-0.8 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, voltage-clamp steps over the range -20 to +50 mV activated an inward calcium current (ICa) and a Ca(2+)-activated chloride current [ICl(Ca)]. ICl(Ca) was blocked by 200 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), or reduction of external chloride. Independent of the presence of potassium, the reversal potential of the SITS-sensitive current varied with extracellular chloride, as predicted for a chloride-selective conductance. The bell-shaped current-voltage relation of ICl(Ca) has a threshold of -20 mV and a peak at +40 mV. No evidence could be found for a Ca(2+)-activated potassium current or a Ca(2+)-activated nonspecific cation current under these conditions. ICl(Ca) contributed to oscillatory inward currents at diastolic potentials in cells superfused by isoproterenol and high Ca2+, suggesting a role for this current in triggered arrhythmias associated with delayed afterdepolarizations. In the normal heart, ICl(Ca) is likely to contribute to rate- and rhythm-dependent repolarization of the cardiac action potential.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4-Aminopyridine; Animals; Caffeine; Calcimycin; Calcium; Calcium Channels; Calcium Chloride; Cells, Cultured; Diacetyl; Dogs; Egtazic Acid; Electrophysiology; Female; Heart; Heart Ventricles; Isoproterenol; Kinetics; Male; Meglumine; Membrane Potentials; Time Factors