Western blot of these precipitated proteins with the same antibody demonstrates that exposure to PVN, for the same period of time that produces functional responses, clearly increased the level of protein tyrosine phosphorylation in cardiac myocytes (Fig. was also mimicked by the phosphotyrosine phosphatase inhibitor pervanadate (PVN). PVN had no effect on basal Ca2+ current or Ca2+ current stimulated by histamine, but it did inhibit Ca2+ current stimulated by -adrenergic receptor activation. Furthermore, the ability of PVN to inhibit -adrenergic stimulation of the Ca2+ current was antagonized by lavendustin A. These results are consistent with the conclusion that in guinea-pig ventricular myocytes -adrenergic inhibition of -adrenergic responses involves a tyrosine kinase-dependent signalling pathway. The fact that methoxamine and PVN antagonized cAMP-dependent responses mediated by -adrenergic, but not H2 histamine, receptor activation suggests that the inhibitory effect of -adrenergic stimulation and tyrosine kinase activity is at the level of the -adrenergic receptor. The ability of -adrenergic receptor (-AR) stimulation to antagonize -adrenergic responses is well documented in noncardiac as well as cardiac preparations. In non-cardiac cells, such effects are associated with 2-AR stimulation (Bylund, 1992). However, in cardiac myocytes, antagonism of -adrenergic responses involves 1-ARs (Buxton & Brunton, 1985, 1986; Boutjdir 1992; Barrett 1993; Iyadomi 1995; Hartmann 1995; Oleksa 1996; Chen 1996; Hool 1997). This is consistent with the fact that cardiac myocytes primarily express 1-, not 2-, ARs (Fedida 1993; Rabbit polyclonal to ZNF268 Terzic 1993). The mechanism responsible for the antagonistic effect of 1-AR stimulation in L-Cycloserine cardiac muscle involves inhibition of -adrenergically stimulated cAMP levels (Watanabe 1977; Buxton & Brunton, 1985, 1986; Barrett 1993; Lemire 1998). The fact that the inhibitory effect is on the cAMP signalling pathway explains why 1-AR stimulation affects a wide range of -adrenergic responses in the heart. This includes -adrenergic regulation of chronotropic effects (Molderings & Schumann, 1989), L-type Ca2+ channel activity (Boutjdir 1992; Chen 1996), CFTR Cl? channel activity (Iyadomi 1995; Oleksa 1996; Hool 1997) and L-Cycloserine contractility (Hartmann 1995). The mechanism by which 1-AR stimulation affects cAMP-dependent responses in cardiac myocytes is not well understood. Biochemical studies have suggested that 1-AR stimulation may either inhibit cAMP L-Cycloserine production (Barrett 1993) or enhance cAMP degradation (Buxton & Brunton, 1985). Functional studies suggest that the former is more likely. Activation of the cAMP-regulated Cl? current by the -AR agonist isoprenaline (Iso) can be antagonized by 1-AR stimulation. However, 1-agonists are unable to inhibit the Cl? current activated by direct stimulation of adenylate cyclase with forskolin, suggesting that 1-AR stimulation exerts its effect upstream of cAMP production (Iyadomi 1995; Oleksa 1996). Consistent with L-Cycloserine this conclusion, 1-agonists are also unable to inhibit persistent activation of the Cl? current in the presence of the non-hydrolysable GTP analogue GTPS (Iyadomi 1995). The signalling pathway linking the 1-AR to its site of action is also unclear. In cardiac muscle, 1-AR stimulation is often associated with the activation of PKC (Fedida 1993; Terzic 1993). However, we previously demonstrated that 1-adrenergic inhibition of the -adrenergically regulated cardiac Cl? current does not involve PKC (Oleksa 1996). Another signalling pathway associated with 1-AR stimulation involves regulation of tyrosine kinase activity (Zhong & Minneman, 1999). Furthermore, we previously reported evidence that basal tyrosine kinase activity inhibits -adrenergic responses in cardiac myocytes (Hool 1998). Therefore, the purpose of this study was to test the hypothesis that 1-AR stimulation inhibits -adrenergic regulation of cardiac responses via a tyrosine kinase-dependent mechanism. To address this question, we evaluated the ability of tyrosine kinase inhibition to block, and phosphotyrosine phosphatase (PTP) inhibition to mimic, the effects of 1-adrenergic stimulation on the L-type Ca2+ current in isolated guinea-pig ventricular myocytes. Our results are consistent with the idea that 1-AR stimulation inhibits -adrenergic responses at least partially through a tyrosine kinase-dependent mechanism that acts at the level of the -AR. Some parts of this work have been published in abstract form (Nulton-Persson 2000). METHODS Cell isolation Single ventricular myocytes were isolated from adult Hartley guinea-pigs using the modification of a method described previously (Hool 1998). The methods used in this study were approved by the L-Cycloserine Institutional Animal Care and Use Committee at Case Western Reserve University. Animals were anaesthetized by intraperitoneal injection of pentobarbital (150 mg kg?1). Hearts were then quickly excised and the coronary arteries perfused via the aorta with a solution containing (mm): 140 NaCl, 5.4 KCl, 2.5 MgCl2, 1.5 CaCl2, 11 glucose and 5.5 Hepes (pH 7.4). The heart was perfused with this solution for 5 min, nominally Ca2+-free solution for 5 min, and then nominally Ca2+ free solution containing 0.5 mg ml?1 collagenase (class B, Boehringer Mannheim) for 30 min. The ventricles were then removed and.