Yu Y, Nair B G, Patel T B
Department of Pharmacology, University of Tennessee, Memphis 38163.
J Cell Physiol. 1992 Mar;150(3):559-67. doi: 10.1002/jcp.1041500317.
We have previously shown that epidermal growth factor (EGF) augments cAMP accumulation in the heart and stimulates cardiac adenylyl cyclase via a G protein mediated mechanism (Nair et al., 1989). More recently, employing an antibody against the carboxy-terminus decapeptide of Gs alpha, we have demonstrated that Gs alpha mediates the effects of EGF on cardiac adenylyl cyclase (Nair et al., 1990). Since the heart comprises of a variety of cell types, the purpose of the studies presented here was to determine whether or not the effects of EGF on adenylyl cyclase were mediated in cardiac myocytes or noncardiomyocytes. Therefore, cultures of ventricular cardiomyocytes and noncardiomyocytes from neonatal rat hearts were established and characterized. Apart from the differences in cellular morphology, cardiomyocytes but not the noncardiomyocytes employed in our studies expressed the alpha- and beta-myosin heavy chain (MHC) mRNA and the beta-MHC protein. Additionally, as described previously, treatment of cardiomyocytes with thyroid hormone increased alpha-MHC mRNA and decreased the expression of beta-MHC mRNA, indicating that the cardiomyocytes employed in our studies were responding in a physiologically relevant manner. EGF in a time-dependent manner increased cAMP accumulation in the cardiomyocytes but not in noncardiomyocytes. Maximum and half-maximum effects were observed at 100 nM and 2 nM concentrations of EGF, respectively. As determined by the presence of immunoreactive EGF receptors and tyrosine phosphorylation of the 170 kDa protein in membranes of cardiomyocytes and noncardiomyocytes, both the cell populations contained functional EGF receptors. Therefore, the differential effects of EGF on cAMP accumulation in the two cell populations appear to be due to differential coupling of the EGF receptors to the adenylyl cyclase system rather than the absence of EGF receptors in noncardiomyocytes. Consistent with our previous findings in isolated membranes and perfused rat hearts, EGF-elicited increase in cAMP accumulation in cardiomyocytes did not involve activation of beta-adrenoreceptors and was abolished by prior treatment of cells with cholera toxin. Overall, our findings demonstrate that EGF-elicited increase in cAMP accumulation in the heart is the reflection of changes in cAMP content of cardiomyocytes and not noncardiomyocytes.
我们之前已经表明，表皮生长因子（EGF）可增强心脏中的cAMP积累，并通过G蛋白介导的机制刺激心脏腺苷酸环化酶（Nair等人，1989年）。最近，我们使用针对Gsα羧基末端十肽的抗体，证明了Gsα介导EGF对心脏腺苷酸环化酶的作用（Nair等人，1990年）。由于心脏由多种细胞类型组成，本文所述研究的目的是确定EGF对腺苷酸环化酶的作用是否在心肌细胞或非心肌细胞中介导。因此，建立并表征了新生大鼠心脏心室心肌细胞和非心肌细胞的培养物。除了细胞形态的差异外，我们研究中使用的心肌细胞而非非心肌细胞表达α和β肌球蛋白重链（MHC）mRNA以及β-MHC蛋白。此外，如先前所述，用甲状腺激素处理心肌细胞会增加α-MHC mRNA并降低β-MHC mRNA的表达，这表明我们研究中使用的心肌细胞以生理相关的方式做出反应。EGF以时间依赖性方式增加心肌细胞中的cAMP积累，但不增加非心肌细胞中的cAMP积累。在100 nM和2 nM浓度的EGF下分别观察到最大和半最大效应。通过免疫反应性EGF受体的存在以及心肌细胞和非心肌细胞膜中170 kDa蛋白的酪氨酸磷酸化确定，两种细胞群体都含有功能性EGF受体。因此，EGF对两种细胞群体中cAMP积累的差异作用似乎是由于EGF受体与腺苷酸环化酶系统的差异偶联，而不是非心肌细胞中不存在EGF受体。与我们之前在分离膜和灌注大鼠心脏中的发现一致，EGF引起的心肌细胞中cAMP积累增加不涉及β-肾上腺素能受体的激活，并且在用霍乱毒素预先处理细胞后被消除。总体而言，我们的研究结果表明，EGF引起的心脏中cAMP积累增加是心肌细胞而非非心肌细胞中cAMP含量变化的反映。
