Abstract
Alpha 1-adrenergic receptors (a1-ARs) are a heterogeneous group of G protein- coupled seven transmembrane receptors that mediate the actions of the endogenous catecholamines, norepinephrine and epinephrine. Three 1a-AR subtypes (a1a, a1b, a1d) have been cloned and classified. cpa-ARs couple to phosphoinositide hydrolysis, adenylyl cyclase, and mitogen activated protein kinase (MAPR) pathways. In some systems, the adenylyl cyclase pathway, including adenosine 3',5'-cyclic monophosphate (cAMP) and protein kinase A (PRA), inhibits activation of extracellular-signal regulated kinase 1/2 (ERR 1/2). However, interaction among these signaling pathways following a1a-AR activation is not well understood. I investigated the coupling of a1a-ARs to various pathways in CHO-Kl cells stably transfected with mouse a1a-ARs, and the interaction between ERK1/2 and norepinephrine-induced cAMP accumulation. cpa-AR activation by norepinephrine increased cytosolic Ca2+ concentration and phosphorylated ERR 1/2 in a time- and concentration-dependent manner. ERR 1/2 phosphorylation was blocked by the MAPR kinase (MER1/2) inhibitor, PD98059, and the a1-AR antagonist, prazosin. A transient elevation in intracellular Ca2+ was required for the a1a-AR stimulation of ERR 1/2 phosphorylation; however, prior activation of protein kinase C (PRC) was not required for ERR1/2 phosphorylation. Norepinephrine also stimulated cAMP accumulation in transfected CHO-R1 cells in a concentration dependent manner via a1a-ARs, which was blocked by the intracellular Ca2+ chelator BAPTA. Norepineplirine-induced ERR 1/2 phosphorylation was inhibited by the adenylyl cyclase activator, forskolin, and was enhanced by the adenylyl cyclase inhibitor, SQ 22536, and the protein kinase A inhibitor, 4-cyano-3-methylisoquinolme. Thus, in transfected CHO-K1 cells, a1a-AR activation activated both the phospholipase C and the adenylyl cyclase signaling pathways. a1a-AR-mediated ERK1/2 phosphorylation was dependent on a rise in intracellular Ca2+ and this pathway was reciprocally regulated by the concomitant activation of adenylyl cyclase, which inhibited ERK1/2 phosphorylation. Thus a1a-AR stimulation of cAMP production may play an important role in regulating ERK1/2 phosphorylation in cell lines and native tissues.|A1a-AR stimulation is coupled to PKC activation, ERK1/2 phosphorylation and cAMP production, which are all associated with regulating cell growth. By using the opa- AR-transfected CHO-K1 cell model, I also investigated the effect of a1a-AR stimulation on cell proliferation in these cells, and the possible mechanisms mediating this effect. Long-term a1a-AR activation by 10 pM norepinephrine (48 h) inhibited cell proliferation as measured both by cell counting and [H]-thymidine incorporation. a1a-AR-mediated inhibition in cell proliferation was not blocked by the MER inhibitor, PD98509, but PD 98509 alone also inhibited cell proliferation in transfected CHO-K1 cells. The PRC activator PMA, which desensitizes PRC fowling long-term stimulation, did not block norepinephrine-induced inhibition of cell proliferation in a1a-AR-transfected CHO-K1 cells, and PMA alone did not have any effect on cell proliferation. The results from these studies showed that a1a-AR activation inhibited cell proliferation in a1a-AR-transfected CHO-K1 cells. These studies suggested that PRC does not play a role in a1a-AR- mediated effect on cell growth, but ERR 1/2 may play a role. cAMP also plays a role in this a1a-AR-mediated growth effect.|A1a-ARs contribute to the regulation of renal function by sympathetic nerves and the a1a-AR subtype has been shown to be present in the renal proximal tubules. However, the functions and signal transduction pathways of a1a-ARs in the renal proximal tubule cells are not well understood. In my project, 1 first developed a renal proximal tubule cell model transfected with epitope hemagglutinin (HA) tagged a1a-AR using the OK. cell line. 1 characterized the signal transduction pathways and cell morphological changes induced by a1a-AR activation, and compared them to those found in the a1a-AR-transfected CHO-K1 cells. After transfection of the mouse a^-AR cDNA into OK cells, I confirmed the expression of the receptor protein by demonstrating the presence of the protein with western blotting and immunoprecipitation against the epitope HA, and receptor density in radioligand saturation binding experiments was also measured. 1 found that transfected OK cells expressed a receptor density of 176 fmol/mg protein, which was similar to the physiological range of receptor density reported by our laboratory in the renal cortex. The transfected ata-ARs are functional, since stimulation of these receptors with phenylephrine induced intracellular Ca2+ mobilization in a concentration dependent manner. Phenylephrine also stimulated ERK1/2 phosphorylation in the a1a-AR-transfected OK cells. Unlike a1a-AR transfected CHO-K1 cells, phenylephrine did not stimulate cAMP accumulation in a1a-AR transfected OK cells. a1a-AR activation induced morphological changes in a1a-AR-transfected CHO-K1 cells, but not in aia-AR-transfected OK cells. These studies suggested that in the renal proximal tubule cells, a1a-AR-induced cellular effects and signal transduction pathways are different from those in the a1a-AR-transfected CHO-K1 cells. The functions of a1a- ARs, as well as the roles of ERK1/2 phosphorylation in the renal proximal tubule cells need to be further investigated, and this a1a-AR-transfected OK cell model provides an excellent tool for these studies.