Zhuo-Wei Hu

Adrenergic Receptors Activate Phosphatidylinositol 3-Kinase in Human Vascular Smooth Muscle Cells ROLE IN MITOGENESIS

Activation of α adrenergic receptors stimulates mitogenesis in human vascular smooth muscle cells (HVSMCs). To examine signaling pathways by which activation of α receptors may induce mitogenesis in HVSMCs, we have found that α receptor stimulated-DNA synthesis and activation of mitogen-activated protein (MAP) kinase are blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). To determine directly if activation of α receptors stimulated PI 3-kinase, in vitro assays of kinase activity were performed in immunocomplexes precipitated by an antibody against the p85α subunit of PI 3-kinase. Noradrenaline stimulated a time- and concentration-dependent activation of PI 3-kinase in the presence of a β adrenergic receptor antagonist. Noradrenaline-stimulated PI 3-kinase activation was blocked by antagonists of α receptors and by pertussis toxin, suggesting that α receptors activate PI 3-kinase via a pertussis toxin-sensitive G protein. Direct activation of protein kinase C by a phorbol ester did not stimulate PI 3-kinase; also, a Ca L-channel blocker did not inhibit noradrenaline-stimulated PI 3-kinase activity. Increased PI 3-kinase activity was detected in both anti-Ras and anti-phosphotyrosine immunoprecipitates from noradrenaline-stimulated HVSMCs. Moreover, noradrenaline stimulated formation of active Ras-GTP complexes. Because blockade of PI 3-kinase by wortmannin inhibited formation of this complex, this result suggests that Ras might be a target of PI 3-kinase. Noradrenaline stimulated tyrosine phosphorylation of the p85 subunit of PI 3-kinase, and a phosphorylated tyrosine protein could be co-immunoprecipitated with anti-p85 of PI 3-kinase. These results demonstrate that stimulation of α receptors activates PI 3-kinase in HVSMCs and that α receptor-activated PI 3-kinase is associated with an increase in active Ras-GTP and activation of tyrosine protein phosphorylation. These pathways may contribute to α receptor-stimulated mitogenic responses including activation of MAP kinase and DNA synthesis in HVSMCs.

Heat shock activates c-Src tyrosine kinases and phosphatidylinositol 3-kinase in NIH3T3 fibroblasts.

There is increasing evidence that cellular responses to stress are in part regulated by protein kinases, although specific mechanisms are not well defined. The purpose of these experiments was to investigate potential upstream signaling events activated during heat shock in NIH3T3 fibroblasts. Experiments were designed to ask whether heat shock activates p60 c-Src tyrosine kinase or phosphatidylinositol 3-kinase (PI 3-kinase). Using in vitro protein kinase activity assays, it was demonstrated that heat shock stimulates c-Src and PI 3-kinase activity in a time-dependent manner. Also, there was increased PI 3-kinase activity in anti-phosphotyrosine and anti-c-Src immunoprecipitated immunocomplexes from heated cells. Heat shock activated mitogen-activated protein kinase (MAPK) and p70 S6 kinase (S6K) in these cells. The role of PI 3-kinase in regulating heat shock activation of MAPK and p70 S6K was investigated using wortmannin, a specific pharmacological inhibitor of PI 3-kinase. The results demonstrated that wortmannin inhibited heat shock activation of p70 S6K but only partially inhibited heat activation of MAPK. A dominant negative Raf mutant inhibited activation of MAPK by heat shock but did not inhibit heat shock stimulation of p70 S6K. Genistein, a tyrosine kinase inhibitor, and suramin, a growth factor receptor inhibitor, both inhibited heat shock stimulation of MAPK activity and tyrosine phosphorylation of MAPK. Furthermore, a selective epidermal growth factor receptor (EGFR) inhibitor, tryphostin AG1478, and a dominant negative EGFR mutant also inhibited heat shock activation of MAPK. Heat shock induced EGFR phosphorylation. These results suggest that early upstream signaling events in response to heat stress may involve activation of PI 3-kinase and tyrosine kinases, such as c-Src, and a growth factor receptor, such as EGFR; activation of important downstream pathways, such as MAPK and p70 S6K, occur by divergent signaling mechanisms similar to growth factor stimulation.

Alpha 1 adrenergic receptor-induced c-fos gene expression in rat aorta and cultured vascular smooth muscle cells.

While growth of blood vessels is important in hypertension, relatively little is known about the contribution of catecholamines. Using isolated rat aorta and cultured smooth muscle cells, we examined adrenergic stimulation of gene expression. Phenylephrine, a selective alpha 1 adrenergic receptor agonist, caused a rapid and transient increase in c-fos mRNA accumulation which was inhibited by prazosin, an alpha 1 receptor antagonist. Similarly, phenylephrine stimulated c-jun and c-myc mRNA accumulation. Chloroethyl-clonidine, a compound which irreversibly blocks alpha 1B receptors, completely blocked the phenylephrine-induced increase in c-fos mRNA. RNase protection experiments demonstrated that rat aorta prominently expressed mRNA for alpha 1B and alpha 1A/D receptors. Phenylephrine-induced c-fos mRNA was partially inhibited by H-7, a protein kinase C inhibitor, and by nifedipine, a Ca2+ channel blocker; these two compounds together had additive effects. In situ hybridization showed that expression of c-fos mRNA induced by phenylephrine was localized to aortas medial layer. These results suggest that alpha 1 receptor-induced increase in c-fos mRNA in aorta is mediated by a chloroethyl-clonidine-sensitive receptor subtype signaling via increasing intracellular Ca2+ concentrations and activating protein kinase C.

Phosphorylation of the cAMP Response Element-binding Protein and Activation of Transcription by α1 Adrenergic Receptors

Activation of α1 adrenergic receptors not only stimulates smooth muscle contraction but also modifies gene expression. We wondered if α1 adrenergic receptors could activate transcription of genes regulated by the cAMP response element-binding protein (CREB). Using Rat1 cells stably transfected with each of the three cloned human α1adrenergic receptor subtypes, norepinephrine strongly stimulated CREB phosphorylation in α1A and α1B but more weakly in α1D-transfected cells. Norepinephrine increased the activity of a somatostatin cAMP-regulated enhancer-chloramphenicol acetyltransferase reporter in these cells. α1 adrenergic receptors are known to activate protein kinase C (PKC) and increase [Ca2+ ] i . Nonetheless, neither GF109203X, a PKC inhibitor, nor BAPTA-AM, a calcium chelator, blocked phosphorylation of CREB induced by norepinephrine. In addition, α1adrenergic receptor-induced CREB phosphorylation was not mediated via the mitogen-activated protein kinase pathway because norepinephrine did not stimulate mitogen-activated protein kinase activity in these cells. Activation of α1 adrenergic receptors increased cAMP accumulation in these cells. Norepinephrine-induced cAMP-regulated enhancer-chloramphenicol acetyltransferase activity was inhibited either by expression of the PKA inhibitory peptide or a dominant negative PKA regulatory subunit mutant. These results demonstrate that α1 adrenergic receptors activate the transcription factor CREB by a PKA-dependent pathway.

Insulin and insulin-like growth factor I differentially induce alpha1-adrenergic receptor subtype expression in rat vascular smooth muscle cells.

Hyperinsulinemia has been implicated as an important risk factor for the development of accelerated cardiovascular disease. We wondered if insulin or IGF-I induced expression of alpha1 adrenergic receptors in vascular smooth muscle cells (VSMCs) which could enhance smooth muscle contraction and cell growth activated by catecholamines. Rat aortic VSMCs were incubated with insulin or IGF-I for various times and expression of alpha1 receptors was detected using [3H]prazosin binding. Both insulin and IGF-I increased alpha1 receptor number; also, these peptides increased expression of the alpha1D receptor gene with no change in expression of the alpha1B receptor gene as detected by RNase protection assays. Using Western blotting, we found that these peptides increased expression of the alpha1D receptor subtype in these cells. Increased expression of the alpha1D receptor mRNA was inhibited by the receptor tyrosine kinase inhibitor genistein and the PI 3-kinase inhibitor wortmannin but was not inhibited by protein kinase C inhibitor H7 or the L-type calcium channel blocker nifedipine. Preincubation of cells with insulin or IGF-I enhanced subsequent norepinephrine stimulation of mitogen activated kinase activity. These results suggest that insulin/IGF-I regulate expression of alpha1 receptors in VSMCs and potentially enhance the effects of catecholamines in settings of hyperinsulinemia.

Doxazosin Inhibits Proliferation and Migration of Human Vascular Smooth-Muscle Cells Independent of α1-Adrenergic Receptor Antagonism

Proliferation and migration of vascular smooth-muscle cells (VSMCs), stimulated by a variety of growth factors, play a critical role in the pathogenesis of vascular diseases. We found unexpectedly that doxazosin, an alpha1-adrenergic-receptor antagonist, inhibits serum-stimulated proliferation of cultured human VSMCs. Subsequent experiments systematically investigated inhibitory effects of doxazosin on mitogenesis stimulated in VSMCs by platelet-derived growth factor (PDGF), epidermal growth factor, and G protein-coupled receptor agonists thrombin and angiotensin II. Doxazosin attenuated the stimulation of DNA synthesis for each of these ligands with median inhibitory concentrations (IC50s) from 0.3 to 1 microM. PDGF-AB (1 nM) increased cell number; doxazosin inhibited this response by 70-80%. Prazosin, a related alpha1-receptor antagonist, had similar but less potent effects on inhibiting mitogenesis in these cells. Doxazosin and prazosin inhibited PDGF-AB-stimulated and insulin-like growth factor (IGF-I)-stimulated migration of VSMCs by approximately 40-50%. These effects of doxazosin were likely unrelated to alpha1-receptor blockade because pretreatment of cells with phenoxybenzamine, an irreversible alpha1 antagonist, did not change the capacity of doxazosin to inhibit of PDGF-stimulated mitogenesis. Also, doxazosin inhibited PDGF-stimulated DNA synthesis in NIH 3T3 cells, which do not express alpha1 receptors. These results suggest that doxazosin is a potent inhibitor of VSMC proliferation and migration through a mechanism unrelated to alpha1-receptor antagonism.

Activation of heat shock protein (hsp)70 and proto-oncogene expression by alpha1 adrenergic agonist in rat aorta with age.

Induction of heat shock proteins (hsp) most likely is a homeostatic mechanism in response to metabolic and environmental insults. We have investigated signal transduction mechanisms involved in alpha1, adrenergic receptor stimulation of hsp7O gene expression in isolated aortas with age. We found that alpha1 adrenergic agonists directly induced hsp70 mRNA in rat aorta in vitro; the alpha1, selective antagonist prazosin blocked this effect whereas chloroethylclonidine, an antagonist which has some selectivity for alpha1B receptors, was ineffective. This response was insensitive to pertussis toxin and was partially blocked by the protein kinase C inhibitor H7. Removal of extracellular calcium attenuated induction of hsp70 mRNA but not the induction of c-fos or c-myc. The induction of hsp70 mRNA by either norepinephrine or by phorbol dibutyrate was blunted in aortas from old (24-27 mo) rats whereas c-fos responses were not diminished in the older vessels. The hsp70 response to elevated temperature (42 degrees C) was not changed with age. Activation of hsp70 expression most likely involves a pertussis toxin insensitive G protein which activates protein kinase C, and requires extracellular calcium. With age, hsp70 gene expression induced by stimulation of alpha1 adrenergic receptors is markedly attenuated, which could modify responses to stress or vascular injury with aging.

Induction of enhanced release of endothelium-derived relaxing factor after prolonged exposure to α-adrenergic agonists : role in desensitization of smooth muscle contraction

Desensitization of α1-adrenergic receptor-mediated contraction occurs in rat aorta after in vitro exposure to α-adrenergic agonists; we previously showed that a component of the desensitization is endothelial cell dependent. Our primary purpose was to examine possible alterations in either the release or action of endothelium-derived relaxing factor (EDRF) in desensitized blood vessels. Rings of rat aorta were desensitized in vitro by exposure to phenylephrine (PE) for 6 h with impaired subsequent ability of PE to induce smooth muscle contraction. PE also induced heterologous desensitization of serotonin-induced contraction, which was blocked by the α1-adrenergic selective antagonist prazosin. Using a “sandwich” bioassay technique, we noted enhanced release of EDRF from the aortic rings that had been previously exposed to PE as compared with controls. The capacity of PE to activate accumulation of inositol monophosphate was impaired in the desensitized blood vessels, both with and without endothelium. Our results suggest that prolonged exposure to α-adrenergic agonists leads to several adaptations in vascular smooth muscle (VSM), including enhanced release of EDRF. Although impaired action of EDRF has been suggested to play a role in diseases such as diabetes and atherosclerosis, our results indicate that release and action of EDRF may be enhanced with prolonged exposure to α-agonists.

Prolonged activation of alpha 1 adrenoceptors induces down-regulation of protein kinase C in vascular smooth muscle.

Sustained exposure of vascular smooth muscle to catecholamines results in desensitization of alpha 1-adrenoreceptor-mediated vascular smooth muscle contraction. The present study was designed to determine the effects of prolonged exposure of blood vessels to catecholamines on protein kinase C (PKC) activity. Incubation of rat aortic smooth muscle with 10 microM norepinephrine (NE) for 4 h resulted in a threefold decrease in sensitivity of the contractile response of rat aortic smooth muscle to the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDBu); this loss in sensitivity was dependent on the presence of endothelium. NE induced a 45% decrease in enzymatic activity of the soluble and particulate forms of PKC. With [3H]PDBu used to label phorbol ester receptor binding sites in the aorta, there was a 34% decrease in [3H]PDBu binding sites in NE-treated blood vessels without change in binding affinity for the ligand. To determine whether this loss in enzymatic activity and [3H]PDBu binding resulted from a decrease in the quantity of the enzyme, Western blot analyses were performed using a monoclonal antibody (MoAb) against PKC. This approach confirmed the presence of an 80-Kd immunoreactive PKC in the soluble fraction of rat aortic smooth muscle and demonstrated a 44% decrease in the amount of PKC in blood vessels after sustained exposure to catecholamines. Our results demonstrate that prolonged activation of alpha-adrenoceptors in blood vessels leads to down-regulation of PKC which may contribute to desensitization of contraction mediated by vasoconstrictors.

Desensitization of α-adrenergic receptor-mediated smooth muscle contraction : role of the endothelium

Desensitization of α-adrenergic receptor-mediated smooth muscle contraction occur in aortas from New England Deaconess Hospital (NEDH) rats harboring pheochromocytoma (PHEO) and following chronic exposure to the α-adrenergic agonist phenylephrine in vitro. Endothelium is known to release an endothelial cell-derived relaxing factor that promotes smooth muscle relaxation. We wondered if the endothelium might contribute to the desensitization of contraction. The role of the endothelium in desensitization was studied using aortic rings with endothelium [E(+)] and with endothelium removed [E(-)]. Maximal phenylephrine (PE)-induced contraction (Emax) for E(+) was 1.7 ± 0.3 g in controls and 0.4 ± 0.1 g in PHEO (p < 0.001), demonstrating desensitization; however, for E(-), Emax was 2.4 ± 0.2 g in PHEO vs. 2.5 ± 0.2 g in controls, demonstrating restoration of maximal contraction when the endothelium was removed. However, sensitivity [-log EC50(M)] to PE in E(-) remained significantly lower in PHEO compared to controls (6.94 ± 0.12 vs. 7.51 ± 0.14, respectively, p < 0.001). Similarly, in aortic ring segments desensitized in vitro with phenylephrine, the maximal contraction in phenylephrine-exposed aortas was 60% of that seen in controls. Removal of the endothelium from the vessels pretreated with phenylephrine fully restored the maximal response and sensitivity of these vessels. Treatment of desensitized vessels with hemoglobin (5 ± 10−5 M) restored the maximal contraction and sensitivity to phenylephrine. When the endothelium was removed prior to chronic exposure to phenylephrine, the sensitivity to phenylephrine decreased while the Emax remained similar to controls. Indomethacin and 8-(sulfophenyl)theophylline had no effect on contraction of the desensitized aortic ring segments. Our results demonstrate that the endothelium plays a major role in α-adrenergic receptor desensitization of blood vessels; this effect of the endothelium is likely mediated by endothelium-derived relaxing factor.