Increased 12-lipoxygenase expression in breast cancer tissues and cells. Regulation by epidermal growth factor

The interaction of growth factors, such as epidermal growth factor (EGF) with their receptors, on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acids, such as arachidonic acid, which can be further metabolized by the lipoxygenase (LO) pathway. Several LO products have been shown to stimulate oncogenes and have mitogenic and chemotactic effects. In this study, we have evaluated the regulation of 12-LO activity and expression in breast cancer cells and tissues. Leukocyte-type 12-LO messenger RNA (mRNA) expression was studied by a specific RT-PCR method in matched, normal, uninvolved and cancer-involved breast tissue RNA samples from six patients. In each of these six patients, the cancer-involved section showed a much higher level of 12-LO mRNA than the corresponding normal section. 12-LO mRNA levels also were greater in two breast cancer cell lines, MCF-7 and COH-BR1, compared with the nontumorigenic breast epithelial cell line, MCF-10F. The growth of the MCF-7 cells was significantly inhibited by two specific LO blockers but not by a cyclooxygenase blocker. Treatment of serum-starved MCF-7 cells with EGF for 4 h led to a dose-dependent increase in the formation of the 12-LO product, 12-hydroxyeicosatetraenoic acid. EGF treatment also increased the levels of the leukocyte-type 12-LO protein expression at 24 h. These results suggest that activation of the 12-LO pathway may play a key role in basal and EGF-induced breast cancer cell growth.     

Cytokine modulation of LDL oxidation by activated human monocytes

There is considerable evidence to suggest that cytokines modulate the pathological cellular events that occur in human atherosclerosis. We sought to determine the effects of T-helper-lymphocyte (TH)-1- and TH2-type cytokines on the ability of human monocytes to oxidize LDL, one of the pathological processes believed to occur in atherosclerosis. The ability of opsonized zymosan (ZOP)-activated human monocytes to oxidize LDL in a 24-hour period was significantly enhanced by pretreatment of the monocytes with the TH2 cytokines, interleukin (IL)-4, or IL-13 compared with untreated monocytes. In contrast, interferon (IFN)-gamma, a TH1 cytokine, inhibited LDL oxidation by activated monocytes. Treatment with IFN-gamma also prevented the IL-4- and IL-13-mediated enhancement of LDL oxidation by ZOP-activated monocytes. Untreated or cytokine-treated unactivated monocytes did not oxidize LDL. The enhancement of LDL oxidation mediated by IL-4 or IL-13 treatment was not due to a mitogenic effect of the cytokines on the monocytes, nor to modulation of superoxide anion (O2-) production. The cytokine regulation of 15-lipoxygenase (LO) in the monocytes was also examined. IL-4 and IL-13 induction of 15-LO mRNA and 15-LO activity in the monocytes was confirmed, as was the previously reported inhibition of induction by IFN-gamma. In summary, IL-4 and IL-13 enhance the ability of activated human monocytes to oxidize LDL, whereas IFN-gamma inhibits the cell-mediated oxidation. The up- and downregulation of activated monocyte-mediated LDL oxidation by these cytokines correlates with the expression of 15-LO activity. Considerable evidence suggests that the progression of atherosclerosis includes events that are immunologically mediated, lending potential physiological relevance to these in vitro observations.     

Mitogenic effect of erythropoietin on cultured aortic myocytes

The administration of recombinant erythropoietin (rHuEpo) to anemic chronic renal failure patients may be associated with an increase in blood pressure, possibly by direct effects on peripheral blood vessels. In the present study, experiments were designed to explore the hypothesis that rHuEpo could enhance vascular resistance through mitogenic effect on vascular smooth muscle cells (VSMCs), and that preexisting hypertension might be a predisposing condition. Cultured VSMCs from the thoracic aortae of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied for DNA synthesis, phospholipase C activity, and cell growth related proto-oncogene expression in the presence of rHuEpo. In cells from both strains, rHuEpo dose-dependently increased DNA synthesis and stimulated phospholipase C activity, as indicated by 3H-thymidine incorporation and 3H-inositol phosphate formation, respectively (EC50 approximately 4 U/ml). Exposure of VSMCs to rHuEpo for various times gradually increased the levels of c-myc and junB and transiently induced c-fos expression, as determined by Northern analysis. rHuEpo-induced DNA synthesis was markedly enhanced in VSMCs from SHR compared to those from WKY. In contrast, rHuEpo-induced phospholipase C activity and proto-oncogene expression did not differ between the two strains. Taken together, these results suggest that rHuEpo may function as a vascular smooth muscle cell growth promoting factor through activation of the phospholipase C cascade and modulation of proto-oncogene expression. It could thereby contribute to vascular hypertrophy and arterial hypertension.     

Prolonged heparin after uncomplicated coronary interventions: a prospective, randomized trial

BACKGROUND: Both ischemic and direct vascular injury (angioplasty) result in the elaboration of proinflammatory substances, including tumor necrosis factor alpha (TNF), which may regulate vascular smooth muscle cell (VSMC) proliferation and promote vessel stenosis. Interleukin-10 (IL-10) is a pleiotropic cytokine with potent antiinflammatory effects in many cells lines. We hypothesized that IL-10 could be used therapeutically to influence vascular remodeling by inhibiting TNF-induced VSMC proliferation. The purposes of this study were (1) to determine whether human myocardium produces endogenous TNF in response to ischemia-reperfusion, (2) to examine the effect of TNF on human arterial smooth muscle proliferation, and (3) to explore the potential therapeutic effect of IL-10 on unstimulated and TNF-stimulated VSMC proliferation. MATERIALS AND METHODS: Right atrial muscle was obtained from patients undergoing elective cardiac surgery. Atrial muscle was subjected to simulated ischemia and reperfusion in vitro and TNF was measured by immunoassay. Human aortic VSMCs were isolated and cultured. Proliferation assays were performed to determine the effect of TNF and IL-10 on VSMC growth. RESULTS: Ischemia-reperfusion resulted in an increase in atrial myocellular TNF (94.5 +/- 15.8 pg/g wet tissue versus control 12.9 +/- 4.4 pg/g wet tissue, P < 0.002). Compared with control, TNF stimulated concentration-dependent VSMC proliferation (P < 0.005). IL-10 alone did not influence VSMC growth. However, following TNF stimulation, IL-10 inhibited VSMC growth at a dose as low as 0.1 pg/ml (P < 0.005). CONCLUSIONS: Ischemia-reperfusion insult results in increased endogenous myocardial TNF accumulation. TNF stimulates VSMC growth which is abrogated by physiologically relevant levels of IL-10. This antiinflammatory cytokine may prove to be an effective therapeutic agent in regulating vessel wall remodeling following both ischemic and direct cardiovascular injury.     

The additive effects of glucose and insulin on the proliferation of infragenicular vascular smooth muscle cells

PURPOSE: Peripheral vascular disease involving the infragenicular arterial tree is common in patients with diabetes mellitus (DM). Accelerated proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of atherosclerosis. Insulin and glucose stimulate VSMC proliferation and are elevated in patients with non-insulin-dependent DM. We have previously described the mitogenic effect of insulin on VSMCs in vitro; the effects of insulin and glucose separately and in combination on the proliferation of VSMCs grown in serum-free media were studied. METHODS: Human infragenicular VSMCs isolated from diabetic patients with end-stage peripheral vascular disease undergoing below-knee amputation were used. Cells from passages 3 to 5 were grown in serum-free media with varying glucose (0.05%, 0.1%, 0.2%, 0.4%, 0.6%, and 0.8%) and insulin (no added insulin, 100 ng/mL, and 1000 ng/mL) concentrations for 6 days. RESULTS: Insulin stimulated VSMC growth at glucose concentrations more than 0.2% (0.4% glucose with no added insulin resulted in 13,073 +/- 336 cells/mL, 0.4% glucose with 100 ng/mL insulin resulted in 16,536 +/- 1175 cells/mL, 0.4% glucose with 1000 ng/mL insulin resulted in 17,500 +/- 808 cells/mL, 0.6% glucose with no added insulin resulted in 14,167 +/- 1062 cells/mL, 0.6% glucose with 100 ng/mL insulin resulted in 18,984 +/- 1265 cells/mL, 0.6% glucose with 1000 ng/mL insulin resulted in 20,450 +/- 1523 cells/mL, 0.8% glucose with no added insulin resulted in 15, 853 +/- 1650 cells/mL, 0.8% glucose with 1000 ng/mL insulin resulted in 26,302 +/- 1919 cells/mL; P <.05 compared with glucose with no added insulin). Glucose stimulated VSMC proliferation up to a concentration of 0.2% (42% and 117% higher growth at 0.1% and 0.2% glucose, respectively, compared with the baseline, P <.05), regardless of the insulin concentration in the media. The greatest growth (26,302 +/- 1919 cells/mL) occurred in the group with the highest concentration of both insulin (1000 ng/mL) and glucose (0.8% glucose; P <.05). CONCLUSION: Both insulin and glucose stimulate the growth of diabetic infragenicular VSMCs. The mitogenic effects of insulin and glucose are additive and may contribute to the development of atherosclerosis in patients with DM.     

Oleic acid and angiotensin II induce a synergistic mitogenic response in vascular smooth muscle cells

Oleic acid and angiotensin II (Ang II) are elevated and may interact to accelerate vascular disease in obese hypertensive patients. We studied the effects of oleic acid and Ang II on growth responses of rat aortic smooth muscle cells (VSMCs). Oleic acid (50 micromol/L) raised thymidine incorporation by 50% at 24 hours and cell number by 55% at 6 days (P<.05). Ang II (10(-11) to 10(-6) mol/L) did not significantly increase thymidine incorporation or VSMC number. Combining Ang II and 50 micromol/L oleic acid doubled thymidine incorporation and VSMC number. Losartan, an angiotensin type 1 (AT1) receptor antagonist, blocked the synergistic interaction between Ang II and oleic acid, whereas the AT2 receptor antagonist PD 123319 did not. Protein kinase C inhibition and downregulation, as well as inhibition of extracellular signal-regulated kinase (ERK) activation by PD 98059, eliminated the rise of thymidine incorporation in response to oleic acid and the synergistic interaction with Ang II. However, the response to 10% fetal bovine serum was unaffected. An antisense oligodeoxynucleotide to ERK-1 and ERK-2 reduced ERK protein expression and activation by 83% and 75%, respectively. Antisense prevented the rise of thymidine incorporation in response to oleic acid and the synergy with Ang II. Antisense reduced but did not prevent increased thymidine incorporation in response to serum. The data indicate that oleic acid and Ang II exert a synergistic mitogenic effect in VSMCs and suggest an important role for the AT1 receptor, PKC, and ERK in this synergy. The observations raise the possibility that a synergistic mitogenic interaction between oleic acid and Ang II accelerates vascular remodeling in obese hypertensive patients.     

Mice deficient for 5-lipoxygenase, but not leukocyte-type 12-lipoxygenase, display altered immune responses during infection with Schistosoma mansoni

Periovular granuloma formation during Schistosoma mansoni infection is a complex, multifaceted immunologic response. Products of arachidonic acid metabolism have been shown to contribute to this response through studies in which general inhibitors of lipoxygenase function reduce granulomatous inflammation. To determine which lipoxygenases are important for granuloma development in schistosomiasis, wild type mice or mice deficient for 5-lipoxygenase (5-LO) or "leukocyte-type" 12-lipoxygenase (12-LO) were infected with S. mansoni and studied for responses to schistosome eggs and egg antigens. At the acute stage of infection, when granuloma formation is usually maximal, 5-LO deficient mice developed smaller granulomas around liver-deposited schistosome eggs compared with wild type or 12-LO deficient mice. 5-LO mice also displayed less antibody-mediated (5 h) and cell-mediated, delayed-type (24 h) hypersensitivity to schistosome egg antigens than did the other two infection groups. In an attempt to determine possible mechanisms for the reduced inflammatory responses, we also measured hepatic mRNA levels of cytokines that have been shown to influence granuloma size (IL-4, IL-10, and IFN-gamma). The mRNA levels for IL-10 were significantly lower in 5-LO-deficient mice, but SEA-stimulated spleen cells did not demonstrate a significant difference in IL-10 production between wild type and 5-LO mice. These data suggest that 5-LO plays a role in host responses to schistosomiasis via a mechanism that cannot be explained solely by changes in expression of these cytokines.     

Interferon gamma up-regulates a novel protein in vascular smooth muscle cells

PURPOSE: By means of the technique of messenger RNA (mRNA) differential display, we previously isolated a partial DNA clone found to be down-regulated at the polytetrafluoroethylene (PTFE) hyperplastic arterial anastomosis compared with the normal artery. The partial DNA gene sequence was found to be homologous with interferon gamma up-regulated protein (IGUP) first found in human psoriatic keratinocytes. We cloned the entire IGUP gene from human vascular smooth muscle cells (VSMCs) to determine its regulation by gamma interferon (gamma-IFN) and other cytokines in cultured human VSMCs. METHODS: By means of polymerase chain reaction, the IGUP gene was amplified from a QUICK-Clone complementary DNA human aorta kit using 5' and 3' oligonucleotide primers to the known IGUP sequence. Immunohistocytochemistry studies compared normal artery and distal anastomotic IH. Human VSMCs were stimulated with 1000 U/mL of gamma-IFN, 5 ng/mL of platelet-derived growth factor BB (PDGF-BB), 3. 2 ng/mL basic fibroblast growth factor, 3.3 ng/mL transforming growth factor beta(TGF-beta), 10 ng/mL of vascular endothelial growth factor, and 10% fetal bovine serum (FBS) for zero, 24, 48 and 72 hours. Western blot analysis of lysates of the stimulated VSMCs was performed to determine up-regulation of IGUP. RESULTS: DNA sequencing confirmed the cloning of the entire coding region of the IGUP gene with 100% homology to the known IGUP DNA sequence. There was strong expression of IGUP in quiescent VSMCs and marked reduction of expression of IGUP in proliferating smooth muscle cells. gamma-IFN was the only cytokine, of the cytokines evaluated, to up-regulate production of IGUP in VSMCs. CONCLUSION: IGUP is a novel protein in VSMCs found to be down-regulated in areas of anastomotic IH, as compared with a normal artery. We have now shown IGUP to be up-regulated only by gamma-IFN in human VSMCs. IGUP may, therefore, be the intermediary for the known gamma-IFN inhibition of human VSMC proliferation.     

Production and secretion of adrenomedullin from vascular smooth muscle cells: augmented production by tumor necrosis factor-alpha

In this study, we demonstrate production and secretion of adrenomedullin (AM) from cultured vascular smooth muscle cells (VSMCs). In addition to endothelial cells (ECs), we found immunoreactive (ir-) AM in culture media of rat and bovine VSMCs in the survey for AM-producing cells. Although the secretion level of ir-AM was at most 1/6 that of rat ECs, all the examined VSMCs were shown to produce AM. By gel filtration, reverse phase high-performance liquid chromatography, and biological characterization, ir-AM in the culture medium was identified to be rat AM of 50 residues. By RNA blot analysis, a positive band of AM mRNA was detected in cultured VSMC at an intensity 3-4 fold higher than that in adrenal gland. Gene expression and production of AM were markedly augmented by tumor necrosis factor-alpha. Based on these data as well as the presence of AM specific receptors on VSMCs, AM secreted from VSMC is deduced to function as an autocrine or paracrine regulator in vascular cell communication.     

Highly selective water and fat imaging applying multislice sequences without sensitivity to B1 field inhomogeneities

1. Homocysteine is an independent risk factor for cardiovascular disease. The mechanisms by which elevated plasma concentrations of homocysteine are related to the pathogenesis of atherosclerosis are not fully understood. Therefore, we examined the effect of homocysteine on cell replication of rat cultured vascular smooth muscle cells (VSMCs) at concentrations similar to those observed in clinical studies. 2. The incorporation of [3H]-thymidine was used as a marker of mitosis. Homocysteine (250-500 microM) was a weak mitogen as compared to platelet-derived growth factor-BB (PDGF-BB, 1 nM) and serum (10%), but it potentiated the mitogenic effect of PDGF-BB four fold at 500 microM. This enhancement of mitogenesis was blunted by the addition of the scavenging enzyme catalase or the antioxidant N-acetyl-L-cysteine. 3. Furthermore, stimulation of VSMC with homocysteine (25-500 microM) decreased the glutathione peroxidase activity of the cells to 50% of control at 500 microM. Inversely, homocysteine enhanced the superoxide dismutase (SOD) activity to 137% of control at 500 microM, but it had no effect on the catalase activity. 4. Homocysteine decreased the activity of bovine purified liver cytosolic glutathione peroxidase in a time- and dose-dependent manner. The maximum decrease was 50%. 5. In summary, homocysteine has a weak mitogenic effect on VSMC, but it dramatically enhances the mitogenic response of PDGF-BB, presumably by disturbing the activity of antioxidant enzymes.     

Effects of lipoxygenase products of arachidonate metabolism on parathyroid hormone secretion

High extracellular Ca2+ (Ca2+ ec) stimulates the formation of inositol phosphates and diacylglycerol and activates phospholipase A2 in porcine parathyroid cells. Ca2+ ec action is also coupled to the formation of arachidonic acid, the precursor of both the cyclooxygenase and lipoxygenase (LO) pathways. We previously reported that LO pathway products might act as second messengers and play a part in regulating PTH secretion by Ca2+ ec. We have now investigated the effects of hydroxyeicosatetranoic acids (HETEs) on PTH secretion. Collagenase-dispersed porcine parathyroid cells were incubated in low [Ca2+] (0.5 mM, maximal stimulation) with or without HETEs for three 15-min periods. 12- and 15-HETEs inhibited PTH secretion in a dose-dependent manner from 10(-12) to 10(-9) M. Maximal inhibition was with 10(-9) M. Since 12- and 15-HETEs are the metabolic reduction products of 12- and 15-HPETEs, we also examined the effect of those precursors on PTH release. 12- and 15-hydroxyperoxyeicosatetranoic acids (HPETEs) were more potent inhibitors of PTH secretion. The threshold concentrations of both HPETEs that inhibited PTH release were lower than those for HETEs: 10(-9) M suppressed PTH secretion. This effect is comparable to that of high [Ca2+] (2 mM). This provides new evidence that products of 12-LO and 15-LO pathways are potent inhibitors of PTH secretion.     

Cigarette smoke extract is a modulator of mitogenic action in vascular smooth muscle cells

Cigarette smoking is associated with an increased incidence of atherosclerotic disease. In this study, we examined the mechanism underlying the growth-modulating effects of cigarette smoke extract (CSE) in confluent vascular smooth muscle cells (VSMCs). The treatment of VSMC by CSE decreased the activities of superoxide dismutase (SOD), catalase and glutathione peroxidase of VSMC in a time-dependent manner. In mitogenesis assays using the confluent cells, CSE was not a direct mitogen for VSMC, but potentiated the stimulatory effect of hydrogen peroxides. The reduction of activities of catalase and glutathione peroxidase was partially prevented by SH-containing compounds. In summary, CSE enhanced the mitogenic effect response of hydrogen peroxides, largely depending on the dysregulation of the activities of SOD, catalase and glutathione peroxidase by CSE.     

Differential expression and biological effects of insulin-like growth factor-binding protein-4 and -5 in vascular smooth muscle cells

Insulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis. The bioactivity of IGF-I is modulated by a group of high affinity, specific binding proteins (IGF-binding proteins; IGFBPs) that are present in the interstitial fluid. Previously, we have reported that porcine VSMCs synthesize and secrete IGF-I and several forms of IGFBPs, including IGFBP-2, IGFBP-4, and IGFBP-5. In this study, we examined the role of autocrine/paracrine secreted IGF-I in controlling the expression of IGFBP-4 and IGFBP-5 as well as the effects of these IGFBPs in modulating the cellular replication response to IGF-I. The concentrations of IGFBP-4 in the conditioned medium increased significantly from <50 ng/ml to 742 +/- 105 ng/ml. This increase was associated with a decrease in the activity of an IGF-I-regulated IGFBP-4 protease. In contrast, the synthesis of IGFBP-5 was inversely correlated with culture density, and its concentration decreased from 792 +/- 91 to 44 +/- 14 ng/ml. IGFBP-5 mRNA in sparse cultures was 3-fold higher compared with those in confluent cultures. This culture density-dependent change in IGFBP-5 mRNA correlated closely with endogenous IGF-I levels. Since treatment of VSMC with exogenous IGF-I increased IGFBP-5 mRNA levels, we neutralized the effect of endogenously secreted IGF-I with an anti-IGF-I antibody to determine if it would alter IGFBP-5 mRNA abundance. This resulted in a 4.4-fold decrease in IGFBP-5 mRNA levels. When added together with IGF-I, exogenous IGFBP-4 inhibited IGF-I-induced DNA synthesis in a concentration-dependent manner. IGFBP-5, on the other hand, potentiated the effect of IGF-I. Therefore, IGFBP-4 and IGFBP-5 appear to be differentially regulated by autocrine/paracrine IGF-I through distinct mechanisms. These two proteins, in turn, play opposing roles in modulating IGF-I action in stimulating VSMC proliferation.     

Transforming growth factor-beta and receptor tyrosine kinase-activating growth factors negatively regulate collagen genes in smooth muscle of hypertensive rats

Previous studies have suggested that differences in vascular smooth muscle cell (VSMC) proliferative responses between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats can be attributed to transforming growth factor-beta (TGF-beta) actions. Because vascular collagen content is reported to be lower in SHR than in WKY rats, in this study we investigated in cell culture whether the differences in collagen content might also be attributed to differential actions of TGF-beta on VSMCs from the two strains. Exposure of VSMCs from WKY to the TGF-beta isoforms -beta1, -beta2, or -beta3 induced rapid, transient elevations in mRNAs encoding collagens alpha1(I), alpha2(I), and alpha1(III); maximum increases were apparent by 2 hours and ranged from twofold [collagen alpha1(III)] to ninefold [collagen alpha1(I)]. Thereafter they returned to near basal levels. When VSMCs from SHR were exposed to these TGF-beta isoforms, only reductions in collagen mRNA levels were observed, persisting for 24 hours. Basic fibroblast growth factor and epidermal growth factor, factors known to stimulate production of the TGF-beta1 isoform in VSMCs, also induced a pattern of gene responses similar to those induced by the TGF-beta isoforms in VSMCs from SHR and WKY rats. The simultaneous presence of TGF-beta did not affect the time course or magnitude of the changes in collagens alpha1(I), alpha2(I), or alpha1(III) mRNA levels in SHR or WKY VSMCs. Examination of the induction of c-myc mRNA and immunoreactive oncoprotein content indicated that c-myc is a likely contributor to the downregulation of the collagen gene activity in both SHR and WKY VSMCs despite the differential regulation of its mRNA by TGF-beta1 in the two VSMC lines. Together these data suggest that in VSMCs from SHR, a number of gene responses to TGF-beta, in addition to cell proliferation, appear to be abnormal compared with WKY rats, and the lower than normal collagen levels observed in the vasculature of SHR may be in part due to abnormalities in TGF-beta responsiveness.     

Mechanism of catecholamine-induced proliferation of vascular smooth muscle cells

BACKGROUND: Catecholamines have been shown to aggravate atherosclerosis in animals and humans, and abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the early stage of atherosclerosis. Catecholamines may be involved in such cell growth. Therefore, a series of experiments using cultured VSMC was performed to elucidate their possible mitogenic effect. METHODS AND RESULTS: We examined the mitogenic effect of catecholamines using rat aortic smooth muscle cells (VSMC) by measuring [3H]thymidine incorporation, checking with flow cytometry, and counting the cell number directly. Furthermore, the catecholamine-activated signal transduction pathway was assessed by measurement of the formation of inositol 1, 4, 5-triphosphate, intracellular Ca2+ concentration, mitogen-activated protein kinase (MAPK) activity, and mitogenic gene expression. Norepinephrine (NE) and phenylephrine stimulated [3H]thymidine incorporation and cell growth. Clonidine and isoproterenol showed little of such effects. Prazosin was more effective than either yohimbine or propranolol in suppressing the mitogenic effect of NE, indicating that catecholamine-induced VSMC proliferation is mediated by alpha 1-adrenoceptors. The alpha 1-adrenoceptor activation was coupled to pertussis toxin-insensitive Gq-protein and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C and MAPK in VSMC. In response to NE, both 42- and 44-kD MAPK were activated and tyrosine was phosphorylated. alpha 1-Adrenoceptor stimulation with NE also caused accumulation of c-fos, c-jun, and c-myc mRNA. Chloroethylclonidine completely blocked the alpha 1-adrenoceptor-mediated mitogenesis. CONCLUSIONS: The effect of catecholamines appears to be mediated via the activation of the chloroethylclonidine-sensitive alpha 1-adrenoceptors that triggers the phosphoinositide hydrolysis and activates the MAPK pathway, leading to DNA synthesis and cell proliferation.