Angiotensin II (ANG II)-induced, activation of phospholipase C (PLC) and Ca(2+)-dependent Cl-channels is an important signal transduction pathway for mesangial cell contraction and growth. Although ANG II receptors are traditionally though to be G protein coupled, recent evidence suggests that they may also mediate protein tyrosine phosphorylation. In cultured rat mesangial cells, 10(-7) MANG II stimulated the tyrosine phosphorylation of PLC-gamma 1 and elevation of intracellular inositol 1,4,5-trisphosphate (IP3) and Ca2+ levels; peak response occurred within 0.5 min. In cell-attached patches, ANG II stimulated the activity of Ca(2+)-dependent, 3- to 4-pS Cl-channels (number of channels x open probability) from 0.063 +/- 0.022 to 0.77 +/- 0.20. Tyrosine kinase inhibition with genistein or herbimycin A blocked all four ANG II-induced responses. We conclude the following. 1) Stimulation of inositol phosphate hydrolysis by PLC, release of IP3-dependent intracellular Ca2+ stores, and activation of Ca(2+)-dependent C1-channels by ANG II are dependent on the tyrosine phosphorylation of PLC-gamma 1.2) This ANG II-induced signal transduction cascade provides a possible mechanism for both the contractile and growth-stimulating effects of ANG II on glomerular mesangial cells.
1. Angiotensin II (AngII)-induced, activation of phospholipase C (PLC) and Ca2+-dependent Cl- channels is an important signal transduction pathway for the regulation of vascular smooth muscle cell (VSMC) and glomerular mesangial cell contraction and growth. While AT receptors are traditionally thought to be G-protein coupled to the beta isoform of PLC, recent evidence suggests that in some tissues AT receptors may also activate the PLC-gamma isoform via tyrosine phosphorylation. 2. By western analysis, we identified PLC-gamma1 in the above cell types. We found that within 3 min of exposure to 10(-7) mol/L AngII, tyrosine phosphorylation of PLC-gamma1 was observed; however, peak response (>3-fold increase) occurred within 0.5 min. In addition, pre-incubation of these cells with the tyrosine kinase inhibitor genistein blocked the tyrosine phosphorylation of PLC-gamma1 by AngII. In contrast, preincubation with the tyrosine phosphatase inhibitor sodium vanadate increased the levels of tyrosine phosphorylation of PLC-gamma1. Similar results were found when intracellular levels of 1,4,5-IP3 were measured after AngII exposure. 3. By using patch clamp techniques on cultured rat mesangial cells exposed to AngII, we found that the release of 1,4,5-IP3-sensitive intracellular Ca2+ stores stimulated low conductance Cl- channels. Preincubation with genistein, abolished the usual 10-fold increase in Cl- channel activity observed with AngII. 4. Therefore, we conclude that in VSMC and glomerular mesangial cells (i) AngII transiently stimulates PLC activity via tyrosine phosphorylation of the gamma1 isoenzyme, (ii) tyrosine phosphorylation of PLC-gamma1 and production of 1,4,5-IP3 in response to AngII is dramatically inhibited by tyrosine kinase inhibition and stimulated by tyrosine phosphatase inhibition, (iii) activation of Ca2+-dependent Cl- channels by AngII-induced release of 1,4,5-IP3-dependent intracellular Ca2+ stores is also abolished by tyrosine kinase inhibition. In summary, this AngII-induced signal transduction cascade provides a possible mechanism for both the contractile and growth-stimulating effects of AngII on VSMC and glomerular mesangial cells.
In vascular smooth muscle cells, the induction of early growth response genes involves the Janus kinase (JAK)/signal transducer and activators of transcription (STAT) and the Ras/Raf-1/mitogen-activated protein kinase cascades. In the present study, we found that electroporation of antibodies against MEK1 or ERK1 abolished vascular smooth muscle cell proliferation in response to either platelet-derived growth factor or angiotensin II. However, anti-STAT1 or -STAT3 antibody electroporation abolished proliferative responses only to angiotensin II and not to platelet-derived growth factor. AG-490, a specific inhibitor of the JAK2 tyrosine kinase, prevented proliferation of vascular smooth muscle cells, complex formation between JAK2 and Raf-1, the tyrosine phosphorylation of Raf-1, and the activation of ERK1 in response to either angiotensin II or platelet-derived growth factor. However, AG-490 had no effect on angiotensin II- or platelet-derived growth factor-induced Ras/Raf-1 complex formation. Our results indicate that: 1) STAT proteins play an essential role in angiotensin II-induced vascular smooth muscle cell proliferation, 2) JAK2 plays an essential role in the tyrosine phosphorylation of Raf-1, and 3) convergent mitogenic signaling cascades involving the cytosolic kinases JAK2, MEK1, and ERK1 mediate vascular smooth muscle cell proliferation in response to both growth factor and G protein-coupled receptors. In vascular smooth muscle cells, the induction of early growth response genes involves the Janus kinase (JAK)/signal transducer and activators of transcription (STAT) and the Ras/Raf-1/mitogen-activated protein kinase cascades. In the present study, we found that electroporation of antibodies against MEK1 or ERK1 abolished vascular smooth muscle cell proliferation in response to either platelet-derived growth factor or angiotensin II. However, anti-STAT1 or -STAT3 antibody electroporation abolished proliferative responses only to angiotensin II and not to platelet-derived growth factor. AG-490, a specific inhibitor of the JAK2 tyrosine kinase, prevented proliferation of vascular smooth muscle cells, complex formation between JAK2 and Raf-1, the tyrosine phosphorylation of Raf-1, and the activation of ERK1 in response to either angiotensin II or platelet-derived growth factor. However, AG-490 had no effect on angiotensin II- or platelet-derived growth factor-induced Ras/Raf-1 complex formation. Our results indicate that: 1) STAT proteins play an essential role in angiotensin II-induced vascular smooth muscle cell proliferation, 2) JAK2 plays an essential role in the tyrosine phosphorylation of Raf-1, and 3) convergent mitogenic signaling cascades involving the cytosolic kinases JAK2, MEK1, and ERK1 mediate vascular smooth muscle cell proliferation in response to both growth factor and G protein-coupled receptors. Previous work by our laboratory (1Marrero M.B. Schieffer B. Paxton W.G. Schieffer E. Bernstein K.E. J. Biol. Chem. 1995; 270: 15734-15738Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 2Marrero M.B. Paxton W.G. Duff J.L. Berk B.C. Bernstein K.E. J. Biol. Chem. 1994; 269: 10935-10939Abstract Full Text PDF PubMed Google Scholar, 3Marrero M.B. Schieffer B. Ma H. Bernstein K.E. Ling B.N. Am. J. Physiol. 1996; 270: C1834-C1842Crossref PubMed Google Scholar, 4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar, 5Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 6Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (651) Google Scholar) on cultured rat aortic vascular smooth muscle cells (VSMC) 1The abbreviations used are: VSMC, vascular smooth muscle cell(s); Ang II, angiotensin II; PDGF, platelet-derived growth factor; MAPK, mitogen-activated protein kinase; JAK, Janus kinase; STAT, signal transducer and activator of transcription; Temed,N,N,N′,N′-tetramethylethylenediamine; DMEM, Dulbecco's modified Eagle's medium; MTS, 3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt. and phenotypically similar glomerular mesangial cells has shown that protein tyrosine phosphorylation plays a critical role in angiotensin II (Ang II)-mediated intracellular signaling cascades. This is true despite the fact that G protein-coupled receptors in general and the Ang II AT1 receptor in particular possess no intrinsic tyrosine kinase activity. It is also now recognized that Ang II can act not only as a vasoactive peptide but also as a growth factor. In particular, Ang II has been shown to stimulate proliferative and hypertrophic growth in VSMC, glomerular mesangial cells, cardiac fibroblasts, and myocytes via AT1 receptor binding (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar, 7Gibbons G.H. Pratt R. Dzau V.J. J. Clin. Invest. 1992; 90: 456-461Crossref PubMed Scopus (631) Google Scholar, 8Itoh H. Mukoyama M. Pratt R.E. Dzau V.J. J. Clin. Invest. 1993; 91: 2268-2274Crossref PubMed Scopus (462) Google Scholar, 9Naftilan A.J. Pratt R.E. Dzau V.J. J. Clin. Invest. 1989; 83: 1419-1424Crossref PubMed Scopus (591) Google Scholar). Like classic growth factors (e.g. platelet-derived growth factor (PDGF) and epidermal growth factor) and some cytokines (e.g. interferons and interleukins) (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar, 8Itoh H. Mukoyama M. Pratt R.E. Dzau V.J. J. Clin. Invest. 1993; 91: 2268-2274Crossref PubMed Scopus (462) Google Scholar, 9Naftilan A.J. Pratt R.E. Dzau V.J. J. Clin. Invest. 1989; 83: 1419-1424Crossref PubMed Scopus (591) Google Scholar, 10Marrero M.B. Bernstein K.E. Schieffer B. J. Mol. Med. 1996; 74: 85-91Crossref PubMed Scopus (38) Google Scholar), Ang II is also capable of stimulating a rapid increase in the mRNA levels of c-fos, an early growth response gene implicated in VSMC proliferation (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar, 7Gibbons G.H. Pratt R. Dzau V.J. J. Clin. Invest. 1992; 90: 456-461Crossref PubMed Scopus (631) Google Scholar, 8Itoh H. Mukoyama M. Pratt R.E. Dzau V.J. J. Clin. Invest. 1993; 91: 2268-2274Crossref PubMed Scopus (462) Google Scholar). However, the Ang II-stimulated intracellular signaling cascades responsible for c-fos induction and therefore proliferation in VSMC have not been well defined. One candidate mitogenic signaling cascade involves the activation of the small GTP-binding protein, Ras, which is traditionally mediated via classic growth factor receptors (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar). Ras activation promotes the formation of a membrane-bound complex with Raf-1 (a serine/threonine protein kinase). Subsequent tyrosine phosphorylation of Raf-1 leads to its activation and the sequential stimulation of several cytoplasmic protein kinases, collectively known as the mitogen-activated protein kinase (MAPK) pathway. This phosphorylation cascade in turn activates a set of regulatory elements leading to the stimulation of early response genes and cellular growth (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar). Our laboratory (5Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) has previously shown that as with classic growth factors, Ang II-induced protein tyrosine phosphorylation promotes the activation of p21ras in VSMC. A second mitogenic cascade that is activated by many cytokine receptors (e.g. interferons and interleukins) involves the JAK (Janus kinase) family of cytoplasmic tyrosine kinases (11Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5028) Google Scholar, 12Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1651) Google Scholar). JAK-mediated tyrosine phosphorylation of STAT (signal transducers and activators of transcription) family members promotes the translocation of these transcription factors to the nucleus, where they bind to specific DNA motifs and induce c-fos gene transcription (11Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5028) Google Scholar, 12Schindler C. Darnell J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1651) Google Scholar, 13Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1266) Google Scholar, 14Winston L.A. Hunter T. Curr. Biol. 1996; 6: 668-671Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In VSMC, our laboratory (6Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (651) Google Scholar) has previously shown that Ang II stimulates the tyrosine phosphorylation of JAK isoforms (JAK2 and TYK2), the tyrosine kinase activity of JAK2, and the tyrosine phosphorylation of STAT isoforms (STAT1, STAT2, and STAT3). Finally, Ang II induces the formation of a complex between JAK2 and the AT1 receptor itself. Our present study examines the role of JAK/STAT and Ras/Raf-1/MAPK signaling cascades in the cellular proliferation mediated by activation of G protein-coupled AT1 receptor and classic growth factor receptors (e.g. PDGF). Ang II plays a crucial role in the regulation of systemic arterial blood pressure, cardiovascular and renal growth, and sodium homeostasis (7Gibbons G.H. Pratt R. Dzau V.J. J. Clin. Invest. 1992; 90: 456-461Crossref PubMed Scopus (631) Google Scholar). Importantly, angiotensin-converting enzyme inhibitors have become a mainstay in the treatment of hypertension, congestive heart failure, cardiac hypertrophy, myocardial infarction, and chronic renal failure (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar). Better definition of Ang II-mediated mitogenic signaling provides the potential for additional specific therapeutic interventions. Tween 20, acrylamide, SDS,N,N′-methylenebisacrylamide, Temed, and nitrocellulose membranes were purchased from Bio-Rad Laboratories. PDGF-BB, molecular weight standards, immunoprecipitin, protein A- and G-agarose, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, trypsin, and all medium additives were obtained from Life Technologies, Inc. Anti-phosphotyrosine (PY20), -Raf-1, -Ras, -PDGF-β receptor, -JAK2, -STAT1, -STAT3, -MEK1, and -ERK1 antibodies were obtained from Santa Cruz Biotechnology, Inc. or Transduction Laboratories. AG-490 was purchased from Calbiochem. Phospho-specific MAPK antibody for detection of catalytically activated ERK1 and ERK2 was obtained from New England BioLabs, Inc. (15Wilmer W.A. Tan L.C. Dickerson J.A. Danne M. Rovin B.H. J. Biol. Chem. 1997; 272: 10877-10881Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The enhanced chemiluminescence kit was obtained from Amersham Corp. Angiotensin II, goat anti-mouse IgG, and all other chemicals were purchased from Sigma. Rat aortic VSMC were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum, 10 mg/ml streptomycin, and 100 units/ml penicillin at 37 °C in a 5% CO2enriched, humidified atmosphere as we have previously described (1Marrero M.B. Schieffer B. Paxton W.G. Schieffer E. Bernstein K.E. J. Biol. Chem. 1995; 270: 15734-15738Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 2Marrero M.B. Paxton W.G. Duff J.L. Berk B.C. Bernstein K.E. J. Biol. Chem. 1994; 269: 10935-10939Abstract Full Text PDF PubMed Google Scholar,5Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 6Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (651) Google Scholar). Cells from passages 5 and 6 were routinely subcultured 1:5 or 1:10 at 7-day intervals, and the medium was changed at 2–3-day intervals. Proliferation was measured using the Cell Titer 96™ AQueousnonradioactive cell proliferation assay (Promega, Inc., Madison, WI) (16Buttke T.M. McCubrey J.A. Owen T.C. J. Immunol. Methods. 1993; 157: 233-240Crossref PubMed Scopus (290) Google Scholar). This assay is based on the cellular conversion of the colorimetric reagent, MTS (3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt), into soluble formazan by dehydrogenase enzymes found only in metabolically active, proliferating cells. MTS in Dulbecco's phosphate-buffered saline (pH 6.0) was mixed with the electron-coupling reagent, phenazine methosulfate. The absorbance of formazan, measured at 490 nm using a 96-well enzyme-linked immunosorbent assay plate reader interfaced with a personal computer (model 3550, Bio-Rad), is directly proportional to the number of living cells in culture. To confirm the accuracy of our MTS proliferation assay, the actual increase in cell number was also directly assessed with a Coulter counter (model ZM, Coulter Corp., Hialeah, FL). VSMC were grown in a 75-mm2 flask to confluence and detached with trypsin-EDTA (0.05% trypsin, 0.53 mol/liter EDTA; Life Technologies Inc.). 20,000 cells were plated into 96-well plates and allowed to settle for 4 h in DMEM supplemented with 10% fetal bovine serum. Prior to experiments, cells were then growth-arrested in serum-deprived DMEM for 24 h (time 0). Cells were then stimulated with 10−7 mol/liter Ang II (Sigma) or 0.33 mmol/liter PDGF (Life Technologies Inc.). After timed ligand exposure, the phenazine methosulfate/MTS mix was added to each well (final volume, 20 μl/100 μl medium) and then incubated for an additional 60 min in (5% CO2 at 37 °C). A 10% SDS solution was then added to stop the reaction, and the absorbance of formazan was measured at 490 nm. VSMC were plated in 96-well plates and maintained in DMEM supplemented with 10% fetal bovine serum as described for the cell proliferation assay above. 24 and 48 h after ligand exposure, cells were pulsed with 1 mCi/ml [3H]thymidine (New England Nuclear, Boston, MA) and then harvested into trichloroacetic acid-precipitable material. Cells were washed with phosphate-buffered saline, incubated in 10% trichloroacetic acid at 4 °C, dissolved at room temperature in 1 mol/liter, and dried on filter paper. The paper was washed three times with phosphate-buffered saline, and then the samples were placed in scintillation liquid and counted on a scintillation counter (Beckman Inc., Palo Alto, CA). Data were plotted as the number of cpm/well. Each experimental data point represents duplicate wells from at least four different experiments. Cells were plated in 96-well plates and growth-arrested in serum-deprived DMEM for 24 h prior to experiments. As described previously (1Marrero M.B. Schieffer B. Paxton W.G. Schieffer E. Bernstein K.E. J. Biol. Chem. 1995; 270: 15734-15738Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 5Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), VSMC were electroporated in 96-well plates using a Multi-Coaxial electrode (model P/N 747, BTX Inc. San Diego, CA) was performed in Ca2+- and Mg2+-free Hanks' balanced salt solution (pH 7.4, 5 mmol/liter KCl, 0.3 mmol/liter KH2PO4, 138 mmol/liter NaCl, 4 mmol/liter NaHCO3, and 0.3 mmol/liter NaHPO4) containing antibodies at a final concentration of 10 mg/ml. Following electroporation, cells were incubated for an additional 30 min at 37 °C (5% CO2), washed once with serum-free DMEM, and then left in serum-free DMEM prior to the experiments. VSMC were stimulated with Ang II or PDGF for timed periods. The immunoprecipitation and Western blotting was performed as described previously (1Marrero M.B. Schieffer B. Paxton W.G. Schieffer E. Bernstein K.E. J. Biol. Chem. 1995; 270: 15734-15738Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 2Marrero M.B. Paxton W.G. Duff J.L. Berk B.C. Bernstein K.E. J. Biol. Chem. 1994; 269: 10935-10939Abstract Full Text PDF PubMed Google Scholar, 3Marrero M.B. Schieffer B. Ma H. Bernstein K.E. Ling B.N. Am. J. Physiol. 1996; 270: C1834-C1842Crossref PubMed Google Scholar, 5Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 6Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (651) Google Scholar). To immunoprecipitate proteins we used the following antibodies: anti-Raf-1 (2 μg/ml), anti-Ras (4 μg/ml), or anti-phosphotyrosine (PY20 clone, 10 μg/ml lysate). The recovered immunoprecipitated proteins were transferred to a nitrocellulose membrane and blotted with anti-JAK2, anti-Raf-1, or anti-PDGF-β receptor or phospho-specific MAPK (New England Biolabs, Inc.) antibodies. The latter antibody recognizes only the catalytically activated forms (phosphorylated on tyrosine residue 204) of p44 and p42 MAPK (ERK1 and ERK2, respectively) (15Wilmer W.A. Tan L.C. Dickerson J.A. Danne M. Rovin B.H. J. Biol. Chem. 1997; 272: 10877-10881Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Finally, proteins were visualized using a horseradish peroxidase conjugated to goat anti-mouse or donkey anti-rabbit IgG and an enhanced chemiluminescence kit. Data were reported as the means ± S.D. for at least four experiments (each in duplicate). Statistical analysis of the raw data was performed by one-way analysis of variance followed by appropriate post-hoc test (Bonferroni) for comparison between groups. Data were analyzed and plotted using SigmaStat™ and SigmaPlot™ (Jandel Scientific, San Rafael, CA). Probability < 0.05 was considered significant. Cellular proliferation, determined by the MTS assay (see “Materials and Methods”), was measured in VSMC after timed exposures to 10−7 mol/liter Ang II or 0.33 mmol/liter PDGF. Both PDGF and Ang II significantly stimulated proliferation within 12 h when compared with cells that had not been exposed to either growth factor or G protein-coupled receptor ligands (Fig.1 A). PDGF-induced proliferation exceeded Ang II-induced proliferative responses. Physiologic cell growth and differentiation mediated by the Ras/Raf-1/MAPK cascade involves the activation of the serine/tyrosine MAPK kinase, MEK1, and the serine/threonine MAPK, ERK1 (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar). Other investigators have recently demonstrated that both Ang II and PDGF are capable of activating ERK1 in VSMC (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar). To evaluate the potential role of the MAPK cascade in VSMC proliferation, antibodies against MEK1 and ERK1 were electroporated into VSMC prior to exposure to Ang II or PDGF. VSMC proliferation in response to Ang II or PDGF was abolished in the presence of anti-MEK1 or -ERK1 antibodies (Fig.1 B). In serum-free negative controls or electroporation experiments with pooled rabbit IgG or sham-absorbed anti-MEK1 or -ERK1 antibodies, no inhibition of Ang II- or PDGF-induced VSMC proliferation was observed (data not shown). In VSMC electroporated with mock antibody (anti-IgG), DNA synthesis measured as [3H]thymidine incorporation increased significantly within 24 h of Ang II or PDGF exposure (Fig.2 A). Also consistent with our proliferation results (Fig. 1 A), [3H]thymidine incorporation was greater after PDGF than after Ang II exposure. We then tested the role of MAPK cascade components in VSMC DNA-synthesis. Indeed, the electroporation of anti-MEK1 or -ERK1 antibodies abolished DNA synthesis in response to either Ang II or PDGF (Fig.2 A). These results suggested that VSMC proliferation and DNA synthesis, in response to both G protein-receptor coupled (i.e. Ang II) and growth factor (i.e. PDGF) receptor ligands, involve the MAPK cascade and are dependent on the activation of MEK1 and ERK1. Previous work by our laboratory (6Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (651) Google Scholar) has shown that the cytosolic tyrosine kinase, JAK2, plays a critical role in Ang II-mediated signaling events, including the activation of STAT proteins. In the present study, we found that Ang II-induced proliferation was virtually abolished by the electroporation of anti-STAT1 or anti-STAT3 (Fig. 1 C). In contrast, there was no statistical difference between PDGF-induced proliferative responses observed in normal VSMC (Fig. 1 A) compared with VSMC electroporated with anti-STAT1 or -STAT3 antibodies (Fig.1 C). The latter observation suggested that blockage of Ang II-induced proliferation was not simply a toxic effect of the electroporated antibodies. In electroporation experiments with sham-absorbed anti-STAT1 or -STAT3 antibodies, no inhibitory effect on Ang II-induced VSMC proliferation was observed (data not shown). Similarly, Ang II-induced [3H]thymidine incorporation was completely prevented in cells electroporated with either anti-STAT1 or -STAT3 antibodies (Fig. 2 B). However, DNA synthesis in response to PDGF was not affected by electroporation of antibodies against STAT1 and STAT3 isoforms. However, when VSMC were electroporated in the presence of a mock antibody (anti-IgG), the typical increase in [3H]thymidine incorporation was observed in response to Ang II or PDGF. Our laboratory (6Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (651) Google Scholar) has previously shown that in VSMC Ang II induces the rapid tyrosine phosphorylation of and activation of the cytoplasmic tyrosine kinase, JAK2. JAK2 activation, in turn, promotes the phosphorylation of STAT1 and STAT3 tyrosine residues. Our above results suggested that STAT1 and STAT3 are necessary and specific for VSMC proliferative responses linked to the G protein-coupled AT1 receptor but not the PDGF-β receptor. Therefore, we investigated the role of JAK2 tyrosine kinase in VSMC proliferation. We were unsuccessful in blocking Ang II- or PDGF-induced VSMC proliferation, DNA synthesis, or auto-tyrosine phosphorylation of JAK2 with the electroporation of commercially available anti-JAK2 polyclonal antibodies (data not shown). Because not all antibodies block or neutralize the biologic activities of the respective antigens, we investigated the effect of AG-490, a specific JAK2 inhibitor (17Dhar A. Shukla S.D. J. Biol. Chem. 1994; 269: 9123-9127Abstract Full Text PDF PubMed Google Scholar,18Meydan N. Grunberger T. Dadi H. Shahar M. Arpaia E. Lapidot Z. Leeder J.S. Freedman M. Cohen A. Gazit A. Levitzki A. Roifman C.M. Nature. 1996; 379: 645-648Crossref PubMed Scopus (848) Google Scholar). AG-490 belongs to the tyrphostin family of tyrosine kinase inhibitors, and these inhibitors inhibit protein tyrosine kinases by binding to the substrate binding site (19Gazit A. Osherov N. Posner I. Yaish P. Poradosu E. Gilon C. Levitzki A. J. Med. Chem. 1991; 34: 1896-1907Crossref PubMed Scopus (248) Google Scholar). Pretreatment of VSMC with 10 μm AG-490 did indeed block Ang II-induced VSMC proliferation (Fig. 3 A), DNA synthesis (Fig. 3 B), and the tyrosine phosphorylation of JAK2 (Fig. 4). AG-490 also blocked PDGF-induced VSMC proliferation (Fig. 3 A), DNA synthesis (Fig. 3 B), and JAK2 tyrosine phosphorylation (Fig. 4). We found that 16 h of pretreatment with AG-490 produced maximal inhibition of Ang II- and PDGF-induced JAK2 tyrosine phosphorylation events while still allowing recovery of VSMC proliferative responses when the AG-490 was removed from the bath. Because PDGF-induced VSMC proliferation and DNA synthesis required JAK2 activity but were unaffected by anti-STAT1 or -STAT3 antibody electroporation, we examined the possibility that JAK2-dependent proliferative responses were mediated through an alternative mitogenic pathway other than the JAK/STAT cascade.Figure 4Effects of JAK2 inhibition on the tyrosine phosphorylation of JAK2 by Ang II and PDGF. Top, VSMC lysates were immunoprecipitated with an anti-JAK2 antibody and then probed with anti-phosphotyrosine antibody. Representative bands corresponding to the molecular mass of JAK2 (135 kDa) are shown from lysates from cells with (right) or without (left) 10 μm AG-490 pretreatment for 16 h prior to timed exposures to Ang II (10−7 mol/liter; upper band) or PDGF (0.33 mmol/liter; lower band).Bottom, VSMC were exposed to serum-free DMEM only (circles) or serum-free DMEM supplemented with the specific JAK2 inhibitor, AG-490 (10 μm) (triangles), for 16 h prior to timed exposure to Ang II (10−7mol/liter) (open symbols) or PDGF (0.33 mmol/liter) (closed symbols). Bands were quantitated by densitometry using a La Cie scanner interfaced with a personal computer. Each band was scanned in two dimensions, and the density was corrected for the background present in the lane. Data represent corrected densities for each time point and are expressed as arbitrary units plotted against time of Ang II or PDGF exposure (mean ± S.E.; n = 3). Results were similar if the addition of antibodies was reversed (i.e. immunoprecipitated with anti-phosphotyrosine antibody and probed with anti-JAK2 antibody) (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Evidence from several groups suggests that JAK2 forms a membrane complex with Ras/Raf-1 and is required for Raf-1 activation in several different nonvascular mammalian cell types (20Winston L.A. Hunter T. J. Biol. Chem. 1995; 270: 30837-30840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 21Wang X.-Y. Fuhrer D.K. Marshall M.S. Yang Y.-C. J. Biol. Chem. 1995; 270: 27999-28002Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 22Xia K. Mukhopadhyay N.K. Inhorn R.C. Barber D.L. Rose P.E. Lee R.S. Narsimhan R.P. D'Andrea A.D. Griffin J.D. Roberts T.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11681-11686Crossref PubMed Scopus (74) Google Scholar). We found that JAK2 inhibition with AG-490 pretreatment blocked both Ang II- and PDGF-induced complex formation between JAK2 and Raf-1 (Fig.5) and the tyrosine phosphorylation of Raf-1 (Fig. 6). We then examined the effect of JAK2 inhibition on Ang II- and PDGF-mediated stimulation of ERK tyrosine phosphorylation. VSMC lysates were probed with a phospho-specific MAPK antibody that recognizes only the catalytically activated forms (phosphorylated on tyrosine residue 204) of p44 and p42 MAPK (ERK1 and ERK2, respectively) (15Wilmer W.A. Tan L.C. Dickerson J.A. Danne M. Rovin B.H. J. Biol. Chem. 1997; 272: 10877-10881Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). AG-490 blocked both ERK1 and ERK2 tyrosine phosphorylation in response to Ang II or PDGF (Fig.7). Finally, we examined the specificity of AG-490 for blocking tyrosine phosphorylation-dependent events known to be induced by Ang II and PDGF (2Marrero M.B. Paxton W.G. Duff J.L. Berk B.C. Bernstein K.E. J. Biol. Chem. 1994; 269: 10935-10939Abstract Full Text PDF PubMed Google Scholar, 5Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 7Gibbons G.H. Pratt R. Dzau V.J. J. Clin. Invest. 1992; 90: 456-461Crossref PubMed Scopus (631) Google Scholar, 8Itoh H. Mukoyama M. Pratt R.E. Dzau V.J. J. Clin. Invest. 1993; 91: 2268-2274Crossref PubMed Scopus (462) Google Scholar). We found that JAK2 inhibition with AG-490 did not prevent PDGF-induced tyrosine autophosphorylation of the PDGF-β receptor itself (Fig.8), nor did it prevent Ang II- or PDGF-induced Ras/Raf-1 complex formation (Fig.9) or phospholipase C-γ1 tyrosine phosphorylation (data not shown). Therefore, this specific JAK2 inhibitor does not block non-JAK cytosolic tyrosine kinases (e.g. pp60c-src) or the intrinsic tyrosine kinase activity of growth factor receptors (e.g. PDGF-β receptor). These results suggested that JAK2 plays a specific role in the tyrosine phosphorylation of Raf-1 and the activation of ERK1 and ERK2, providing a mechanism for cross-talk between two diverse mitogenic pathways, namely the JAK/STAT and MAPK cascades.Figure 6Effects of JAK2 inhibition on the tyrosine phosphorylation of Raf-1 by Ang II and PDGF. Top, VSMC lysates were immunoprecipitated with an anti-phosphotyrosine antibody and then probed with anti-Raf-1 antibody. Representative bands corresponding to the molecular mass of Raf-1 (72 kDa) are shown from lysates from cells with (right) or without (left) 10 μm AG-490 pretreatment for 16 h prior to timed exposures to Ang II (10−7 mol/liter; upper band) or PDGF (0.33 mmol/liter; lower band).Bottom, VSMC were exposed to serum-free DMEM only (circles) or serum-free DMEM supplemented with the specific JAK2 inhibitor, AG-490 (10 μm) (triangles), for 16 h prior to timed exposure to Ang II (10−7mol/liter) (open symbols) or PDGF (0.33 mmol/liter) (closed symbols). Bands were quantitated by densitometry using a La Cie scanner interfaced with a personal computer. Each band was scanned in two dimensions, and the density was corrected for the background present in the lane. Data represent corrected densities for each time point and are expressed as arbitrary units plotted against time of Ang II or PDGF exposure (mean ± S.E.; n = 3). Results were similar if the addition of antibodies was reversed (i.e. immunoprecipitated with anti-Raf-1 antibody and probed with anti-phosphotyrosine antibody) (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Effects of JAK2 inhibition on the tyrosine phosphorylation of ERK by Ang II and PDGF. Top, VSMC lysates were probed with a phospho-specific MAPK antibody (New England Biolabs, Inc.) that recognizes only the catalytically activated forms (phosphorylated on tyrosine residue 204) of p44 and p42 MAPK (ERK1 and ERK2, respectively) (15Wilmer W.A. Tan L.C. Dickerson J.A. Danne M. Rovin B.H. J. Biol. Chem. 1997; 272: 10877-10881Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Representative bands corresponding to the molecular masses of ERK1 and ERK2 are shown from lysates from cells with (lower band) or without (upper band) 10 μm AG-490 pretreatment for 16 h prior to timed exposures to Ang II (10−7 mol/liter; right) or PDGF (0.33 mmol/liter; left). Bottom, VSMC were exposed to serum-free DMEM only (circles) or serum-free DMEM supplemented with the specific JAK2 inhibitor, AG-490 (10 μm) (triangles), for 16 h prior to timed exposure to Ang II (10−7 mol/liter) (open symbols) or PDGF (0.33 mmol/liter) (closed symbols). Bands were quantitated by densitometry using a La Cie scanner interfaced with a personal computer. Each band was scanned in two dimensions, and the density was corrected for the background present in the lane. Data represent corrected densities for each time point and are expressed as arbitrary units plotted against time of Ang II or PDGF exposure (mean ± S.E.; n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Effects of JAK2 inhibition on the autotyrosine phosphorylation of the PDGF-β receptor by PDGF. Top, VSMC lysates were immunoprecipitated with an anti-phosphotyrosine antibody and then probed with anti-PDGF-β receptor antibody. Representative bands corresponding to the molecular mass of PDGF-β receptor (180 kDa) are shown from lysates from cells with (right) or without (left) 10 μm AG-490 pretreatment for 16 h prior to timed exposures to PDGF (0.33 mmol/liter). Bottom, VSMC were exposed to serum-free DMEM only (circles) or serum-free DMEM supplemented with the specific JAK2 inhibitor, AG-490 (10 μm) (triangles), for 16 h prior to timed exposure to PDGF (0.33 mmol/liter). Bands were quantitated by densitometry using a La Cie scanner interfaced with a personal computer. Each band was scanned in two dimensions, and the density was corrected for the background present in the lane. Data represent corrected densities for each time point and are expressed as arbitrary units plotted against time of Ang II or PDGF exposure (mean ± S.E.; n = 3). Results were similar if the addition of antibodies was reversed (i.e. immunoprecipitated with anti-PDGF-β receptor antibody and probed with anti-phosphotyrosine antibody) (data not shown).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Effect of JAK2 inhibition on the Ang II- and PDGF-induced Ras/Raf-1 complex formation. VSMC lysates were immunoprecipitated with an anti-Ras antibody and then probed with anti-Raf-1 antibody. Representative experiments show Ras/Raf-1 complex formation in lysates from cells with (right) or without (left) 10 μm AG-490 pretreatment for 16 h prior to timed exposures to 10−7 mol/liter Ang II (n = 3) or 0.33 mmol/liter PDGF (n = 3). The Raf-1 protein standard is shown on the far left.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Several groups have previously shown that two mitogenic cascades, the JAK/STAT and Ras/Raf-1/MAPK, are stimulated by the AT1receptor in VSMC (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar, 6Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (651) Google Scholar, 14Winston L.A. Hunter T. Curr. Biol. 1996; 6: 668-671Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 23Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1995; 270: 19059-19065Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 24Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar). Both cascades link the binding of ligands to cell surface receptors, with intracellular signaling elements that promote nuclear transcription events resulting in cellular growth (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar). Our laboratory (6Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (651) Google Scholar) has shown that Ang II stimulates the tyrosine phosphorylation and activation of JAK2 and subsequently the tyrosine phosphorylation of STAT isoforms in VSMC. Bhat et al. (23Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1995; 270: 19059-19065Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 24Bhat G.J. Thekkumkara T.J. Thomas W.G. Conrad K.M. Baker K.M. J. Biol. Chem. 1994; 269: 31443-31449Abstract Full Text PDF PubMed Google Scholar) have also demonstrated in cultured neonatal fibroblasts that Ang II induces STAT protein phosphorylation, translocation of STAT proteins into the nucleus, and initiation of early response gene transcription. Our laboratory (5Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) has shown that in VSMC Ang II stimulates the proto-oncogene, p21ras. Activated Ras then forms a membrane-bound complex with Raf-1 (a serine/threonine protein kinase), leading to the activation of Raf-1 by tyrosine phosphorylation (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar). Raf-1 then phosphorylates and activates MEK1, which in turn leads to the activation of ERK1. Because G protein-coupled receptors lack intrinsic tyrosine kinase activity, the activation of these mitogenic signaling cascades requires the recruitment of cytosolic tyrosine kinases, such as pp60c-src, MEK1, and JAK2. Previous work by our laboratory (5Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) has shown that blocking of pp60c-srcwith electroporated anti-pp60c-src antibodies prevented Ang II-induced formation of the Ras/Raf-1 membrane complex in VSMC. Our present study shows that inhibition of JAK2 with AG-490 and inhibition of MEK1 with antibody electroporation prevents VSMC proliferation and DNA synthesis in response to Ang II. Together, these observations indicate that the G protein-coupled AT1receptor stimulates major mitogenic signaling pathways in VSMC via tyrosine phosphorylation, in particular the JAK/STAT and the Ras/Raf-1/MAPK cascades. In contrast to the G protein-coupled AT1 receptor, classic growth factor receptors that possess intrinsic tyrosine kinase activity (e.g. PDGF and epidermal growth factor receptors) are thought not to require cytosolic tyrosine kinases to mediate downstream proliferative signaling events (7Gibbons G.H. Pratt R. Dzau V.J. J. Clin. Invest. 1992; 90: 456-461Crossref PubMed Scopus (631) Google Scholar, 8Itoh H. Mukoyama M. Pratt R.E. Dzau V.J. J. Clin. Invest. 1993; 91: 2268-2274Crossref PubMed Scopus (462) Google Scholar). Consistent with this premise, our laboratory (2Marrero M.B. Paxton W.G. Duff J.L. Berk B.C. Bernstein K.E. J. Biol. Chem. 1994; 269: 10935-10939Abstract Full Text PDF PubMed Google Scholar, 5Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) has previously demonstrated that inhibition of cytosolic pp60c-src tyrosine kinase activity prevents Ang II- but not PDGF-induced stimulation of phospholipase C-γ1 and Ras/Raf-1 complex formation in VSMC. In contrast, JAK2 inhibition with AG-490 does not block Ang II- or PDGF-induced Ras/Raf-1 complex formation or phospholipase C-γ1 tyrosine phosphorylation. In addition, AG-490 did not prevent PDGF-β receptor autophosphorylation in response to PDGF. However, in the present study we find that inhibition of the JAK2 tyrosine kinase virtually abolishes VSMC proliferation in response to PDGF. Our results indicate that JAK2 plays a crucial role in the VSMC proliferation mediated by both G protein-coupled (i.e. Ang II) and classic growth factor (i.e. PDGF) receptor ligands. Surprisingly, electroporation of VSMC with anti-STAT1 or -STAT3 antibodies did not prevent PDGF-induced proliferation or DNA synthesis. Therefore, the JAK2-dependent VSMC proliferation that we observe in response to PDGF is likely not mediated via the traditional JAK-induced tyrosine phosphorylation of STAT1 or STAT3 transcription factors. Several groups have shown that growth hormone-, interferon-, and interleukin-induced activation of early growth response genes (e.g. c-myc, c-fos, and c-jun), cell proliferation, Ras/JAK2/Raf-1 complex formation, and Raf-1 kinase activity are dependent on JAK2 in several nonvascular mammalian cell types (20Winston L.A. Hunter T. J. Biol. Chem. 1995; 270: 30837-30840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 21Wang X.-Y. Fuhrer D.K. Marshall M.S. Yang Y.-C. J. Biol. Chem. 1995; 270: 27999-28002Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 22Xia K. Mukhopadhyay N.K. Inhorn R.C. Barber D.L. Rose P.E. Lee R.S. Narsimhan R.P. D'Andrea A.D. Griffin J.D. Roberts T.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11681-11686Crossref PubMed Scopus (74) Google Scholar). Indeed, in the present study we find that both Ang II- and PDGF-induced JAK2/Raf-1 complex formation, Raf-1 tyrosine phosphorylation, and ERK1 and ERK2 kinase activity are dependent on JAK2 activity. Therefore, our data provide a key molecular link between the mitogenic JAK/STAT and Ras/Raf-1/MAPK cascades in VSMC. In summary, our present study emphasizes the important role played by the JAK/STAT and Ras/Raf-1/MAPK cascades in mediating VSMC proliferation in response to both G protein-coupled AT1receptors and classic growth factor receptors. We have shown that JAK2 activation by both G protein-coupled and growth factor receptors provides a convergent signaling element for these two diverse mitogenic cascades in VSMC. Our present and past studies (1Marrero M.B. Schieffer B. Paxton W.G. Schieffer E. Bernstein K.E. J. Biol. Chem. 1995; 270: 15734-15738Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 2Marrero M.B. Paxton W.G. Duff J.L. Berk B.C. Bernstein K.E. J. Biol. Chem. 1994; 269: 10935-10939Abstract Full Text PDF PubMed Google Scholar, 3Marrero M.B. Schieffer B. Ma H. Bernstein K.E. Ling B.N. Am. J. Physiol. 1996; 270: C1834-C1842Crossref PubMed Google Scholar, 4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar, 5Schieffer B. Paxton W.G. Chai Q. Marrero M.B. Bernstein K.E. J. Biol. Chem. 1996; 271: 10329-10333Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 6Marrero M.B. Schieffer B. Paxton W.G. Heerdt L. Berk B.C. Delafontaine P. Bernstein K.E. Nature. 1995; 375: 247-250Crossref PubMed Scopus (651) Google Scholar) suggest that Ang II-induced VSMC proliferation requires protein tyrosine phosphorylation via JAK2, MEK1, and pp60c-src, which in turn are necessary for the activation of several diverse mitogenic factors, specifically STAT proteins, Raf-1, ERK1, and phospholipase C-γ1. More importantly, the inhibition of these individual signaling molecules prevents VSMC proliferation. Current clinical therapeutic interventions for the prevention or regression of maladaptive cardiovascular growth (e.g. hypertension, congestive heart failure, cardiac hypertrophy, atherosclerosis, angioplasty injury) include the inhibition of G protein-coupled AT1 receptors (e.g. AT1 receptor antagonist and losartan) or their respective ligand (e.g. angiotensin-converting enzyme inhibitors) (4Bernstein K.E. Marrero M.B. Trends Cardiovasc. Med. 1996; 6: 179-187Crossref PubMed Scopus (27) Google Scholar, 7Gibbons G.H. Pratt R. Dzau V.J. J. Clin. Invest. 1992; 90: 456-461Crossref PubMed Scopus (631) Google Scholar, 8Itoh H. Mukoyama M. Pratt R.E. Dzau V.J. J. Clin. Invest. 1993; 91: 2268-2274Crossref PubMed Scopus (462) Google Scholar, 25Timmermans P.B.M. Wong P.C. Chiu A.T. Herblin W.F. Benfield P. Carini D.J. Lee R.J. Wexler R.R. Saye J.A.M. Smith R.D. Pharmacol. Rev. 1993; 45: 205-251PubMed Google Scholar). Our better understanding of the two mitogenic signaling pathways investigated in the present study presents potential new and specific targets for future therapeutic interventions in various cardiovascular diseases associated with VSMC proliferation. We thank Dr. Craig J. Ling and Cynthia A. Ling for editorial assistance.
In A6 distal nephron cells, short-circuit current (Isc) was increased by basolateral exposure to prostaglandin E2 (PGE2; peak response at 1 microM). The effect was only partially abolished by either apical amiloride, an Na+ channel blocker, or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a Cl- channel blocker. In apical cell-attached patches, we observed a 7-pS Cl- channel with a linear current-voltage relationship, a reversal potential near resting membrane potential, and open probability > 0.5. The channel was blocked by diphenylamine-2-carboxylate, glibenclamide, and NPPB but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The frequency of observed Cl- channel activity increased 7-fold with 10-min exposure to PGE2 and 3.7-fold with longer (10-50 min) exposure to PGE2. The PGE2-induced increase in Cl- channel activity was due primarily to an increase in the number of functional channels. The following conclusions were made: 1) activation of apical, 7-pS Cl- channels in A6 cells accounts for the PGE2-induced increase in the amiloride-insensitive Isc, and 2) 7-pS Cl- channel activation was mediated via an increase in channel density without substantial effects on channel kinetics.
Platelet-derived growth factor (PDGF)-induced Ca2+ signaling mechanisms were examined in cultured rat glomerular mesangial cells. PDGF-BB stimulated the tyrosine phosphorylation of phospholipase C (PLC)-gamma 1, the formation of a PLC-gamma 1/PDGF-beta receptor membrane complex, and the generation of intracellular inositol 1,4,5-trisphosphate (IP3). Preincubation with a tyrosine kinase inhibitor (genistein) abolished these PDGF-induced responses. Activation of 1-pS Ca2+ channels in cell-attached patches by intrapipette PDGF-BB was also abolished by tyrosine kinase inhibition. In the absence of PDGF-BB, channels were activated in cell-attached patches exposed to intrapipette thapsigargin (IP3-independent releaser of intracellular Ca2+ stores) and in excised inside-out patches exposed to increasing “cytoplasmic” Ca2+ (10(-8) to 10(-6) M). In cell-attached patches, channel activation by PDGF-BB was abolished when extracellular Ca2+ was < 1 mM. In glomerular mesangial cells 1) PDGF-BB stimulates tyrosine phosphorylation of PLC-gamma 1, PDGF-beta receptor/PLC-gamma 1 membrane complex formation, IP3 production, and 1-pS Ca2+ channel activity; 2) all four PDGF-induced responses are abolished by tyrosine kinase inhibition; 3) PDGF receptor-operated Ca2+ channels are sensitive to both intra- and extracellular Ca2+.
We used patch-clamp methods to investigate the effects of basolateral endothelin-1 (ET-1) on the amiloride-sensitive Na+ channel in A6 distal nephron cells. One hundred picomolar ET-1 decreased channel activity via an increase in mean time closed (P < 0.01, n = 10). Channel inhibition by pM ET-1 was mimicked by an ET-B receptor agonist (P < 0.05, n = 7) and was prevented by ET-B antagonists (P = 0.14, n = 10) but not by an ET-A antagonist (P < 0.05, n = 4). With the inhibitory ET-B receptor blocked, higher doses of ET-1 (10 nM) actually increased channel activity through an increase in mean time open (P < 0.001, n = 12). The current-voltage relationship and the number of channels were not changed by basolateral ET-1 exposure. We conclude that 1) basolateral ET-1 regulates amiloride-sensitive Na+ channels; 2) binding of picomolar ET-1 to ET-B receptors inhibits, whereas the binding of nanomolar ET-1 to a different ET receptor (likely ET-A) stimulates, channel activity; and 3) these dose-dependent, distal nephron responses provide a potential mechanism for the in vivo natriuresis and antinatriuresis observed in response to "subpressor" and "pressor" concentrations of ET-1, respectively.
To investigate the effects of luminal adenosine on amiloride-sensitive Na+ channels, we applied the cell-attached patch-clamp technique to A6 distal nephron cells. Exposure to luminal 30 nM adenosine increased number of channels x open probability (NP0) from 0.38 +/- 0.08 to 0.77 +/- 0.09 (means +/- SE; P < 0.01, n = 17). Luminal exposure to an A1-receptor antagonist (30 nM 8-cyclopentyl-1,3-dipropylxanthine) abolished (P = 0.17, n = 11), whereas an A1 agonist (30 nM N6-cyclohexyladenosine) reproduced (P < 0.02, n = 6) the stimulatory effect of 30 nM adenosine. In contrast, higher concentrations of luminal adenosine (1 or 10 microM) decreased NP0 from 0.65 +/- 0.09 to 0.24 +/- 0.10 (P < 0.02, n = 11) and from 0.80 +/- 0.11 to 0.19 +/- 0.03 (P < 0.01, n = 8), respectively. Channel inhibition by high-dose luminal adenosine was abolished by an A2 antagonist (30 microM 3,7-dimethyl-1-propargylxanthine; P = 0.2, n = 10) and mimicked by an A2 agonist (100 nM CGS-21680 hydrochloride; P < 0.0005, n = 8). We conclude that 1) purinergic regulation of distal nephron Na+ channels is mediated by stimulatory apical A1 receptors and inhibitory apical A2 receptors; 2) basal urinary adenosine concentrations (in nM) would stimulate Na+ reabsorption, whereas higher urinary concentrations (in microM), e.g., renal ischemia and elevations in filtered NaCl load, would increase Na+ excretion; and 3) urinary adenosine may be involved in feedback regulation of distal nephron Na+ transport.
We used the cell-attached patch clamp technique to investigate the interaction of exogenous prostaglandins (PG), intracellular [Ca2+]i, and protein kinase C (PKC) on the high selectivity, 4 pS Na+ channel found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) cultures grown on collagen supports with 1.5 microM aldosterone. Application of 0.5 microM PGE2 to the basolateral membrane decreased mean NP0 (number of channels times the open probability) for apical Na+ channels by 46.5% (n = 9). There was no consistent change in NP0 after apical 0.5 microM PGE2 (n = 12) or after apical or basolateral 0.5 microM PGF2 alpha (n = 8). Release of [Ca2+]i stores with 0.25 microM thapsigargin (n = 7), or activation of apical membrane PKC with apical 0.1 microM 4 beta-phorbol-12-myristate-13-acetate (n = 5) or 10 microM 1-oleyl-2-acetylglycerol (n = 4) also decreased NP0. Depletion of [Ca2+]i stores (0.25 microM thapsigargin pretreatment) (n = 7) or inhibition of apical PKC (100 microM D-sphingosine pretreatment) (n = 8) abolished the inhibitory effects of basolateral PGE2. Conclusions: (a) apical Na+ transport in rabbit CCT principal cells is modulated by basolateral PGE2; (b) the mechanism involves release of IP3-sensitive, [Ca2+]i stores; and (c) Ca(2+)-dependent activation of apical membrane PKC, which then inhibits apical Na+ channels.
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