Biomedical subjects

P Zahradka

Publications and source records attributed to P Zahradka.

At least 19 recordsLinked to original sources

PubMed2001

A novel organ culture method to study intimal hyperplasia at the site of a coronary artery bypass anastomosis.

BACKGROUND: Intimal hyperplasia or restenosis at the site of a coronary artery bypass anastomosis contributes to early graft failure, and growth factor release in response to construction of the anastomotic site strongly influences this process. Due to the difficulties in studying restenosis after coronary artery bypass graft surgery, we have tested whether an organ culture model we have developed can simulate the early events associated with intimal hyperplasia. METHODS: End-to-side anastomosis of porcine radial artery to porcine coronary artery were constructed. The vessels were trimmed and incubated under standard tissue culture conditions for 14 days. Appropriate controls were treated similarly. The vessels were frozen, cryosectioned, and immunostained for the expression of the proliferation marker proliferating cell nuclear antigen (PCNA). A proliferative index (PCNA positive nuclei/total nuclei) was calculated for comparative purposes. RESULTS: Limited PCNA staining was observed in noncultured vessel segments (0.046+/-0.045). A slight increase in this index was observed in vessels that had been placed into culture without manipulation (0.230+/-0.141) and in vessels subjected to an arteriotomy (0.462+/-0.249). However, the most significant increase was obtained after construction of an anastomosis (4.98+/-6.66). No change in total cell number was evident over the course of the experiment or in relation to the treatment. CONCLUSIONS: Culture conditions and incision slightly stimulate cell proliferation in porcine coronary artery segments when compared with basal conditions of a native artery. In contrast, construction of an anastomosis increases proliferation 108-fold. Therefore, surgical manipulation of arterial conduits during construction of an anastomotic site is the primary trigger for intimal hyperplasia, independent of dissection and incision of the vessel. Furthermore, these data indicate the organ culture model we have developed will be useful for examining the cellular and molecular mechanisms that mediate intimal hyperplasia at the site of a coronary artery bypass graft anastomosis.

Anastomosis, Surgical↗

PubMed2001

Sarpogrelate inhibits serotonin-induced proliferation of porcine coronary artery smooth muscle cells: implications for long-term graft patency.

BACKGROUND: Serotonin can induce proliferation of vascular smooth muscle cells. We assessed the ability of a specific serotonin receptor antagonist, sarpogrelate, to inhibit proliferation of cultured porcine coronary artery smooth muscle cells. METHODS: Cell proliferation and mitotic activity were measured using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide. To determine the effect of sarpogrelate on DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and protein synthesis, radioactive incorporation of 3H-thymidine, 3H-uridine, and 3H-phenylalanine, respectively, was used. Synthesis of DNA was also assessed by flow cytometry with propidium iodide as a fluorochrome. RESULTS: Serotonin, platelet-derived growth factor, endothelin, and angiotensin II all induced proliferation of porcine coronary artery smooth muscle cells. Sarpogrelate specifically inhibited the serotonin-induced cytokine trigger but did not influence platelet-derived growth factor-, endothelin-, or angiotensin II-induced cell proliferation. Sarpogrelate inhibited the serotonin-induced increase in intracellular free ionized calcium concentration, prevented mitogen-activated protein kinase activation, and down-regulated expression of the protooncogenes c-fos and c-jun. Sarpogrelate acted at the G1 phase of the cell cycle. CONCLUSIONS: These data suggest that sarpogrelate could be used as a therapeutic agent to inhibit serotonin-induced neointimal hyperplasia and improve patency of coronary artery bypass grafts.

Animals↗

PubMed2001

Bradykinin receptor antagonists attenuate neointimal proliferation postangioplasty.

Bradykinin has been linked to the development of restenosis in response to vascular injury. We therefore examined the effect of bradykinin on vascular smooth muscle cell growth and neointimal formation in organ culture. Bradykinin stimulated both RNA and DNA synthesis (by 175%) in smooth muscle cells from either porcine or human coronary arteries and increased cell number in a concentration-dependent manner. Both p42/44 mitogen-activated protein kinase (MAPK) and p38 kinase were also activated. Treatment with [Hyp(3),Tyr(Me)(8)]bradykinin, a B(2) receptor agonist, stimulated thymidine incorporation by 146%, whereas B(1)-selective Lys-des-Arg(9)-bradykinin had no effect. Addition of the B(2) antagonist HOE-140 reduced the stimulation by 56%, whereas B(1)-selective des-Arg-HOE-140 had no significant effect. Similarly, HOE-140 attenuated angioplasty-induced neointimal formation in organ culture with an efficacy approaching 100% inhibition. These experiments suggest that bradykinin promotes smooth muscle proliferation after vascular injury, presumably via B(2) receptor-dependent activation of MAPK family pathways, and may explain the negative outcome of angiotensin converting enzyme inhibitor therapy on restenosis in nonrodent models.

Angioplasty↗

PubMed1999

Angiotensin II receptor antagonists prevent neointimal proliferation in a porcine coronary artery organ culture model.

OBJECTIVES: Angiotensin II (AngII) generation in response to vascular injury has long been assumed to influence neointimal proliferation contributing to restenosis. This concept has been supported by evidence that ACE inhibition and AT1 receptor blockade effectively limits restenosis in the rat. On the other hand, ACE inhibition has proven ineffective in clinical trails. The present study examines the response of the porcine coronary artery after angioplasty in vitro and compares the actions of an ACE inhibitor to AngII receptor antagonists. METHODS AND RESULTS: Captopril, an ACE inhibitor, and the AngII receptor antagonists, losartan and PD123319, were evaluated for their ability to attenuate neointimal proliferation in a porcine organ culture model of coronary restenosis. The neointima was significantly increased by 300% after angioplasty compared to non-angioplasty controls. The AT1 receptor antagonist, losartan, produced a significant reduction in neointimal index at 10(-5) mol/l, while its in vivo metabolite, EXP3174, reduced neointimal proliferation at 10(-6) mol/l. PD123319, a selective antagonist of the AT2 receptor, also restricted neointimal proliferation at 10(-5) mol/l. Treatment with captopril (10(-6) mol/l) increased the neointimal proliferation by approximately 200% after angioplasty. CONCLUSIONS: Direct blockade of AngII receptors effectively inhibits cell proliferation and restenosis post-angioplasty in vitro. ACE inhibition, exclusive of flow, does not attenuate proliferative restenosis. These data suggest that AngII contributes to neointimal proliferation and validates the concept that receptor antagonists could contribute to the therapeutic management of restenosis.

Analysis of Variance↗

PubMed1999

Insulin-like growth factor-I (IGF-I)-dependent activation of pp42/44 mitogen-activated protein kinase occurs independently of IGF-I receptor kinase activation and IRS-1 tyrosine phosphorylation.

The proliferation and metabolism of H4IIE hepatoma cells is apparently mediated through the insulin receptor. These cells, however, also have high-affinity binding sites for insulin-like growth factor-I (IGF-I). Addition of insulin to H4IIE cells increased RNA synthesis, DNA synthesis and cell number. IGF-I, on the other hand, was ineffective at concentrations equivalent to the lowest effective insulin dose, although stimulation was observed with concentrations 100-fold higher. Similar results were obtained when glucose uptake was measured. Western blot analysis demonstrated that tyrosine phosphorylation patterns produced by insulin and IGF-I differed. In particular, phosphorylation of insulin receptor substrate-1 (IRS-1) was evident after treatment with insulin, but not after treatment with IGF-I. Correspondingly, insulin, but not IGF-I, stimulated receptor tyrosine kinase activity. In contrast with these results, both insulin and IGF-I induced mitogen-activated protein (MAP) kinase phosphorylation and activity at a concentration of 10 nM. The correlation between insulin-dependent and IGF-I-dependent MAP kinase activation was confirmed by Western blot analysis of phosphorylated MAP kinase kinase (MEK). These results suggest that phosphorylation of IRS-1 is essential for both cell proliferation and glucose metabolism, but is uncoupled from the MAP kinase cascade. Furthermore, stimulation of MEK and MAP kinase is independent of receptor tyrosine kinase activity.

Animals↗

PubMed1999

Inhibition of serotonin-induced vascular smooth muscle cell proliferation by sarpogrelate.

Antiproliferative behavior of sarpogrelate (Anplag, MCI-9042, (+/-)-1-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]-3-(dimethylamino)-2-pro pyl hydrogen succinate hydrochloride), a serotonin 2A (5-HT2A) receptor antagonist, was established using radioactive incorporation of [(3)H]thymidine, [(3)H]uridine, and [(3)H]phenylalanine in cultured rat aortic smooth muscle cells in response to a 5-HT-induced cytokine trigger. Fluorescence-activated cell sorting was used to confirm these observations. 5-HT-induced DNA, RNA, and protein synthesis were inhibited maximally at a concentration of 1 microM sarpogrelate. Although other cytokines such as platelet-derived growth factor and endothelin also induced DNA, RNA, and protein synthesis in rat aortic smooth muscle cells, cell proliferation was not influenced by sarpogrelate, even at large pharmacological concentrations (10 microM). Sarpogrelate's antiproliferative actions were found to be more potent than ketanserin. These data indicate that sarpogrelate operates as a specific inhibitor of 5-HT-mediated cell proliferation and is a good candidate for preventing serotonin-induced neointimal hyperplasia.

Animals↗

PubMed1998

Modulation of the vascular smooth muscle angiotensin subtype 2 (AT2) receptor by angiotensin II.

The angiotensin subtype 2 (AT2) receptor is scarce in most adult vascular tissues except after injury. Since angiotensin II (AngII) is released upon injury, we examined the possibility that AngII governs AT2 receptor expression in smooth muscle cells (SMC). A polyclonal antiserum, raised to a peptide corresponding to the AT2 receptor C-terminus, recognized a approximately 45-kDa protein after transfection of cos-7 cells with AT2 receptor cDNA. Detection of a approximately 65-kDa band in extracts of SMC indicated that the AT2 receptor was glycosylated. Treatment of SMCs with AngII increased AT2 receptor levels fourfold over 24 h. This response was abrogated by losartan, but not by PD123319, indicating AT1 receptor involvement. AngII-dependent increases in AT2 receptor levels were also prevented by LY294002, an inhibitor of phosphatidyinositol 3-kinase, but not by rapamycin. These results indicate AngII influences AT2 receptor expression through the AT1 receptor via a signaling pathway that includes PI3K.

Angiotensin II↗

PubMed1998

Expression and regulation of the insulin-like growth factor-1 receptor by growing and quiescent H4IIE hepatoma.

Recent evidence that insulin-like growth factor-1 (IGF-1) influences certain properties of H4IIE hepatoma cells independent of insulin led us to examine whether H4IIE cells express IGF-1 receptors. Competitive binding experiments demonstrated IGF-1, but not insulin or IGF-II, could compete with [125I]IGF-1. Chemical crosslinking detected a protein with an apparent mass of 175 kDa and its identity as the IGF-1 receptor alpha-subunit was confirmed by Western blotting. The apparent molecular mass of this protein decreased to 135 kDa following deglycosylation. Immunofluorescence microscopy verified that both insulin and IGF-1 receptors were present, although measurement of IGF-1 receptor quantity revealed they were less abundant than insulin receptors. Binding of IGF-1 was low in growing cells and higher in a quiescent cell population. Scatchard analysis confirmed that receptor density was increased in non-growing H4IIE cells while there was no apparent difference in receptor affinity. Western blot analysis and RT-PCR revealed that both protein and mRNA levels were elevated as cell growth ceased. Interestingly, addition of insulin to quiescent H4IIE cells, which stimulates cell proliferation, further increased IGF-1 receptor protein levels with a peak at 12-24 h. Distinct modes of regulating IGF-1 receptor expression are indicated.

Animals↗

PubMed1998

Ca2+ mobilization in adult rat cardiomyocytes by angiotensin type 1 and 2 receptors.

The role of angiotensin II (AngII) in the regulation of heart function under normal and pathological conditions has been well documented. Although two types of AngII receptors (AT1 and AT2 receptors) are found in equal proportions in the rat heart, most studies have focused primarily on AT1 receptor-coupled events. In this study, the contribution of both types of AngII receptors to cardiac function was evaluated by measuring intracellular calcium ([Ca2+]i) levels at ambient temperature in freshly isolated adult rat ventricular cardiomyocytes. Exposure of cardiomyocytes to AngII (0.01 to 10 microM) resulted in an immediate and sustained increase in [Ca2+]i in a concentration-dependent manner. The increase in [Ca2+]i in cardiomyocytes by AngII was blocked by either losartan or compound PD123319 (1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)- 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid), non-peptide antagonists of the AT1 and AT2 receptors, respectively. The specificity of the action of these antagonists was verified by their inability to alter the basal levels of [Ca2+]i as well as KCl- or ATP-induced increases in [Ca2+]i. AngII was also observed to initiate spontaneous beating activity in cardiomyocytes, which was prevented by both losartan and compound PD123319 in a concentration-dependent manner (0.01 to 10 microM). These data indicate that the activation of both AT1 and AT2 receptors may stimulate a signalling pathway that influences [Ca2+]i and spontaneous beating activity in cardiomyocytes.

Adenosine Triphosphate↗

PubMed1998

Repression of phosphoenolpyruvate carboxykinase gene activity by insulin is blocked by 3-aminobenzamide but not by PD128763, a selective inhibitor of poly(ADP-ribose) polymerase.

Expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced by 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase. Synthesis of PEPCK mRNA is repressed by insulin, but remains detectable in H4IIE hepatoma cells exposed simultaneously to both 3-aminobenzamide and insulin. This capability of 3-aminobenzamide to block the inhibitory actions of insulin suggests that ADP-ribosylation is required for the regulation of PEPCK gene expression by insulin. Furthermore, neither changes in chromatin condensation nor cell growth status were linked to these events. The inability of 3,4-dihydro-5-methylisoquinolinone (PD128763), a selective inhibitor of poly(ADP-ribose) polymerase, to impede insulin-dependent repression of PEPCK gene expression, however, indicated that 3-aminobenzamide does not operate by inhibiting poly(ADP-ribosyl)ation. The potential involvement of mono(ADP-ribosyl)ation, a process that is also inhibited by 3-aminobenzamide, in the regulation of PEPCK gene activity was then evaluated. Analysis of poly(ADP-ribose) polymerase activity and poly(ADP-ribosyl)ation confirmed that there were no significant changes in response to insulin, while microsomal mono(ADP-ribosyl)transferase activity was elevated approximately fourfold. An increase in protein hydroxylamine-sensitive mono(ADP-ribosyl)ation was observed following insulin treatment. The sensitivity of the mono(ADP-ribosyl)transferase activity to 3-aminobenzamide but not PD128763 makes it plausible that mono(ADP-ribosyl)ation rather than poly(ADP-ribosyl)ation contributes to the regulation of PEPCK gene expression.

ADP Ribose Transferases↗

PubMed1998

Molecular cloning of rat cardiac sarcolemmal Ca2+/Mg2+ ectoATPase (Myoglein).

Rat cardiac sarcolemmal Ca2+/Mg2+ ectoATPase (Myoglein), a membrane-bound enzyme requiring millimolar concentrations of Ca2+ or Mg2+ for maximal hydrolysis of ATP, has been purified to apparent homogeneity. Tryptic digestion and amino acid sequencing was used to design an oligonucleotide probe for screening a rat heart cDNA library; this produced a partial cDNA clone (pND2.1), and sequencing of a 400 base pair portion revealed a 100% homology to human platelet CD36. Northern blotting with pND2.1 detected a 3.1 kb transcript in rat heart but not in other tissues. Interspecies expression analysis (cardiac tissue total RNA blot probed with pND2.1) detected a approximately 2.0 kb transcript in canine, rabbit and porcine heart, whereas transcripts of a 4.1 kb, approximately 3.0 kb and 2.1 kb were observed in human cardiac tissue. A rat genomic DNA Southern blot, probed with pND2.1, indicated that there was a single copy of the gene in the rat genome. Expression of the pND2.1 cDNA in E. coli produced an 89 kDa polypeptide recognized by anti-human CD36 antibody but not by anti-rat Ca2+/Mg2+ ectoATPase antibody. It is concluded that rat cardiac Ca2+/Mg2+ ectoATPase is tightly associated with a protein highly homologous to the adhesion molecule CD36.

Adenosine Triphosphatases↗

PubMed1998

FGF-2-induced negative inotropism and cardioprotection are inhibited by chelerythrine: involvement of sarcolemmal calcium-independent protein kinase C.

Fibroblast growth factor-2 (FGF-2), administered to the isolated rat heart by perfusion and under constant pressure, is protective against ischemia-reperfusion (I-R). Here we have investigated whether FGF-2 cardioprotection: (a) is dependent on flow modulation; (b) is linked to effects on contractility; (c) is mediated by protein kinase C (PKC); and (d) is linked to PKC and/or mitogen activated protein kinase (MAPK) associated with the sarcolemma. The isolated rat heart was used as a model. Under conditions of constant flow FGF-2 induced significant improvement in recovery of contractile function during I-R. Under constant perfusion pressure, FGF-2 induced a negative inotropic effect (15% decrease in developed pressure). Chelerythrine, a specific PKC inhibitor, prevented both the FGF-2-induced negative inotropic effect before ischemia, and cardioprotection during I-R. FGF-2 induced a chelerythrine-preventable, five-fold increase in sarcolemmal calcium-independent PKC activity. It also increased the association of PKC subtypes -epsilon and -delta with sarcolemmal membranes, detected by Western blotting, as well as, for PKC delta, by immunolocalization. FGF-2 increased the association of PKC epsilon with the membrane fraction of adult cardiomyocyte in culture, confirming that it can affect PKC signaling in cardiomyocytes directly and in a manner similar to its effects in situ. Finally, FGF-2 induced increased active MAPK at sarcolemmal as well as cytosolic sites. Active sarcolemmal MAPK remained elevated when the FGF-2-induced protection was prevented by chelerythrine. In conclusion, we have provided evidence that cardioprotection by FGF-2 is independent of flow modulation. PKC activation mediates both the FGF-2-induced negative inotropic effect before ischemia and the cardioprotective effect assessed during reperfusion, suggesting a cause and effect relationship. Furthermore, FGF-2 cardioprotection is linked to targeting of sarcolemmal sites by calcium-independent PKC.

Alkaloids↗

PubMed1997

Angiotensin II activates phosphatidylinositol 3-kinase in vascular smooth muscle cells.

Phosphatidylinositol 3-kinase (PI3K) is an important component of the signal transduction systems activated by tyrosine kinase receptors. It has not been established, however, whether PI3K is also an essential mediator for G protein-coupled receptors. The potential involvement of PI3K in G protein-linked angiotensin II (Ang II)-dependent signaling was assessed in a primary cell culture system of porcine coronary artery smooth muscle cells (SMCs). Treatment of quiescent SMCs with Ang II (10(-5) to 10(-8) mol/L) resulted in a dose-dependent activation of PI3K when assayed in vivo and in vitro. The Ang II receptor antagonists losartan and PD123319 were used to establish that Ang II stimulates PI3K through the Ang II type-1 (AT1) receptor. Immunofluorescent microscopy revealed that Ang II (10(-6) mol/L) stimulated the translocation of p85, the regulatory subunit of PI3K, from the perinuclear region to distinct foci throughout the cell within 15 minutes. Western blot analysis of p85 subcellular distribution demonstrated that p85 concentrations were also increased within 15 minutes in the membrane fraction and concomitantly decreased in the cytoskeletal and nuclear fractions. These changes in PI3K location and activity were paralleled by increased tyrosine phosphorylation of p85. A potential correlation between angiotensin-mediated PI3K activation and SMC growth was found using LY294002, a specific inhibitor of PI3K, which blocked the increase in DNA and RNA synthesis as well as cellular hyperplasia generated by Ang II (10(-6) mol/L) stimulation of quiescent SMCs. These data indicate that PI3K may operate as a mediator of vascular SMC growth after stimulation with Ang II.

Angiotensin II↗

PubMed1997

Immunodetection of activated mitogen-activated protein kinase in vascular tissues.

Mitogen-activated protein kinase (MAPK) is a key modulator of cytoplasmic-nuclear signal transmission, and for this reason measurement of MAPK activity has become very popular. Monitoring of MAPK activity may be particularly relevant to the cardiovascular system where it has already been shown that the stimulation of cardiomyocytes and smooth muscle cells by stretch and by growth factors activates MAPK. Since both growth factors and mechanical stress are causal agents for certain pathologies, enhanced MAPK activity may be a good predictor of disease progression. A variety of methods have been designed to measure the activation of this enzyme including an in vitro assay coupled to either gel electrophoresis or binding to P81 paper, an activity gel assay to detect p42/44 isoforms and, more recently, monitoring MAPK phosphorylation using immunoblot detection. The validity of the latter method is based on the correlation between MAPK activity and the degree of phosphorylation. The antibodies have also been of use in the detection of MAPK translocation in cell monolayers. In this report, we discuss the advantages and disadvantages of all MAPK detection methods and demonstrate an additional application for the MAPK antibodies using an in vitro restenosis model. In addition, the utility of MAPK measurements to smooth muscle pathophysiology and vascular injury (as a predictor of injury) has been assessed.

Animals↗

PubMed1997

Coronary artery smooth muscle in culture: migration of heterogeneous cell populations from vessel wall.

A method for establishing primary cultures of smooth muscle cells (SMCs) from the porcine coronary artery without either microdissection and/or enzymatic dispersion was developed using selective migration of cells from coronary explants in vitro. This culture method relies on the heterogeneity of cell types and differences in their migration and adherence ability to separate SMC from contaminating fibroblasts or endothelial cells. The cell type was determined by immunohistochemical staining with monoclonal antibodies to SM alpha-actin, SM myosin, h-caldesmon and von Willebrand factor. The first wave of migration (1-7 days) consisted of a mixture of fibroblasts and SMCs. Only SMCs were present in the second wave of migration (7-14 days). Endothelial cells, which exhibited a lower capacity for migration and adherence, were restricted to the third wave of migration (14-21 days). Cells obtained from the second wave of migration exhibited the characteristic single-layered, aligned, hill-and-valley pattern of SMCs when confluent. Quiescence was attained 4-5 days after removal of serum, as established by [3H]-thymidine incorporation. Stimulation of the quiescent SMCs with 20% FBS resulted in a synchronous re-entry into the cell-cycle with S phase reached 15-18 h later. The SMCs prepared using this protocol thus exhibit the structural markers and capacity to undergo phenotypic modulation that are characteristic of SMCs in vivo. This approach to establishing primary cultures of SMCs offers the advantage of selecting for the subpopulation of cells capable of migration in response to injury or growth factor stimulation.

Animals↗

PubMed1996

Stimulation of protein synthesis by phosphatidic acid in rat cardiomyocytes.

Phosphatidic acid (PA) was observed to stimulate protein synthesis in adult cardiomyocytes in a time- and concentration-dependent manner. The maximal stimulation in protein synthesis (142 +/- 12% vs 100% as the control) was achieved at 10 microM PA within 60 min and was inhibited by actinomycin D (107 +/- 4% of the control) or cycloheximide (105 +/- 6% of the control). The increase in protein synthesis due to PA was attenuated or abolished by preincubation of cardiomyocytes with a tyrosine kinase inhibitor, genistein (94 +/- 9% of the control), phospholipase C inhibitors 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate or carbon-odithioic acid O-(octahydro-4,7-methanol-1H-inden-5-yl (101 +/- 6 and 95 +/- 5% of the control, respectively), protein kinase C inhibitors staurosporine or polymyxin B (109 +/- 3 and 93 +/- 3% of the control), and chelators of extracellular and intracellular free Ca2+ EGTA or BAPTA/AM (103 +/- 6 and 95 +/- 6% of the control, respectively). PA at different concentrations (0.1 to 100 microM) also caused phosphorylation of a cell surface protein of approximately 24 kDa. In addition, mitogen-activated protein kinase was stimulated by PA in a concentration-dependent manner; maximal stimulation (217 +/- 6% of the control) was seen at 10 microM PA. These data suggest that PA increases protein synthesis in adult rat cardiomyocytes and thus may play an important role in the development of cardiac hypertrophy.

Animals↗

PubMed1996

Insulin is required for angiotensin II-mediated hypertrophy of smooth muscle cells.

The prevalence of hypertension and atherosclerosis among subjects with hyperinsulinemia supports the premise of a direct metabolic link between insulin and angiotensin II at the cellular level. In the present study, the effect of insulin on the angiotensin II-induced growth of A10 smooth muscle cells (SMC) was investigated. Treatment of quiescent A10 cells with angiotensin II caused an increase in RNA synthesis, proto-oncogene c-fos mRNA levels and cell size dependent upon pretreatment with insulin. The insulin requirement was independent of its actions as a growth factor, since a pre-treatment of at least 24 h with insulin was essential for growth stimulation by angiotensin II. Using RT-PCR, insulin was shown to regulate AT2 receptor expression in both quiescent and differentiating cells. These data suggest the AT2 receptor, which mediates the growth effects of angiotensin II in A10 cells, may be the critical target for the effect of insulin.

Angiotensin II↗

PubMed1996

Inhibition of RNA synthesis by bradykinin involves both the B1 and B2 receptor subtypes.

The efficacy of angiotensin converting enzyme inhibitors in the treatment of heart disease is due in part to the accumulation of bradykinin (BK). Since BK can exert its effect by influencing cell proliferation, we chose to study the effect of BK on the growth of A10 vascular smooth muscle cells. Ligand binding studies to determine which BK receptor subtypes are present on A10 cells showed that both B1 and B2 receptors were present in approximately equal numbers. Examination of RNA synthesis demonstrated that BK inhibits uridine incorporation in a dose-dependent manner. This decrease in RNA synthesis was blocked by both B1 and B2 receptor antagonists, as well as by addition of indomethacin, a cyclooxygenase inhibitor. The latter result suggested that prostaglandins mediate the biological actions of BK. Consequently, we examined the direct effect of two prostaglandins, PGE2 and PGI2 (prostacyclin), on A10 cells. PGE2 caused a decrease in RNA synthesis, thus mimicking the effect of BK, while PGI2 did not. Therefore, the inhibition of RNA synthesis in A10 vascular smooth muscle cells by BK requires both B1 and B2 receptor subtypes and this action of BK is apparently mediated by de novo synthesis of prostaglandins.

Animals↗