Biomedical subjects

B C Berk

Publications and source records attributed to B C Berk.

At least 109 records · Page 6Linked to original sources

PubMed1993

The biology of angiotensin II receptors.

Angiotensin II is very important in the regulation of blood pressure. This small peptide binds to cell surface receptors, initiating a wide diversity of physiologic responses. There are two major subtypes of angiotensin II receptors referred to as AT1 and AT2. In this article we describe the cloning and the biochemical characterization of the AT1 receptor. Antibodies against this receptor have been used to define its tissue distribution. The AT1 receptor is a member of the seven transmembrane spanning class of receptors. It initiates a complex series of signaling events, including activation of membrane phospholipases and intracellular kinases. In the human a single AT1 receptor protein mediates virtually all the effects of angiotensin II, suggesting that tissue specificity of angiotensin II must be due to organ-specific intracellular signaling.

Amino Acid Sequence↗

PubMed1993

Phosphorylation of Na(+)-H+ antiporter is not stimulated by phorbol ester and acidification in granulocytic HL-60 cells.

During differentiation of HL-60 cells into granulocyte-like cells, mRNA and protein levels for the Na(+)-H+ antiporter increased 10- to 15-fold. However, functional activity, as measured by recovery from an acid load [intracellular pH (pHi) 6.5] increased by only about twofold. In addition, basal pHi (measured in the absence of bicarbonate) increased from 7.15 to 7.26, suggesting an alteration in the antiporter's "set point" during HL-60 cell differentiation. To gain insight into the role of the Na(+)-H+ antiporter in HL-60 cell differentiation, we studied mRNA expression of the NHE-1, NHE-3, and NHE-4 isoforms. Only the NHE-1 isoform mRNA increased during differentiation. Because it has recently been shown that the antiporter is regulated by phosphorylation, we next studied NHE-1 protein phosphorylation during HL-60 cell differentiation. Differentiation by exposure to 1 microM retinoic acid for 6 days caused a 15-fold increase in the synthesis of the NHE-1 protein. However, immunoprecipitation of 32P-labeled antiporter showed a decrease in band intensity. These data indicate that during HL-60 cell differentiation, there was a net decrease in the phosphorylation of NHE-1 despite an increase in pHi. Nonetheless, recovery from an acid load (pHi 6.51) was significantly more rapid in differentiated than control cells: 62 +/- 6 vs. 38 +/- 8 mmol H+.min-1.1 cells-1, respectively. However, acid loading decreased antiporter phosphorylation by twofold in differentiated and undifferentiated HL-60 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Carrier Proteins↗

PubMed1993

Fluid shear stress stimulates platelet-derived growth factor expression in endothelial cells.

Fluid flow and the associated shear stress play a critical role in vascular growth and remodeling. Recent data suggest that increased endothelial cell expression of platelet-derived growth factor (PDGF) A- and B-chain by flow may participate in these events. In the present study, we examined the mechanism for flow-induced PDGF expression, focusing on protein kinase C (PKC). Bovine aortic endothelial cells were exposed to flow (shear stress = 30 dyn/cm2) in a parallel-plate flow chamber. Increases in PDGF B-chain, but not PDGF A-chain, were observed within 3 h, maximal within 6 h (13-fold increase), and sustained for 24 h. PKC appeared to be involved because phorbol 12-myristate 13-acetate induced PDGF B-chain mRNA. Activation of PKC alone, however, was insufficient to induce PDGF mRNA because the selective PKC activator, 1-oleoyl-2-acetyl-sn-glycerol, did not induce PDGF expression. A PKC-independent pathway was suggested by the fact that inhibition of PKC (downregulation with phorbol 12,13-dibutyrate or exposure to staurosporine) failed to block PMA or flow-induced PDGF B-chain expression. These results demonstrate flow-induced PDGF B-chain expression in endothelial cells that appears to be mediated, in part, by a PKC-independent pathway.

Animals↗

PubMed1993

Probucol decreases neointimal formation in a swine model of coronary artery balloon injury. A possible role for antioxidants in restenosis.

BACKGROUND: Restenosis after percutaneous transluminal coronary angioplasty is the major limitation of the long-term success of this procedure. The process of restenosis is similar to an accelerated form of atherosclerosis. Thus, therapeutic interventions that limit the progression and initiation of atherosclerosis may be beneficial in the treatment of restenosis. One such intervention is the antioxidant drug probucol, which has demonstrated benefit in animal models of atherosclerosis. METHODS AND RESULTS: Twenty-six female domestic swine were divided into three study groups (control, n = 9; low-dose probucol, n = 9; high-dose probucol, n = 8) before oversized balloon injury of the left anterior descending and left circumflex coronary arteries. Probucol (1 g/d, low-dose group; 2 g/d, high-dose group) was administered 2 days before balloon injury and was continued until the swine were killed 2 weeks after balloon injury. Morphometric analysis of the injured arteries included the intimal area (square millimeters), maximal intimal thickness (millimeters), and residual lumen (ratio of luminal to intimal plus luminal area). Treatment with high-dose probucol significantly reduced neointimal formation compared with control animals (decreases of 36% in intimal area, P = .007; 20% in maximal intimal thickness, P = NS; and an increase of 15% in residual lumen, P = .02). CONCLUSIONS: The major finding of this study is that the antioxidant drug probucol reduces neointimal formation after oversized balloon injury in a swine model of restenosis. This suggests that active oxygen species may play a role in restenosis.

Animals↗

PubMed1993

Na(+)-H+ exchange inhibitors decrease neointimal formation after rat carotid injury. Effects on smooth muscle cell migration and proliferation.

The presence of multiple growth stimuli at the sites of vascular injury following angioplasty suggests that therapies targeted toward common growth pathways will be more effective than therapies that inhibit only a single growth factor. We tested this hypothesis using amiloride and ethyl isopropyl amiloride (EIPA), which are inhibitors of the Na(+)-H+ exchanger, whose activity is required in many cells for proliferation and migration. In the rat carotid injury model, EIPA (100 micrograms/h for 15 days) significantly decreased intimal area and the ratio of intimal to medial area, whereas amiloride (25 micrograms/h) showed an inhibitory trend that was similar to that observed for captopril (80 mg/kg per day) and heparin (25 U/h). EIPA and amiloride inhibited rat vascular smooth muscle cell DNA synthesis, with IC50 values of 8.8 and 82.2 microM, respectively. Using platelet-derived growth factor as a chemoattractant, EIPA caused a concentration-dependent inhibition of migration (IC50, approximately 60 microM). Because amiloride and EIPA have nonspecific effects on cellular function (especially inhibition of tyrosine kinases), we sought to characterize the specific role of the Na(+)-H+ exchanger in vascular smooth muscle cell proliferation and migration. We generated a Na(+)-H+ exchanger-deficient mutant cell line [RNHE(-)]. Studies with these cells suggested that the inhibitory effects of EIPA and amiloride were mediated only in part via Na(+)-H+ exchange because (1) RNHE(-) cells grew well at pH 6.8 to 7.5 in bicarbonate-containing medium, and (2) there was no difference in migration in response to platelet-derived growth factor in the RHNE(-) cells. In summary, these data indicate that amiloride and EIPA inhibit neointimal formation in the rat carotid after injury. However, the mechanism of inhibition is likely to involve cellular events other than Na(+)-H+ exchange, such as an effect on tyrosine kinases.

Amiloride↗

PubMed1992

Angiotensin II stimulates the pp44 and pp42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells.

Vasoconstrictors such as angiotensin II (ang II) stimulate vascular smooth muscle cell growth and share many signal transduction mechanisms with growth factors. Recently, growth factors have been shown to stimulate mitogen-activated protein (MAP) kinases, a family of serine/threonine protein kinases which phosphorylate pp90rsk, a cytosolic kinase that phosphorylates ribosomal S6 protein. We examined the effect of ang II on MAP kinase activity and phosphorylation. Ang II stimulated MAP kinase activity by 4-fold after 5 min exposure and also increased tyrosine phosphorylation of 42 kDa (74 +/- 41%) and 44 kDa (263 +/- 85%) proteins, shown to be pp42mapk and pp44mapk by Western blot analysis using a MAP kinase antibody. These results suggest that ang II-stimulated protein synthesis is mediated by a MAP kinase dependent pathway.

Angiotensin II↗

PubMed1992

The Na/K/2Cl cotransporter is increased in hypertrophied vascular smooth muscle cells.

Hypertrophy of vascular smooth muscle cells (VSMC) is a pathogenic feature of hypertension which may contribute to abnormal vessel tone and function. As a consequence of the increase in cell size associated with hypertrophy, it is likely that alterations in the mechanisms that regulate VSMC intracellular volume occur. Because the Na+/H+ exchanger plays an important role in volume regulation and because we previously observed long term alterations in Na+/H+ exchange and pHi in response to angiotensin-II-induced (ang II) hypertrophy, we studied cell-acidifying mechanisms. To do this, we measured alkaline recovery from NH4Cl-mediated alkalinization, using the fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. VSMC were growth-arrested (0.4% calf serum for 24 h) or hypertrophied (100 nM ang II in 0.4% calf serum for 24 h). Ang II-treated cells exhibited a 107% increase in alkaline recovery over control cells (13.86 +/- 1.87 versus 6.68 +/- 1.01 mmol H+/min/liter cells). The increase in alkaline recovery was not a result of increased Cl-/HCO-3 exchange becaue it was not HCO-3 dependent nor inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid. Studies with bumetanide and the sterically inhibited substrate N(CH3)4+ showed that the alkaline recovery was mediated by NH4+ transport via the Na/K/2Cl cotransporter. Ang II-treated cells exhibited a 334% increase in bumetanide-sensitive alkaline recovery over control cells (9.16 +/- 1.90 versus 2.11 +/- 1.46 mmol H+/min/liter cells). Ang II-treated cells also exhibited a 90% increase in bumetanide-sensitive 86Rb uptake over control cells. These findings demonstrate that Na/K/2Cl cotransport activity is specifically induced in ang II-hypertrophied VSMC and establish this transporter as a component of the hypertrophic growth response.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1992

Na+/H+ antiporter gene expression increases during retinoic acid-induced granulocytic differentiation of HL60 cells.

During differentiation of human leukemic HL60 cells into granulocytes, sustained increases in intracellular pH and Na+/H+ antiporter activity have been observed. In the present study we report that retinoic acid (RA)-induced granulocytic differentiation of HL60 cells causes an approximately 18-fold increase in the steady-state mRNA levels for the Na+/H+ antiporter. This was due to an increase in the rate of Na+/H+ antiporter gene transcription as measured by nuclear run-on analysis. Antiporter protein synthesis increased by seven-fold during RA-induced granulocytic differentiation of HL60 cells as measured by immunoprecipitation of 35S-methionine-labeled proteins with the RP1-c28 Na+/H+ antiporter antibody. No increase in antiporter mRNA was observed in response to etretinate, an analogue of retinoic acid, which did not induce differentiation. Thus, Na+/H+ antiporter gene expression is associated with RA-induced granulocytic differentiation of HL60 cells. The present findings and our previous data (Rao et al., 1991) demonstrate that Na+/H+ antiporter gene expression is a generalized feature of HL60 cell differentiation.

Carrier Proteins↗

PubMed1992

5-HT2 receptor mRNA is overexpressed in cultured rat aortic smooth muscle cells relative to normal aorta.

Proliferation of smooth muscle cells in arteries is associated with contractile hypersensitivity to serotonin (5-HT). A possible explanation is that smooth muscle cells express increased numbers of phospholipase C (PLC)-coupled 5-HT receptors (5-HTR), which could mediate contractile and mitogenic signals via phosphatidylinositol turnover. To test this hypothesis, we performed a molecular characterization of 5-HTR subtypes in normal aorta and passaged rat aortic smooth muscle cells (RASM) in culture. Northern blot analysis revealed that growth-arrested cultured cells expressed 5-HT2R mRNA at 50-fold greater levels than aorta. 5-HT1CR mRNA was not detected in either case. 5-HT stimulated intracellular Ca2+ mobilization (fivefold peak increase) and c-fos mRNA induction (10-fold peak increase); both responses were strongly inhibited by selective 5-HT2R antagonists. Specific agonists for the 5-HT1AR, 5-HT1BR, and 5-HT1DR failed to induce c-fos mRNA. Although 5-HT (10 microM) increased [3H]thymidine incorporation (28% relative to 10% calf serum), it was a weak mitogen for cultured RASM based on cell counts. Thus there is high level expression of 5-HT2R mRNA by cultured RASM relative to aorta, and the 5-HT2R appears to be the only 5-HTR subtype mediating early growth signals in these cells. These data suggest that, following arterial injury in vivo, smooth muscle cells may overexpress the 5-HT2R, resulting in 5-HT contractile hypersensitivity and increased responsiveness to other growth factors.

Animals↗

PubMed1992

Flow-induced calcium transients in single endothelial cells: spatial and temporal analysis.

Endothelial cells (EC) are uniquely situated to respond to hemodynamic forces. Because flow-mediated release of endothelial-derived relaxing factors is associated with increased EC intracellular calcium ([Ca2+]i), we sought to determine the effects of fluid shear stress on EC [Ca2+]i. Cells were subjected to flow in parallel-plate flow chambers and glass capillary tubes, and single cell [Ca2+]i was measured using fura-2. Upon initiation of flow (shear stress of 30 dyn/cm2), [Ca2+]i increased within 30 s to a peak value (approximately 4 times basal) and then decreased slowly to a plateau (approximately 2 times basal) that persisted for greater than 5 min. A striking finding was that the increases in [Ca2+]i were nonhomogeneous; the nuclear region and a periplasma membrane region were higher than the cytosol. After flow cessation, the increase in [Ca2+]i could be elicited repeatedly by resumption of flow. Removing extracellular Ca2+ did not eliminate the response. In contrast to EC, rat aortic smooth muscle cells showed no flow-mediated increase in [Ca2+]i. The complexity of EC [Ca2+]i response to flow suggests regulation of [Ca2+]i by several mechanisms that may serve a role in both short- and long-term EC responses to flow.

Animals↗

PubMed1992

Development and characterization of a cloned rat pulmonary arterial smooth muscle cell line that maintains differentiated properties through multiple subcultures.

BACKGROUND: Pulmonary hypertension is associated with abnormal pulmonary arterial contractility and growth. The mechanisms for these abnormalities are largely unknown. To study these processes, we sought to develop an in vitro system. Even though cultured aortic and pulmonary artery smooth muscle cells (SMCs) have been of considerable value in studying smooth muscle biology, one drawback of this system has been that these cells often lose differentiated properties in an unpredictable manner when they are passaged in culture. In addition, there appear to be significant differences in physiological and pathological responses between the systemic and pulmonary circulations, many of which could be directly related to the smooth muscle. We therefore established a cloned population of rat pulmonary arterial SMCs (PASMCs) that maintain differentiated properties through multiple subcultures. METHODS AND RESULTS: PASMCs were obtained initially by enzymatic dissociation from pulmonary arteries of adult Sprague-Dawley rats. From these cells, clones were isolated. The cloned cells retained expression of functional surface receptors for angiotensin II, norepinephrine, and alpha-thrombin and high levels of the smooth muscle isoforms of alpha-actin, myosin heavy chain, myosin regulatory light chain, and alpha-tropomyosin mRNAs even after multiple passages. The cells could also be transfected and processed exogenous transcripts in a smooth muscle-specific fashion. CONCLUSIONS: These cloned PASMCs retain many differentiated characteristics and should be valuable for future studies of pulmonary vascular smooth muscle cell biology.

Animals↗

PubMed1992

Na+, K(+)-adenosine triphosphatase regulation in hypertrophied vascular smooth muscle cells.

Vascular smooth muscle cell hypertrophy is a normal compensatory state that may play a pathogenic role in hypertension. Angiotensin II stimulates a hypertrophic response in cultured vascular smooth muscle cells. As part of the growth response, angiotensin II rapidly activates the Na(+)-H+ exchanger, increasing Na+ influx. Because Na+, K(+)-ATPase is the major cellular mechanism for regulating intracellular Na+, we studied the effects of angiotensin II-induced hypertrophy on Na+, K(+)-ATPase expression and activity. Angiotensin II caused rapid increases in both steady-state Na+, K(+)-ATPase activity (ouabain-sensitive 86Rb uptake) and intracellular [Na+]. Angiotensin II also caused a sustained increase in Na+, K(+)-ATPase at 24 hours with a 73% increase in maximal 86Rb uptake per milligram protein and a fourfold increase in Na+, K(+)-ATPase alpha-1 messenger RNA levels. Thus, angiotensin II hypertrophy was associated with rapid increases in Na+, K(+)-ATPase activity due to increased Na+ entry and sustained increases due to a specific increase in Na+, K(+)-ATPase expression. These data demonstrate dynamic regulation of Na+, K(+)-ATPase at the functional and molecular level and suggest that similar compensatory mechanisms should be present in vivo. Alterations in such compensatory pathways may be fundamental to the pathogenesis of hypertension.

Angiotensin II↗

PubMed1992

JE mRNA accumulates rapidly in aortic injury and in platelet-derived growth factor-stimulated vascular smooth muscle cells.

The early response to vascular injury is characterized by migration of inflammatory cells, including monocytes, and platelets to the damaged vessel wall. These inflammatory cells may serve as a source of growth factors and cytokines that stimulate vascular smooth muscle cell (VSMC) migration and proliferation associated with intimal hyperplasia. JE is a platelet-derived growth factor (PDGF)-inducible "early" gene that encodes a monocyte chemoattractant and, as such, could play an important role in inflammation. We now report that JE mRNA levels are increased in intact aorta after balloon injury. The time course of this increase, with maximal levels at 4 hours, is similar to that seen in PDGF-treated cultured rat aortic VSMCs. The accumulation of JE mRNA in cultured VSMCs is accompanied by a marked increase in the secretion of JE protein. The elevation of JE mRNA levels in VSMCs shows specificity for PDGF, because angiotensin II, alpha-thrombin, and epidermal growth factor fail to increase JE mRNA levels. In contrast to 3T3 fibroblasts, the accumulation of JE mRNA in VSMCs in response to PDGF is predominantly due to an increase in JE mRNA stability. The accumulation of JE mRNA in VSMCs stimulated by PDGF appears to occur via a novel pathway(s) independent of Ca2+ mobilization, Na(+)-H+ exchange, protein kinase C activation, or elevation in cAMP levels. These findings suggest that VSMCs may take part in the early inflammatory response after injury through the production of JE, a potent monocyte chemoattractant. Finally, our data suggest that JE may be a marker for PDGF-specific effects on VSMCs, both in vitro and in vivo. Thus, in addition to direct effects on VSMC growth and migration, PDGF may play a role in the early inflammatory response after vascular injury by inducing chemoattractants, such as that encoded by JE.

Angiotensin II↗

PubMed1992

Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression.

Vascular smooth muscle cells (VSMCs) proliferate in response to arterial injury. Recent findings suggest that, in addition to platelet-derived growth factors, growth factors from inflammatory cells and endothelial cells at the site of injury may contribute to VSMC proliferation. We hypothesized that a common mechanism by which endothelial cells and inflammatory cells stimulate VSMC growth could be the active oxygen species (i.e., O2-, H2O2, and .OH) generated during arterial injury. Using xanthine/xanthine oxidase to generate active oxygen species, we studied the effects of these agents on VSMC growth. Xanthine/xanthine oxidase (100 microM xanthine and 5 microunits/ml xanthine oxidase) stimulated DNA synthesis in growth-arrested VSMCs by 180% over untreated cells. Administration of the scavenging enzymes superoxide dismutase and catalase demonstrated that H2O2 was primarily responsible for xanthine/xanthine oxidase-induced VSMC DNA synthesis. H2O2 directly increased VSMC DNA synthesis and cell number (maximal at 200 microM) but decreased DNA synthesis of endothelial cells and fibroblasts. This effect was protein kinase C independent: sphingosine, a potent protein kinase C inhibitor, failed to block H2O2-induced VSMC DNA synthesis. H2O2 (200 microM) stimulated c-myc and c-fos mRNA levels by fourfold and 20-fold, respectively, as compared with quiescent levels. In contrast to DNA synthesis, H2O2 induction of c-myc and c-fos mRNA was primarily protein kinase C dependent. These findings show that H2O2 specifically increases VSMC DNA synthesis and suggest a role for this oxidant in intimal proliferation, especially after arterial injury.

Animals↗

PubMed1992

Bovine angiotensin converting enzyme cDNA cloning and regulation. Increased expression during endothelial cell growth arrest.

Angiotensin converting enzyme (ACE) is a zinc-containing dipeptidase that converts angiotensin I to angiotensin II, a powerful vasoconstrictor and smooth muscle growth factor. ACE activity has been shown to be dynamically regulated by hormones, ACE inhibitors, and endothelial cell growth state. To study how ACE expression is regulated, we isolated and sequenced the bovine ACE gene using both ACE-specific cDNA and genomic clones. Bovine ACE cDNA encodes a single polypeptide of 1,306 residues with a molecular mass of 150 kd. Bovine ACE is approximately 80% homologous to that of other species. It contains two homologous domains of equal size. Alignment of ACE sequences from bovine, human, mouse, and rabbit reveals that during evolution both domains have been highly conserved. We used the bovine ACE cDNA to study regulation of ACE gene expression during density-dependent growth arrest. As endothelial cells became growth-arrested (6 days after confluence), there was a 12-fold increase in ACE activity and a 90% decrease in DNA synthesis. Immunocytochemically detectable ACE markedly increased in growth-arrested cells. The increase in ACE was due to increased ACE gene expression, as assayed by RNase protection, which showed a 20-fold increase in ACE-specific mRNA. The present study shows that bovine ACE is highly regulated by endothelial cell growth state at the level of protein and mRNA expression. Such dynamic regulation may have important consequences for angiotensin II production during endothelial cell proliferation after arterial injury.

Amino Acid Sequence↗

PubMed1991

Na+/H+ antiporter gene expression during monocytic differentiation of HL60 cells.

During differentiation of human promyelocytic HL60 cells into monocytes there are sustained increases in intracellular pH and Na+/H+ antiporter activity. Here we show that increased transcription and expression of the gene for the Na+/H+ antiporter precedes phorbol 12-myristate 13-acetate (PMA)-induced HL60 cell differentiation. PMA increased steady-state Na+/H+ antiporter mRNA levels approximately 50-fold within 8 h (at which time less than 15% of cells had differentiated). This increase was due to an increased transcriptional rate as determined by nuclear run on. Immunoprecipitation of [35S]methionine-labeled Na+/H+ antiporter using an antiporter fusion protein antibody (RP1-c28) showed an equivalent increase in Na+/H+ antiporter protein synthesis. The synthetic diacylglycerol, 1-oleolyl-2-acetylglycerol, an activator of protein kinase C, which unlike PMA did not cause differentiation, failed to induce Na+/H+ antiporter mRNA. Furthermore, inhibition of PMA-induced differentiation by either sphingosine or cycloheximide prevented accumulation of Na+/H+ antiporter mRNA. Together, these findings strongly suggest a close association of Na+/H+ antiporter induction with HL60 cell differentiation. The HL60 cell system is a promising model to study the mechanisms of Na+/H+ antiporter gene regulation and its function in differentiation.

Carrier Proteins↗

PubMed1991

Uric acid stimulates vascular smooth muscle cell proliferation by increasing platelet-derived growth factor A-chain expression.

Recent data suggest that uric acid is generated locally in the vessel wall by the action of xanthine oxidase. This enzyme, activated during ischemia/reperfusion by proteolytic conversion of xanthine dehydrogenase, catalyzes the oxidation of xanthine, thereby generating free radicals and uric acid. Because of the potential role of ischemia/reperfusion in vascular disease, we studied the effects of uric acid on rat aortic vascular smooth muscle cell (VSMC) growth. Uric acid stimulated VSMC DNA synthesis, as measured by [3H]thymidine incorporation, in a concentration-dependent manner with half-maximal activity at 150 microM. Maximal induction of DNA synthesis by uric acid (250 microM) was approximately 70% of 10% calf serum and equal to 10 ng/ml platelet-derived growth factor (PDGF) AB or 20 ng/ml fibroblast growth factor. Neither uric acid precursors (xanthine and hypoxanthine) nor antioxidants (ascorbic acid, glutathione, and alpha-tocopherol) were mitogenic for VSMC. Uric acid was mitogenic for VSMC but not for fibroblasts or renal epithelial cells. The time course for uric acid stimulation of VSMC growth was slower than serum, suggesting induction of an autocrine growth mechanism. Exposure of quiescent VSMC to uric acid stimulated accumulation of PDGF A-chain mRNA (greater than 5-fold at 8 h) and secretion of PDGF-like material in conditioned medium (greater than 10-fold at 24 h). Uric acid-induced [3H]thymidine incorporation was markedly inhibited by incubation with anti-PDGF A-chain polyclonal antibodies. Thus uric acid stimulates VSMC growth via an autocrine mechanism involving PDGF A-chain. These findings suggest that generation of uric acid during ischemia/reperfusion contributes to atherogenesis and intimal proliferation following arterial injury.

Animals↗

PubMed1991

Thrombin-stimulated events in cultured vascular smooth-muscle cells.

Thrombin is present in high concentrations at sites of clots and may have important post-clotting effects on adjacent vascular tissue. This may be particularly important for vascular smooth-muscle cells (VSMC), whose growth and contractility are altered following atherosclerotic-associated thromboses. To study the cellular signal events by which thrombin exerts its actions, the effects of purified human alpha-thrombin were examined in cultured rat aortic VSMC. alpha-Thrombin stimulated a biphasic change in intracellular pH (pHi), causing an early rapid acidification, followed by a sustained alkalinization. The increase in pHi was dependent on extracellular Na+ and inhibited by 5'-(NN-dimethyl)amiloride, consistent with mediation by Na+/H+ exchange. alpha-Thrombin rapidly increased free intracellular [Ca2+] ([Ca2+]i). The increase in [Ca2+]i was secondary to activation of phospholipase C, as demonstrated by increases in InsP3 (226%) and InsP2 (387%) and decreases in polyphosphoinositides at 15 s. Expression of the mRNA for the proto-oncogene c-fos was induced by alpha-thrombin. Stimulation of c-fos mRNA was not dependent on alterations in pHi, but required a rise in [Ca2+]i. Despite many growth-related signals shared by alpha-thrombin with platelet-derived growth factor, alpha-thrombin failed to stimulate [3H]thymidine incorporation or cell division, although there was a maximal increase of 52% in protein synthesis. The data suggest that there are cellular signal events not activated by alpha-thrombin which are required for proliferation of these aortic VSMC.

Animals↗