Biomedical subjects

T A Brock

Publications and source records attributed to T A Brock.

At least 37 records · Page 2Linked to original sources

PubMed1996

Vascular endothelial growth factor, a multifunctional polypeptide.

Angiogenesis, the sprouting of new blood vessels from pre-existing vessels, is a complex, multicellular phenomenon involving capillary endothelial cell (EC) proliferation, migration, and tissue infiltration. The elucidation of the biochemical and molecular factors which control angiogenesis is fundamental to our understanding of normal blood vessel development, as well as of the pathogenesis of abnormal blood vessel formation. Angiogenesis is associated with numerous physiological processes, including embryogenesis, wound healing, organ regeneration, and the female reproductive cycle. However, abnormal angiogenesis also plays a major role in the pathogenesis of tumor growth, rheumatoid arthritis, atherosclerosis and various retinopathies. The cellular and molecular mechanisms underlying both physiological and pathophysiological angiogenesis are only now beginning to be understood. Vascular endothelial growth factor was initially discovered as an unidentified tumor-derived factor which increased microvascular permeability (vascular permeability factor, VPF). Subsequently, it was determined that the protein exhibited mitogenic effects on endothelial cells, but not other cell types. Multiple receptor subtypes have been described which may in part explain the multiplicity of biological actions that have been ascribed to VEGF/VPF in the literature. In this overview, we briefly summarize what is currently known about VEGF and VEGF receptor biology, as well as VEGF receptor signal transduction mechanisms in endothelial cells.

Amino Acid Sequence↗

PubMed1995

Interaction of the Flt-1 tyrosine kinase receptor with the p85 subunit of phosphatidylinositol 3-kinase. Mapping of a novel site involved in binding.

We have examined the interactions of the p85 regulatory subunit of phosphatidylinositol 3-kinase with the endothelium-specific Flt-1 receptor tyrosine kinase using the yeast two-hybrid system. We find that both the amino- and carboxyl-terminal SH2 domains of p85 bind to Flt-1. We have performed site-directed mutagenesis on the carboxyl-terminal tail of the Flt-1 receptor in order to identify the site(s) that is responsible for the p85 interactions. A single tyrosine to phenylalanine change at position 1213 inhibits the binding of both p85 SH2 domains. Phosphopeptide mapping of the wild type and mutant protein expressed in insect cells verifies that this amino acid is a target for autophosphorylation. The amino acids following this tyrosine are VNA and thus define a novel binding site for p85.

Amino Acid Sequence↗

PubMed1995

Peroxynitrite stimulates vascular smooth muscle cell cyclic GMP synthesis.

Peroxynitrite stimulated the synthesis of cyclic GMP by rat aortic smooth muscle in a time- and dose-dependent manner. Peak formation of cyclic GMP occurred at 1 min with 100 microM peroxynitrite and was inhibited by oxyhemoglobin. Peroxynitrite was less potent than nitric oxide in stimulating cyclic GMP synthesis. Peroxynitrite also enhanced endothelial-dependent cyclic GMP synthesis, via generation of a long-lived substance, which was prevented by inhibition of glutathione synthesis. These data show that peroxynitrite stimulates cyclic GMP synthesis, inferring production of low yields of nitric oxide or associated derivatives. Additionally, vascular exposure to peroxynitrite potentiates endothelial-dependent activation of guanylate cyclase.

Animals↗

PubMed1995

Calcium-sensitive chloride channels in vascular smooth muscle cells.

Chloride (Cl-) channels were characterized in vascular smooth muscle cells (VSMC) using radioisotope flux and patch-clamp electrophysiological techniques. Transmembrane 125iodine (125I) efflux from subcultured (Passage 1-5) rat aortic VSMCs was used as an indicator of Cl- movements to study the relationship between intracellular calcium concentration ([Ca2+]i) and Cl- channel activity. Angiotensin II (Ang II) (10(-7) M) and adenosine 5'-triphosphate (ATP) (10(-4) M) induced rapid increases (9.7- and 14.9-fold, respectively) in 125I efflux rates. We found that both Ang II- and ATP-stimulated 125I efflux and [Ca2+]i increases were completely abolished after brief incubation (20 microM, 20 min) with the acetoxymethyl ester of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), a membrane-permeable Ca2+ chelator. However, when external EGTA was used to blunt agonist-stimulated Ca2+ influx, 125I efflux was still increased in response to Ang II and ATP. These data suggest that Ca2+ release from intracellular sites is sufficient to activate Cl- channels in response to Ang II and ATP. Using standard patch-clamp electrophysiological techniques, we found that Ang II, a Ca(2+)-mobilizing agonist, stimulated outward Cl- currents (gCl = 75 pS) in cell-attached (C/A) patches of primary and subcultured VSMCs. Collectively, these data suggest that Ang II and other vasoconstrictor agents stimulate Cl- channel activity via increases in [Ca2+]i. Cl- channel activation may help to depolarize the VSMC membrane leading to increased Ca2+ influx during agonist stimulation.

Animals↗

PubMed1994

Superoxide and peroxynitrite in atherosclerosis.

The role of reactive oxygen species in the vascular pathology associated with atherosclerosis was examined by testing the hypothesis that impaired vascular reactivity results from the reaction of nitric oxide (.NO) with superoxide (O2-), yielding the oxidant peroxynitrite (ONOO-). Contractility studies were performed on femoral arteries from rabbits fed a cholesterol-supplemented diet. Cholesterol feeding shifted the EC50 for acetylcholine (ACh)-induced relaxation and impaired the maximal response to ACh. We used pH-sensitive liposomes to deliver CuZn superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) to critical sites of .NO reaction with O2-. Intravenously injected liposomes (3000 units of SOD per ml) augmented ACh-induced relaxation in the cholesterol-fed group to a greater extent than in controls. Quantitative immunocytochemistry demonstrated enhanced distribution of SOD in both endothelial and vascular smooth muscle cells as well as in the extracellular matrix. SOD activity in vessel homogenates of liposome-treated rabbits was also increased. Incubation of beta very low density lipoprotein with ONOO- resulted in the rapid formation of conjugated dienes and thiobarbituric acid-reactive substances. Our results suggest that the reaction of O2- with .NO is involved in the development of atherosclerotic disease by yielding a potent mediator of lipoprotein oxidation, as well as by limiting .NO stimulation of vascular smooth muscle guanylate cyclase activity.

Acetylcholine↗

PubMed1994

Calcium-mobilizing agonists stimulate anion fluxes in cultured endothelial cells from human umbilical vein.

The goal of the present studies was to determine whether anion fluxes are involved in thrombin- and histamine-activated signal transduction pathways in human umbilical vein endothelial cells (HUVECs). 125Iodine (125I) efflux techniques were used to test the sensitivity of anion fluxes to increases in [Ca2+]i and activation of protein kinase C. HUVECs exhibited constant 125I efflux rates under basal conditions. Administration of thrombin or histamine stimulated an increase in 125I efflux rates which returned to control values after approximately 1-2 min. Since both agonists stimulate increases in [Ca2+]i, we tested the hypothesis that 125I efflux was sensitive to changes in [Ca2+]i. When HUVECs were exposed to ionomycin or thapsigargin, the 125I efflux rate increased and remained elevated for several minutes. In subsequent experiments, HUVECs were incubated with the cell permanent Ca2+ chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, to buffer changes in [Ca2+]i. This treatment reduced both basal and thrombin-stimulated 125I efflux. However, when Ca2+ was removed from the efflux buffer and replaced with EGTA, peak thrombin-stimulated 125I efflux remained unchanged. This anion efflux was also sensitive to activation of protein kinase C since phorbol 12-myristate 13-acetate and phorbol, 12,13-dibutyrate blunted thrombin-mediated increases in 125I efflux. Preincubation of HUVECs with protein kinase C inhibitor peptide [19-36] antagonized the phorbol ester-mediated decrease in thrombin-stimulated 125I efflux. We suggest that 125I efflux in HUVECs represents a Ca(2+)-sensitive anion conductance and that intracellular Ca2+ release, but not extracellular Ca2+ influx, is sufficient to initiate channel activity.

Anions↗

PubMed1993

Inhibition of vascular permeability factor (vascular endothelial growth factor) with antipeptide antibodies.

Vascular permeability factor (VPF), also known as vascular endothelial cell growth factor (VEGF), is a 34- to 43-kDa dimeric protein synthesized and secreted by a variety of tumor and normal cells. At nanomolar concentrations, VPF causes an increase in microvascular permeability and is thought to be responsible for enhanced permeability of tumor blood vessels and for the fluid accumulation associated with solid and ascites tumors. In addition, VPF/VEGF is a mitogen for endothelial cells and may play an important role in maintaining vascular endothelium and in promoting tumor angiogenesis. Antibodies were raised against a series of synthetic peptides derived from the predicted human VPF amino acid sequence. The antibodies were assayed for their ability to bind native and denatured/reduced VPF. Antibodies to peptides from the N- and C-termini bound both denatured/reduced and native VPF; antibodies directed to internal segments (e.g., amino acids 27-48 and 85-101) strongly bound denatured/reduced VPF but were substantially less effective at binding native VPF. These results suggest that the N- and C-termini are exposed regions of the protein in solution. Individually, antibodies to the N- and C-termini each partially blocked VPF permeability activity, and, in combination, blocked nearly 100% of this activity. Also, the N- and C-terminal antibodies blocked the VPF-mediated stimulation of both endothelial cell growth and increase in free cytosolic calcium.

Amino Acid Sequence↗

PubMed1993

Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), has recently been shown to increase cytosolic free calcium in endothelial cells. In the present study, we investigated the coronary vascular effects of recombinant human and native guinea pig VEGF/VPF in isolated canine coronary arteries in the presence and absence of intimal endothelium, indomethacin, and NG-monomethyl-L-arginine, a competitive nitric oxide synthase inhibitor. Addition of recombinant VEGF/VPF (1-660 pM) in coronary arteries that had been previously contracted with prostaglandin F2 alpha induced a slow, dose-dependent relaxation, reaching a maximum of -59.1 +/- 6.7% (mean +/- SE, n = 19). Mechanical disruption of the intimal endothelium completely abolished the observed relaxation. No direct vascular effect of recombinant VEGF/VPF on the endothelium-disrupted coronary arteries was noted. Pretreatment of endothelium-intact coronary arteries with 5 microM of indomethacin did not alter the observed relaxation (-57.3 +/- 7.0%, n = 18), whereas pretreatment with either NG-monomethyl-L-arginine or 10 microM of genistein, a known inhibitor of tyrosine kinase, significantly inhibited the relaxation. Addition of native VEGF/VPF (1-100 pM) also induced an endothelium-dependent relaxation in the isolated coronary arteries. Heating of recombinant VEGF/VPF (70 degrees C, 25 min) or prior incubation with a specific antibody raised against a VEGF/VPF peptide completely abolished the relaxation. Finally, recombinant VEGF/VPF stimulated a slow rise in cytosolic free calcium in cultured human endothelial cells that was qualitatively similar to that of native VEGF/VPF.(ABSTRACT TRUNCATED AT 250 WORDS)

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↗

PubMed1991

Mas oncogene receptor coupling and peptide specificity in Balb 3T3 and vascular smooth muscle cells.

The mas oncogene receptor has been reported to confer angiotensin (Ang) responsiveness in NG115-401L neuronal cell line. To test if mas oncogene encodes an Ang receptor in peripheral tissue, Balb 3T3 and rat aortic vascular smooth muscle cells (VSMC) were cotransfected with a plasmid containing the mas oncogene (pSM422) and a plasmid expressing a selectable marker (pRSV-Neo). Transfected cells (Balb/mas and VSMC/mas) expressed the appropriate 2.4 Kb mas transcript, which was not present in parental cells. Both Balb/mas and VSMC/mas cells acquired Ang II and Ang III responsiveness as documented by Ang-stimulated increased [Ca2+]i. The ED50 for these peptides were relatively high (4 - 6 x 10(-5) M). Ang III was approximately two times more potent than Ang II in stimulating 45Ca efflux from Balb/mas cells, and its effect was not blocked by Sar1, Ile8-Ang II. In contrast, substance P and a substance P analogue ([D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P) behaved as agonists, resulting in the stimulation of 45Ca efflux and [Ca2+]i in Balb/mas cells without affecting control cells. The rank order potency for stimulating 45Ca efflux in Balb/mas cells was substance P analogue much greater than Ang III, substance P greater than Ang II. In summary, the authors show that although Ang III can stimulate biochemical events in mas transfected cells, which are known to be essential for Ang receptor signal transduction in other cell types, ie, [Ca2+]i and pHi transients, as well as inositol triphosphate formation, it did that at supraphysiological concentrations of the peptide.

3T3 Cells↗

PubMed1991

Tumor-secreted vascular permeability factor increases cytosolic Ca2+ and von Willebrand factor release in human endothelial cells.

Vascular permeability factor (VPF), a tumor-secreted heparin-binding protein (Mr approximately 38,000), is responsible for increased vessel permeability and fluid accumulation associated with tumor growth. Vascular permeability factor also promotes the growth of human umbilical vein endothelial cells (EC) and bovine pulmonary ECs in vitro. It is shown for the first time that guinea pig VPF (half-maximal and maximal dose approximately 0.4 and 22 pmol/l (picomolar), respectively), as well as human VPF, are potent stimuli for human ECs resulting in [Ca2+]i increases (maximal three- to fourfold) and inositol triphosphate (IP3) formation. Unlike the maximal responses to thrombin and histamine, the [Ca2+]i response to a maximal VPF dose was preceded by a characteristic 10- to 15-second delay. Guinea pig VPF also selectively increased [Ca2+]i in cultured aortic and pulmonary artery ECs, but not aortic smooth muscle cells, human fibroblasts, or neutrophils. Affinity-purified rabbit antibody (raised to a synthetic peptide representing VPF N-terminal amino acids 1 to 24) adsorbed all vessel permeability-increasing activity, EC growth-promoting activity, and specifically all activity responsible for increasing EC [Ca2+]i. Similar to other mediators that increase [Ca2+]i in cultured ECs, VPF also induced a 200% increase in von Willebrand factor release. Together these data indicate that VPF acts directly on ECs and that rapid cellular events in its in vivo/in vitro actions are likely to involve phospholipase C activation, [Ca2+]i increase, and von Willebrand factor release.

Calcium↗

PubMed1990

Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells.

Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.

Adenosine Triphosphate↗

PubMed1990

Osmotic activation of Na(+)-H+ exchange in human endothelial cells.

Regulation of intracellular pH (pHi) via a Na(+)-H+ exchange-dependent mechanism was studied in cultured human umbilical vein endothelial cells (HEC) using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein, as well as measuring 22Na influx. Basal pHi of HEC incubated in a bicarbonate-free Na+ medium was 6.99 +/- 0.03. In HEC that had been acid-loaded using nigericin or a NH4Cl prepulse, pHi recovery occurred via a Na(+)-dependent mechanism that was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). The potency of amiloride derivatives to inhibit 22Na influx was EIPA greater than 5-(N,N-dimethyl)amiloride greater than amiloride [Ki (extracellular Na = 30 mM) = 17 nM, 150 nM, and 8.8 microM, respectively]. EIPA-sensitive 22Na influx in acid-loaded HEC was a saturable function of the external Na+ concentration (0-130 mM), exhibiting an approximate Km and Vmax of 19.70 +/- 0.14 mM and 34.01 +/- 2.2 nmol.10(6) cells-1.min-1, respectively. H+ efflux was also dependent on external Na+ and blocked by EIPA. At resting pHi, HEC Na(+)-H+ exchange was slightly stimulated by increases in medium osmolality. However, when HEC were acid-loaded in the presence of hypertonic (sucrose) medium, Na(+)-H+ exchange activity (22Na influx or pHi recovery) increased markedly. Overall, these data indicate that pHi in cultured HEC can be regulated by a Na(+)-H+ exchanger and that its activity can be markedly influenced by osmolality at acidic pHi.

Amiloride↗

PubMed1990

Glomerular endothelial cells respond to calcium-mobilizing agonists with release of EDRF.

To determine whether glomerular endothelial cells (GEN) may play a role in the local control of glomerular function by releasing endothelium-derived relaxing factor (EDRF), the effect of several agonists on GEN cytosolic calcium concentration ([Ca2+]i) and GEN EDRF release was determined. Bradykinin, ATP, thrombin, and platelet-activating factor (PAF) all increased [Ca2+]i in GEN in a concentration-dependent manner, whereas serotonin, acetylcholine, phenylephrine, and endothelin-1 were without effect. Coincubation of glomerular mesangial cells (GMC) with GEN augmented mesangial cell guanosine 3',5'-cyclic monophosphate (cGMP) content five- to sixfold, Bradykinin elicited a further concentration-dependent increase in GMC cGMP content in the presence but not absence of GEN. The GEN-dependent bradykinin-stimulated GMC cGMP accumulation was abolished by hemoglobin and methylene blue, blunted by gossypol, and augmented by superoxide dismutase. Other agonists capable of augmenting GEN [Ca2+]i also stimulated GMC cGMP accumulation in the presence but not in the absence of GEN. Thus cultured GEN release a factor that stimulates cGMP accumulation in adjacent mesangial cells which has the pharmacological characteristics of EDRF.

Animals↗

PubMed1990

Endothelin receptor-coupling mechanisms in vascular smooth muscle: a role for protein kinase C.

Endothelin (ET), a peptide that is released from cultured endothelial cells, is a potent vasoconstrictor that induces characteristically long-lasting contractions. We used the A10 vascular smooth muscle cell (VSMC) line to probe mechanisms underlying ET-induced contractions. Intracellular Ca2+ ([Ca2+]i) and pH were monitored in A10 monolayers using the fluorescent dyes Fura-2 and 2,7-bis-carboxyethyl-5,6-carboxyfluorescein, respectively. Synthetic porcine ET induced rapid and transient increases in [Ca2+]i (EC50 value, 0.75 nM; maximum, approximately 6-fold above basal). External Ca2+ removal did not block the ability of ET (0.5 or 50 nM) to increase initial [Ca2+]i, although [Ca2+]i returned to prestimulus levels faster as compared with that seen in the presence of external Ca2+. Total cell 45Ca2+ content decreased within 30 sec and remained below prestimulus values for at least 20 min (34 +/- 2% decrease after 5 min, n = 3) in ET-stimulated VSMC. ET stimulated a transient rise in inositol trisphosphate formation in [3H]myo-inositol labeled VSMC, peaking in 30 sec (62 +/- 20% increase, n = 3). In contrast, ET-stimulated diacylglycerol formation in [3H]arachidonic acid-labeled VSMC was sustained and biphasic, exhibiting two peaks at 15 sec (41 +/- 16% increase) and at 5 min (75 +/- 7% increase, n = 3). ET (50 nM) also induced an intracellular alkalinization of 0.17 +/- 0.02 (n = 10) pH units above basal.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗

PubMed1989

Subtype-specific increase in G-protein alpha-subunit mRNA by interleukin 1 beta.

The guanine nucleotide regulatory proteins (G-proteins) which are substrates for ADP-ribosylation by pertussis toxin (alpha i-1, alpha i-2, alpha i-3 and alpha o) transduce a variety of hormonal signals. Endothelial cells express mRNA for three alpha i subtypes although the level of alpha i-1 mRNA is very low. Interleukin 1 beta (IL 1 beta), a pleiotropic inflammatory mediator which stimulates a complex series of responses in human endothelial cells leading to increased coagulation and platelet adhesion, increases expression of one subtype of alpha i (alpha i-2) mRNA in human endothelial cells as determined by Northern blot analysis without affecting the level of mRNA for other alpha-subunits. These studies show that mRNA levels for alpha i subtypes are independently regulated, suggesting that there may be subtype specificity in the cell's requirements for the Gi class of signal-transducing proteins.

Adenosine Diphosphate Ribose↗

PubMed1989

Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization.

Cultured endothelial cells release a potent vasoconstrictor peptide, endothelin. Cumulative addition of synthetic endothelin to isolated rabbit aortic rings elicited a concentration-dependent increase in contractile tension which was endothelium-independent. In cultured rabbit vascular smooth muscle cells loaded with the fluorescent dye fura 2, endothelin induced a concentration-dependent increase in [Ca2+]i over the range of 0.01 to 100 nM. Moreover, in the absence of extracellular Ca2+, endothelin could still induce an increase in [Ca2+]i. In addition, endothelin stimulated 45Ca2+ efflux from preloaded vascular smooth muscle cells in the presence and absence of extracellular Ca2+, as well as stimulating 45Ca2+ influx in a concentration-dependent manner. Measurement of inositol phosphates in [3H]-myoinositol-labelled vascular vascular trisphosphate. Unlabelled endothelin inhibited (125I)-endothelin binding to cultured rabbit vascular smooth muscle cells in a concentration-dependent manner. Binding was not inhibited by other vasoactive hormones or calcium channel ligands, suggesting cell surface receptors specific for endothelin. We conclude that one of the initial membrane events in the action of endothelin is to induce phospholipase C-stimulated PIP2 hydrolysis and that this signalling mechanism is initiated by endothelin/receptor interaction at the plasma membrane.

Animals↗

PubMed1989

GTP gamma S increases thrombin-mediated inositol trisphosphate accumulation in permeabilized human endothelial cells.

Ca2+-mobilizing agonists stimulate phospholipase C-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol trisphosphate (IP3) formation in pulmonary as well as in peripheral vascular endothelial cells (EC). In general, it is believed that receptor-phospholipase C interactions involve a guanine nucleotide regulatory (G) protein. This interaction can be inhibited by Bordetella pertussis toxin in certain cells. Here we report that pertussis toxin catalyzes the [32P]ADP ribosylation of a Mr = 41,000 protein in human umbilical vein EC. However, prior EC treatment with pertussis toxin (250 ng/ml for 20 h) does not inhibit thrombin-induced Ca2+ flux or IP3 formation, despite markedly attenuating the radiolabeling of the Mr = 41,000 protein (less than 5% control). Treatment of digitonin-permeabilized human umbilical vein EC with GTP gamma S, a stable GTP analog, or AIF4-, but not with GDP beta S, stimulates IP3 accumulation. However, GDP beta S inhibits GTP gamma S-induced IP3 accumulation. Although thrombin alone is not very effective in elevating IP3 levels in permeabilized EC, thrombin and GTP gamma S act in a synergistic fashion to increase IP3 accumulation. Overall, these observations are interpreted to indicate that a pertussis toxin-insensitive G protein is a key intermediate in the signaling pathway linking thrombin receptors to phospholipase C in human umbilical vein EC.

Calcium↗