Vanadate increases cytosolic free calcium in rat aortic smooth muscle cells

Although several studies have shown that vanadate evokes vasoconstriction whether it elevates cytosolic free calcium, [Ca²⁺]_i, in vascular smooth muscle (VSM) cells has not been investigated. The present study shows that acute additions of low concentrations of vanadate (10–200) to cultured aortic smooth muscle cells (ASMC) produced a rapid and a concentrationdependent increase in [Ca²⁺]_i with an EC₅₀ (mean ± SEM) value of 42 ± 11 μM. Inclusion of vanadate (200 μM) led to a significant increase (p < 0.05) in the peak [Ca²⁺]_i level to 190 ± 23 nM from a basal level of 102 ± 2 nM. At concentrations > 200 μM, vanadate caused quenching of fura-2 fluorescence. For example, addition of 1 mM vanadate led to an apparent decrease in fluorescence by about 50 % (due to a quenching effect), followed by a transient rise. H₂O₂, which is used in the preparation of peroxide forms of vanadate, pervanadate (PV), also produced a rise in [Ca²⁺]_i. These data suggest that vanadate promotes vascular tone by elevating [Ca²⁺]_i in ASMC. However, [Ca²⁺]_i measurements made with higher concentrations of vanadate and PV, using the fura-2 method, must be interpreted with caution.     

Serotonin-induced cytosolic free calcium transients in cultured vascular smooth muscle cells

Serotonin induced a transient elevation in the levels of cytosolic calcium in cultured rat vascular smooth muscle cells. Ketanserin, a selective antagonist of serotonin 2 receptors, dose-dependently inhibited the elevation of cytosolic calcium induced by serotonin, and ultimately unmasked a serotonin-induced decrease in the levels of cytosolic calcium. These observations show that serotonin has direct and dual effects, that is, it increases and decreases cytosolic free calcium concentrations in vascular smooth muscle cells, in culture. Knowledge of such events is important because serotonergic inhibitors may prove to be useful drugs for treating clinical hypertension and vasospastic disorders.     

Platelet-derived growth factor regulates K-Cl cotransport in vascular smooth muscle cells

Platelet-derived growth factor (PDGF), apotent serum mitogen for vascular smooth muscle cells (VSMCs), plays animportant role in membrane transport regulation and in atherosclerosis. K-Cl cotransport (K-Cl COT/KCC), the coupled-movement of K and Cl, isinvolved in ion homeostasis. VSMCs possess K-Cl COT activity and theKCC1 and KCC3 isoforms. Here, we report on the effect of PDGF on K-ClCOT activity and mRNA expression in primary cultures of rat VSMCs. K-ClCOT was determined as the Cl-dependent Rb influx and mRNA expression bysemiquantitative RT-PCR. Twenty four-hour serum deprivation inhibitedbasal K-Cl COT activity. Addition of PDGF increased total proteincontent and K-Cl COT activity in a time-dependent manner. PDGFactivated K-Cl COT in a dose-dependent manner, both acutely (10 min)and chronically (12 h). AG-1296, a selective inhibitor of the PDGFreceptor tyrosine kinase, abolished these effects. Actinomycin D andcycloheximide had no effect on the acute PDGF activation of K-Cl COT,suggesting posttranslational regulation by the drug. Furthermore, PDGFincreased KCC1 and decreased KCC3 mRNA expression in a time-dependentmanner. These results indicate that chronic activation of K-Cl COTactivity by PDGF may involve regulation of the two KCC mRNA isoforms,with KCC1 playing a dominant role in the mechanism of PDGF-mediated activation.

     

Chloride-dependent calcium transients induced by angiotensin II in vascular smooth muscle cells

Cl^– is essential for the vasoconstrictive response to angiotensin II (ANG II). In vascular smooth muscle cells (VSMC), we determined whether ANG II-induced transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is Cl^– dependent. After incubating the cells at different extracellular Cl^– concentration ([Cl^–]_e) for 40 min, the ANG II-induced Ca²⁺ transients at 120 meq/l Cl^– were more than twice those at either 80 or 20 meq/l Cl^–. Replacing Cl^– with bicarbonate or gluconate yielded similar results. In addition, after removal of extracellular Ca²⁺, ANG II-induced as well as platelet-derived growth factor-induced Ca²⁺ release exhibited Cl^– dependency. The difference of Ca²⁺ release with high vs. low [Cl^–]_e was not affected by acutely altering [Cl^–]_e 1 min before administration of ANG II when [Cl^–]_i was yet to be equilibrated with [Cl^–]_e. Pretreatment of a Cl^– channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid, increased ANG II-induced Ca²⁺ release and entry at 20 meq/l Cl^– but did not alter those at 120 meq/l Cl^–. However, after equilibration, a reduced [Cl^–]_e did not affect thapsigargin-induced Ca²⁺ release, suggesting that Cl^– may not affect the size of intracellular Ca²⁺ stores. Nevertheless, at high [Cl^–], the peak increase of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] induced by ANG II was approximately sixfold that at low [Cl^–]. Thus the Cl^–-dependent effects of ANG II on Ca²⁺ transients may be mediated, at least in part, by a Cl^–-dependent Ins(1,4,5)P₃ accumulation in VSMC. anion; inositol 1,4,5-trisphosphate; Ca²⁺ release     

Platelet-derived growth factor is a chemoattractant for vascular smooth muscle cells

In previous experiments (Grotendorst et al, 1981), we showed that platelet-derived growth factor promotes the migration of smooth muscle cells in vitro. Using a "checkerboard" analysis, we now establish that platelet-derived growth factor (PDGF) acts as a true chemoattractant for cultured aortic smooth muscle cells. Other growth factors such as epidermal growth factor, fibroblast growth factor, and insulin are not chemoattractants. The chemotactic response occurs before the initiation of DNA synthesis and is not affected by inhibition of DNA synthesis. Chemotaxis occurs at levels of PDGF lower than required for mitogenesis. RNA and protein synthesis are required for the chemotactic response. As found previously in bacteria and leucocytes, we find that methylation reactions are required for the chemotactic response. The possibility is discussed that PDGF acts in vivo at sites of vascular injury to attract smooth muscle cells from the medial layer to the luminal surface, and is involved in the early stages of the formation of atherosclerotic plaques.     

Increase in cytosolic calcium and phosphoinositide metabolism induced by angiotensin II and [Arg]vasopressin in vascular smooth muscle cells

Effects of angiotensin II and [Arg]vasopressin on cytosolic free Ca2+ concentration ([Ca2+]i) and phosphoinositide metabolism were studied in cultured aortic smooth muscle cells obtained from Wistar-Kyoto rats and their spontaneously hypertensive substrain. [Ca2+]i was measured using the fluorescent Ca2+ indicator quin2. No clear differences in basal [Ca2+]i were detected between cells derived from the two strains. High concentrations of angiotensin II (greater than or equal to 10 nM) and [Arg]vasopressin (greater than or equal to 100 nM) elicited large and rapid increases in [Ca2+]i, followed by a rapid return to control values. Low concentrations of these peptides (less than or equal to 1.0 nM) elicited small and slow increases in [Ca2+]i that persisted for minutes. These responses were blocked by specific antagonists for each of these peptides. Only high concentrations of angiotensin II caused [Ca2+]i increases in "Ca2+-free" medium, which suggested that high concentrations of angiotensin II could release Ca2+ from intracellular pools. A high concentration of angiotensin II and [Arg]vasopressin elicited progressive accumulations of inositol phosphates. Only high concentrations of angiotensin II caused inositol phosphate accumulation in Ca2+-free medium. Maximal accumulation of inositol phosphate elicited by angiotensin II and [Arg]vasopressin was found to be additive. A desensitization to the effects of both peptides on Ca2+ mobilization occurred despite the continued accumulation of inositol phosphates. These observations indicated that angiotensin II and [Arg]vasopressin interacted with independent receptors, both of which are linked to phosphoinositide breakdown and Ca2+ mobilization.     

Effects of prostaglandins on the cytosolic free calcium concentration in vascular smooth muscle cells

The effects of prostaglandin (PG) F2 alpha and 9,11-epithio-11,12-methanothromboxane A2 (STA2), a stable analogue of thromboxane A2, on the cytosolic free calcium concentration ([Ca2+]i) in vascular smooth muscle cells were studied with a new fluorescent Ca2+ indicator fura 2. PGF2 alpha and STA2, which are strong vasoconstrictors, caused rapid phasic and subsequent tonic increases in [Ca2+]i. PGF2 alpha caused dose-dependent elevation of [Ca2+]i not only in control solution but also in the calcium-free solution. A first stimulation with PGF2 alpha caused dose-dependent decrease in the response of [Ca2+]i to a second stimulation with PGF2 alpha. Pretreatment with 13-Azaprostanoic acid, a receptor level antagonist of thromboxane A2 inhibited the increase of [Ca2+]i induced by STA2. These results suggest that PGF2 alpha induces calcium mobilization followed by smooth muscle contraction through its specific receptors.     

Regional and mechanistic differences in platelet-derived growth factor-isoform-induced alterations in cytosolic free calcium in porcine vascular smooth muscle cells

Three specific platelet-derived growth factor (PDGF) isoforms are thought to bind with differing affinities to two distinct PDGF receptors which undergo activation following dimerization. Recent evidence has been presented that marked differences exist between the ability of PDGF-AA versus PDGF-AB and PDGF-BB to stimulate alterations in second messengers in cultures of vascular smooth muscle cells (VSMC), a result which was thought to be due to low numbers of the A-type receptor in this cell type (Sachinidis, A., Locker, R., Vetter, W., Tatje, D., and Hoppe, J. (1990) J. Biol. Chem. 265, 10238-10243, 1990). In particular, PDGF-BB and PDGF-AB but not PDGF-AA could elicit alterations in cytosolic free calcium (Ca2+i). However, because these studies were performed on large cell populations using biochemical assays of PDGF activity, a minor PDGF-AA-Ca(2+)-responsive population of cells might go undetected. To test this possibility, VSMC were isolated from either thoracic or abdominal pig aorta, and alterations in Ca2+i were monitored using Multiparameter Digitized Video Microscopy following stimulation with PDGF isoforms alone, or either before or after exposure of VSMC to 5 mM EGTA. PDGF-AA-responsive cells were found to exist only in cultures of thoracic VSMC, caused oscillations in Ca2+i, represented 20% of the PDGF-BB-responsive cells, and were subsequently responsive to PDGF-BB. PDGF-BB elicited monophasic alterations in Ca2+i in both thoracic and abdominal VSMC. Prior addition of EGTA inhibited PDGF-AA but not PDGF-BB-induced alterations in Ca2+i. Addition of EGTA during PDGF-AA-induced Ca2+i oscillations inhibited subsequent oscillations in Ca2+i, while addition of EGTA at the peak of the PDGF-BB Ca2+ response resulted in a more rapid return of Ca2+i to prestimulation levels. These data suggest that regional differences in the distribution of PDGF-A- and B-type receptor exists in vivo, and that activation of the A- and B-type PDGF receptors results in distinct alterations in Ca2+i.     

Effects of valinomycin on calcium mobilization in vascular smooth muscle cells induced by angiotensin II

The effect of the specific potassium (K+) ionophore valinomycin on increase in intracellular calcium concentration [( Ca2+]i) was studied in vascular smooth muscle cells (VSMC). Valinomycin at more than 10(-9) M dose-dependently suppressed phasic increase in [Ca2+]i in VSMC induced by angiotensin II (AII) in both control and Ca2+-free solution, indicating that it suppressed the release of Ca2+ from intracellular Ca2+ stores. Nicorandil and cromakalim, which are both K+ channel openers, also suppressed the increases in [Ca2+]i induced by AII in the Ca2+ free solution. However, valinomycin did not suppress AII-induced production of inositol 1,4,5-trisphosphate (IP3), which is known to mediate the release of Ca2+. These results indicate that decrease of intracellular K+ induced by valinomycin suppressed the release of Ca2+ from intracellular Ca2+ stores induced by IP3.     

Platelet-derived growth factor induces c-fms and scavenger receptor genes in vascular smooth muscle cells.

Vascular smooth muscle cells proliferate and transform to foam cells in the process of atherosclerosis. In the present study, we demonstrated that platelet-derived growth factor (PDGF)-BB induced expression of proto-oncogene c-fms in vascular smooth muscle cells, which normally do not express c-fms, isolated from either human umbilical artery or rabbit aorta. No effect of the protein kinase C activator, phorbol ester, was demonstrated on mRNA expression of c-fms. In contrast, the scavenger receptor activity was induced by both PDGF-BB and phorbol ester. These results indicate that two characteristic genes of monocyte-macrophages were induced by PDGF-BB via the different pathways, and suggest that PDGF-BB plays an important role in initiating phenotypic conversion of smooth muscle cells to macrophage-like cells.     

Regulation of atrial natriuretic factor receptors by angiotensin II in rat vascular smooth muscle cells

Atrial natriuretic factor (ANF) is actively involved in the control of blood pressure and fluid homeostasis as a physiological antagonist of the renin-angiotensin system. To evaluate a possible interaction between ANF and angiotensin II (Ang-II) receptors, we investigated the effect of long term pretreatment (18 h) of rat cultured vascular smooth muscle cells with Ang-II. Binding of 125I-labeled ANF and cyclic GMP production induced by ANF were measured. After preincubation of the cells with Ang-II (1, 10, and 100 nM), the number of ANF binding sites (Bmax) was decreased by 30, 59, and 71%, respectively, with a slight decrease of the Kd values. Sar1-Ile8-Ang-II (100 nM), a specific Ang-II receptor antagonist, totally inhibited the down-regulation induced by Ang-II (10 nM). Moreover, the regulatory effect of Ang-II on ANF receptors appeared more slowly as compared to ANF homologous receptor regulation. Ang-II pretreatment did not desensitize but increased cyclic GMP production elicited by ANF, implying that only the number of non-guanylate cyclase-coupled receptors was affected. These findings, which were not observed with 100 nM of epinephrine, norepinephrine, histamine, serotonin, and Arg-vasopressin, demonstrate a specific and functional link between ANF and Ang-II receptors. This study also shows that the regulation of ANF receptors is heterogeneous, providing new evidence of multiple classes of ANF receptors.     

Receptor binding activity and cytosolic free calcium response by synthetic endothelin analogs in cultured rat vascular smooth muscle cells

Using a variety of synthetic analogs of porcine endothelin (pET), we have studied the effects of these analogs on receptor binding activity and cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMC). Removal of C-terminal Trp21 residue, truncated derivatives pET(1-15) and (16-21), substitution of disulfide bond, Cys(3-11) or Cys(1-15), by Cys (Acm), all resulted in a complete loss of receptor binding activity and [Ca2+]i response, while N-terminal elongation of Lys-Arg residues, but not oxidation of Met7 residue, decreased receptor binding activity and [Ca2+]i response. [Cys1-15,Cys3-11]pET was far more potent than [Cys1-11,Cys3-15]pET in receptor binding and [Ca2+]i response. These data indicate that the C-terminal Trp21 as well as the proper double cyclic structure formed by the intramolecular disulfide bonds of the pET molecule are essential for receptor binding and subsequent [Ca2+]i increase in rat VSMC.     

Platelet-derived growth factor and transforming growth factor-beta regulate plasminogen activator inhibitor-1 synthesis in vascular smooth muscle cells

Bovine vascular smooth muscle cells (SMC) were examined for production of plasminogen activator inhibitor-1 (PAI-1) which may play a key role in regulating the fibrinolytic system. Growth-arrested SMC released active PAI (101 arbitrary units (AU)/10(6) cells/24 h) and a latent form of PAI (880 AU/10(6) cells/24 h) into the conditioned medium (CM). The levels of PAI were significant since 880 AU of PAI could inhibit approximately 1 microgram of tissue plasminogen activator. The extracellular matrix of SMC also contained PAI activity; however, the level was 17-fold less than that observed in the CM. SMC-PAI was a rapid inhibitor of tissue plasminogen activator (kass greater than 10(7) M-1 S-1) and was identified as a 45-kDa protein immunologically related to endothelial cell PAI-1. PAI-1 comprised 20 and 30%, respectively, of the newly synthesized protein detected in the CM and extracellular matrix of SMC. The SMC growth modulators, platelet-derived growth factor and transforming growth factor-beta, induced PAI-1 activity and protein synthesis by 2- and 3-fold, respectively, in a dose- and time-dependent manner. The increases in PAI-1 activity and protein synthesis were ascribed to elevated levels of PAI-1 mRNA as judged by Northern blot analysis of total RNA prepared from control and platelet-derived growth factor- and transforming growth factor-beta-treated cells. Increases in PAI-1 mRNA levels were evident 1 h after growth factor treatment and were maximal after 4 h. PAI-1 mRNA levels were unaffected by cycloheximide treatment. The results indicate that SMC synthesize and release PAI-1 which could regulate the normal fibrinolytic environment of the arterial wall. During atherosclerosis or after vascular injury increases in platelet-derived or locally produced mitogens may stimulate further PAI-1 synthesis and generate a prothrombotic state.     

Functional angiotensin II receptors in cultured vascular smooth muscle cells

To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (AII) were identified and characterized using the radioligand 125I-angiotensin II. Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific 125I-AII binding similar to that seen with homogenates of the intact rat mesenteric artery. In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 +/- 0.2 nM and a total binding capacity of 81.5 +/- 5.0 fmol/mg protein (equivalent to 4.5 x 10(4) sites per cell). Angiotensin analogues and antagonists inhibited 125I-AII binding to cultured VSMC in a potency series similar to that observed for the vascular AII receptor in vivo. Nanomolar concentrations of native AII elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sar1,Ile8-AII. Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 125I-AII binding sites or hormonal responsiveness. Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle.     

Platelet-derived growth factor receptors direct vascular development independent of vascular smooth muscle cell function

Complete loss of platelet-derived growth factor (PDGF) receptor signaling results in embryonic lethality around embryonic day 9.5, but the cause of this lethality has not been identified. Because cardiovascular failure often results in embryonic lethality at this time point, we hypothesized that a failure in cardiovascular development could be the cause. To assess the combined role of PDGF receptor α (PDGFRα) and PDGFRβ, we generated embryos that lacked these receptors in cardiomyocytes and vascular smooth muscle cells (VSMC) using conditional gene ablation. Deletion of either PDGFRα or PDGFRβ caused no overt vascular defects, but loss of both receptors using an SM22α-Cre transgenic mouse line led to a disruption in yolk sac blood vessel development. The cell population responsible for this vascular defect was the yolk sac mesothelial cells, not the cardiomyocytes or the VSMC. Coincident with loss of PDGF receptor signaling, we found a reduction in collagen deposition and an increase in MMP-2 activity. Finally, in vitro allantois cultures demonstrated a requirement for PDGF signaling in vessel growth. Together, these data demonstrate that PDGF receptors cooperate in the yolk sac mesothelium to direct blood vessel maturation and suggest that these effects are independent of their role in VSMC development.