Endothelin-mediated stimulation of DNA synthesis in vascular smooth muscle cells

Effects of endothelin on DNA synthesis were investigated in two clones of vascular smooth muscle cells, 1YB4 and A7r5. The peptide stimulated DNA synthesis in both clones with apparent EC50 of less than 1 ng/ml. More than 17 h was required before initiating endothelin-stimulated DNA synthesis. The platelet-derived growth factor at a concentration which had no effects by itself on DNA synthesis enhanced the effect of low concentrations of endothelin. A calcium antagonist, nifedipine, inhibited endothelin-induced DNA synthesis. These data suggest that endothelin stimulates DNA synthesis in vascular smooth muscle cells through nifedipine-sensitive mechanisms that can be modulated by platelet-derived growth factor.     

Adenosine as a natural prostaglandin antagonist in vascular smooth muscle

Adenosine has actions on smooth muscle similar to those of prostaglandin (PG) antagonists. Like some PG antagonists it is a phosphodiesterase inhibitor and seems to interfere with calcium effects. It has agonist/antagonist interactions with theophylline, a PG antagonist. In rat mesenteric vascular smooth muscle adenosine blocked responses to noradrenaline which depend on release of intracellular calcium but not those to potassium ions which depend on calcium entry from extracellular fluid. Partial inhibition of endogenous PG synthesis by indomethacin enhanced the adenosine effect. In preparations in which vascular reactivity had been abolished by indomethacin and then partly restored by 1 or 5 ng/ml PGE2, adenosine also inhibited responses to noradrenaline: the curve for the 5 ng/ml PGE2 concentration was to the right of and parallel to the 1 ng/ml curve consistent with a competitive interaction between adenosine and PGE2. Similar interactions between adenosine and PGE2 were shown in human lymphocytes in which activation also depends on calcium release. These findings suggest how calcium-dependent metabolic responses may be controlled and indicate further reasons for caution in the interpretation of cyclic AMP experiments.     

Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells

Recently, a potent vasoconstrictor peptide, endothelin (EDT), was isolated from vascular endothelial cells. We examined its effect on rat vascular smooth muscle cells (VSMCs). EDT induced the elevation of intracellular calcium, which was dependent on extracellular calcium and inhibited by a calcium-channel antagonist in a competitive manner. EDT caused a rapid and transient increase in the c-fos and c-myc mRNA levels and stimulated the DNA synthesis of VSMCs in a dose-dependent manner. This effect of EDT on the proliferation of VSMCs might be related to the development of atherosclerosis.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Ca2+ requirement of prostanoid but not of superoxide production by rat Kupffer cells

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 microM free extracellular Ca2+ and greater than or equal to 100 microM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.     

Differential regulation of smooth muscle contraction in rabbit internal anal sphincter by substance P and bombesin.

Substance P and bombesin induce contraction of isolated IAS smooth muscle cells by different intracellular mechanisms. The cells contracted in a dose dependent manner to both peptides. The kinetics of contraction were different. Substance P induced contraction peaked at 30 seconds and declined in a time dependent manner while bombesin induced contraction peaked at 30 seconds and was maintained for up to 8 minutes. The absence of extracellular calcium in the medium (0 calcium and 2 mM EGTA) had no affect on substance P induced contraction while it blocked bombesin induced contraction. Substance P induced contraction was blocked by the calmodulin antagonist W7 (10(-9)M) and was not affected by the PKC antagonist H7 (10(-6)M). Bombesin induced contraction was blocked by the PKC antagonist H7 and was not affected by the calmodulin antagonist W7. Our data indicate that substance P induces a transient contraction utilizing intracellular calcium and a calmodulin dependent pathway, while bombesin induces a sustained contraction utilizing calcium from extracellular sources and a calmodulin independent pathway.     

Effects of nicotine on phenotypic modulation and initiation of DNA synthesis in cultured arterial smooth muscle cells

During the early stages of atherogenesis, as well as during in vitro cultivation, smooth muscle cells modulate from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and the formation of an extensive rough endoplasmic reticulum and a large Golgi complex; it leads to decreased contractility and the commencement of cell growth and secretion of extracellular matrix components. In this paper, the effects of nicotine on adult rat arterial smooth muscle cells cultivated in vitro were studied by transmission electron microscopy and 3H-thymidine autoradiography. The results show that the drug speeded the initial rate of transition of the cells from contractile to synthetic phenotype in primary culture. Further, it stimulated the initiation of DNA synthesis in growth-arrested secondary cultures. Its effect was independent of other mitogens and additive to that of serum. The influences of nicotine, both on the modulation of the smooth muscle phenotype and the initiation of DNA synthesis, occurred at concentrations lower than those obtained in the blood after smoking and could contribute to the role of smoking as a risk factor for atherosclerosis.     

The effects of calmodulin antagonist drugs on isolated sarcoplasmic reticulum from malignant hyperpyrexia susceptible swine

1. Because calcium antagonist drugs increase contracture in both control and malignant hyperpyrexia susceptible (MHS) skeletal muscle, the effect of these drugs on the sarcoplasmic reticulum (SR) was investigated. 2. The calmodulin antagonist drugs inhibited the Ca2+ dependent ATPase activity and the ATP-dependent Ca2+ uptake, and accelerated the efflux of Ca2+ from isolated SR preparations from both control and MHS skeletal muscle. These effects of calmodulin antagonist drugs on SR Ca2+ transport functions were consistent with their in vitro pharmacological effects on control and MHS muscle.     

An extracellular role for calmodulin-like activity in cell proliferation.

1. Addition of extracellular pure pig brain calmodulin was found to modulate DNA synthesis and cell proliferation in K562 human leukaemic lymphocytes. At lower cell densities calmodulin significantly stimulated [3H]thymidine uptake; at higher densities it decreased it. 2. A protein biochemically indistinguishable from calmodulin was detected in the cell-conditioned media of rapidly dividing K562 cells. The concentration of calmodulin-like activity found in the conditioned media of these and a range of other normal and neoplastic cells (250-1636 ng/ml) was of the same order as would stimulate DNA synthesis in subconfluent cells. 3. Amounts of extracellular calmodulin-like activity and immunoreactivity varied during cell growth from low to high density, a peak of extracellular calmodulin preceding DNA synthesis in synchronized K562 cells. Extracellular calmodulin concentrations did not correlate with the presence of lactate dehydrogenase in the medium. 4. Inhibition of extracellular calmodulin activity by calmodulin antagonist immobilized on agarose beads, or by antibody to calmodulin, significantly decreased DNA synthesis. 5. These data strongly suggest that calmodulin or a very closely related protein can influence mitosis through an extracellular mechanism.     

Coupling between inositol phosphate formation and DNA synthesis in smooth muscle cells stimulated with neurokinin A

The two mammalian neuropeptides substance P (SP) and neurokinin A (NKA) have been demonstrated to stimulate DNA synthesis in connective tissue cells, suggesting that peripheral neurons may play a role in development and tissue regeneration. In this study we have tried to identify intracellular messengers required for SP- and NKA-induced DNA synthesis. SP and NKA, as well as platelet-derived growth factor (PDGF) stimulated formation of inositol phosphates in smooth muscle cells (SMC), whereas no effect on inositol phosphates formation occurred in response to nonmitogenic neuropeptides. Pretreatment of the cells with pertussis toxin markedly decreased DNA synthesis induced by NKA. This toxin inhibits formation of inositol phosphates by acting on a regulatory G-protein. Calcium and calmodulin antagonists also inhibited NKA-induced DNA synthesis. These results imply that the mitogenic signal(s) produced by activated neuropeptide receptors involves formation of inositol phosphate and activation of a calcium/calmodulin dependent process. We further report that other neuropeptides occurring in peripheral neurons, i.e., vasoactive intestinal polypeptide, calcitonin gene-related peptide, neuropeptide Y, somatostatin, or cholecystokinin, are without growth-stimulatory effect on cultured SMC.     

The role of calcium in DNA synthesis and development of mouse preimplantation embryos in vitro.

The effect of calcium upon embryonic growth was studied using cultured mouse preimplantation embryos. Both morphological development of the embryos and embryo DNA synthesis were shown to be dependent on the Ca2+ concentration in the medium in which the embryos were grown. Reduction of the calcium concentration below 10(-5) M completely blocked cell division and blastocyst formation in the cultured embryos, but only moderately inhibited embryo DNA synthesis. Trifluoperazine, a calmodulin antagonist, strongly inhibited the Ca(2+)-dependent DNA synthesis in the embryos. On the other hand, the drug only slightly affected the morphological development of the embryos. These results demonstrate that calcium independently affects two different aspects of the embryo development, i.e. DNA synthesis and cell division. It is suggested that the former effect is calmodulin-dependent, while the latter involves the calcium-dependence of metabolite transport through the cell membranes.     

Effect of trifluoperazine on DNA and LDL-receptor synthesis in smooth muscle cells exposed to hypercholesterolemic medium in vitro

We recently demonstrated that calmodulin and/or protein kinase C may play a crucial role in cholesterol-induced atherogenesis in experimental animal model system. The present study, which was undertaken to elucidate the effect of trifluoperazine (known as a potent inhibitor of calmodulin and protein kinase C) on DNA and LDL-receptor synthesis of aortic smooth muscle cells exposed to hypercholesterolemic medium, revealed that (a) trifluoperazine at a concentration of 25 microM caused an approximately threefold increase in the [35S]methionine-incorporated LDL-receptor protein as compared with values found in control cells; (b) the drug at concentrations greater than or equal to 0.1 microM caused inhibition of DNA synthesis as compared with values found in control cells. These results demonstrate that the preventive effect of trifluoperazine on the atherogenic activity of smooth muscle cells may be due to its ability to increase LDL-receptors synthesis as well as concomitant inhibitory action on DNA synthesis of smooth muscle cells exposed to hypercholesterolemic medium.     

Parathyroid hormone causes a transient rise in intracellular ionized calcium in vascular smooth muscle cells

The effect of parathyroid hormone on intracellular calcium concentration in vascular smooth muscle cells in culture was studied. Human PTH 1-34 (hPTH (1-34)) caused a transient rise in intracellular calcium in a dose-dependent manner at physiological concentrations. The effect of PTH was mimicked by dibutyryl cyclic AMP and inhibited by a PTH receptor antagonist. The effect of PTH was increased in parallel with extracellular calcium concentration and a sustained response was observed when extracellular calcium was 2 mM or higher. The PTH action was blocked by nisoldipin, a calcium antagonist, but not by ouabain, a Na, K-ATPase inhibitor. These data indicate that PTH increases intracellular calcium through its receptor via opening calcium channels. A possible role of this effect in the regulation of vascular tone is also discussed.     

Effects of calcium-antagonists and calmodulin inhibitors on DNA synthesis in cultured rat vascular smooth muscle cells

To investigate the role of intracellular Ca2+ in the mechanism of cellular proliferation of vascular smooth muscle cells (VSMC), the effects of Ca2+-antagonists and calmodulin (CaM) inhibitors on DNA synthesis stimulated by serum-derived growth factors were studied in cultured VSMCs derived from rat aorta. DNA synthesis assessed by incorporation of [3H]thymidine into the cells was significantly stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF) or fetal bovine serum (FBS), of which the effects were dose-dependently inhibited by a variety of Ca2+-antagonists, such as verapamil, diltiazem and nicardipine. Trifluoperazine and W-7, both specific CaM inhibitors, similarly inhibited DNA synthesis stimulated by EGF, PDGF or FBS in a dose-dependent manner, whereas W-5, a less specific CaM inhibitor, was minimally effective. These data suggest that the Ca2+-CaM system plays an important role in the mechanism of growth factor-induced DNA synthesis in VSMCs.     

Comparison of the effect of ATP on intracellular calcium ion dynamics between rat testicular and cerebral arteriole smooth muscle cells

Adenosine 5'-triphosphate (ATP), when released from neuronal and non-neuronal tissues, interacts with cell surface receptors produces a broad range of physiological responses. The goal of the present study was to examine the issue of whether vascular smooth muscle cells respond to ATP. To this end, the dynamics of the intracellular concentration of calcium ions ([Ca(2+)](i)) in smooth muscle cells in testicular and cerebral arterioles was examined by laser scanning confocal microscopy. ATP produced an increase in [Ca(2+)](i) in arteriole smooth muscle cells. While P1 purinoceptor agonists had no effect on this process, P2 purinoceptor agonists induced a [Ca(2+)](i) increase and a P2 purinoceptor antagonist, suramin, completely inhibited ATP-induced [Ca(2+)](i) dynamics in both arteriole smooth muscle cells.In testicular arterioles, Ca(2+) channel blockers and the removal of extracellular Ca(2+), but not thapsigargin pretreatment, abolished the ATP-induced [Ca(2+)](i) dynamics. In contrast, Ca(2+) channel blockers and the removal of extracellular Ca(2+) did not completely inhibit ATP-induced [Ca(2+)](i) dynamics in cerebral arterioles. Uridine 5'-triphosphate caused an increase in [Ca(2+)](i) only in cerebral arterioles and alpha,beta-methylene ATP caused an increase in [Ca(2+)](i) in both testicular and cerebral arterioles.We conclude that testicular arteriole smooth muscle cells respond to extracellular ATP via P2X purinoceptors and that cerebral arteriole smooth muscle cells respond via P2X and P2Y purinoceptors.     

Ca2+/calmodulin is involved in growth factor-induced retinoblastoma gene product phosphorylation in human vascular endothelial cells.

In human vascular endothelial cells, both growth factor-induced DNA synthesis and retinoblastoma gene product (RB) phosphorylation are absolutely dependent on extracellular Ca2+, and are potently inhibited by an active calmodulin antagonist, W-7, but not an inactive analogue, W-12. A reduction in the extracellular Ca2+ or an addition of W-7 as late as 8 h after growth factor stimulation still inhibits both RB phosphorylation and DNA synthesis to the full extent. However, once RB phosphorylation occurs 12-16 h after addition of the growth factors, it is not reversed by subsequent Ca2+ reduction or W-7. These results suggest the existence of a Ca2+/calmodulin-dependent process relatively late in the mitogenic signalling cascade, at a step proximal to RB phosphorylation reaction itself.     

Antisense oligonucleotides to stromelysin mRNA inhibit injury-induced proliferation of arterial smooth muscle cells.

Smooth muscle cell migration and proliferation are important events in the formation of intimal lesions associated with atherosclerosis and restenosis following balloon angioplasty. To make this possible, the smooth muscle cell has to change from a contractile to an activated repair cell with capacity to synthesize DNA and extracellular matrix components. There is now considerable evidence that the extracellular matrix has important functions in modulating the phenotypic properties of smooth muscle cells, but less is known about the role of the matrix metalloproteinases. The present study investigates the role of stromelysin in the modulation of rat aortic smooth muscle cell morphology and function following mechanical injury in vitro and in vivo. Antisense mRNA oligonucleotides were used to investigate the role of stromelysin expression in injury-induced phenotypic modulation and the subsequent migration and proliferation of vascular smooth muscle cells. Cultured rat aortic smooth muscle cells and balloon-injured rat carotid arteries were used as experimental models. Light- and electron microscopy were used to follow changes in smooth muscle cell phenotype and lesion formation and incorporation of 3H-thymidine to detect DNA synthesis. Injury-induced DNA synthesis and migration in vitro were inhibited by 72% and 36%, respectively, by adding stromelysin antisense oligonucleotides to the medium prior to injury. In primary cultures, 67% of the smooth muscle cells treated with stromelysin antisense were retained in a contractile phenotype as judged by analysis of cell fine structure, compared to 15% untreated cells and 40% in cells treated with mismatched oligonucleotides. Examination of the carotid arteries one week after balloon injury likewise demonstrated a larger fraction of contractile cells in the inner parts of the media in vessels treated with antisense oligonucleotides compared to those treated with mismatched oligonucleotides. The neointima was also distinctly thinner in antisense-treated than in mismatched-treated and control arteries at this time. These findings indicate that stromelysin mRNA antisense oligonucleotides inhibited phenotypic modulation of rat arterial smooth muscle cells and so caused a decrease in migration and proliferation and neointima formation in response to vessel wall injury.     

Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells

The effects of adenosine and two analogs, L-phenylisopropyladenosine (L-PIA) and 5'-N-ethylcarboxamidoadenosine (NECA), on cAMP production and on platelet-derived growth factor (PDGF)-stimulated initiation of DNA synthesis in growth-arrested cultures of rat arterial smooth muscle cells (SMC) were studied. The intracellular cAMP concentration was dose-dependently enhanced by micromolar concentrations of adenosine and its analogs, with the potency order NECA greater than adenosine greater than L-PIA. The effect was antagonized, in a competitive manner, by the adenosine receptor antagonist 8-phenyltheophylline (8-PT). The stimulatory effect of adenosine was enhanced by 3 microM dipyridamole an adenosine-uptake blocker. DNA synthesis was inhibited in a parallel manner, showing the same potency order. The inhibition was antagonized by 8-PT. Forskolin, a diterpene with the ability to stimulate the catalytic unit of adenylate cyclase and thereby cAMP formation, potentiated the effects of micromolar concentrations of NECA and L-PIA. Forskolin, by itself, stimulated cAMP production and inhibited DNA synthesis. The forskolin-stimulated increase in cAMP was inhibited by L-PIA at nanomolar concentrations. L-PIA in the nanomolar concentration range also stimulated DNA synthesis when initiation was stimulated with suboptimal concentrations of PDGF. These findings suggest the presence of adenosine receptors of both the A1- and A2-subtype on SM-mediating bidirectional changes of cAMP and DNA synthesis.     

A novel ETA antagonist (BQ-123) inhibits endothelin-1-induced phosphoinositide breakdown and DNA synthesis in rat vascular smooth muscle cells.

The effects of a novel cyclic pentapeptide (BQ-123), an endothelin (ET) antagonist selective for the ETA receptor subtype, on phosphoinositide breakdown and DNA synthesis stimulated by ET-1 were studied in cultured rat vascular smooth muscle cells (VSMC). BQ-123 competitively inhibited the binding of [125I]ET-1 to VSMC with the apparent Ki of 4 x 10(-9) M. BQ-123 dose-dependently inhibited formation of inositol-1,4,5-trisphosphate and [3H]thymidine uptake stimulated by ET-1. These data suggest that the ET-1-induced DNA synthesis in VSMC is mainly mediated by ETA receptor subtype.