Cerebral Vascular Smooth Muscle Cells Research Articles

Peroxynitrite induces apoptosis in canine cerebral vascular muscle cells: possible relation to neurodegenerative diseases and strokes

Considerable evidence is accumulating to suggest that in vivo formation of free radicals in the brain, such as peroxynitrite (ONOO −), and programmed cell death (i.e. apoptosis) play important roles in neurodegeneration and stroke. However, it is not known whether ONOO − can induce apoptosis in cerebral vascular smooth muscle cells (CVSMCs). The present study was designed to determine whether or not canine CVSMCs undergo apoptosis following treatment with ONOO −. Direct exposure of canine CVSMCs to ONOO − induced apoptosis in a concentration-dependent manner, as confirmed by means of fluorescence staining, TdT-mediated dUTP nick-end labeling and comet assays. Peroxynitrite treatment resulted in an elevation of [Ca 2+] i in the CVSMCs. Peroxynitrite-induced apoptosis may thus be brought about by activation of Ca 2+-dependent endonucleases. Although the precise mechanisms by which peroxynitrite induces apoptosis need to be further investigated, the present findings could be used to suggest that ONOO − formation in the brain may play important roles in neurodegenerative processes and strokes via detrimental actions on cerebral microvessels and blood flow.

Expression of the nuclear factor-κB and proto-oncogenes c-Fos and c-Jun are induced by low extracellular mg 2+ in aortic and cerebral vascular smooth muscle cells: possible links to hypertension, atherogenesis, and stroke

Proto-oncogene (c-fos, c-jun) and nuclear factor-kappa B (NF-κB) expression, as well as DNA synthesis, in aortic and cerebral vascular smooth muscle cells (VSMCs) were upregulated by a decrease in extracellular magnesium ions ([Mg 2+] o). Upregulation of these transcriptional factors was inversely proportional to the [Mg 2+] o and occurred over the pathophysiologic range of serum Mg 2+ found in patients presenting with hypertension, ischemic heart disease, and stroke. Removal of extracellular Ca 2+ ([Ca 2+] o), use of nifedipine or protein kinase C (PKC) inhibitors prevented the upregulation of the proto-oncogenes and DNA synthesis in VSMCs. These data show that [Mg 2+] o may be an important, heretofore, overlooked natural modulator of proto-oncogene and NF-κB expression in VSMCs and that Ca 2+ and PKC may play critical roles in induction of c-fos and c-jun in VSMCs induced by a decrease in [Mg 2+] o. These results point to a role for low serum Mg 2+ in potential development of hypertension, atherogenesis, vascular disease, and stroke.

Voltage-gated K+ channels in rat small cerebral arteries: molecular identity of the functional channels.

Voltage-gated potassium (KV) channels represent an important dilator influence in the cerebral circulation, but the composition of these tetrameric ion channels remains unclear. The goals of the present study were to evaluate the contribution of KV1 family channels to the resting membrane potential and diameter of small rat cerebral arteries, and to identify the alpha-subunit composition of these channels using patch-clamp, molecular and immunological techniques. Initial studies indicated that 1 micromol l(-1) correolide (COR), a specific antagonist of KV1 channels, depolarized vascular smooth muscle cells (VSMCs) in pressurized (60 mmHg) cerebral arteries from -55 +/- 1 mV to -34 +/- 1 mV, and reduced the resting diameter from 152 +/- 15 microm to 103 +/- 20 microm. In patch clamped VSMCs from these arteries, COR-sensitive KV1 current accounted for 65 % of total outward KV current and was observed at physiological membrane potentials. RT-PCR identified mRNA encoding each of the six classical KV1 alpha-subunits, KV1.1-1.6, in rat cerebral arteries. However, only the KV1.2 and 1.5 proteins were detected by Western blot. The expression of these proteins in VSMCs was confirmed by immunocytochemistry and co-immunoprecipitation of KV1.2 and 1.5 from VSMC membranes suggested KV1.2/1.5 channel assembly. Subsequently, the pharmacological and voltage-sensitive properties of KV1 current in VSMCs were found to be consistent with a predominant expression of KV1.2/1.5 heterotetrameric channels. The findings of this study suggest that KV1.2/1.5 heterotetramers are preferentially expressed in rat cerebral VSMCs, and that these channels contribute to the resting membrane potential and diameter of rat small cerebral arteries.

Low extracellular magnesium ions induce lipid peroxidation and activation of nuclear factor-kappa B in canine cerebral vascular smooth muscle: possible relation to traumatic brain injury and strokes

The present study was designed to test the hypothesis that administration of low extracellular levels of magnesium ions ([Mg 2+] o) to primary cultured cerebral vascular smooth muscle cells will cause lipid peroxidation, degradation of IκB-α, and activation of nuclear transcription factor kappa B (NF-κB) in cultured cerebral vascular smooth muscle cells. Low [Mg 2+] o (0, 0.15, 0.3 and 0.48 mM) resulted in concentration-dependent rises in malondialdehyde (MDA) in as little as 3 h after exposure to low [Mg 2+] o, rising to levels 3–12×normal after 18–24 h; the lower the [Mg 2+] o, the higher the MDA level. Using electrophoretic mobility shift assays and specific antibodies, low [Mg 2+] o caused two DNA-binding proteins (p50, p65) to rise in nuclear extracts in a concentration-dependent manner. High [Mg 2+] o (i.e. 4.8 mM) downregulated p50 and p65. Using a rabbit antibody, IκB phosphorylation (and degradation) was stimulated by low [Mg 2+] o (in a concentration-dependent manner) and inhibited by a low concentration of the NF-κB inhibitor, pyrrolidine dithiocarbamate. These new biochemical and molecular data indicate that low [Mg 2+] o, in concentrations found in the blood of patients, after traumatic brain injury (TBI) and diverse types of strokes, can elicit rapid lipid peroxidation and activation of NF-κB in cerebral vascular smooth muscle cells. The present results, when viewed in light of other recently published data, suggest that low [Mg 2+] o-induced lipid peroxidation and activation of NF-κB play important roles in TBI and diverse types of strokes.

Catalase prevents elevation of [Ca 2+] i induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke and brain pathology

Several studies have suggested that alcohol-induced brain injury is associated with generation of reactive oxygen species (ROS). The recent findings, that antioxidants (Vitamin E and pyrrolidine dithiocarbamate (PDTC)) prevent intracellular Ca 2+ ([Ca 2+] i) overload in cerebral vascular smooth muscle cells, induced by alcohol, demonstrate indirectly that ROS formation is related to cerebral vascular injury. The present experiments were designed to test the hypothesis that catalase, an hydrogen peroxide (H 2O 2) scavenging enzyme, can prevent or ameliorate alcohol-induced elevation of [Ca 2+] i. Preincubation of cultured canine cerebral vascular smooth muscle cells with catalase (20–1000 units/ml) didn’t produce any apparent changes from controls in resting levels of [Ca 2+] i after 1–3 days. Exposure of the cerebral vascular cells to culture media containing 10–100 mM ethanol resulted in significant rises in [Ca 2+] i ( p<0.01). Although exposure of these cells to a low concentration of catalase (20 units/ml) failed to prevent the increased level of [Ca 2+] i induced by ethanol, concomitant addition of higher concentrations of catalase (100–1000 units/ml) and ethanol (10–100 mM) inhibited or ameliorated the rises of [Ca 2+] i induced by ethanol either at 24 h or at 3 days, in a concentration-dependent manner. Catalase, in the range of 100–200 units/ml, inhibited ∼50% of the [Ca 2+] i increases caused by ethanol in the first 24 h. Catalase at a concentration of 1000 units/ml inhibited completely excessive [Ca 2+] i accumulation. The present results when viewed in light of other recently published data suggest that H 2O 2 generation may be one of the earliest events triggered by alcohol in alcohol-induced brain-vascular damage, neurobehavioral actions and stroke.

Hypoxia Activates a 238-pS KCa Channel in Cerebral Arterial Vascular Smooth Muscle Cells by a Mechanism That Involves Reduction of 20-HETE Level

Hypoxia Activates a 238-pS KCa Channel in Cerebral Arterial Vascular Smooth Muscle Cells by a Mechanism That Involves Reduction of 20-HETE Level

Reduction of bFGF-induced smooth muscle cell proliferation and endothelin receptor mRNA expression by mevastatin and atorvastatin

The anti-atherosclerosis mechanisms of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) occur via both cholesterol-dependent and cholesterol-independent mechanisms. The present study used aortic and cerebral vascular smooth muscle cells (SMC) from rat to investigate whether atorvastatin and mevastatin affect basic fibroblast growth factor (bFGF)-induced SMC proliferation and the mRNA expression of endothelin A (ET A) and endothelin B (ET B) receptors. Cell proliferation was assessed by MTT and real-time PCR was used to quantify ET A and ET B receptor mRNA. bFGF-induced concentration and time dependent SMC proliferation and up-regulation of the mRNA expression of ET A and ET B receptors. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors inhibited bFGF-induced proliferation of SMC ( P<0.01). In aortic SMC atorvastatin and mevastatin significantly inhibited bFGF-induced mRNA expression of endothelin ET A and ET B receptors ( P<0.05). Although in cerebral SMC the inhibitory effect of the statins was comparable in size with that seen in aortic SMC, only reached borderline significance ( P=0.06) for ET A receptor mRNA but not for ET B. The findings suggested a direct effect of statins on the vascular wall beyond their well-known lipid lowering effect in anti-atherosclerosis. Furthermore, the specific antagonists of ET A and ET B receptors (FR139317 and BQ788, respectively) significantly inhibited bFGF-induced SMC proliferation ( P<0.001). The results suggested that endothelin receptors and the mevalonate pathway were involved in bFGF-induced SMC proliferation.

Ethanol induces rapid lipid peroxidation and activation of nuclear factor-kappa B in cerebral vascular smooth muscle: relation to alcohol-induced brain injury in rats

The present study was designed to test the hypothesis that acute administration of alcohol (ethanol) to primary cultured cerebral vascular smooth muscle cells will cause lipid peroxidation, inhibition of IκB phosphorylation, and inhibition of nuclear transcription factor-kappa B (NF-κB). Ethanol (10, 25, 100 mM) resulted in concentration-dependent rises in malondialdehyde in as little as 30–45 min after exposure to the alcohol, rising to levels 2.5–10× normal after 18–24 h. Using EMSA assays and specific antibodies, ethanol caused three DNA-binding proteins (p50, p65, c-Rel) to rise in nuclear extracts in a concentration-dependent manner. Using a rabbit antibody, IκB phosphorylation (and degradation) was stimulated by ethanol (in a concentration-dependent manner) and inhibited by a low concentration of the NF-κB inhibitor, pyrrolidine dithiocarbamate. These new biochemical and molecular data indicate that ethanol, even in physiologic concentrations, can elicit rapid lipid peroxidation and activation of NF-κB in cerebral vascular muscle cells. The present results when viewed in light of other recently published data suggest that ethanol-induced lipid peroxidation and activation of nuclear transcription factors probably play important roles in alcohol-induced brain-vascular damage, neurobehavioral actions and stroke.

Connexin45 gap junction channels in rat cerebral vascular smooth muscle cells.

Cells in blood vessel walls express connexin (Cx)43, Cx40, and Cx37. We recently characterized gap junction channels in rat basilar artery smooth muscle cells and found features attributable not only to these three connexins but also to an unidentified connexin, including strong voltage dependence and single channel conductance of 30-40 pS. Here, we report data consistent with identification of Cx45. Immunofluorescence using anti-human Cx45 and anti-mouse Cx45 antibodies revealed labeling between alpha-actin-positive cells, and RT-PCR of mRNA from arteries after endothelial destruction yielded amplicons exhibiting 90-98% identity with mouse Cx45 and human Cx45. Dual-perforated patch clamping was performed after exposure to oligopeptides that interfere with docking of Cx43, Cx40, or Cx45. Cell pairs pretreated with blocking peptides for Cx43 and Cx40 exhibited strongly voltage-dependent transjunctional conductances [voltage at which voltage-dependent conductance declines by one-half (V1/2) = +/-18.9 mV] and small single channel conductances (31 pS), consistent with the presence of Cx45, whereas cell pairs pretreated with blocking peptide for Cx45 exhibit weaker voltage-dependent conductances (V1/2 = +/-37.9 mV), consistent with block of Cx45. Our data suggest that Cx45 is transcribed, expressed, and forms functional gap junction channels in rat cerebral arterial smooth muscle.

Importance of magnesium ions in development of tolerance to ethanol: studies on cultured cerebral vascular smooth muscle cells, type-2 astrocytes and intact rat brain

This study was designed to examine the roles of intracellular free magnesium ion concentration ([Mg2+]i) in ethanol-induced intoxication and development of tolerance in cultured canine cerebral vascular smooth muscle cells and astrocytes as well as intact rat brain. The basal, resting level of [Mg2+]i in cerebrovascular cells was 732.5 ± 82.4 μM. Exposure of cultured canine cerebral vascular smooth muscle cells to ethanol (10 and 25 mM) for 24 h reduced the concentrations of [Mg2+]i to 521.1 ± 59.6 μM, and 308.2 ± 37.8μM, respectively. However, exposure of these cultured vascular cells to the same concentrations of ethanol, after initial pretreatment with ethanol for 24 h, failed to interfere with the levels of [Mg2+]i. Measurement of [Mg2+]i at 48 h and 72 h indicated that the decreased levels of [Mg2+]i induced by ethanol at 24 h treatment returned toward baseline. Similar experiments were performed in cultured type-2 astrocytes isolated from neonatal rat brain. The basal level of [Mg2+]i in type-2 astrocytes was about 125 μM. Incubation of these cells with 10 mM ethanol for 10 min resulted in a 27% reduction in the level of [Mg2+]i, whereas incubation with 25 mM ethanol resulted in almost a 50% reduction in [Mg2+]i. The decreased levels of [Mg2+]i lasted around 30 min, until the measurement finished. Continuous incubation of these cultured astrocytes, with ethanol (either 10 mM or 25 mM), for more than 24 h, indicated that the concentrations of [Mg2+]i in type-2 astrocytes were equivalent to those at basal, resting levels. In vivo31P-NMR spectroscopy, performed on intact rat brains, indicated that an initial administration of 4 mg/kg ethanol (∼20–25 mM blood alcohol level) resulted (after 20–40 min of exposure) in severe deficits in whole brain [Mg2+]i (550 ± 33 μM to 358 ± 24 μM). Repeated injections of ethanol (4 mg/kg) over the next 24–72 h resulted in progressively diminishing effects on brain [Mg2+]i. These experimental data indicate that chronic ethanol treatment can induce a tolerance to depletion of [Mg2+]i in cerebrovascular smooth muscle cells, type-2 astrocytes as well as intact rat brain. The results suggest that [Mg2+]i might play a major role in alcohol-induced tolerance in the brain.

Antioxidants prevent depletion of [Mg 2+] i induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke

Low serum concentrations of Mg 2+ ions have been reported, recently, in patients with coronary disease, atherosclerosis, and stroke as well as in patients with cerebral hemorrhage. The aim of the present study was to determine whether potent antioxidants [α-tocopherol and pyrrolidine dithiocarbamate (PDTC)] can prevent or ameliorate intracellular Mg 2+ ([Mg 2+] i) depletion associated with cerebral vascular injury induced by alcohol. Exposure of cultured canine cerebral vascular smooth muscle cells to alcohol (10–100 mM) for 24 h induced marked depletion in [Mg 2+] i (i.e., ∼ 30–65%, depending upon alcohol concentration). Treatment of the cultured cells with either PDTC (0.1 μM) or α-tocopherol (15 μM) for 24 h, alone, failed to interfere with basal [Mg 2+] i levels. However, preincubation of the cells with either α-tocopherol or PDTC for 24 h completely inhibited the depletion of [Mg 2+] i induced by exposure to 10–100 mM ethanol. These results indicate that α-tocopherol and PDTC prevent decreases in [Mg 2+] i produced by ethanol. Moreover, these new results suggest that such protective effects of α-tocopherol and PDTC on cerebral vascular cells might be useful therapeutic tools in prevention and amelioration of cerebral vascular injury and stroke in alcoholics.

Importance of extracellular Ca 2+ and intracellular Ca 2+ release in ethanol-induced contraction of cerebral arterial smooth muscle

The present study was designed to investigate the roles of extracellular Ca 2+ ([Ca 2+] 0) influx and intracellular free Ca 2+ ([Ca 2+] i) release in ethanol-induced contractions of isolated canine cerebral arteries and primary cultured, cerebral vascular smooth muscle cells. Ethanol (20–200 mM) produced significant contractions in isolated canine basilar arterial rings in a concentration-dependent manner. Removal of [Ca 2+] 0 and pretreatment of canine basilar arterial rings with verapamil (an antagonist of voltage-gated Ca 2+ channels), thapsigargin (a selective antagonist of the sarcoplasmic reticulum Ca 2+ pump), caffeine plus ryanodine (a specific antagonist of ryanodine-sensitive Ca 2+ release), or heparin (an inositol 1,4,5,-trisphosphate [InsP 3]-mediated Ca 2+ release antagonist) markedly attenuated (~50%–80%) ethanol-induced contractions. The absence of [Ca 2+] 0 and preincubation of primary single smooth muscle cells obtained from canine basilar arteries with verapamil, thapsigargin, heparin, or caffeine plus ryanodine markedly attenuated (~50%–80%) the transient and sustained elevations in [Ca 2+] i induced by ethanol. Results of the present study suggest to us that both Ca 2+ influx through voltage-gated Ca 2+ channels and Ca 2+ release from intracellular stores (both InsP 3 sensitive and ryanodine sensitive) are required for ethanol-induced contractions of isolated canine basilar arteries.

Extracellular magnesium regulates nuclear and perinuclear free ionized calcium in cerebral vascular smooth muscle cells: possible relation to alcohol and central nervous system injury

Quantitative digital imaging microscopy, confocal laser scanning microscopy (CLSM), and multiple molecular fluorescent probes were utilized to test the hypothesis that cerebral vascular muscle cell nuclear ([Ca 2+] n), perinuclear ([Ca 2+] pn), and cytoplasmic free calcium ([Ca 2+] i) levels are regulated by the concentration of extracellular free magnesium ions ([Mg 2+] o). Primary cultured canine cerebral vascular smooth muscle cells were loaded with either fura-2/AM, indo-1/AM, or fluo-3/AM, and the subcellular Ca 2+ responses to stepwise reduction in [Mg 2+] o (i.e., from 1.36 to 0.17 mM) were analyzed over time. With normal 1.36 mM [Mg 2+] o-containing incubation media, basal mean [Ca 2+] i was 89.6±15 nM. Lowering [Mg 2+] o to 1.07, 0.88, 0.48, and 0.17 mM resulted in rapid (<4 min) increments in [Ca 2+] i going to 213±43, 368±67, 471±77, and 642±98 nM, respectively; the longer the exposure time (up to 30 min) to lowered [Mg 2+] o, the higher the [Ca 2+] i. Restoration of [Mg 2+] o to normal caused decreases in [Ca 2+] i to 215.9±42.3 nM, but only complete removal of [Ca 2+] o returned [Ca 2+] i to basal levels. Results show that basal [Ca 2+] pn (282±92 nM) exceeds basal cytoplasmic Ca 2+ (61±27.8 nM) and [Ca 2+] n (20±7.6 nM). However, reduction of normal [Mg 2+] o to 0.48 mM resulted in dramatic, rapid rises in all subcellular compartments, where [Ca 2+] pn (1503±102 nM)>cytoplasmic Ca 2+ (688±49 nM)≡[Ca 2+] n (674±12 nM). Nuclear Ca 2+ rose dramatically (e.g., 35–40 times basal levels). Both verapamil (1 μM) and Ni 2+ (5 mM) prevented, completely, the rises in Ca 2+ in all compartments, suggesting that Mg 2+-dependent Ca 2+ accumulation may be dependent on nuclear, endoplasmic reticulum–Golgi, and cytoplasmic L-type voltage membrane-regulated Ca 2+ channels. The normally low [Ca 2+] n suggests that Ca 2+ does not transport passively across the nuclear membrane in cerebral vascular smooth muscle cells. These results may help to explain much of the impact of hypomagnesemic states on cerebral–central nervous system pathobiology, and, particularly, alcohol-induced strokes.

Sex steroid hormones exert biphasic effects on cytosolic magnesium ions in cerebral vascular smooth muscle cells: possible relationships to migraine frequency in premenstrual syndromes and stroke incidence

Clinically, it is known that: (1) magnesium (Mg) supplementation relieves premenstrual problems (e.g., migraine, bloating and edema) occurring in the late luteal phase of the menstrual cycle; and (2) migraine syndromes, particularly in women, are associated with deficits in brain and serum ionized Mg levels. We investigated whether concentrations of sex steroid hormones, found in the serum during the menstrual cycle of women, are associated with changes in the levels of cytosolic free magnesium ions ([Mg 2+] i) in single cultured canine cerebral vascular smooth muscle cells. The resting level of [Mg 2+] i in these cells was 645 ± 89 μM before exposure to sex steroid hormones. Exposure of these vascular cells to a low concentration of estrogen (10 pg/ml) failed to interfere with the levels of [Mg 2+] i. However, exposure to estrogen, at concentrations ranging from 40 to 200 pg/ml, induced significant loss of [Mg 2+] i in a concentration-dependent manner. At a concentration of 200 pg/ml estrogen, the level of [Mg 2+] i decreased ∼30% in comparison with controls. Progesterone produced biphasic effects on the levels of [Mg 2+] i, depending on its concentration. Exposure of the cultured cells to a low concentration of progesterone (0.5 ng/ml) resulted in an increased level of [Mg 2+] i (from 690 ± 50 μM to 753 ± 56 μM, p < 0.05). However, when these cells were exposed to higher concentrations of progesterone (i.e., from 5.0 to 20 ng/ml), the cellular levels of [Mg 2+] i were decreased significantly. The higher the estrogen or progesterone concentration, the lower the levels of [Mg 2+] i. In contrast, testosterone, a male hormone, didn’t produce any significant alteration in [Mg 2+] i levels in these cerebral vascular smooth muscle cells. These data indicate that low, physiological concentrations of female sex hormones, estrogen and progesterone, help cerebral vascular smooth cells sustain normal concentrations of [Mg 2+] i, which are beneficial to vascular function, whereas high levels of estrogen and progesterone deplete, significantly, [Mg 2+] i in cerebral vascular smooth muscle cells, possibly resulting in cerebrovasospasms and reduced cerebral blood flows related to premenstrual syndromes, migraine and stroke risk. Our findings could provide new insight into the mechanism whereby migraine occurs frequently in the late luteal phase in the premenstrual syndrome. In addition, our results demonstrate that female sex steroids but not testosterone (in physiologic concentrations) can exert direct effects on [Mg 2+] i in cerebral vascular cells.

Extracellular pH, Ca(2+) influx, and response of vascular smooth muscle cells to 5-hydroxytryptamine.

Cerebral vascular smooth muscle cells (VSMCs) contract on extracellular pH (pH(o)) increases and relax on pH(o) decreases. These changes in tone are believed to result from changes in [Ca(2+)](i), although the responsible mechanisms are not fully understood. VSMCs also contract in response to 5-hydroxytryptamine (5-HT), which increases [Ca(2+)](i) via both Ca(2+) release and influx. We hypothesized that examining effects of pH(o) decreases on 5-HT-induced [Ca(2+)](i) changes would allow us to identify mechanisms whereby pH(o) influences tone. Accordingly, we compared [Ca(2+)](i) increases in cerebral VSMCs, evoked by 5-HT, with increases evoked by increased pH(o) and examined 5-HT-dependent [Ca(2+)](i) increases at normal and decreased pH(o). We monitored [Ca(2+)](i,), using the Ca(2+)-sensitive dye fura 2, in cultured rat cerebral VSMCs obtained by enzymatic digestion of middle cerebral arteries and their branches (passages 1 to 3) grown on glass coverslips and superfused with physiological saline. Increasing pH(o) from 7.3 to 7.8 increased [Ca(2+)](i), and these increases were prevented in Ca(2+)-free solutions. Decreasing pH(o) from 7.3 to 6.9 did not alter [Ca(2+)](i) unless [Ca(2+)](i) was first raised by treatment with 5-HT (10 micromol/L). 5-HT resulted in biphasic [Ca(2+)](i) increases characterized by transient peaks blocked by the Ca(2+)-ATPase inhibitor thapsigargin (10 nmol/L) and prolonged plateaus blocked by the Ca(2+) channel blocker Ni(2+) (1 mmol/L). Acidification did not alter the transient peaks but significantly reduced 5-HT-induced Ca(2+) influx. We conclude that increasing pH(o) induces Ca(2+) influx in rat cerebral VSMCs and decreasing pH(o) inhibits 5-HT-stimulated Ca(2+) entry but not intracellular Ca(2+) release.

Swelling‐activated cation channels mediate depolarization of rat cerebrovascular smooth muscle by hyposmolarity and intravascular pressure

1. Increases in intravascular pressure depolarize vascular smooth muscle cells. Based on the attenuating effects of Cl- channel antagonists, it has been suggested that swelling-activated Cl- channels may be integral to this response. Consequently, this study tested for the presence of a swelling-activated Cl- conductance in both intact rat cerebral arteries and isolated rat smooth muscle cells. 2. A 50 mosmol l-1 hyposmotic challenge (300 to 250 mosmol l-1) constricted rat cerebral arteries. This constriction contained all the salient features of a pressure-induced response including smooth muscle cell depolarization and a rise in intracellular Ca2+ that was blocked by voltage-operated Ca2+ channel antagonists. The hyposmotically induced depolarization was attenuated by DIDS (300 microM) and tamoxifen (1 microM), a response consistent with the presence of a swelling-activated Cl- conductance. 3. A swelling-activated current was identified in cerebral vascular smooth muscle cells. This current was sensitive to Cl- channel antagonists including DIDS (300 microM), tamoxifen (1 microM) and IAA-94 (100 microM). However, contrary to expectations, the reversal potential of this swelling-activated current shifted with the Na+ equilibrium potential and not the Cl- equilibrium potential, indicating that the swelling-activated current was carried by cations and not anions. The swelling-activated cation current was blocked by Gd3+, a cation channel antagonist. 4. Gd3+ also blocked both swelling- and pressure-induced depolarization of smooth muscle cells in intact cerebral arteries. 5. These findings suggest that swelling- and pressure-induced depolarization arise from the activation of a cation conductance. This current is inhibited by DIDS, tamoxifen, IAA-94 and gadolinium.

Low [Mg(2+)](o) induces contraction of cerebral arteries: roles of tyrosine and mitogen-activated protein kinases.

The present study was designed to investigate the mechanism of action of low extracellular magnesium ion concentration ([Mg(2+)](o)) on isolated canine basilar arteries and single cerebral vascular smooth muscle cells from these arteries. Low-[Mg(2+)](o) medium (0-0.6 mM) produces endothelium-independent contractions in isolated canine basilar arteries in a concentration-dependent manner; the lower the concentration of [Mg(2+)](o), the stronger the contractions. The low-[Mg(2+)](o) medium-induced contractions are significantly attenuated by pretreatment of the arteries with low concentrations of either SB-203580, U-0126, PD-98059, genistein, or an Src homology 2 (SH2) domain inhibitor peptide. IC(50) levels obtained for these five antagonists are consistent with reported inhibitor constant (K(i)) values for these tyrosine kinase and mitogen-activated protein kinase (MAPK) antagonists. Low-[Mg(2+)](o) medium (0-0.6 mM) produces transient intracellular calcium ion concentration ([Ca(2+)](i)) peaks followed by a slow, sustained, and elevated plateau of [Ca(2+)](i) in primary single smooth muscle cells from canine basilar arteries. Low-[Mg(2+)](o) medium induces rapid and stable increases in [Ca(2+)](i); these increases are inhibited markedly in the presence of either SB-203580, U-0126, PD-98059, genistein or a SH2 domain inhibitor peptide. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the low-[Mg(2+)](o)-induced contractions or the elevation of [Ca(2+)](i). The present study suggests that activation of several cellular signaling pathways, such as protein tyrosine kinases (including the Src family) and MAPK, appears to play important roles in low-[Mg(2+)](o)-induced contractions and the elevation of [Ca(2+)](i) in smooth muscle cells from canine basilar arteries.

Antioxidants prevent elevation in [Ca 2+] i induced by low extracellular magnesium in cultured canine cerebral vascular smooth muscle cells: possible relationship to Mg 2+ deficiency-induced vasospasm and stroke

Low serum concentrations of Mg 2+ ions have been reported, recently, in patients with coronary disease, atherosclerosis and stroke as well as in patients with cerebral hemorrhage. The aim of the present study was to determine whether potent antioxidants [α-tocopherol and pyrrolidine dithiocarbamate (PDTC)] can prevent or ameliorate intracellular Ca 2+ ([Ca 2+] i) overload associated with cerebral vascular injury induced by low extracellular free Mg 2+ ([Mg 2+] o). Exposure of cultured canine cerebral vascular smooth muscle cells to low [Mg 2+] o (0.15–0.6 mM) vs. normal [Mg 2+] o (1.2 mM) for either 10 min or 2 h induced concentration-dependent rises in [Ca 2+] i. Treatment of the cultured cells with either PDTC (0.1 μM) or α-tocopherol (15 μM) for 24 h, alone, failed to interfere with basal [Ca 2+] i levels. However, preincubation of the cells with either α-tocopherol or PDTC for 24 h completely inhibited the elevation of [Ca 2+] i induced by exposure to low [Mg 2+] o, not only for 10 min, but also for 2 h. These results indicate that α-tocopherol and PDTC prevent rises in [Ca 2+] i produced by low [Mg 2+] o, which probably result from low [Mg 2+] o-induced lipid peroxidation of cerebral vascular smooth muscle cell membranes. Moreover, these new results suggest that such protective effects of α-tocopherol and PDTC on cerebral vascular cells might be useful therapeutic tools in cerebral vascular injury associated with low [Mg 2+] o and accumulation of [Ca 2+] i.

Sphingomyelinase and ceramide analogs induce contraction and rises in [Ca(2+)](i) in canine cerebral vascular muscle.

Studies were designed to investigate effects of neutral sphingomyelinase (N-SMase) and ceramide analogs as well as phosphorylcholine on vascular tone and Ca(2+) mobilization in isolated canine cerebral arterial smooth muscle. N-SMase (0.001-0.1 U/ml) provoked a gradual but sustained vasoconstriction of arterial rings in a concentration-related manner that was endothelium independent. Incubation of denuded arterial rings in Ca(2+)-free medium or pretreatment with verapamil in extracellular Ca(2+) resulted in a reduction of the N-SMase-evoked constriction. Exposure of arterial rings to 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA)-AM did not, however, result in a reduction of N-SMase-induced constriction. Both staurosporine and bisindolymaleimide I attenuated N-SMase-induced contractions to 66% and 72% of control, respectively. N-SMase caused gradual and sustained rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) in primary cultured cerebral vascular smooth muscle cells. Pretreatment of these cultured cells with nimodipine and verapamil caused a steady decline in N-SMase-induced rises in [Ca(2+)](i). Exposure of the cells to Ca(2+)-free solution reversed the [Ca(2+)](i)-induced rise triggered by N-SMase to the resting baseline. Both C(8) and C(16) ceramide (10(-9)-10(-6) M), but not phosphorylcholine, constricted denuded canine arterial rings in a concentration-related manner and elevated [Ca(2+)](i). Our results suggest that the sphingomyelin-signaling pathway, via a probable release of ceramide molecules, may play an important role in regulation of cerebral arterial wall tone.

Extracellular magnesium regulates effects of vitamin B6, B12 and folate on homocysteinemia-induced depletion of intracellular free magnesium ions in canine cerebral vascular smooth muscle cells: possible relationship to [Ca2+]i, atherogenesis and stroke

Homocysteine (HC) at concentrations of from 0.05 to 1.0 mM caused dose-dependent loss of [Mg2+]i in cultured cerebral vascular smooth muscle cells (VSMC), whereas cysteine and methionine (its metabolic products) failed to interfere with changes in [Mg2+]i. HC, methionine and cysteine did not produce any changes in [Ca2+]i. Lowering [Mg2+]o to 0.3 mM resulted in elevation of [Ca2+]i and loss of [Mg2+]i. Depletion of [Mg2+]i, induced by HC, was potentiated by low Mg2+. Preincubation of these cells with vitamin B6, vitamin B12, folic acid, alone, did not alter [Ca2+]i or [Mg2+]i. Likewise, concomitant addition of vitamin B6, vitamin B12, or folic acid, together with HC (1 mM) did not change the reduction in [Mg2+]i induced by HC. However, concomitant addition of HC and the three vitamins inhibited completely the loss of [Mg2+]i. Exposure of these cells to each vitamin, alone, or combination of the three vitamins failed to interfere with reduction in [Mg2+]i induced by low [Mg2+]i, but it did suppress the rise in [Ca2+]i. Interestingly, in the presence of low [Mg2+]o, the vitamin combination did not retard depletion of [Mg2+]i. The present findings are compatible with the hypothesis that an increased serum HC concentration causes abnormal metabolism of Mg2+ in cerebral VSMC, thus priming these cells for HC-induced atherogenesis, cerebral vasospasm and stroke. Our results suggest the need for the three B-vitamins, together with normal physiological levels of Mg2+, in order to prevent [Mg2+]i depletion and occlusive cerebral vascular diseases induced by homocysteinemia.