Canine Cerebral Vascular Smooth Muscle Research Articles

Alcohol-induced apoptosis of canine cerebral vascular smooth muscle cells: role of extracellular and intracellular calcium ions

Exposure of canine cerebral vascular smooth muscle cells (VSMCs) to ethanol (10, 25 and 100 mM) for 1, 3 and 5 days induced apoptosis with its typical characteristics of nuclear shrinkage, condensation, and DNA breakage as well as formation of apoptotic bodies observed by fluorescence staining, terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling and comet assays. Such effects of alcohol on cerebral VSMCs were time- and concentration-dependent. The threshold ethanol concentration for induction of the apoptotic process was found to be 10 mM. Extracellular and intracellular Ca 2+ chelators, i.e. ethylglycol-bisβ-aminoethylether- N, N, N′ N′-tetraacetic acid (EGTA, 5 mM) and 1,2-bis(2-aminophenoxy)-ethane- N, N, N′, N′-tetra-acetic acid AM (BAPTA, 10 −6 M), respectively, ameliorated greatly the number of cerebral VSMCs which underwent apoptosis. Verapamil, however, failed to inhibit apoptosis of cerebral VSMCs. From these new findings, we suggest that alcohol-induced apoptosis may contribute to alcohol-induced brain-vascular damage and stroke. In addition, our findings point to potential caution for humans who imbibe two or more standard drinks per day or who undergo ‘binge drinking’.

Cocaine induces apoptosis in cerebral vascular muscle cells: potential roles in strokes and brain damage

Cocaine abuse is known to induce different types of brain-microvascular damage and many adverse cerebrovascular effects, including cerebral vasculitis, intracranial hemorrhage, cerebral infarction and stroke. A major physiological event leading to these pathophysiological actions of cocaine could be apoptosis. Whether cocaine can cause brain-microvascular pathology and vascular toxicity by inducing apoptosis of cerebral vascular smooth muscle cells is not known. This study, using several different methods to discern apoptosis, was designed to investigate if primary cultured canine cerebral vascular smooth muscle cells can undergo apoptosis when treated with cocaine. After treatment with cocaine (10 −6–10 −3 M) for 12–24 h, the death rates of cerebral vascular smooth muscle cells increased in a concentration-dependent manner compared with controls. Morphological analysis of cerebral vascular smooth muscle cells using confocal fluoresence microscopy showed that the percentage of apoptotic cerebral vascular smooth muscle cells increased after cocaine (10 −6–10 −3 M) treatment in a concentration-dependent manner. TUNEL assays also showed positive results for cerebral vascular smooth muscle cells treated with cocaine. These results clearly demonstrate that cerebral vascular smooth muscle cells can undergo rapid apoptosis in response to cocaine in a concentration-dependent manner. Cocaine-induced apoptosis may thus play a major role in brain-microvascular damage, cerebral vascular toxicity and strokes.

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.

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.

Antioxidants prevent ethanol-induced contractions of canine cerebral vascular smooth muscle: relation to alcohol-induced brain injury

The present study was designed to test the hypothesis that α-tocopherol (Vit. E) and pyrrolidine dithiocarbamate (PDTC) might exert direct effects on alcohol-induced contractions of canine basilar cerebral arteries. After precontraction of arterial ring segments with ethanol, PDTC (10 −8–10 −6 M) and Vit. E (10 −6–10 −4 M) induced concentration-dependent relaxations of cerebral arteries, compared to untreated controls. The effective concentrations producing approximately 50% of the maximal relaxation responses (EC 50 values) were about 2.48±0.09 × 10 −7 M for PDTC, and 1.87±0.10 × 10 −5 mM for Vit. E, respectively. Preincubation of these arterial rings with EC 50’s of PDTC or Vit. E for 40 min attenuate markedly the contractions produced by alcohol, at concentrations of 1–400 mM. However, both PDTC and Vit.E do not relax equi-potent precontractions induced by either KCl or prostaglandin F 2α (PGF 2 α ) or inhibit their contractions. These data suggest that alcohol-induced contractions of cerebral arteries are mediated via excitation-contraction coupling pathways different from those used by KCl or receptor-mediated agonists such as PGF 2 α . The present results, when viewed in light of other recently published data, suggest that antioxidants may prove useful in the amelioration and treatment of alcohol-induced brain damage and strokes.

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.

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.

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.

Methanol Elevates Cytosolic Calcium Ions in Cultured Canine Cerebral Vascular Smooth Muscle Cells: Possible Relation to CNS Toxicity

LI, W., T. ZHENG, J. WANG, B. T. ALTURA AND B. M. ALTURA. Methanol elevates cytosolic calcium ions in cultured canine cerebral vascular smooth muscle cells: Possible relation to CNS toxicity. ALCOHOL 18(2/3) 221–224, 1999.—Acute exposure of cultured canine cerebral vascular smooth muscle cells to methanol (10–400 mM) results in concentration-dependent elevation of the concentration of intracellular free calcium ion ([Ca 2+] i) as measured with the fluorescent indicator, fura-2, and digital imaging microscopy. The resting level of [Ca 2+] i in the cerebral vascular smooth muscle cells was 89.3 ± 5.3 nM. Exposure of these cells to 10 mM methanol for only 5 min resulted in significant elevation in [Ca 2+] i (i.e., to 105.7 ± 4.6) ( p < 0.05). Methanol (10 mM) is a concentration found in the blood of victims demonstrating early CNS toxicity. Other, higher concentrations of methanol rapidly raised [Ca 2+] i upwards of 60% over basal resting levels. These result suggest that methanol-induced cerebral vasospasm is a consequence of large rises in intracellular Ca 2+. These events could play a crucial role in methanol-induced cerebral edema, brain hemorrhage, and cerebral and retinal infarcts, eventuating in severe deficits in brain blood flow and the known, subsequent CNS disturbances.

Pyrrolidine dithiocarbamate prevents ethanol-induced elevation of [Ca 2+] i in cultured canine cerebral vascular smooth muscle cells

Pyrrolidine dithiocarbamate (PDTC) has been shown to block nuclear transcription factor (NF-κB) activation induced by a wide range of stimuli in different cell lines. NF-κB is a common element of the promoter region of inflammatory cytokines which can be stimulated by ethanol. Recently, we have shown that PDTC can ameliorate cerebrovascular damage, brain cortical damage, leukocyte adhesion and rolling, and stroke induced by ethanol. We, therefore, tested the effects of preincubation with PDTC on alcohol-induced changes in intracellular free calcium ions ([Ca 2+] i) in cultured canine cerebral smooth muscle cells. These vascular cells, chronically treated with ethanol (10–100mM) for 24 and 72 h, exhibited concentration-dependent rises in [Ca 2+] i. PDTC (0.1 μM) itself failed to influence resting levels of [Ca 2+] i in these vascular muscle cells. PDTC (0.1 μM) pretreatment, however, inhibited completely the elevations in [Ca 2+] i induced by chronic ethanol (10–100 mM). The present results suggest that ethanol-induced production of reactive oxygen species and elevation of [Ca 2+] i in cultured canine cerebral vascular smooth muscle cells triggers induction of transcription factor NF-κB, which could play an important role in alcohol-induced brain damage and stroke.

C 2-ceramide attenuates prostaglandin F 2 α-induced vasoconstriction and elevation of [Ca 2+] i in canine cerebral vascular smooth muscle

Sphingolipids have emerged as important components of signal transduction pathways involved in a variety of cellular processes. In the present study, we examined the effects of C 2-ceramide, a cell-permeable sphingolipid, on contraction of canine cerebral vascular smooth muscle and intracellular free Ca 2+ ([Ca 2+] i). C 2-ceramide (10 −8–10 −4 M) alone did not elicit any significant changes in either basal tension or resting levels of [Ca 2+] i in canine cerebrovascular muscle. However, C 2-ceramide (10 −7–10 −4 M) attenuated prostaglandin F 2 α (PGF 2 α )-induced contractions in isolated canine cerebrovascular smooth muscle rings. C 2-ceramide (10 −5 M) inhibited the secondary phasic rise of [Ca 2+] i evoked by PGF 2 α in cultured canine cerebral vascular smooth muscle cells, resulting in decreases in the elevation in [Ca 2+] i. NO inhibitors ( l-NNA, l-NMMA), an inhibitor of prostanoid synthesis (indomethacin), an inhibitor of opiate actions and several inhibitors of the pharmacologic actions of various vasoactive amines all failed to interfere with the vasorelaxant response of C 2-ceramide. Our results suggest that the sphingomyelin signaling pathway may play an important regulatory role in cerebral arterial wall tone.

α-Tocopherol prevents ethanol-induced elevation of [Ca 2+] i in cultured canine cerebral vascular smooth muscle cells

Exposure of cultured canine cerebral vascular smooth muscle cells to ethanol (10–400 mM) for 1–5 days results in concentration-dependent elevation in resting intracellular free calcium ([Ca 2+] i) levels. Preincubation of these cultured vascular cells with α-tocopherol (20 μM), alone, did not produce any apparent changes from control resting levels of [Ca 2+] i. However, after concomitant addition of α-tocopherol (20 μM) and ethanol (10–400 mM), the rises of [Ca 2+] i induced by ethanol were attenuated markedly. These results suggest that alcohol-induced lipid peroxidation of cerebral vascular muscle cell membranes triggers membrane entry of extracellular Ca 2+, which could play an important role in ethanol-induced cerebrovasospasm, brain ischemia and stroke. Moreover, these new results support the concept recently advanced to suggest that α-tocopherol-induced amelioration of membrane lipid alterations of cerebral vascular cells can prevent ethanol-induced excessive accumulation of [Ca 2+] i.

Staurosporine and H7 attenuate ethanol-induced elevation in [Ca 2+] i in cultured canine cerebral vascular smooth muscle cells

Chronic exposure of cultured canine cerebral vascular smooth muscle cells to ethanol (10–400 mM) for 1–5 days resulted in significant concentration-dependent elevation in resting intracellular free calcium ([Ca 2+]i) levels. Preincubation of these cultured vascular cells with inhibitors of protein kinase C (PKC), staurosporine and H7, induced no apparent changes from the control resting levels of [Ca 2+] i. However, the increases of [Ca 2+] i due to ethanol treatment were attenuated markedly by staurosporine and H7. Our data suggest that activation of PKC plays an important role in ethanol's action in producing a sustained rise in [Ca 2+] i in cerebral vascular smooth muscle cells. Activation of PKC could thus play a crucial role in the pathogenesis of alcohol-induced cerebral ischemia and stroke.

Chronic treatment of cultured cerebral vascular smooth cells with low concentration of ethanol elevates intracellular calcium and potentiates prostanoid-induced rises in [Ca 2+] i: Relation to etiology of alcohol-induced stroke

The influence of chronic treatment of cultured canine cerebral vascular smooth muscle cells, with low concentrations of ethanol, on the intracellular concentrations of free calcium ([Ca 2+] i) was studied by use of the fluorescent indicator, fura-2, and digital imaging microscopy. The resting level of [Ca 2+] i in the cerebral vascular smooth muscle cells was 89 ± 3.2 nM. Exposure of these cells to 10 and 25 mM ethanol for 5 days resulted in significant elevation of [Ca 2+] i (mean rises to 208 ± 11.4 and 307 ± 14.0 nM, respectively), and potentiated the transient rise in [Ca 2+] i induced by 10 −7 M PGF 2α. However, exposure of these cerebral cells to a high-concentration ethanol (100 mM) resulted in only a slight increase of [Ca 2+] i (106 ± 6.9 nM) and lack of effects on the [Ca 2+] i response to PGF 2α. Irrespective of the different ethanol treatments, the subcellular distribution of [Ca 2+] i was heterogeneous in all the cells tested. Our data suggest that chronic exposure of cerebral vascular smooth muscle cells to ethanol, particularly at low concentrations, results in dramatic increases in [Ca 2+] i and the responses of these vascular smooth muscle cells to prostanoids. These results support an hypothesis whereby ethanol induces stroke by causing spasm and rupture of cerebral blood vessels as a consequence of large rises in intracellular Ca 2+.

Low levels of serum ionized magnesium are found in patients early after stroke which result in rapid elevation in cytosolic free calcium and spasm in cerebral vascular muscle cells

Ninety-eight patients admitted to the emergency rooms of three urban hospitals with a diagnosis of either ischemic stroke or hemorrhagic stroke exhibited early and significant deficits in serum ionized Mg 2+ (IMg 2+), but not total Mg, as measured with a unique Mg 2+-sensitive ion-selective electrode. Twenty-five percent of these stroke patients exhibited >65% reductions in the mean serum IMg 2+ found in normal healthy human volunteers or patients admitted for minor bruises, cuts or deep lacerations. The stroke patients also demonstrated significant elevation in the serum ionized Ca 2+ (ICa 2+)/IMg 2+ ratio, a sign of increased vascular tone and cerebrovasospasm. Exposure of primary cultured canine cerebral vascular smooth muscle cells to the low concentrations of IMg 2+ found in the stroke patients, e.g. 0.30–0.48 mM, resulted in rapid and marked elevations in cytosolic free calcium ions ([Ca 2+] i) as measured with the fluorescent probe, fura-2, and digital image analysis. Coincident with the rise in [Ca 2+] i, many of the cerebral vascular cells went into spasm. Reintroduction of normal extracellular Mg 2+ ion concentrations failed to either lower the [Ca 2+] i overload or reverse the rounding-up of the cerebral vascular cells. These results suggest that changes in Mg 2+ metabolism play important roles in stroke syndromes and in the etiology of cerebrovasospasm associated with cerebral hemorrhage.

Acute cocaine results in rapid rises in intracellular free calcium concentration in canine cerebral vascular smooth muscle cells: possible relation to etiology of stroke

Effects of cocaine on intracellular free calcium concentration ([Ca 2+] i) in cultured canine cerebral vascular smooth muscle cells were studied using digital imaging microscopy and the calcium molecular fluorescent indicator, fura-2. Acute treatment of cerebral vascular smooth muscle cells with cocaine HCl, from a low concentration of 10 −9 M up to 10 −5 M, induced significant increases of [Ca 2+] i. Irrespective of the changes in [Ca 2+] i, the subcellular distribution of [Ca 2+] i appeared heterogeneous in both normal and cocaine-treated cells. These results suggest that cocaine induces cerebral vasospasm by a rapid elevation of [Ca 2+] i in vascular smooth muscle cells; these ionic events could play a crucial role in the pathogenesis of cocaine-induced cerebral ischemia and stroke.

Alcohols induce rapid depletion of intracellular free Mg 2+ in cerebral vascular muscle cells: relation to chain length and partition coefficient

Acute effects of a series of alcohols (methanol, ethanol, n-butanol) on intracellular free magnesium concentration ([Mg 2+] i in canine cerebral vascular smooth muscle cells was studied using mag-fura-2 and digital imaging microscopy. In 1.2 mM [Mg 2+] o, basal [Mg 2+] i was 500 ± 30 μM. Exposure of cells to a low concentration (25 mM) of ethanol, but not methanol, for only 30 s resulted in significant loss of [Mg2 2] i. Exposure to 100 mM methanol, ethanol, and butanol for 30 s resulted in a relative order of potency for [Mg 2+] i depletion, where butanol ⪢ ethanol > methanol. The heterogeneous and relative subcellular compartmented concentrations of [Mg 2+] i, where perinuclear > nuclear ⪢ peripheral (cytosolic) region, was not significantly altered by the alcohols. The degree of cellular depletion of [Mg 2+] i; was directly a function of each alcohol's partition coefficient and chain length. The latter is suggestive of the probability that alcohols promote intracellular depletion of Mg 2+ by partitioning in membranes and disordering lipid bilayers.