INTRODUCTION

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EVER SINCE VERY EARLY STUDIES on the cerebral circulation, it has been suggested that cerebrovasospasm is a trigger in cerebral ischemia and stroke (2, 20). However, the etiology of cerebrovasospasm remains elusive. The myogenic tone of cerebral vessels is strongly dependent on the plasma concentration of extracellular Mg²⁺ concentration ([Mg²⁺]_o) (7, 10) and is believed to play an important role in the autoregulation of cerebral blood flow (7, 13). Collectively, these studies suggest that a decreased content of plasma Mg²⁺ will increase cerebrovascular tone and may induce vasospasmic responses; the end results could be development of cerebral ischemia, infarction, and stroke (6, 7, 10). In this context, it has been shown in vivo, with the use of direct intravital microscopy, that perfusion of the cortical microcirculation with low cerebrospinal fluid [Mg²⁺]_o results in spasm and rupture of postcapillary venules (5). By use of specific Mg²⁺-selective electrodes, it has been reported that 98 stroke (both ischemic and hemorrhagic) patients out of 105 patients from three urban hospitals exhibited low levels of serum ionized Mg²⁺ on admission to the emergency room (12).

Multiple signaling pathways may participate in mechanisms of peripheral vasoconstriction (26). Ca²⁺ is a major determinant of contractile force in all types of muscles. The initiation of contraction in vascular smooth muscle is believed to initiate from a rise of free cytosolic [Ca²⁺]. Protein tyrosine kinases have been suggested as being important signal-transduction pathways in the regulation of tone and intracellular Ca²⁺ concentration ([Ca²⁺]_i) of vascular smooth muscle (21). Activated and autophosphorylated receptor tyrosine kinases recruit Src homology 2 (SH2) domain-containing adaptor proteins and play a role in agonist-induced activation of Ras (31). The mitogen-activated protein kinase (MAPK) kinase [MAPK/extracellular signal-regulated kinase (ERK) kinase], or MEK, a cytosolic nonreceptor protein kinase, is in the family of tyrosine kinases (32). MAPKs, substrates of MEK, serve to relay, amplify, and integrate diverse signals, thus allowing a cell to coordinate a physiological response.

We have recently found that low, defined serum ionized [Mg²⁺]_o levels result in a rapid concentration-dependent rise in [Ca²⁺]_i in cultured cerebral arterial smooth muscle concomitant with contraction of these isolated primary vascular smooth muscle cells (12). Several recent clinical trials have demonstrated the therapeutic usefulness of administration of Mg²⁺ in the treatment of stroke (27). However, the mechanisms whereby low [Mg²⁺]_o alters cerebral arterial vascular tone may be complex and are less well defined. Our present study aimed to test the hypothesis that the mechanical effects of low-[Mg²⁺]_o physiological medium on cerebral arteries are attributable, in large measure, to activation of protein tyrosine kinases and recruitment of SH2 domain adaptor proteins and MAPK and that these enzyme pathways, collectively, regulate influx of extracellular Ca²⁺ concentration, resulting in modulation of [Ca²⁺]_i.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

General procedures. Rings of canine basilar arteries were obtained from male mongrel dogs (18-22 kg), after pentobarbital sodium anesthesia (40 mg/kg iv), and placed in normal Krebs-Ringer bicarbonate solution at pH 7.4 containing (in mM) NaCl 118, KCl 4.7, KH₂PO₄ 1.2, MgSO₄ 1.2, CaCl₂ 2.5, dextrose 10, and NaHCO₃ 25 (9). The rings were 3-4 mm in length. The segments were mounted on stainless steel pins under 2-g resting tension in isolated organ baths, attached to force transducers (Grass model FT 03), and connected to Grass model 7 polygraphs. The organ baths containing normal Krebs-Ringer bicarbonate solution were gassed continuously with 95% O₂-5% CO₂ and warmed to 37°C (pH 7.4). Tissues were allowed to equilibrate for at least 90 min before data collection. At the start of an experiment, rings were exposed for 30-45 min to 80 mM KCl, and this was repeated every 30-45 min until contractile responses were stable (2-3 times). Successful removal of endothelium was assessed by showing that acetylcholine (10⁸-10⁶ M) failed to relax segments precontracted by 10⁶ M prostaglandin F₂, whereas it did relax the endothelium-intact segments (37, 41). When tissues were pretreated by various drugs, the drugs were applied for at least 15 min before the concentration-response curves were obtained.

Intracellular Ca²+ measurement and cell culture. Primary smooth muscle cells from canine basilar arteries for image analysis experiments were seeded on glass coverslips (12-mm diameter; ~1 × 10⁴ cells/coverslip) and used 2-3 days postseeding (39). Monolayers of the smooth muscle cells grown on the coverslips were loaded with 2.0 µM fura 2-AM and 0.12% pluronic F-127 (60 min, 37°C), and the experimental procedures for [Ca²⁺]_i measurements were carried out as described previously (24, 39) with the use of fura 2-AM. The resulting images were then used to calculate [Ca²⁺]_i in smooth muscle cells. [Ca²⁺]_i was calculated according to the following equation (19)
A dissociation constant (K_d) of 224 nM was used for the fura 2-Ca²⁺ complex (19, 39). B is the ratio of fluorescence intensity of fura 2 to fura 2-Ca²⁺ complex excited at 380 nm (24, 39); R is the ratio of fluorescence intensity of fura 2 excited at 340 nm to fluorescence intensity of fura 2 at 380 nm; R_min is R at 0 mM [Ca²⁺]; R_max is R at saturating [Ca²⁺]. Particular care was taken to minimize photobleaching of the dye. Experiments were carried out in total darkness, and exposure to excitation light was <2 s in all experiments.

Drugs. The following pharmacological agents were purchased from Sigma Chemical (St. Louis, MO): acetylcholine HCl, daidzein, EGTA, genistein, naloxone HCl, and propranolol HCl. Atropine sulfate was bought from MANN (New York, NY). U-0126 was purchased from Promega (Madison, WI). SB-203580 was bought from Tocris Cookson (Ballwin, MO). Cimetidine HCl and diphenhydramine HCl were received from Smith Kline & French Laboratories (Welwyn Garden City, Herts, UK). DMSO, PD-98059, and an SH2 domain inhibitor peptide were purchased from Calbiochem (La Jolla, CA). Phentolamine methanesulfonate was purchased from CIBA Pharmaceutical (Summit, NJ). Methysergide maleate was received from Sandoz Pharmaceuticals (Hanover, NJ). All other organic and inorganic chemicals were obtained from Fisher Scientific (Fair Lawn, NJ) and were of the highest purity.

Calculations and statistical analysis. The contractile responses (in g) and [Ca²⁺]_i are expressed as means ± SE. Statistical evaluation of the results was carried out by the Newman-Keuls test and ANOVA with the use of Scheffe's contrast test. The results were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Low [Mg²+]_o and contraction of canine basilar arteries. The typical, representative concentration-response curves of low-[Mg²⁺]_o medium (0-0.6 mM)-induced contractions in canine basilar arteries, with and without endothelium, are illustrated in Fig. 1A. [Mg²⁺]_o-deficient medium produces a rapid contractile response (phasic component) that is followed by a prolonged and sustained increase in vessel tension (tonic component). As shown in Fig. 1, A and B, such arterial contractions, induced by low-[Mg²⁺]_o medium, demonstrate concentration-dependent and endothelium-independent responses. There are significantly greater developed tensions in low-[Mg²⁺]_o medium-treated endothelium-denuded segments; the lower the concentration of [Mg²⁺]_o, the stronger the contraction (Fig. 1, A and B; P < 0.01).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent contractile responses to extracellular Mg2+ concentration ([Mg2+]o) deficiency in isolated, endothelium-intact (+E) and endothelium-denuded (E) canine basilar arterial rings. A: vertical bar, tension in g; horizontal bar, time in min. B: each point represents mean ± SE, expressed as tension (in g) developed by Mg2+-free medium; no. of experiments is 6 each. # P < 0.05 and * P < 0.01.

A protein tyrosine kinase antagonist and an SH2 domain inhibitor attenuate low-[Mg²+]_o-induced contractions. As demonstrated in Fig. 2, A and B, the contractile responses of denuded canine basilar arteries to low-[Mg²⁺]_o medium are markedly reduced in the presence of genistein (an antagonist of protein tyrosine kinases) or an SH2 domain inhibitor peptide in a concentration-dependent manner but not in the presence of daidzein, an inactive homolog of genistein (29). The concentrations producing 50% of the maximal inhibitory effects (IC₅₀ values) of genistein and the SH2 domain inhibitor peptide are 6.3 ± 0.3 × 10⁵ and 0.9 ± 0.1 × 10⁶ M, respectively. Mean values for low- [Mg²⁺]_o-induced contractions in the presence of genistein, an SH2 domain inhibitor peptide, and daidzein are shown in Fig. 2C.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Inhibitory effects of genistein and an Src homology 2 (SH2) domain inhibitor peptide on contractile responses of endothelium-denuded canine basilar arterial rings to low-[Mg2+]o medium. Preincubation time of these antagonists (A-C) was 15 min. A: vertical bar, tension in g; horizontal bar, time in min. A and C: concentrations of genistein, daidzein, and SH2 domain inhibitor peptide used herein are 6.5 × 105, 6.5 × 105, and 106 M, respectively. B and C: each point represents peak value and mean ± SE, expressed as tension (in g) developed by Mg2+-free medium; n = 8. # P < 0.05, * P < 0.01, and ** P < 0.001.

Effects of a protein tyrosine kinase antagonist and an SH2 domain inhibitor on low-[Mg²+]_o-induced elevations in [Ca²⁺]_i. Figure 3A demonstrates that low-[Mg²⁺]_o medium produces rapid [Ca²⁺]_i peaks followed by steady-state [Ca²⁺]_i plateaus in primary single smooth muscle cells from canine basilar arteries. Such elevations in [Ca²⁺]_i illustrate, in an inverse-concentration-dependent manner, that the lower the [Mg²⁺]_o in the medium, the greater the increase in [Ca²⁺]_i. Figure 3, B and C, illustrates that preincubation of primary single smooth muscle cells, from canine basilar arteries, with either genistein or an SH2 domain inhibitor peptide, but not daidzein, effectively attenuates both the rapid and the sustained increments in [Ca²⁺]_i induced by low-[Mg²⁺]_o medium. Such inhibitory effects of these two antagonists show a concentration-dependent manner. The calculated IC₅₀ values of genistein and an SH2 domain inhibitor peptide for such attenuation of the increases in [Ca²⁺]_i are 6.0 ± 0.2 × 10⁵ and 0.8 ± 0.03 × 10⁶ M, respectively, which is consistent with the contractile responses induced by low-[Mg²⁺]_o medium under the same conditions. Mean peak [Ca²⁺]_i values, in the absence and presence of the antagonists, are shown in Fig. 3D.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent intracellular Ca2+ concentration ([Ca2+]i) increments in single smooth muscle cells from canine basilar arteries induced by low-[Mg2+]o medium are modified by genistein and SH2 domain inhibitor peptide. Preincubation time of antagonists (B-D) was 15 min. A and B: vertical bar, [Ca2+]i in nM; horizontal bar, time in min. B and D: concentrations of genistein, daidzein, and SH2 domain inhibitor peptide used herein are 6.5 × 105, 6.5 × 105, and 106 M, respectively. C and D: each point represents peak value and mean ± SE, expressed as [Ca2+]i in nM; n = 15. # P < 0.05, * P < 0.01, and ** P < 0.001.

MAPK kinase and MAPK antagonists attenuate low-[Mg²+]_o-induced contractions. As shown in Fig. 4A, pretreatment of endothelium-denuded canine basilar arterial segments with SB-203580, a highly specific antagonist of p38 MAPK (22); U-0126, a potent, selective antagonist of MEK1/MEK2 (17); or PD-98059, a selective antagonist of MAPK kinase (MAPKK) (1), significantly inhibits low-[Mg²⁺]_o-induced contractions (both rapid and stable components). The inhibitory effects of these three antagonists show concentration-dependent effects (Fig. 4B). The calculated IC₅₀ values for SB-203580, U-0126, and PD-98059 for such contractions are 2.0 ± 0.3 × 10⁶, 0.7 ± 0.1 × 10⁶, and 8.7 ± 0.3 × 10⁶ M, respectively. Mean values for various low-[Mg²⁺]_o (0-0.6 mM)-induced contractile tension developments in the presence of SB-203580, U-0126, and PD-98059 are demonstrated in Fig. 4C.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Inhibitory effects of U-0126, PD-98059, and SB-203580 on contractile responses of endothelium-denuded canine basilar arterial rings to low-[Mg2+]o medium. Preincubation time of antagonists (A-C) was 15 min. A: vertical bar, tension in g; horizontal bar, time in min. A and C: concentrations of U-0126, PD-98059, and SB-203580 used herein are 2 × 106, 106, and 105 M, respectively. B and C: each point represents peak value and mean ± SE, expressed as tension (in g) developed by Mg2+-free medium; n = 6. # P < 0.05, * P < 0.01, and ** P < 0.001.

Effects of MAPKK and MAPK antagonists on low-[Mg²⁺]_o-induced elevations in [Ca²+]_i. As shown in Fig. 5, A and B, both the low-[Mg²⁺]_o-induced rapid and steady-state elevation of [Ca²⁺]_i are effectively suppressed by preincubation of the cells with either SB-203580, U-0126, or PD-98059. The calculated IC₅₀ values of SB-203580, U-0126, and PD-98059 for such attenuation of the increases in [Ca²⁺]_i are 1.6 ± 0.1 × 10⁶, 0.5 ± 0.1 × 10⁶, and 8.2 ± 0.2 × 10⁶ M, respectively, which are in close agreement to those of the low-[Mg²⁺]_o-induced arterial contractions under the same conditions (see Fig. 4B). Calculated mean peak [Ca²⁺]_i values are shown in Fig. 5C.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Increments in [Ca2+]i in single smooth muscle cells from canine basilar arteries, induced by low-[Mg2+]o medium, are modified by U-0126, PD-98059, and SB-203580. Preincubation time of antagonists (A-C) was 15 min. A: vertical bar, [Ca2+]i in nM; horizontal bar, time in min. A and C: concentrations of U-0126, PD-98059, and SB-203580 used herein are 2 × 106, 106, and 105 M, respectively. B and C: each point represents peak value and mean ± SE, expressed as [Ca2+]i in nM; n = 12. * P < 0.01 and ** P < 0.001.

Failure of several specific pharmacological antagonists to attenuate or interfere with low-[Mg²+]_o-induced contractions. Incubation of canine basilar arterial rings with a wide variety of specific amine, opiate, and prostanoid antagonists (i.e., diphenhydramine 10⁶ M, cimetidine 10⁵ M, phentolamine 10⁶ M, methysergide 10⁶ M, propranolol 10⁵ M, atropine 10⁶ M, naloxone 10⁵ M, and indomethacin 10⁵ M, 5.0 µM) failed to either attenuate or interfere with contractions induced by 0 mM [Mg²⁺]_o (n = 6 each, data not shown). Likewise, these antagonists failed to attenuate the rises in [Ca²⁺]_i produced by 0 mM [Mg²⁺]_o (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study demonstrates that decrements in extracellular Mg²⁺ result, in a concentration-dependent manner, in a rapid elevation in contractile tension development similar to that reported previously in helically cut strips of rat aortic smooth muscle (3, 4). Interestingly, the low [Mg²⁺]_o used herein (i.e., 0.3-0.6 mM) has been found recently in the serum of patients with hypertension, cerebral infarction, hemorrhagic stroke, and ischemic heart diseases with the use of new specific Mg²⁺-selective electrodes (6, 8, 11, 12).

Protein tyrosine kinases have been shown to be involved in the contraction of rabbit cerebral arterial smooth muscle either by activation of receptors or by opening of Ca²⁺ channels (21). All protein tyrosine kinases of the Src family share highly conserved amino acid sequences termed SH2. SH2 sequences bind phosphorylated proteins implicated in normal cellular signaling and transportation (18). However, little knowledge is currently available concerning the actions of tyrosine kinases in cerebral arteries. In the present study, the ability of genistein (an antagonist of protein tyrosine kinase) and an SH2 domain inhibitor peptide, but not daidzein (an inactive homolog of genistein), to impair extracellular Mg²⁺ deficiency-induced contractions may implicate an involvement of tyrosine kinase activation (phosphorylation, including Src) in vascular contractile responses of cerebral smooth muscle to low [Mg²⁺]_o. We used daidzein, a structurally similar but inactive form of genistein, in the present study as a control agent to test the selectivity of tyrosine kinase antagonists, especially genistein. The calculated IC₅₀ values reported herein for genistein and the SH2 domain inhibitor peptide are 6.3 ± 0.3 × 10⁵ and 0.9 ± 0.1 × 10⁶ M, respectively, which are in a range similar to the reported inhibitor constant (K_i) values of genistein for protein tyrosine kinase (1.2 × 10⁵ M) (30) and SH2-SH3/phosphoprotein interaction (0.67 × 10⁶ M) (18). Similarly, other investigators have demonstrated that tyrosine kinase activation is important in serotonin-induced vascular contraction (34); in addition, carbamylcholine-, norepinephrine-, and epinephrine-induced contractions in vascular smooth muscle were significantly reduced by several different tyrosine kinase inhibitors, i.e., genistein, geldanomycin, and tyrphostin (16, 34). Importantly, tyrphostin (30 mM) and genistein (100 mM) significantly attenuated erythrocyte lysate-induced contraction in rabbit cerebral arteries, and genistein markedly inhibited KCl-induced contraction in these same vessels (21). Furthermore, the Src-selective tyrosine kinase inhibitor, PP1, is able to block contractions of guinea pig gastric longitudinal smooth muscle induced by thrombin receptor-activating peptide, TFLLR-NH₂ (TF), and epidermal growth factor (EGF)-urogastrone (42). These studies provide the foundation for the assertion that, in addition to their mitogenic activities, tyrosine kinase(s) and Src itself may play some important roles in agonist-induced smooth muscle contraction.

The involvement of tyrosine kinase, including the Src family, is reinforced by the present findings, in that genistein and an SH2 domain inhibitor peptide suppress both the low-[Mg²⁺]_o-induced rapid and the stable increments in [Ca²⁺]_i in single canine basilar smooth muscle cells at calculated IC₅₀ values of 6.0 ± 0.2 × 10⁵ M for genistein and 0.8 ± 0.03 × 10⁶ M for the SH2 domain inhibitor, which are in nice agreement with those of the low-[Mg²⁺]_o-induced arterial contractions under the same conditions and are consistent with previously published K_i values for these two antagonists (18, 29). These data suggest that contraction of the arteries to low [Mg²⁺]_o may be mediated, at least partially, by an elevation in [Ca²⁺]_i in canine basilar arterial smooth muscle cells. This conclusion is well supported by several lines of experimental data reported recently by other investigators: 1) platelet-derived growth factor BB elicits Ca²⁺ influx in human cultured vascular smooth muscle cells via a tyrosine kinase-dependent mechanism (14), 2) genistein can inhibit the activity of L-type Ca²⁺ channels in vascular smooth muscle cells from rat portal vein (25), 3) serotonin-evoked Ca²⁺ release from the sarcoplasmic reticulum in vascular smooth muscle cells is blocked by genistein (28), and 4) it has been proposed that tyrosine phosphorylation by both nonreceptor and receptor tyrosine kinases could be an important mechanism by which voltage-operated channels (VOCs) are regulated in vascular muscle (35, 36). In this context, evidence has been brought forth that such VOCs are regulated by Mg²⁺ in cerebral vascular muscle (7, 40).

One commonly used tyrosine kinase-dependent pathway is the MAPK pathway. Multiple MAPK pathways exist, but generally they can be subdivided into three groups: the ERK, the p38 kinase, and the c-jun NH₂-terminal kinases. The enzymes primarily responsible for both tyrosyl and threonyl phosphorylation of MAPK are the MAPK/ERK kinases, or MEK (34). As a tyrosine kinase, MEK might be a logical candidate to be activated by low [Mg²⁺]_o stimulation. The important observation presented herein is that SB-203580, a highly specific antagonist of p38 MAPK (22); U-0126, a potent and selective antagonist of MEK1/MEK2 (17); and PD-98059, a specific MAPKK antagonist (1), produce significant concentration-dependent attenuation of extracellular Mg²⁺ deficiency-induced contractions in endothelium-denuded canine basilar arteries. The calculated IC₅₀ values for SB-203580, U-1026, and PD-98059 are 2.0 ± 0.3 × 10⁶, 0.7 ± 0.1 × 10⁶, and 8.7 ± 0.3 × 10⁶ M, respectively. These values are consistent with the reported K_i values for U-0126 (~0.53 µM) (17), SB-203580 (~0.1-1.0 µM) (22), and PD-98059 (~2.0-7.0 µM) (1) for 50% inhibition of MEK1/MEK2, p38 MAPK, and MAPKK, respectively. Similarly, several reported recent studies indicate that 1) PD-98059 significantly inhibited vasopressin and KCl-induced elevated tone in rat middle cerebral arteries (23); 2) PD-98059 reduced contraction to 5-hydroxytrypamine in rat aorta, rat mesenteric arteries, and rat tail arteries (34); and 3) the contractile actions of thrombin receptor-activating peptide (TF) and EGF on guinea pig gastric longitudinal smooth muscle were attenuated by PD-98059 (42). These previous findings implicate the MAPK pathway in modulation of vascular smooth muscle contractility. Our present results suggest that activation of both MAPKK and MAPK pathways in cerebral arterial smooth muscle cells plays roles in these low-[Mg²⁺]_o contractile responses.

It has been shown previously that angiotensin II (ANG II)-induced increases of [Ca²⁺]_i in smooth muscle cells can result in activation of MAPK in rat aortic smooth muscle cells (32); ANG II-stimulated [Ca²⁺]_i increases in smooth muscle cells from spontaneously hypertensive rats could be significantly reduced by PD-98059 (33), and cumulative addition of exogenous Ca²⁺-increased myogenic tone of rat middle cerebral arteries was inhibited significantly by PD-98059 (23). This may be a pathway by which low [Mg²⁺]_o leads to increases of [Ca²⁺]_i in smooth muscle cells from canine basilar arteries and activates MAPKK and MAPK in the smooth muscle cells. In this context, our present findings indicate that SB-203580, U-0126, and PD-98059 significantly inhibited the low [Mg²⁺]_o-induced concomitant rise in [Ca²⁺]_i in single cells from canine basilar smooth muscle at calculated IC₅₀ values of 1.6 ± 0.1 × 10⁶, 0.7 ± 0.1 × 10⁶, and 8.2 ± 0.2 × 10⁶ M, respectively, which are in close agreement with those of the low [Mg²⁺]_o-induced arterial contractions under the same conditions; these values are consistent with previously reported K_i values for these three antagonists (1, 17, 22). These results could thus be used to support the above-mentioned contention that MAPKK and MAPK are indeed involved in low-[Mg²⁺]_o contractile responses.

Because decrements in [Mg²⁺]_o can reduce the intracellular Mg²⁺ concentration ([Mg²⁺]_i) in cerebral arterial smooth muscle cells (24), low [Mg²⁺]_o may directly act either on membrane tyrosine kinases or may first decrease [Mg²⁺]_i, then induce an increase in [Ca²⁺]_i, and further activate cytosolic tyrosine kinases and MAPK. The latter mechanism may be more suitable for the present study. It is also possible that Mg²⁺ might be affecting K⁺ channels. For example, Zhang et al. (40) have shown in rat cerebrovascular smooth muscle cells that [Mg²⁺]_i can modulate the properties of large-conductance Ca²⁺-dependent K⁺ channels. Our laboratory has shown, using patch-clamp techniques, that [Mg²⁺]_o can affect K⁺ channels in both rat cerebral endothelial cells and canine cerebral vascular muscle cells (Ref. 15 and unpublished data). Lastly, our results might be used to speculate that, like the recently proposed "Ca²⁺ receptor," a Mg²⁺ receptor might exist in cerebral vascular muscle cells. A variety of specific pharmacological antagonists of known endogenously formed vasoconstrictors did not inhibit or attenuate the low-[Mg²⁺]_o-induced contractions and elevation of [Ca²⁺]_i, which suggests no involvement of endogenous vasoconstrictors in such low-[Mg²⁺]_o contractile actions.

In summary, several points are noteworthy regarding the potential physiological significance of our present study. First, low-[Mg²⁺]_o medium, found in serum of human subjects under pathophysiological conditions (6, 8, 11, 12), induces contractions in canine cerebral arteries in an endothelium-independent manner. Second, such low-[Mg²⁺]_o medium-induced contractions can be significantly attenuated by tyrosine kinase antagonists, genistein, and U-0126 as well as PD-98059, an antagonist of p38 MAPK, SB-203580, and an SH2 domain inhibitor peptide. Third, and concomitantly, the increase in [Ca²⁺]_i in single cells from canine cerebral arterial smooth muscle induced by the low-[Mg²⁺]_o medium can be suppressed by the above-mentioned antagonists, also in concentrations associated with their specific inhibitory actions on their target enzymes. Overall, these results support the importance of tyrosine kinases including the Src family and MAPK pathways to low-[Mg²⁺]_o-associated cerebral vascular contraction. The present studies thus could help to shed new light on the etiologies of cerebral ischemia, cerebrovasospasm, and diverse cerebral vascular-stroke disease states associated with low serum levels of [Mg²⁺]_o (6, 8, 11, 12) and could be of considerable help in pinpointing potential new avenues for pharmacological and therapeutic intervention, particularly in Mg²⁺-deficient states. Lastly, the approach used herein, if applied to the intact cerebral microcirculation, may provide useful clues to the role of [Mg²⁺]_o and its cellular signaling pathways in regulation of cerebral blood flow.