INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

INCREASING EVIDENCE INDICATES that acute "binge drinking" of ethanol by human subjects (e.g., ingestion of >80 g in <24 h) is associated epidemiologically with an ever-growing number of strokes and sudden deaths. Binge drinking has been related to intracerebral and subarachnoid hemorrhage as well as cerebral infarction (for reviews, see Refs. 1, 3, and 21). This recent recognition of an association between ethanol and strokes has lead to an increased interest in the action of ethanol on the cerebral circulation. Recent observations of the in situ cerebral microcirculation and a variety of isolated mammalian cerebral arteries indicate that acute treatment with ethanol produces prolonged constrictions in cerebral blood vessels (1-3, 6, 18), which suggests that such spasmogenic actions of ethanol are involved in the hypoxic, ischemic, and hemorrhagic actions of alcohol in the brain.

Multiple signaling pathways may participate in mechanisms of peripheral vasoconstriction. Several studies (2, 6-8, 27, 28, 48, 50, 51) indicate that increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) occur when arteries and single vascular smooth muscle cells are exposed to ethanol. This [Ca²⁺]_i increase could occur by a direct interaction with the contractile proteins and/or effects of Ca²⁺ acting as a cofactor for some cellular signal pathway(s), such as protein kinase C (PKC) (44) or phosphatidylinositol 3-kinases (PI3Ks) (36). PKC, a family of Ca²⁺-sensitive and Ca²⁺-insensitive phospholipid-dependent protein kinases that are present in high concentrations in vascular smooth muscle (26), has been shown to play an important role in cellular signal transduction. Several observations (24, 45) raise the possibility that activation of PKC isoforms might be involved in vasoconstriction induced by ethanol. However, there is no direct evidence that PKC is associated with ethanol-induced cerebral vasoconstriction. PI3Ks are a group of enzymes that act as direct biochemical links between a novel phosphatidylinositol pathway and receptor tyrosine kinases that appear to modulate Ca²⁺ channels through activity of these PI3Ks (11). Recently, PI3Ks and one PI3K product, phosphatidylinositol 3,4,5-trisphosphate (PIP₃), have received more attention because both PIP₃ and PI3Ks have been suggested to act as second messengers (38). However, there is no direct evidence that PI3Ks are associated with ethanol-induced cerebral vasoconstriction.

How does ethanol induce vascular injury in the brain? Over the past two decades, our laboratory has attempted to gain insight into ethanol-related cerebral vascular problems (for a review, see Ref. 1). With these points in mind, we designed the present study to determine whether specific PKC and PI3K antagonists would inhibit contractile responses of cerebral arteries to ethanol and whether these actions are associated with reduction in levels of [Ca²⁺]_i, thereby giving us insight into the potential contribution of these two cellular signaling pathways to ethanol-induced cerebrovasospasm.

	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 (weight 18-22 kg) after administration of pentobarbital sodium anesthesia (40 mg/kg iv) as described previously (48). The rings were placed in a normal Krebs-Ringer bicarbonate solution at pH 7.4 containing (in mM) 118 NaCl, 4.7 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 2.5 CaCl₂, 10 dextrose, and 25 NaHCO₃ (4). The rings were 3-4 mm in length. The segments were mounted on stainless steel pins under 2 g of resting tension in isolated organ baths, attached to force transducers (model FT 03, Grass), and connected to polygraphs (model 7, Grass). 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 beginning of an experiment, rings were exposed for 30-45 min to 80 mM KCl, and this was repeated every 30-45 min until responses were stable (two or three times). When tissues were pretreated with various drugs, each drug was applied for at least 15 min before the concentration-response curves were obtained. All of the animal experimental procedures were approved by our institutional Animal Care and Use Committee.

[Ca²+]_i measurement. Although it is known that smooth muscle cells can convert rapidly from a contractile to a synthetic phenotype under culture conditions, the transient [Ca²⁺]_i response data of primary smooth muscle cells obtained from canine basilar arteries and the contractile responses of isolated canine basilar arteries are in good agreement. Primary smooth muscle cells from canine basilar arteries for image-analysis experiments were seeded on glass coverslips (12-mm diameter, ~1 × 10⁶ cells/coverslip) and were used 2-3 days postseeding (49, 50). Monolayers of the smooth muscle cells, grown on the coverslips, were loaded with 2.0 µM fura 2-acetoxymethyl ester (AM) and 0.12% pluronic acid F-127 (60 min, 37°C), and the experimental procedures for [Ca²⁺]_i measurements were carried out as described previously using fura 2-AM (49, 50). Fluorescence ratios (R) were obtained by dividing the 340-nm image by the 380-nm image. No image misalignments occurred when these two ratiometric images were superimposed. The resulting images were then used to calculate [Ca²⁺]_i in smooth muscle cells using external standards containing 2.54 mM Ca²⁺ and 0 mM Ca²⁺ plus 10 mM EGTA for maximum and minimum fluorescence ratios (R_max and R_min, respectively) of the 340-nm and 380-nm images (49, 50). [Ca²⁺]_i was calculated according to the following equation (19, 49, 50)
A dissociation constant (K_d) of 224 nM was used for the fura 2-Ca²⁺ complex (9, 49, 50). B is the ratio of fluorescence intensity of fura 2 to the fura 2-Ca²⁺ complex excited at 380 nm. 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 (St. Louis, MO): acetylcholine hydrochloride, bisindolylmaleimide I hydrochloride, EGTA, and propranolol hydrochloride. Atropine sulfate was bought from Mann Research Laboratory (New York, NY). Cimetidine hydrochloride and diphenhydramine hydrochloride were received from Smith Kline and French Laboratories (Welwyn Garden City, Herts, UK). DMSO, Gö-6976, and wortmannin were purchased from Calbiochem (La Jolla, CA). Phentolamine methanesulfonate was purchased from Ciba Pharmaceutical (Summit, NJ). Methysergide maleate was purchased from Sandoz Pharmaceuticals (Hanover, NJ). LY-294002 was purchased from Biomol Research Labs (Plymouth Meeting, PA). 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 response (in g), percentage of maximal KCl-induced contraction, and [Ca²⁺]_i were expressed as means ± SE. Statistical evaluation of the results was carried out using analysis by the Newman-Keuls test and ANOVA using Scheffé's contrast test. The results were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ethanol-induced elevations in [Ca²+]_i are inhibited by PKC antagonists. Although not shown, intact versus endothelium-denuded cerebral arterial rings failed to manifest any significant differences in contractile concentration-response curves to ethanol (n = 36; P > 0.05). Only intact vessel rings were used in the present study. To our knowledge, a concentration of 20 mM ethanol can be considered as representative of moderate ethanol intake, because it can be found in the blood of most humans after oral ingestion of only 1-2 oz of ethanol (6, 7, 25). Accordingly, ingestion of 90-200 mM ethanol should be considered as heavy to very heavy ethanol intake, because 88 mM ethanol is known to be found in the blood of humans with ethanol-induced strokelike episodes (25). Therefore, we used a concentration range of 20-200 mM ethanol in the present study.

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View larger version (24K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent inhibitory effects of protein kinase C (PKC) antgonists on intracellular Ca2+ concentration ([Ca2+]i) changes in single smooth muscle cells from canine basilar arteries induced by varying concentrations of ethanol. A and C: concentrations of Gö-6976 and bisindolylmaleimide I (BIS) used were 106 and 5 × 107 M, respectively; preincubation time was 15 min. B and C: points represent peak values and means ± SE; n = 14-16. C: #P < 0.05; *P < 0.01; **P < 0.001.

PKC antagonists attenuate ethanol-induced contractions. As shown in Fig. 2, A and B, pretreatment of intact canine basilar arteries with Gö-6976 or bisindolylmaleimide I significantly attenuates ethanol-induced contractions (both phasic and tonic components) in a concentration-dependent manner. The IC₅₀ values for Gö-6976 and bisindolylmaleimide I are (6.36 ± 0.28) × 10⁷ M and (3.02 ± 0.12) × 10⁷ M, respectively, which are consistent with the reduced elevation in [Ca²⁺]_i induced by ethanol in the presence of these antagonists under the same conditions (Fig. 1, A and B). Mean values for vasoconstrictions induced by varying concentrations of ethanol in the presence of bisindolylmaleimide I and Gö-6976 are shown in Fig. 2C. According to our previous in vivo studies, ethanol administered either intraperitoneally, intravenously, or orally in doses to yield blood alcohol levels of ~11, 22, 44, and 88 mM induces ~10, 20, 31, and 35% reduction, respectively, in diameters of rat cortical arteries and branches of basilar arteries (2, 3, 6, 7). Also, we have some unpublished experimental data on in situ branches of canine basilar arteries that show that systemic administration of ethanol yielding blood levels of ~11, 22, 44, and 88 mM produces ~12, 25, 35, and 45% reductions in diameters of the arterial vessels (Gebrewold, Altura, and Altura; unpublished studies). We therefore believe that the in vitro results shown in the present study using isometric tension techniques are not too different from the in vivo rat and dog brain data.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent inhibitory effects of PKC antagonists on contractile responses of endothelium-intact canine basilar arterial smooth muscle induced by varying concentrations of ethanol. A and C: concentrations of Gö-6976 and BIS used were 106 and 5 × 107 M, respectively; preincubation time was 15 min. B and C: points represent peak values and means ± SE; n = 8. C: #P < 0.05; *P < 0.01; **P < 0.001.

Ethanol-induced rises in [Ca²+]_i are inhibited by PI3K antagonists. Figure 3A shows that preincubation of the cells with 5 × 10⁷ M wortmannin or 10⁵ M LY-294002 [both are selective antagonists of PI3Ks (10, 14, 36, 43)] effectively inhibits both the transient [Ca²⁺]_i peak and the secondary plateau of [Ca²⁺]_i induced by ethanol (to lower steady states) in basilar arterial smooth muscle cells. The inhibitory activity of these two antagonists shows clear concentration-dependent effects (Fig. 3B). The calculated IC₅₀ values for wortmannin and LY-294002 are (4.38 ± 0.14) × 10⁷ M and (2.29 ± 0.06) × 10⁶ M, respectively. Mean peak [Ca²⁺]_i values obtained for varying concentrations of ethanol in the presence of wortmannin or 10⁵ M LY-294002 are shown in Fig. 3C.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent inhibitory effects of phosphatidylinositol 3-kinase (PI3K) antagonists on [Ca2+]i changes in single smooth muscle cells from canine basilar arteries induced by varying concentrations of ethanol. A and C: concentrations of wortmannin and LY-294002 used were 106 and 5 × 106 M, respectively; preincubation time was 15 min. B and C: points represent peak values and means ± SE; n = 11-14. C: #P < 0.05; *P < 0.01; **P < 0.001.

PI3K antagonists attenuate ethanol-induced contractions. Figure 4, A and B, illustrates that the presence of wortmannin or LY-294002 attenuates contractile responses (both phasic and tonic components) of intact canine basilar arteries to ethanol in a concentration-dependent manner. The calculated IC₅₀ values for wortmannin and LY-294002 are (5.36 ± 0.25) × 10⁷ M and (3.18 ± 0.15) × 10⁶ M, respectively (Fig. 4B), which are in close agreement with the IC₅₀ values found for the reduced [Ca²⁺]_i levels produced by ethanol in the presence of these antagonists under the same conditions (Fig. 3, A and B). Mean values for varying concentrations of ethanol-induced contractions in the presence of wortmannin or LY-294002 are shown in Fig. 4C.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Contractile responses of endothelium-denuded canine basilar arteries to varying concentrations of ethanol were modified by PI3K antagonists. A and C: concentrations of wortmannin and LY-294002 used were 106 and 5 × 106 M, respectively; preincubation time was 15 min. B and C: points represent peak values and means ± SE; n = 8. C: #P < 0.05; *P < 0.01; **P < 0.001.

Effects of PKC and PI3K antagonists on ethanol and phenylephrine-precontracted canine basilar arterial segments. After achieving full contractile responses of isolated canine basilar arterial rings to 200 mM ethanol, we noted that administration of 10⁶ M Gö-6976, 5 × 10⁷ M bisindolylmaleimide I, 10⁶ M wortmannin, or 5 × 10⁶ M LY-294002 led to a reduction of the ethanol contractions to ~50-75% of the initial level (Figs. 5A and 6A). The addition of identical concentrations of Gö-6976, bisindolylmaleimide I, wortmannin, or LY-294002 to the medium brought about significant relaxation in phenylephrine (PE)-precontracted isolated cerebral arteries (Figs. 5B and 6B). The effects of these antagonists on ethanol-precontracted cerebral arterial segments (Figs. 5A and 6A) are much stronger than those of equipotent PE-precontracted segments (Figs. 5B and 6B).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Inhibitory effects of 106 M Gö-6976 and 5 × 107 M BIS on isolated canine basilar arterial rings precontracted by 200 mM ethanol (A) and 0.2 µM phenylephrine (PE) (B) as functions of time for relaxation and tension. Data expressed as means ± SE; n = 6 each. #P < 0.05; *P < 0.01; **P < 0.001 compared with control.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Inhibitory effects of 106 M wortmannin and 5 × 106 M LY-294002 on isolated canine basilar arterial rings precontracted by 200 mM ethanol (A) and 0.2 µM PE (B) as functions of time for relaxation and tension. Data expressed as means ± SE; n = 6 each. #P < 0.05; *P < 0.01; **P < 0.001 compared with control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study aimed to determine whether activation of PKC and PI3Ks are involved in contractile responses of cerebral arteries to ethanol and whether these effects are associated with alteration in [Ca²⁺]_i, thereby giving us some insight into the potential contribution of these two cellular signaling pathways to ethanol-induced cerebrovasospasm.

PKC has been implicated in the control of a variety of intracellular activities including vascular smooth muscle contraction (22, 44). Its role in regulation of vascular tone is in large measure strengthened by the identification of PKC as a physiological target for diacylglycerol, which is a second messenger in several cellular signal transduction pathways (22). Evidence has accumulated to suggest that PKC may play an important role in regulation of cerebrovascular tone under physiological and pathological conditions (17, 33). An increase in PKC activity of cerebral arteries in response to vasoconstrictor agonists has been recently reported (17, 33). Several studies have also demonstrated that ethanol-induced vasoconstriction in the rat aorta may be inhibited by PKC antagonists (24, 45). However, to our knowledge, there is no experimental evidence indicating PKC involvement in ethanol-induced contractile responses in cerebral vascular smooth muscle. In this context, our present observations clearly indicate that Gö-6976 (a selective antagonist of Ca²⁺-dependent PKC- and PKC-I isozymes) and bisindolylmaleimide I (a specific PKC antagonist) markedly attenuate the ethanol-induced contractile responses of canine basilar arterial segments, which suggests the probable involvement of PKC activation in such arterial contractions. This contention is supported by the IC₅₀ values found herein experimentally. Several investigations have indicated that similar or higher concentrations of these two antagonists have been used to inhibit PKC in diverse preparations (16, 30, 32, 53). However, it must be entertained that in some cases the 15- to 54-fold differences between IC₅₀ and inhibition constant (K_i) values could be reflective of the possibility that alternate pathways or targets may be involved. Additionally, it is possible that the in situ enzymes may be covalently modified and that unique isoforms may be involved.

The involvement of PKC isoforms in the ethanol-induced contraction pathway is reinforced by the present finding that in single smooth muscle cells from canine basilar arteries preincubated with PKC antagonists (Gö-6976 and bisindolylmaleimide I), ethanol produces slower and smaller increments in [Ca²⁺]_i compared with untreated control cells (Fig. 4A). Similarly, other investigators have demonstrated that 1) inhibition of PKC activity with either staurosporine or chelerythrine can inhibit availability and long opening of L-type Ca²⁺ channels in A7r5 cells (35); 2) inhibition of PKC activity blunts the relative increase in cytosolic free Ca²⁺ in rabbit afferent arterioles in response to angiotensin II (40); 3) PKC-induced constriction of pressured rat cerebral arteries is associated with a decrease in [Ca²⁺]_i, suggesting an increase in the Ca²⁺ sensitivity of the contractile process (17); and 4) low extracellular magnesium-induced contractions and increases in [Ca²⁺]_i of rat aortic and canine cerebral arterial smooth muscles are attenuated markedly by PKC inhibitors (46, 47).

The growing importance of PI3Ks in signal transduction has been pointed out during the past three years (13). A number of proteins have been shown to be associated with PI3Ks after stimulation of cells, generally as a result of tyrosine-phophorylated proteins associating via the PI3K p85 SH₂ domains (15). We demonstrate herein for the first time that two potent antagonists of PI3Ks, wortmannin and LY-294002, significantly suppress ethanol-induced contractions in canine basilar arteries and the concomitant elevation of [Ca²⁺]_i in canine basilar single arterial smooth muscle cells, indicating the likely involvement of products of PI3Ks (e.g., phosphatidylinositol 3,4-bisphosphate and PIP₃) in such vessel contractions. This contention gains further support from the experimentally derived IC₅₀ values. The IC₅₀ value for LY-294002 was (3.18 ± 0.15) × 10⁶ M, which is consistent with the reported K_i value for this antagonist for PI3Ks, which is 1.4 × 10⁶ M (43). Although the IC₅₀ value for wortmannin was (5.36 ± 0.25) × 10⁷ M, which is higher than the reported K_i value, similar or even higher concentrations of this antagonist have been used to inhibit PI3Ks in human and animal smooth muscle cells (10, 14). Our present findings are consistent with and well supported by experimental data reported recently by other investigators for some other vasoactive substances: 1) angiotensin II may stimulate the Ca²⁺ channels of vascular smooth muscle cells through activity of PI3Ks and protein tyrosine kinases (41); 2) wortmannin attenuates the contraction of guinea pig gastric longitudinal smooth muscle induced by thrombin and epidermal growth factor (53); 3) the amplitude of contractions of the rat aorta induced by KCl, PE, and prostaglandin F₂ is decreased by wortmannin (42); 4) addition of wortmannin or LY-294002 to HepG2 cells inhibits the release of intracellular Ca²⁺ induced by coactivation of phospholipase C and PI3K (38); and 5) addition of wortmannin or LY-294002 to canine cerebral vascular smooth muscle cells inhibits the increases in [Ca²⁺]_i induced by low extracellular Mg²⁺ concentration (47). PI3Ks are composed of the p85 regulatory subunit and the p110 catalytic subunit, which contains a p21^ras-binding domain (39). The p21^ras-binding domain plays an important role in a variety of signaling pathways for cellular growth, differentiation, and transformation (37). Recently, p21^ras was reported to stimulate Ca²⁺ channels (29); it therefore may be involved in the Ca²⁺ influx stimulated by ethanol via activation of PI3Ks. However, additional mechanisms for ethanol-stimulated elevation and contraction must also be considered. It was shown previously that activation of PI3Ks leads to synthesis of PI3K products (e.g., PIP₃) that may bind to and activate atypical PKC isozymes (31, 34), and PIP₃ may also indirectly stimulate PKC through activation of "small G proteins" such as Rac (20). Furthermore, PIP₃ may directly act on Ca²⁺ channels (11).

It was noted that the antagonists of PKC and PI3Ks used herein produced relaxant effects (albeit much smaller effects compared with those against ethanol) on 0.2 µM equipotent PE-precontracted isolated canine cerebral arteries. In our opinion, this does not indicate that the signaling pathways activated by ethanol are not specific. The pathways coupling PE and ethanol to contraction may have some common elements, which are probably downstream from the site at which ethanol initiates contraction. Similar results have been reported by other investigators for PE and other vasoactive substances (42, 46, 47, 53).

In summary, our present study suggests that 1) ethanol-induced cerebral arterial contractions can be significantly attenuated in a concentration-related manner by PKC antagonists and PI3K antagonists, 2) the increase in [Ca²⁺]_i in cerebral arterial smooth muscle cells induced by ethanol can be suppressed by the above-mentioned antagonists also in a concentration-related manner, and 3) the antagonists of PKC and PI3Ks used in this study act on canine cerebral vascular muscle cells in concentrations associated with specific inhibitory actions on target enzymes. On the basis of the above and previous findings, several points are noteworthy regarding the initiation of ethanol contractions of cerebral vascular smooth muscle: 1) Ca²⁺-mediated activation and translocation of certain PKC isozymes (probably Ca²⁺ dependent) and activation of PI3Ks may occur, 2) PKC- and PI3K-stimulated increases in [Ca²⁺]_i may occur in the smooth muscle cells of canine cerebral arteries, 3) PKC-Ca²⁺ sensitization and phosphorylation of myosin light chain kinases may occur, and, finally, 4) activation of actin and interaction with myosin may be followed by contraction. Overall, these results provide considerable evidence for the importance of PKC and PI3Ks in ethanol-associated cerebral vascular contraction. We believe that these two signal-transduction pathways may play important roles in the etiology of ethanol-induced stroke and that the approach taken in this study may prove useful in the development of new prophylactic and therapeutic tools for prevention and amelioration of alcohol-induced strokes.