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European Journal of Pharmacology - Molecular Pharmacology Section, 246 (1993) 299-301

Rapid communication

Cocaine induces rapid loss of intracellular free Mg2+ in cerebral vascular smooth muscle cells

Burton M. Altura, Aimin Zhang, Toni P.-O. Cheng and Bella T. Altura

Departments of Physiology, Medicine and Anatomy and Cell Biology,
State University of New York Health Science Center at Brooklyn,
450 Clarkson Avenue, Brooklyn, NY, U.S.A.

 

Acute exposure of cultured canine cerebral vascular smooth muscle cells to low concentrations of cocaine HCl (10-9 to (10-7 M) resulted in significant, rapid (1 min) loss of intracellular free Mg ions ([Mg2+]i); these reductions (12-25%) in [Mg2+] were reversible upon exposure to normal, Mg2+-containing physiological salt solution. These findings help to provide a rational basis for why cocaine can result in cerebrovasospasm and stroke.

 

Mg2+ (cytosolic, free); Cocaine strokes, Mag-fura-2

Use of cocaine is associated with an ever-growing number of aneurysmal subarachnoid hemorrhages, intracerebral hemorrhages, brain edema and occlusion-type strokes in human subjects (for reviews, see Levine et al., 1991; Sloan et al., 1991). Cocaine can result in serious central nervous system damage and major alterations in fetal brain structure, with subsequent neurologic, cognitive and behavioral dysfunctions.

While it is certain that cocaine facilitates strokes in the brain, and that it is a risk for stroke in individuals who have cerebrovascular disease, it is by no means certain how these strokes are brought about (for reviews, see Levine et al., 1991; Sloan et al., 1991). Using in vivo 31P-nuclear magnetic resonance spectroscopy on rats, we found that systemic administration of cocaine HCl resulted in concentration-dependent, significant and progressive deficits in whole brain intracellular free Mg2+ ([Mg2+]). (Altura and Gupta, 1992). Intracellular pH ([H+]i) also fell in a progressive manner but only after a significant fall in brain [Mg2+]i. Brains of rats that exhibited stroke-like events, upon autopsy, demonstrated continued intracellular acidosis associated with progressive loss of phosphocreatine and elevation of inorganic phosphate (Pi) up until death. Recently, we found that low concentrations (i.e., 10-1 to 10-7 M) of cocaine HCl induce contraction of intact and isolated cerebral vascular smooth muscle cells from a variety of mammals (Huang et al., 1990; He et al., 1993), including sub-human primates which is associated with a rapid rise (within 1-5 min) in intracellular free Ca2+ ions ([Ca2+]i) (He et al., 1993). We, therefore, tested the hypothesis that cocaine-induced reduction in cerebral vascular smooth muscle [Mg2+]i might set into motion the cocaine-induced cerebrovasospasm resulting in hypoxia, ischemia and stroke observed in the in vivo 31P-NMR studies.

Experiments were carried out on single cultured primary cerebral vascular smooth muscle cells obtained from sodium pentobarbital-anesthetized (40 mg/kg) male mongrel dogs (18-21 kg) using digital imaging microscopy with the fluorescent probe mag-fura-2, according to previously established methods (Zhang et al., 1992). The cells were cultured in Dulbecco's modified Eagle's medium at 37°C in a humidified atmosphere composed of 95% air/5% CO2. The cells were loaded with mag-fura-2 (Molecular Probes, Eugene, OR) by incubating them with 5 µ mag-fura-2/AM in the culture media for 60 min under 95% air/5% CO2. To improve loading efficiency, 0.12% pluronic acid F-127 (Sigma Chemicals St. Louis, MO) was used in the loading media. The labeled cells were washed for 1 and 5 min with HEPES buffer solution (in mM; NaCl 118, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, HEPES 5 and glucose 10) containing 0, 10-9), 10-7 and 10-5 M cocaine HCl. The pH was adjusted to pH 7.4 with NaOH. Measurement of [Mg2+]i, was performed using a TN 85090 Fluorplex Image Analyzer (Tracor Northern, Madison WI). Images of mag-fura-2 fluorescence at 510 nm emissions were obtained with 335 and 370 nm excitation wavelengths using a silicon intensified target (SIT) camera. Fluorescence ratios (R335/370) were obtained by dividing the 335 image by the 370 image.

An in vitro calibration method was used to calculate [Mg2+]i of single cerebral vascular smooth muscle cells employing 0 and 10 mM buffered MgSO4 standard solutions. From these standards solutions, the maximum (Rmax) and minimum (Rmin) fluorescence ratios of the 335-nm and 370-images were generated. (Mg2+]i, was calculated according to the following equation (Raju et al., 1989):

[Mg2+]i = Kd x B x (R - Rmin)/(Rmax - R)

A Kd of 1.5 mM was used for the mag-fura-2-complex (Raju et al., 1989). B is the ratio of fluorescence intensity of mag-fura-2 to the Mg bound mag-fura-2 at 370 nm. Particular care was taken to minimize photo-bleaching of the dye. Experiments were done in total darkness, and exposure to excitation light was less than 2 min in duration in all experiments. Cocaine HCl was obtained from The National Institute of Drug Abuse (Research Triangle Park, NC).

Where appropriate, means ± S.E.M.s were calculated and compared for statistical significance by paired t-tests and ANOVA using Scheffe's contrast test for multiple comparisons.

With no cocaine, the basal level of [Mg2+]i was 480 ± 20 and 510 ± 30 µM at 1 and 5 min, respectively (Table 1), with a heterogeneous distribution of [Mg2+]i. Exposure to 10-9 M cocaine for only 1 min resulted in a 12% reduction in [Mg2+]i; at 5 min the [Mg2+]i level was reduced 22%. Although not shown, a number of the cells exhibited reductions to 275-300 µM (Mg2+ii. Addition of 10-7 M cocaine to these cultured cells resulted in 15 and 24% reductions at 1 and 5 min, respectively. Addition of 10-5 M cocaine failed to result in any further reduction in [Mg2+]i.

 

TABLE 1

Effects of cocaine on [Mg2+]i, in cultured canine cerebral vascular smooth muscle cells.

Cocaine

[Mg2+]i(µM)

 

(M)



1 min

5 min




0

480±20

510±30

10-9

420±20 *

400±20 *

10-7

410±20 *

390±30 *

10-5

430±35

390±35 *

0

480±50

535 ±50

(at end)

 

 


n=33
* Significantly diff. from 0-cocaine (P < 0.05).

 

Reintroduction of, and incubation (for 15 min) with, normal HEPES buffer containing 0-cocaine restored normal [Mg2+]i.

To our knowledge, this is the first demonstration that very low concentrations of cocaine HCl can result in rapid reduction in [Mg2+]i in single cells. These data, thus, show that cocaine is capable of producing by a direct mechanism (in the absence of nervous elements, circulating hormones or blood), significant and time-dependent depletion of [Mg2+]i from cerebral vascular smooth muscle cells. In view of these observations, it would be important to determine if similar depletion of [Mg2+]i is promoted by cocaine and 'crack' cocaine in other cell types in the brain (e.g., endothelial, glial and neuronal cells). The fact that [Mg2+]i fell often below 325 µM is potentially quite important, since most of the 325 Mg2+-regulated cellular enzyme systems operate at [Mg2+]i between 400- 1000 µM (Garfinkel and Garfinkel, 1985).

Our findings are consistent with a vasospastic response in cerebral microvessels leading to vascular occlusion and/or intracerebral bleeding set into motion by a loss of cerebral vascular smooth muscle [Mg2+], (Huang et al., 1990; Altura and Gupta, 1992). It is known that Mg2+ normally either gates or has an action on Ca2+ entry and intracellular release in vascular muscle cells (Zhang et al., 1992). Thus, depletion of [Mg2+]i by cocaine, as shown here, would allow entry and intracellular release of Ca2+ , which was, indeed, found recently (He et al., 1993), causing cerebral vasospasm. The progressive rise in brain [H+]i, and [Pi], and the associated loss in PCr, in cocaine-stroked animals, noted recently (Altura and Gupta, 1992) are consistent with this hypothesis.

 

Acknowledgement

This study was supported in part by a Research Grant AA-08674 from N.I.A.A.A. to B.M.A.

 

References

Altura, B.M. and R.K Gupta, 1992, Cocaine induces intracellular free Mg deficits, ischemia and stroke as observed by in-vivo 31P-NMR of the brain, Biochim. Biophys. Acta (Biomembr.) 1111, 271.

Garfinkel, L. and D. Garfinkel, 1985, Magnesium regulation of the glycolytic pathway and the enzymes involved. Magnesium 4, 60.

He, G.-Q., A- Zhang, B.T. Altura and B.M. Altura, 1993, Cocaine induced cerebrovasospasm and its mechanism of action, J. Pharmacol. Exp. Ther., in press.

Huang, Q.-F., A. Gebrewold, B.T. Altura and B.M. Altura, 1990, Cocaine-induced cerebral vascular damage ran be ameliorated by Mg2+ in rat brain, Neurosei. Lett. 109, 113.

Levine, S.R., J.C.M., Brust, N. Futrell, L.M. Brass, D. Blake, P. Fayad, L.R. Schultz, C.H. Miliken, K.L Ho and K.M.A. Welch, 1991, A comparative study of the cerebrovascular complications of cocaine: alkaloidal versus hydrochloride, a review, Neurology 41, 1173.

Raju, R., E. Murphy, L.A. Levy, R.D. Hall and R.E. London, 1989, A fluorescent indicator for measuring cytosolic free magnesium, Am. J. Physiol. 256, C540.

Sloan, M.A., S.J. Kitter, D. Rigamonti and T.R. Price, 1991, Occurrence of stroke associated with use/abuse of drugs, Neurology 41, 1358.

Zhang, A., T.P.-O. Cheng, B.T. Altura and B.M. Altura, 1992, Extracellular magnesium regulates intracellular free Mg2+ in vascular smooth muscle cells, Pfügers Arch. 421, 391.

Correspondence to: Prof. B.M. Altura, Dept. of Physiology - Box 31, SUNY-Health Science Center @ Brooklyn, 450 Clarkson Avenue, Brooklyn, N.Y. 11203 USA. Tel. (718) 270-2618; Fax: (718) 270-3103.


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