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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