Burton M. Altura^1,2,3, Asefa Gebrewold¹, Bella T. Altura^1,3, And Raj K. Gupta⁴

Departments of Physiology¹ and Medicine², and The Center for Cardiovascular and Muscle Research³, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, New York 11203 and Department of Physiology and Biophysics⁴, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461

Received 4/2/97; Accepted 5/5/97; On-line 5/15/97

3. METHODS

Male Wistar rats, weighing 135-180 g, were anesthetized lightly with pentobarbital sodium (Nembutal, 3 g/100 g, i.m.). After induction of anesthesia, each rat was placed in a General Electric Omega 400 WB spectrometer with a 9.4T vertical bore magnet utilizing double tuned ³¹P/¹H RF coils (4). The animal was carefully accommodated in the NMR probe (with head pointing down) so that all of the brain was contained within the RF coil. In order to make certain that the brain was positioned properly, we also obtained proton images using S50 gradient coils. After obtaining control ³¹P-NMR spectra (prior to cocaine administration), each animal was removed from the NMR probe, a femoral vein was cannulated, and either MgCl₂ was administered (10 µmoles/min) at a constant infusion rate followed 45 min later by i.p. injection of cocaine HCl (5, followed by 30 mg/kg 2 hr. later) or a comparable volume of 0.9% NaCl. Each cocaine-or saline-injected animal was then returned to the NMR probe and repeat ³¹P-NMR spectra were obtained at various intervals of time (e.g., 3-120 min, or up until death had occurred). Control, naive animals were administered the cocaine HCl in the absence of Mg²⁺ infusion. Animals were autopsied upon death or sacrificed to determine whether subarachnoid or intracerebral bleeding had occurred. All animals identified as having hemorrhagic stroke exhibited, upon autopsy, 1-3 ml of blood in the subarachnoid space and/or brain.

The chemical shift difference between the alpha- and ß-phosphoryl group resonances of ATP (delta_alphaß), along with a knowledge of the apparent K_d of MgATP (50 µmol/1 at pH 7.2, 37°C) under intracellular ionic conditions, was used to determine the concentration of [Mg²⁺]_i (4,10):

The K_d^MgATP was corrected for varying pH as needed. delta_alphaß^MgATP = 1340 Hz and delta_alphaß^ATP = 1748 Hz were utilized for calculations.

Intracellular pH (pH_i) was measured from the ³¹P-NMR spectra by use of the following equation (4):

where V_p is the ³¹P Larmor frequency in MHz and delta_obs is the chemical shift difference between the Pi and P-creatine (PCr) resonances in Hz.

The [PCr]/[ATP] and [Pi]/[ATP] concentration ratios were calculated from the ratios of integrated areas and corrected for partial saturation of resonance intensities (4). Ion selective electrodes (ISEs) were utilized to measure plasma ionized Mg²⁺, H⁺, K⁺ Na⁺, and Ca²⁺ (11). Total plasma Mg was measured with a Kodak Ektachem DT60 Analyzer (11). Where appropriate, mean values ± S.E. were calculated and compared using paired or unpaired Student's t-test,; and ANOVA for multiple comparisons. Chi-square tests (and regression analyses) were also used. A P-value less than 0.05 was considered significant.

The animal experiments were conducted in accord with the highest standards of human aminal care and that the appropriate approval of the experiments has been obtained from the university committee dealing with this issue.