David H. Ross

Morphine and Ethanol: Selective Depletion of Regional Brain Calcium

Administration of morphine or ethanol to rats produces a decrease in regional brain calcium in vivo. This effect is selectively antagonized by the stereospecific narcotic antagonist naloxone. Reserpine and the dopamine-acetaldehyde conjugate salsolinol also produce a depletion of regional brain calcium, but only the salsolinol depletion is antagonized by naloxone. Experiments with naloxone provide evidence for two calcium-sensitive pools in the central nervous system.

Morphine induced calcium depletion in discrete regions of rat brain.

The in vivo administration of a single dose of morphine produces a decrease of tissue calcium in the rat brain. This decrease is observed to be linear, dose‐dependent, time‐dependent and to occur to an equal degree in 8 discrete brain regions. This effect of morphine is blocked by naloxone and exhibits a high degree of sterospecificity. The reserpine induced decrease of brain calcium was not antagonized by naloxone. Differentiation of this response using reserpine and naloxone indicates the possibility of calcium pools in the central nervous system.

THE LEVELS OF LABILE INTERMEDIARY METABOLITES IN MOUSE BRAIN FOLLOWING RAPID TISSUE FIXATION WITH MICROWAVE IRRADIATION

The levels of several labile glycolytic and organic phosphate metabolites in mouse brain were determined following rapid inactivation with 2450 MHz microwave irradiation. The levels of ATP in mouse brain following a 0·25 s exposure in a 6 kW microwave oven was found to be 2·416 ± 0·061. Whole brain levels of 8 labile intermediary metabolites in 0·4 s irradiated samples were comparable to those reported using the previously‐described methods of freeze‐blowing or whole‐animal immersion. Analysis of these same metabolites in 4 gross areas of brain did not reveal any anoxic changes betwen superficial and deeper brain areas. The advantages of the mcrowave irradiation inactivation technique for regional brain studies of labile intermediary metabolites is discussed.

SELECTIVE ACTION OF ALCOHOLS ON CEREBRAL CALCIUM LEVELS

I t is generally agreed that while alcohol and opiates produce tolerance and physical dependence, there has been no consistent demonstration of evidence for commonality of action between these two agents at the biochemical level. In consideration of the steps that lead to induction of tolerance and physical dependence, it would appear necessary to explore the changes that may be occurring in the region of the synaptic junction. One parameter has been recently examined based on its role in neuronal function and requirements for membrane integrity. It has been demonstrated that significant changes in cerebral calcium content may be readily measured after treatment in vivo with ethanol, morphine, or the tetrahydroisoquinoline alkaloid salsolino1.l Salsolinol is the alkaloid conjugate formed nonenzymatically from dopamine and acetaldehyde and has been postulated to play a role in the pharmacology of alcoholism.? The depletion of calcium by these three drugs was prevented by the opiate antagonist naloxone, whereas depletion of calcium by reserpine was unaffected by naloxone pretreatment. Further investigation into the effects of opiates on calcium content revealed that the depletion was dose and time dependent and stereospecific; the inactive isomer dextrophan showed no calcium-depleting effects. Through use of reserpine and naloxone, evidence was also obtained that indicates at least two distinct pools of calcium available for depletion, one of which was morphine sensitive, and the other was reserpine sensitive.’ We have suggested that the activities of these two agents, opiates and ethanol, may be directed toward a common locus of action in producing their effects on the nervous system. The experiments reported in this paper extend our investigations into the common action of morphine and ethanol and provide further evidence for the neurochemical role of salsolinol after alcohol ingestion.

Rate of inactivation of adenyl cyclase and phosphodiesterase: Determinants of brain cyclic AMP

Abstract In order to assess the effects of time requirements of different tissue inactivation methods, concentrations of cyclic adenosine monophosphate in rat brain were determined. Fixation of tissues was obtained by the following methods: decapitation with removal of brain and freezing in liquid nitrogen; decapitation into liquid nitrogen; whole animal immersion in liquid nitrogen; 1.5 kW maximal field strength microwave irradiation for 8 seconds; and, 5 kW maximal field strength microwave irradiation for 2 seconds. Results of these studies indicate that as the time is reduced for inactivation of brain adenyl cyclase and phosphodiesterase, levels of cyclic adenosine monophosphate become progressively lower. This same correlation is also evident in studies of regional brain concentrations of cyclic adenosine monophosphate after 1.5 kW and 5 kW microwave inactivation. It is concluded that 5 kW maximal field strength microwave exposure is the most rapid means of enzyme inactivation permitting a more accurate estimation of endogenous cyclic adenosine monophosphate concentrations. Its use offers rapid inactivation with minimization of trauma and facilities the study of regional metabolites through ease of dissection.

Characterization of a high-affinity Mg2+-independent Ca2+-ATPase from rat brain synaptosomal membranes.

Abstract: A high‐affinity Mg2+‐independent Ca2+‐ATPase (Ca2+‐ATPase) has been differentiated from the Mg2+‐dependent, Ca2+‐stimulated ATPase (Ca2+, Mg2+‐ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+‐ATPase was found to be 1.33 μM with a Km for ATP of 19 μM and a Vmax of 33 nmol/mg/min. Using Ca‐ATP as a substrate, the Km for Ca‐ATP was found to be 0.22 μM. Unlike Ca2+, Mg2+‐ATPase, Ca2+‐ATPase was not inhibited by N‐ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. UnLike Ca2+, Mg2+‐ATPase activity, ATP‐dependent Ca2+ up take was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+‐ATPase described. Ca2+‐ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+‐ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+‐ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high‐affinity Mg2+‐independent Ca2+‐ATPase may be a separate form from Ca2+, Mg2+‐ATPase. The capacity of Mg2+‐independent Ca2+‐ATPase to hydrolyze GTP suggests this protein may be involved in GTP‐dependent activities within the cell.

Calcium content and binding in synaptosomal subfractions during chronic morphine treatment.

Chronic exposure to morphine in mice produced an increase in Ca2+ content of synaptosomes, synaptic plasma membranes (SPM), and synaptic vesicles. Ca2+ binding capacity was significantly reduced in tolerant SPM fractions. Naloxone significantly reversed the increased calcium content and reduced binding capacity of SPM when administered to 72-h-treated mice. Scatchard analysis of binding curves reveals three distinct classes of Ca2+ binding sites. During tolerance, the high- and low-affinity sites exhibit a reduced capacity to bind calcium, which may be reversed by in vivo and in vitro administration of naloxone. The increase in SPM and synaptic vesicle calcium content may reflect adaptive changes in the cell membrane during tolerance development, which may contribute to changes in neurotransmitter and second messenger function.

Effects of in vivo ethanol administration on Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ uptake activity in synaptosomal membranes

Abstract1.Acute administration of ethanol (4 g/kg, i.p.) to mice inhibits the sequestration of calcium into endoplasmic reticulum-like organelles in synaptosomal membranes.2.Ethanol administration inhibits both Ca2+-stimulated adenosine triphosphate hydrolysis and ATP-dependent calcium uptake in the vesicles at time of loss of righting reflex.3.At recovery of righting reflex, the Ca2+-ATPase activity returns to normal levels, while the ATP-dependent uptake remains inhibited.4.The effect of ethanol is specific for the sequestration (active transport) of calcium since calcium binding to synaptic membranes is not altered.5.Alteration in mechanisms responsible for synaptosomal buffering of cytosolic Ca2+ levels by in vivo ethanol may contribute to altered transmitter release rates following ethanol adminstration.

Inositol 1,4,5-trisphosphate induced mobilization of Ca2+ from rat brain synaptosomes

Inositol 1,4,5-trisphosphate (IP3) was found to release Ca2+ from presynaptic nerve endings (synaptosomes) made permeable with saponin. ATP-dependent Ca2+ uptake was carried out until equilibrium was reached. Addition of IP3 produced a rapid release of Ca2+, which was complete within 60 sec, followed by Ca2+ reaccumulation to the original level in 5–7 min. Cholinergic receptor stimulation with muscarine also produced a similar Ca2+ release from synaptic endoplasmic reticulum. Ca2+ release by IP3 was not detectable in the absence of the mitochondrial inhibitors oligomycin or sodium azide. Reaccumulation of Ca2+ was prevented by the presence of vanadate, a potent inhibitor of Ca2+/Mg2+ ATPase. Half maximal and near complete release of Ca2+ took place at 0.4 μM and 3 μM IP3 concentrations, respectively. These studies demonstrate for the first time IP3 mobilization of Ca2+ from endoplasmic reticulum within synaptic plasma membranes.

Selective control of calcium levels by naloxone

Abstract Acute treatment with morphine sulfate produces a selective loss of calcium from synaptosomal particulate fractions of rat brain. No changes in sodium, potassium or magnesium content were observed for myelin, synaptosomal particulate or mitochondrial fractions. Acute opiate treatment (90 min.) while causing calcium loss, produced no changes in regional brain content for sodium, potassium or magnesium. Naloxone, in the presence of morphine, reversed the calcium loss in both regional brain areas and synaptosomal particulate fractions. An hypothesis is offered that naloxone may bind to synaptosomal membranes protecting a morphine sensitive calcium pool, or may reverse the calcium loss seen after opiate agonist treatment.