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A series of X-ray pole figures were obtained from oriented and unoriented rigid polyvinyl chloride sheets. From these, information about crystallite orientation in uniaxially and biaxially drawn sheets stretched at 90°C was obtained. Uniaxial orientation produced two crystallite orientations with the extent of orientation increasing with draw ratio; for biaxial samples the chain direction (c) was distributed uniformly in the plane of the sheet, with the planar orientation improving as draw ratio increased. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 528–535, 2007

Chronic exposure to morphine in mice produced an increase in Ca(2+) content of synaptosomes, synaptic plasma membranes (SPM), and synaptic vesicles. Ca(2+) 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 Ca(2+) 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.

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.

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 microM with a Km for ATP of 19 microM 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 microM. 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+ uptake 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.

The effect of a single administration of morphine sulfate (15 mg/kg, s.c. or 30 mg/kg, i.p., 30 min) on Ca2+-stimulated Mg2+-dependent ATPase activity was investigated in synaptosomal plasma membranes (SPM) prepared from rat cortex. Morphine produced a significant decrease in Ca2+,Mg2+-ATPase activity in synaptosomal fractions (SPM 1 + 2) known to contain a high density of opiate receptors and calmodulin-dependent Ca2+,Mg2+-ATPase. However, in another subpopulation (SPM 3) that contains fewer opiate receptors and less enzyme activity, no such decrease in the enzyme activity was observed after the opiate administration. The decrease in Ca2+,Mg2+-ATPase activity seen in SPM 1 + 2 was specifically antagonized by the opiate antagonist naloxone hydrochloride (2 mg/kg, s.c.) when given 15 min before morphine administration. Mg2+-ATPase was not altered either by morphine or by a naloxone-morphine combination. These findings give further evidence for the role of intracellular Ca2+ in mediating many of the acute effects of opiates.

The calcium content of membranes may regulate a number of key neuronal processes. Studies are described in which acute and chronic administration of ethanol was shown to alter calcium binding to synaptosomal plasma Membranes.

Rats receiving the dihydropyridine Ca++ agonist BAY K8644 (0.1-3 mg/kg SC) displayed increasing loss of body temperature. At the highest dose tested (3 mg/kg) rats exhibited decreased motor activity, ataxia, increased vocalization upon handling and increased auditory sensitivity. Nimodipine (1 mg/kg SC) produced antagonism of this response when used as pretreatment at 15 and 30 minutes. The phenylalkylamine, verapamil (5 mg/kg) and the benzothiazepine diltiazem (10 mg/kg) did not alter BAY K8644-induced hypothermia. None of the three Ca++ channel antagonists produced changes in body temperature at the antagonist doses used. BAY K8644 (3 mg/kg SC) produced stimulation of Ca++/Mg++ ATPase activity by 31% in hypothalamus but not in cortex or cerebellum. This stimulation of enzyme activity was selectively prevented by nimodipine but not verapamil or diltiazem. No changes in enzyme activity were observed when Ca++ channel antagonists were used alone. These studies demonstrate that the Ca++ agonist BAY K8644 produces receptor mediated hypothermia which is dihydropyridine receptor dependent. Activation of Ca++ ATPase in the hypothalamus suggests that activation of dihydropyridine receptors may be coupled to Ca++ transport systems in this brain region and may reinforce the Ca++ set point theory of thermoregulation.

The in vivo effect of the mu agonist morphine and antagonist naloxone on [3H]nimodipine receptor binding in rat brain regions has been investigated. Morphine administration (15 mg/s.c.) for thirty minutes produced a 19% decrease in [3H]nimodipine receptor binding (Bmax 158.2 fmol to 128.9 fmol) in cortex and 29% decrease in cerebellum (65.3 fmol to 46.0 fmol). Lesser changes were observed in hippocampal and striatal regions with no changes in hypothalamus and brain stem. All effects were completely antagonized by naloxone pretreatment (1 mg/kg). The studies suggest that opiates in vivo can alter [3H]nimodipine binding to the Ca2+ channel receptor protein. These findings agree with the previously observed decreases in Ca2+ influx in nerve ending preparations and inhibition of ICa2+ following opiate treatment and suggest opiates reduce Ca2+-dependent neurotransmitter release by altering the Ca2+ channel receptor protein in an allosteric fashion.

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.

Ca2+ transport activity in synaptosomal membranes has been identified as having two major components: Ca2+-stimulated ATP hydrolysis and ATP-dependent CA2+ uptake. Both processes exhibit similar affinities for Ca2+ and operate maximally under identical buffer conditions. Subcellular fractionation studies revealed the Ca2+/Mg2+ ATPase and ATP-dependent CA2+ uptake activities to be highest in synaptic plasma membrane fractions 1 and 2, with lesser activity in synaptic vesicles and mitochondria. Progressive treatment with Triton X-100 activated, then decreased Ca2+/Mg2+ ATPase, Mg2+ ATPase and Ca2+ ATPase. ATP-dependent Ca2+ uptake was progressively decreased by similar treatment with Triton X-100. These studies illustrate that Ca2+ ATPase and ATP-dependent Ca2+ uptake may provide two important mechanisms for buffering of cytosolic Ca2+ at the nerve terminal. These systems may function to rapidly sequester cytosolic Ca2+ following a rise during depolarization and then extrude Ca2+ from the terminal against a concentration gradient. This regulation of cytosolic Ca2+, represented by two processes of the type seen in other plasma membranes, may play critical roles in calcium homeostasis in nerve cells.

The effects of a kappa opiate agonist have been evaluated on [3H]nimodipine binding to dihydropyridine receptors for 'L'-type Ca2+ channels in rat brain regions. Administration of U50-488H (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1,-pyrolidinyl-cyclohexyl benzeneacetamide) produced a 28% decrease in Bmax in cortex and a 23% decrease in cerebellum. No changes were seen in the affinity (Kd) for [3H]nimodipine binding sites. Slight changes in hippocampal and striatal binding capacities were observed with no changes seen in hypothalamus and brainstem. The kappa antagonist MR2266 effectively reversed in vivo all changes in [3H]nimodipine binding without producing any effect alone. These studies suggest that kappa opiate receptors may be directly coupled to L-type calcium channels as evidenced by [3H]nimodipine binding studies and may account for the findings that kappa opiate agonists inhibit neurotransmitter release by allosterically interfering with the Ca2+ channel protein in brain membranes.

An ATP-dependent mechanism for Ca2+ uptake in human platelet membrane fractions has been identified and characterized. Ca2+ uptake into a membrane fraction is shown to be stimulated at low concentrations of ATP and Ca2+ and to require magnesium ions. Initial rate kinetics, using Eadie-Scatchard analysis, indicated a single class of calcium uptake sites in the presence of ATP, with a Kd for free [Ca2+] of 0.145 microM. Ca2+ uptake in the presence of several ATP concentrations demonstrates that ATP binds to at least two sites, representing high and low affinities of 3.21 and 80.1 microM, respectively. The neuroleptic drug fluphenazine inhibited ATP-stimulated calcium uptake (IC50 = 55 microM), suggesting this ATP-dependent Ca2+ uptake system may provide a useful ion-transport model with which to study neuroleptic therapy in humans.

A high affinity Ca2+/Mg2+ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca2+ (≤1 μM) believed to approximate the range of Ca2+ in the synaptosomal cytosol (0.1 to 5.0 μM). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca2+-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca2+-stimulated ATP hydrolysis was characterized by a reduction inVmax for Ca2+. Levorphanol pretreatment reduced the Hill coefficient (HN) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca2+ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca2+/Mg2+ ATPase in synaptic membranes. Levorphanol (10 μM), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca2+-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca2+-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inVmax for both Ca2+ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca2+-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca2+/Mg2+ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca2+ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca2+ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca2+ following inhibition of Ca2+/Mg2+ ATPase may underlie some of the pharmacological effects of opiate drugs.

Chronic ethanol administration inhibits ATP-dependent Ca++ uptake in a preparation of synaptic membranes prepared from mice following 1, 4 and 7 days of ethanol exposure in a liquid diet. Addition of calmodulin (2.5 micrograms) to membranes from mice receiving the control diet produced a slight stimulation of ATP dependent Ca++ uptake. Membranes from ETOH treated mice exhibited reduced capacity to take up Ca++ in ATP-dependent fashion. When calmodulin was added to membranes isolated from mice receiving ETOH on Days 1, 4 and 7 ATP-dependent Ca++ uptake was significantly stimulated (p less than 0.01) compared to (1) ETOH treated membranes in absence of calmodulin, and (2) control membranes. Behavioral tolerance as estimated by bar holding technique was found to be 25, 65 and 91 percent complete for Days 1, 4 and 7 respectively. These studies demonstrate that continued exposure of mice to ethanol via consumption of an ethanol containing liquid diet inhibits one of the mechanisms involving the cytosolic buffering of intracellular Ca++ in nerve terminals. This biochemical effect seen in parallel with the development of tolerance to ethanol impairment of bar holding suggests that increased cytosolic Ca++ may aid in central nervous system adaptation to ethanol.

Previous studies have demonstrated that chronic low-dose administration of ethanol in the drinking fluid of rats produces an increase in content of calcium on synaptic membranes. In the present study, ethanol was studied in vitro and in vivo for its effects on Ca2+ binding to a high affinity receptor site on synaptic membranes. Ethanol (1-50 micrometer) significantly inhibits the binding of Ca2+ in non-competitive fashion, together with inhibiton of a cooperative mechanism for Ca2+ binding. Ruthenium red and neuraminidase were used to probe surface glycoprotein sites as receptors for Ca2+. Use of these probes together with ethanol in vitro or in vivo suggests that increases in Ca2+ receptor sites after chronic ethanol exposure are related to an increase in sialic acid exposure, possibly as a result of conformational changes in membrane protein. These findings are consistent with the hypothesis that chronic low-dose exposure to ethanol causes changes in proteins of synaptic membranes, which may in turn regulate the cellular adaptation to ethanol.

We report here characterization of calmodulin-stimulated Ca2+ transport activities in synaptic plasma membranes (SPM). The calcium transport activity consists of a Ca2+-stimulated, Mg2+-dependent ATP hydrolysis coupled with ATP-dependent Ca2+ uptake into membraneous sacs on the cytosolic face of the synaptosomal membrane. These transport activities have been found in synaptosomal subfractions to be located primarily in SPM-1 and SPM-2. Both Ca2+-ATPase and ATP-dependent Ca2+ uptake require calmodulin for maximal activity (KCm for ATPase = 60 nM; KCm for uptake = 50 nM). In the reconstituted membrane system, KCa was found to be 0.8 microM for Ca2+-ATPase and 0.4 microM for Ca2+ uptake. These results demonstrate for the first time the calmodulin requirements for the Ca2+ pump in SPM when Ca2+ ATPase and Ca2+ uptake are assayed under functionally coupled conditions. They suggest that calmodulin association with the membrane calcium pump is regulated by the level of free Ca2+ in the cytoplasm. The activation by calmodulin, in turn, regulates the cytosolic Ca2+ levels in a feedback process. These studies expand the calmodulin hypothesis of synaptic transmission to include activation of a high-affinity Ca2+ + Mg2+ ATPase as a regulator for cytosolic Ca2+.

The effects of dibutyryl cyclic AMP (dbcAMP) and dibutyryl cyclic GMP (db-cGMP) were tested on Ca2+-ATPase, Mg2+-ATPase, and (Ca2++ Mg2+)-ATPase activities in lysed synaptosomes prepared from whole rat brains (minus cerebellum). At concentrations from 0.1 to 2.0 mM, db-cGMP produced a selective, concentration-dependent increase in Ca2+-ATPase activity. Both db-cGMP and db-cAMP slightly reduced Mg2+-ATPase activity, whereas neither compound had concentration-dependent effects on (Ca2++ Mg2+)-ATPase activity. These findings suggest that the Mg2+-independent, Ca2+-ATPase activity in rat brain is regulated by a cyclic GMP-dependent process. Further, the data provide evidence that the Ca2+-ATPase activity in lysed synaptosomal membranes represents an enzyme that is distinguishable from both the Mg2+- and (Ca2++ Mg2+-ATPase.

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.

The effects of morphine sulfate on rectal temperature and on Ca++-stimulated Mg++ATPase activity in crude synaptosomal fraction (P2) of cortex, hypothalamus and cerebellum were investigated in rat. Morphine (3–15 mg/kg, SC) produced hyperthermia at 30–120 min after the drug administration. The Ca++/Mg++ ATPase activity in hypothalamus and cortex was decreased while there was no change in Mg++ATPase activity. The enzyme activity in cerebellum was not affected. The opiate antagonist naloxone hydrochloride (5 mg/kg, SC) antagonized the effect of morphine on rectal temperature and Ca++/Mg++ATPase activity. The effects of different calcium channel antagonists (nimodipine 1 mg/kg, verapamil 2,5 mg/kg and diltiazem 10 mg/kg, SC) on the changes induced by morphine were also investigated. These antagonists not only antagonized morphine hyperthermia, but also the inhibitory effect of morphine on Ca++/Mg++ATPase activity in hypothalamus. The calcium channel agonist BAY K8644 (3 mg/kg, SC) produced hypothermia and also stimulation of Ca++/Mg++ATPase activity in hypothalamus. Naloxone failed to alter these effects of BAY K8644. These studies demonstrate that Ca++ transport in hypothalamus, as indicated by Ca++/Mg++ATPase activity, plays an important role in thermoregulation and thermoregulatory changes induced by opiates.

Pharmacological actions of opiate drugs are characterized by analgesia, respiratory depression and sedation or CNS depression. These parameters together with changes in body temperature may be observed after a single dose of the opiate agonist. However, repeated administration of the drug at a constant dose or the necessity to increase the dose to achieve the same degree of response produces an effect commonly referred to as tolerance. This effect, although easily produced by opiate drugs, is clearly one of the least understood events in all of narcotic research.

The effects of alpha 1- and alpha 2-adrenergic agonists, viz., phenylephrine and clonidine, respectively, were studied on rat liver plasma membrane Ca++-ATPase. Phenylephrine produced a 23% inhibition of enzyme activity at 5 microM. Prazosin, an alpha 1 antagonist, completely prevented the effect of phenylephrine. Clonidine produced a comparable inhibition of Ca++-ATPase, but was not reversed by the antagonist yohimbine, suggesting a lack of functionally significant alpha 2 receptors as previously reported. The results support the role of high-affinity Ca++-ATPase in liver plasma membranes in the control of cytosolic free Ca++ levels through regulation by alpha 1-adrenergic receptors. In vitro and acute ethanol exposure produced inhibition of plasma membrane Ca++-ATPase. In addition, ethanol treatment significantly reversed the inhibitory effect of phenylephrine on Ca++-ATPase. Chronic ethanol exposure for four weeks increased Ca++-ATPase activity over control and increased enzyme activity in the presence of phenylephrine. These results demonstrate that ethanol alters the alpha-adrenergic receptor interaction with Ca++-ATPase resulting in reduced receptor regulation of cytosolic Ca++ levels. These changes may prevent the liver from maintaining Ca++ levels for second messenger functions, such as glycolysis and gluconeogenesis.

Treatment of rat brain synaptosomal lysate with N-ethylmaleimide (NEM) was found to stimulate ATP-dependent Ca2+-uptake. This Ca2+-uptake stimulation was blocked by dithioerythritol (DTE), mitochondrial inhibitors oligomycin and sodium azide, but not by vanadate, an inhibitor of plasma membrane Ca2+ pump. Maximal stimulation of Ca2+-uptake was observed at a NEM/protein ratio of 0.1 mumole/0.5-1.0 mg. On fractionation, it was found that NEM did not affect synaptic plasma membrane Ca2+-uptake, but almost doubled it in synaptic mitochondria.

Administration of the opiate U-50,488H (3-20 mg/kg s.c.), a selective kappa receptor agonist, produced a dose-dependent decrease of rectal temperature in rats. This hypothermic effect of U-50,488H was accompanied by an enhanced activity of Ca2+/Mg2+ ATPase in crude synaptosomal (P2) fractions obtained from hypothalamus but not from cortex or cerebellum. Mg2+ ATPase activity in these regions was not altered by U-50,488H (15 mg/kg s.c.). Naloxone (5 mg/kg) partially and MR2266 (5 mg/kg) completely reversed the temperature and enzyme changes. Pretreatment with the calcium channel blockers nimodipine (1 mg/kg s.c.), diltiazem (10 mg/kg s.c.) and verapamil (2.5 mg/kg s.c.) potentiated the hypothermic effect of U-50,488H as well as the stimulation of Ca2+/Mg2+ ATPase in hypothalamus. These observations suggest that kappa agonists may produce opiate receptor mediated hypothermia through changes in intracellular Ca2+ levels in the hypothalamus.

Dihydropyridine (DHP) receptors appear to be coupled to voltage-sensitive calcium channels that mediate Ca(++) flux in neural tissue. A number of toxins known to interact with these channels induce tremors and seizures and modulate the ability of DHP compounds to alter the gating properties of Ca(++) channels. It is therefore likely that tremorgenic myco-toxins, an economically important group of fungal toxins, may modulate Ca(++) channels either directly or by their ability to act at DHP receptors. Tremorgenic doses of aflatrem, cyclopiazonic acid, and verruculogen increase the number and decrease the affinity of DHP receptors in rat cortex. These results suggest that the Ca(++) channel and its associated receptors are important targets for several classes of fungal toxins.

— 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.

The effect of the opiate antagonists naloxone and MR2266 on ethanol-induced hypothermia and changes in Ca 2+ -stimulated Mg 2+ -ATPase activity in brain regions were investigated in the present study. Administration of different doses of ethanol (0.5–2 g/kg, IP) produced a dose-dependent hypothermia. Ca 2+ /Mg 2+ -ATPase activity in the hypothalamus was stimulated at 30 min and 2 hr after ethanol (2 g/kg, IP) treatment. In cortex, enzyme activity was inhibited by ethanol at 30 min with no change seen at 2 hr. Naloxone (7.5 mg/kg, SC) at a dose which did not affect body temperature or enzyme activity, partially inhibited ethanol-induced hypothermia and enzyme activity at the earliest time (30 min) but not at 2 hours. The opiate Kappa antagonist MR2266 (5 mg/kg, SC), however, significantly protected against ethanol hypothermia and enzyme activation measured at 30–120 min. This evidence suggests that ethanol-induced hypothermia and subsequent activation changes of Ca 2+ /Mg 2+ -ATPase in the hypothalamus may be regulated by opiate Kappa receptors, and that Ca 2+ ions play an important role in mediating the effects of ethanol.

— 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 results are discussed in terms of a specific effect of opiate drugs and the role of calcium in opiatereceptor interactions.

1. Acute administration of ethanol (2.5 gm/kg, i.p.) to rats inhibits the cytosolic buffering of Ca2+ in nerve terminals. 2. Ca2+ ATPase and ATP-dependent Ca2+ uptake are both inhibited 30 min after a single dose of ethanol. 3. Chronic ethanol administration (6%, 14 days) did not inhibit Ca2+ ATPase but significantly stimulated ATP-dependent Ca2+ uptake. 4. Lubrol WX treatment of acute ethanolicmembranes reverses the inhibition of Ca2+ ATPase seen following ethanol. 5. Lubrol WX treatment of chronic ethanolic membranes prevents the increase in ATP-dependent Ca2+ uptake seen in ethanolic membranes. 6. Both acute and chronic ethanol-induced changes in Ca2+ transport within nerve teminals may involve lipid-dependent parameters of the membrane which may underlie neuronal adaptation.

1. 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.

The effects of alpha 1 and alpha 2-adrenergic receptor ligands on Ca2+/Mg2+-ATPase have been studied using synaptosomal plasma membranes isolated from rat brain cortex. Both phenylephrine and clonidine inhibited Ca2+/Mg2+-ATPase, in a concentration-dependent fashion. IC50 values for half-maximal inhibition for phenylephrine and clonidine were 29 microM and 18 microM, respectively. The inhibitory effect of phenylephrine was reversed by the alpha antagonist prazosin while yohimbine and rauwolscine reversed the inhibition of enzyme activity by clonidine. The two antagonist subtypes were effective only against the respective agonist subtypes, demonstrating distinct subtype preferences. Analysis of the kinetics of enzyme inhibition indicate both agonists to be noncompetitive. Some evidence suggests that yohimbine may exhibit mixed agonist/antagonist properties which depend on [Ca2+]. The present study provides biochemical evidence to support auto receptor alpha-adrenergic receptor regulation of neurotransmitter release.

The mechanisms by which ethanol produces its effects on the central nervous system are both diverse and complicated. An increasing amount of evidence, however, suggests that many of the primary and secondary effects of this chemical may be related to its interaction with the cell membrane (Hunt, 1975). During the initial phase of ethanol absorption, there is a depression of motor function, leading to overall depression of the central nervous system. Continued administration of ethanol leads to a tolerance of this sedative-hypnotic effect so that, over time, the animal or human no longer responds to the same concentration of ethanol with characteristic depression.

Enzyme markers associated with calcium and glycoprotein metabolism may be correlated with the sensitivity and preference to alcohol in rodent strains.

Synaptosomal membranes accumulate 3–6 times more Ca2+ in the presence of ATP (50–1000 μM) than basal Ca2+ accumulation (-ATP). The location of this Ca2+ accumulation appears to reside on the cytosolic face of the synaptosome since lysed synaptosomes accumulate 4-times more Ca2+ than intact synaptosomes. The inclusion of mitochondrial inhibitors, oligomycin (0.7 μg/ml), sodium azide (100 μM) and dinitrophenol (100 μM) differentiate mitochondrial from nonmitochondrial Ca2+ accumulation under conditions that are [Ca2+]- and ATP-dependent. In the presence of low concentrations of ATP (200 μM), the Ca2+ accumulation process remains independent of the presence of mitochondrial inhibitors when Ca free 2+ =2.5 μM. When Ca free 2+ is increased to 6.8 μM, mitochondrial inhibitors differentiate mitochondrial from nonmitochondrial accumulation. These studies suggest that optimal conditions for the measurement of Ca2+ accumulating mechanisms in synaptosomal membranes depend on both [Ca2+] and ATP. Use of these assay conditions provide evidence that ATP-dependent Ca2+ uptake may be a viable mechanism for the regulation of synaptosomal Ca2+ levels.

The phospholipid requirement for Ca2+-stimulated, Mg2+-dependent ATP hydrolysis (Ca2+/Mg2+-ATPase) and Mg2+-stimulated ATP hydrolysis (Mg2+-ATPase) in rat brain synaptosomal membranes was studied employing partial delipidation of the membranes with phospholipase A2 (Hog pancreas), phospholipase C (Bacillus cereus) and phospholipase D (cabbage). Treatment with phospholipase A2 caused an increase in the activities of both Ca2+/Mg2+-ATPase and Mg2+-ATPase whereas with phospholipase C treatment both the enzyme activities were inhibited. Phospholipase D treatment had no effect on Ca2+/Mg2+-ATPase but Mg2+-ATPase activity was inhibited. Inhibition of Mg2+-ATPase activity after phospholipase C treatment was relieved with the addition of phosphatidylinositol-4,5-bisphosphate (PIP2) and to a lesser extent with phosphatidylinositol-4-phosphate (PIP) and phosphatidylcholine (PC). Phosphatidylserine (PS), phosphatidic acid (PA), PIP and PIP2 brought about the reactivation of Ca2+/Mg2+-ATPase. Phosphatidylinositol (PI) and PA inhibited Mg2+-ATPase activity. Kms for Ca2+ (0.47 microM) and Mg2+ (60 microM) of the enzyme were found to be unaffected after treatment with the phospholipases.

Levorphanol inhibits Ca ++ binding to synaptic membranes in a dose dependent fashion (0.5–10nM) and induces release of calcium previously bound to membranes. The inhibition is noncompetitive and exhibits a K i of 9 × 10 −9 M. Naloxone (10 nM) blocks this inhibition while dextrophan (100nM) is without effects. The Ca ++ binding exhibits a high affinity (K D =0.84 μ M) and a saturable capacity of 350 picomles/mg protein. The binding curve is sigmoid with a Hill coefficient of 2.2, while in the presence of levorphanol (0.5 to 50nM), the curve becomes hyperbolic with a Hill coefficient of 1.1. The data suggest the calcium and opiate receptor binding sites are in close proximity and may be functionally linked through sub-unit interactions.

Preparations of lysed synaptosomes exhibit a high affinity Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ accumulation activity, with a Km for Ca2+ congruent to 0.5 microM, close to the cytosolic concentration of Ca2+. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1-20 microM), both Ca2+/Mg2+ ATPase and ATP-dependent CA2+ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5-1.0 microM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca2+ channels or Na+-Ca2+ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca2+ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca2+ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.

Lysed synaptosomal membranes were prepared from brain cortices of HA/ICR Swiss mice, and the ATP-stimulated Ca2+ uptake, Ca2+-stimulated Mg2+-dependent ATPase activity, and the Ca2+-stimulated acyl phosphorylation of these membranes were studied. The Km values for free calcium concentrations ([Ca2+]f) for these processes were 0.50 microM, 0.40 microM, and 0.31 microM, respectively. Two kinetically distinct binding sites for ATP were observed for the ATP-stimulated Ca2+ uptake and the Ca2+-stimulated Mg2+-ATPase activity. The high-affinity Km values for ATP for these two processes were 16.3 microM and 28 microM, respectively. These results indicate that the processes studied operate in similar physiological concentration ranges for the substrates [Ca2+]f and ATP under identical assay conditions and, further, that these processes may be functionally coupled in the membrane.

The novel kappa agonist U50-488H in vitro produced a concentration-dependent decrease (0.25-25 microM) in [3H]nimodipine binding in neuronal P2 fractions [corrected] from rat brain cortex. Kinetic analysis indicates the decrease in binding results from a reduced Bmax with no change in affinity (Kd). The kappa antagonist, MR2266, blocked the decrease in [3H]nimodipine binding to membrane fractions. At equimolar concentrations (25 microM), morphine in vitro had no effect on [3H]nimodipine binding, while U50-488H demonstrated potent inhibition. Further kinetic analysis indicates that the IC50 for U50-488H is 0.5-0.7 microM with a KI by a Dixon plot of 1.5-1.7 microM [corrected]. These results suggest that kappa opiate receptors may be coupled to dihydropyridine receptors and as a result modulate Ca++ entry and neurotransmitter release in brain neurons.

Recently, the discovery of endogenous opiate peptides has reawakened new interest in the possibility that an endogenous chemical may mediate actions of alcohol and lead to ethanol dependence.

An indium–arsenide detector array installed at the focal plane of a grating spectrometer provides the time history of the intensity of each output line of multiline HF chemical lasers. The construction and calibration of this instrument are discussed, and representative data are presented. Line competition measurements are shown for a 200-W supersonic-diffusion-type HF laser; individual line outputs fluctuate more than total multiline output. The spectral effects of forward/reverse-wave competition on an unstable ring resonator operating at 350 W are detailed. Simultaneous single-line gain measurements on a multiline HF medium are demonstrated. Other applications of this useful instrument are discussed.

To the Editor:— The hospital I serve has been able to successfully solve a problem that has been perplexing many hospital administrators, medical staffs, and communities. It is the challenge of admitting patients on the most equitable basis. The situation confronting most hospital administrators is the demand for hospitalization in the face of bed shortages. Because of these demands, most administrators have found themselves in the harassing position of meeting pressures brought to bear on themselves, on their board of trustees, and on the hospital's medical staff by community members who seek to influence their own or their friends' early hospital admission. If the community member making such an imperative request is the wife of a member of the board of trustees, the brother of an officer of the medical staff, a friend who has donated $5,000 to the hospital in its building campaign, or a person of no prominence