Biomedical subjects

D H Ross

Publications and source records attributed to D H Ross.

At least 19 recordsLinked to original sources

PubMed1989

Influence of ethanol on calcium, inositol phospholipids and intracellular signalling mechanisms.

Studies have implicated Ca++ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca++ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca++/Mg++-ATPase, the high affinity membrane Ca++ pump. Current research suggests that a subtype of the voltage-dependent Ca++ channel, the dihydropyridine-sensitive Ca++ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases 45Ca++-influx and [3H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca++/Mg++-ATPase activity in neuronal membranes. Changes in Ca++ channel and Ca++/Mg++-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca++ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca++/Mg++-ATPase activity. The increased sensitivity of three Ca++-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.

Animals↗

PubMed1989

Microwave induced stimulation of 32Pi incorporation into phosphoinositides of rat brain synaptosomes.

Exposure of synaptosomes to microwave radiation at a power density of 10 mW/sq cm or more produced stimulation of the 32Pi-incorporation into phosphoinositides. The extent of 32Pi incorporation was found to be much more pronounced in phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2) as compared to phosphatidylinositol (PI) and phosphatidic acid (PA). Other lipids were also found to incorporate 32Pi but no significant changes in their labeling were seen after exposure to microwave radiation. Inclusion of 10 mM lithium in the medium reduced the basal labeling of PIP2, PIP and PI and increased PA labeling. Li+ also inhibited the microwave stimulated PIP2, PIP and PI labeling but had no effect on PA labeling. Calcium ionophore, A23187, inhibited the basal and microwave stimulated 32Pi labeling of PIP and PIP2, stimulated basal labeling of PA and PI and had no effect on microwave stimulated PA and PI labeling. Calcium chelator, EGTA, on the other hand, had no effect on basal labeling of PA and PI, stimulated basal PIP and PIP2 labeling but did not alter microwave stimulated labeling of these lipids. Exposure of synaptosomes to microwave radiation did not alter the chemical concentration of phosphoinositides indicating that the turnover of these lipids was altered. These results suggest that low frequency microwave radiation alter the metabolism of inositol phospholipids by enhancing their turnover and thus may affect the transmembrane signalling in the nerve endings.

Animals↗

PubMed1988

The effect of kappa agonist U50-488H on [3H]nimodipine receptor binding in rat brain regions.

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.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1988

In vivo morphine decreases [3H]nimodipine receptor binding in rat brain regions.

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.

Animals↗

PubMed1988

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

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.

Adenosine Triphosphate↗

PubMed1987

A novel kappa agonist inhibits [3H]nimodipine binding to a Ca++ channel receptor protein in rat brain.

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.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1987

Stimulation of synaptosomal ATP-dependent Ca2+-uptake by N-ethylmaleimide.

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.

Adenosine Triphosphate↗

PubMed1987

Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes.

A high affinity Ca2+/Mg2+ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca2+ (less than or equal to microM) believed to approximate the range of Ca2+ in the synaptosomal cytosol (0.1 to 5.0 microM). 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 in Vmax 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 microM), 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 in Vmax 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1987

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 microM and 3 microM IP3 concentrations, respectively. These studies demonstrate for the first time IP3 mobilization of Ca2+ from endoplasmic reticulum within synaptic plasma membranes.

Adenosine Triphosphate↗

PubMed1987

Alpha-adrenergic receptor regulation of Ca2+/Mg2+-ATPase in brain synaptic membranes.

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.

Animals↗

PubMed1987

Alterations in alpha-adrenergic and muscarinic cholinergic receptor binding in rat brain following nonionizing radiation.

Microwave radiation produces hyperthermia. The mammalian thermoregulatory system defends against changes in temperature by mobilizing diverse control mechanisms. Neurotransmitters play a major role in eliciting thermoregulatory responses. The involvement of adrenergic and muscarinic cholinergic receptors was investigated in radiation-induced hyperthermia. Rats were subjected to radiation at 700 MHz frequency and 15 mW/cm2 power density and the body temperature was raised by 2.5 degrees C. Of six brain regions investigated only the hypothalamus showed significant changes in receptor states, confirming its pivotal role in thermoregulation. Adrenergic receptors, studied by [3H]clonidine binding, showed a 36% decrease in binding following radiation after a 2.5 degrees C increase in body temperature, suggesting a mechanism to facilitate norepinephrine release. Norepinephrine may be speculated to maintain thermal homeostasis by activating heat dissipation. Muscarinic cholinergic receptors, studied by [3H]quinuclidinyl benzilate binding, showed a 65% increase in binding at the onset of radiation. This may be attributed to the release of acetylcholine in the hypothalamus in response to heat cumulation. The continued elevated binding during the period of cooling after radiation was shut off may suggest the existence of an extra-hypothalamic heat-loss pathway.

Animals↗

PubMed1986

Interaction of kappa receptor agonists with Ca2+ channel antagonists in the modulation of hypothermia.

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.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1986

Phospholipid requirement of Ca2+-stimulated, Mg2+-dependent ATP hydrolysis in rat brain synaptic membranes.

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.

Animals↗

PubMed1986

Activation of dihydropyridine receptors differentially regulates temperature responses in rat.

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.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1986

Opiate receptor mediated hyperthermic responses in rat following Ca++ channel antagonists.

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.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1986

Chronic ethanol administration inhibits calmodulin-dependent Ca++ uptake in synaptosomal membranes.

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.

Adenosine Triphosphate↗

PubMed1986

Effects of opiates on high-affinity Ca2+,Mg2+-ATPase in brain membrane subfractions.

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.

Animals↗

PubMed1985

Activation of central muscarinic receptors inhibit Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ transport in synaptic membranes.

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.

Animals↗