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Biomedical subjects

F T Crews

Publications and source records attributed to F T Crews.

At least 37 records · Page 2Linked to original sources
PubMed1998

Species differences in regional patterns of 3H-8-OH-DPAT and 3H-zolpidem binding in the rat and human brain.

The rat has proven to be a valuable preclinical model for characterizing effects of psychotrophic drugs and for identifying new psychotherapeutic agents in pharmacological screens. However, substantial differences have been described between the rat and human brain in regard to the neuroanatomical distribution of some drug and neurotransmitter receptor binding sites. To assess the utility of the rat as a model for the neuroanatomical topography of 5-HT1A and type 1 benzodiazepine (BDZ) receptors in humans, the distribution of binding sites for 3H-8-OH-DPAT (5-HT1A agonist) and 3H-zolpidem (type 1 BDZ agonist) was compared with autoradiography in select regions of the rat and human brain. Concordance in the binding patterns for the two ligands was observed in several brain regions for the two species. However, substantial differences were also found in the topography of binding sites for the ligands in the rat and human brain. High 3H-8-OH-DPAT binding was seen in the dorsal raphe nucleus and hippocampal formation in both the rat and human brain. However, species differences were observed in the relative distribution of ligand binding among hippocampal subregions. In the cerebral cortex, the laminar distribution of 3H-8-OH-DPAT binding sites was notably different for rats and humans. In humans, outer cortical layers were most densely labeled with 3H-8-OH-DPAT, whereas in the rat cortex, the highest binding was in the inner layers. A striking difference between rats and humans was observed for 3H-8-OH-DPAT binding in the lateral septal nucleus, which was densely labeled in the rat but weakly labeled in humans. Substantial differences between rats and humans were also observed for 3H-zolpidem binding. In the rat brain, high densities of binding sites were found in the medial septum, inferior colliculus, and substantia nigra reticulata. These regions showed very low 3H-zolpidem binding in the human brain. Intermediate binding was seen in the rat cerebral cortex, and low binding was found in the hippocampus. By contrast, in humans, cerebral cortical regions were the most densely labeled of all regions studied, and certain hippocampal subregions exhibited relatively high binding. The striking neuroanatomical differences in 3H-8-OH-DPAT and 3H-zolpidem binding observed between rats and humans suggest that different functional consequences may be produced within specific brain regions after administration of drugs that influence 5-HT1A and type 1 BZD receptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1998

Ethanol tolerance and synaptic plasticity.

Current concepts of the mechanisms underlying many of the pharmacological effects of ethanol on the CNS involve disruption of ion channel function via the interaction of ethanol with specific hydrophobic sites on channel subunit proteins. Of particular clinical importance is the development of tolerance and dependence to ethanol, and it is likely that adaptive changes in synaptic function in response to ethanol's actions on ion channels play a role in this process. In this article, Judson Chandler, Adron Harris and Fulton Crews discuss potential mechanisms of ethanol-induced changes in synaptic function that might provide a cellular basis for ethanol tolerance and dependence. It is proposed that multiple mechanisms are involved that include both transcriptional and post-translational modifications in NMDA and GABAA receptors.

Cytoskeletal Proteins↗
PubMed1998

Uncoupling of muscarinic cholinergic phosphoinositide signals in senescent cerebral cortical and hippocampal membranes.

Muscarinic-cholinergic signals in brain are mediated in part through the hydrolysis of phosphoinositides (PtdIns) by phospholipase C (PLC). To test the hypothesis that muscarinic PtdIns signals change during aging, membranes were prepared from the cerebral cortex and hippocampus of young (4-6 months old), middle aged (8-10 months old) and senescent (24-26 months old) Fisher 344 rats. Carbachol dose-dependently increased [3H]-PtdIns hydrolysis in both brain regions in all three age groups, however, in senescent rats the maximal response was decreased to 69.26 +/- 4.33% (p < 0.01) in cortex and to 48.29 +/- 2.55% (p < 0.01) in hippocampus of young rat values. In contrast to the decrease in carbachol-stimulated phosphoinositide hydrolysis, calcium-stimulated phosphoinositide hydrolysis was not altered. GTP gamma S also dose-dependently increased [3H]-PtdIns hydrolysis in membranes from all three age groups through G-protein-PLC activation. Similar to carbachol, GTP gamma S-activated [3H]-PtdIns hydrolysis was reduced approximately 40% in senescent rats membranes. Muscarinic receptor (mAChR) density, as determined by [3H]-QNB binding decreased slightly in cortical membranes, but not in hippocampal membranes. These data suggest that muscarinic stimulated [3H]-PtdIns responses are decreased in senescent brain primarily due to an uncoupling of the receptor-G-protein and/or G-protein-PLC link, although decreases in receptor density may also contribute to reduced muscarinic [3H]-PtdIns signaling.

Aging↗
PubMed1998

Induction of Fos-like proteins and ultrasonic vocalizations during ethanol withdrawal: further evidence for withdrawal-induced anxiety.

The ethanol withdrawal syndrome includes anxiety as a prominent symptom. Because the extent that specific regions of brain are critical to the generation of this emotional state is unknown, Fos-like immunoreactivity (Fos-LI) was used to associate specific regions of the rat brain with the anxiety component of the ethanol withdrawal syndrome exacerbated by an air puff challenge in rats. Chronic ethanol liquid diet was administered intragastrically for 4 days or by having the rats consume the diet for 14 days. During withdrawal from either treatment protocol, Fos-LI was induced most prominently in forebrain areas, although the midbrain and hindbrain were also represented. Included in these Fos-LI positive regions were many cortical regions, septum, accumbens, claustrum, amygdala, paraventricular nucleus of the thalamus and hypothalamus, hippocampus, locus coeruleus, and central gray. Fos-LI expression differed mostly in intensity between the two treatment and withdrawal protocols, with the gastric protocol producing the greatest Fos-LI induction in most brain regions. The threshold for air puff-induced ultrasonic vocalizations was decreased, and the number of vocalizations was increased and the period of vocalization was extended. These behavioral data indicate that aversively motivated responding in rats during ethanol withdrawal can be readily quantified with the ultrasonic vocalizations test without precipitating convulsive activity. Furthermore, a comparison of the effects of the air puff challenge versus withdrawal on Fos-LI indicated that the behavioral state induced in these two situations share functional neuroanatomical features. Some regions--such as the accumbens core, medial septum, subregions of the amygdala, hippocampus, substantia nigra, and cerebellum--exhibited little Fos-LI during withdrawal and also did not exhibit strong increases after the addition of the air puff challenge. However, other regions-such as the cerebral cortex (medial prefrontal, frontal, cingulate and ventrolateral orbital, claustrum, and tenia tecta), hypothalamus, and locus ceoruleus- exhibited Fos-LI at levels higher than that seen after either the ethanol withdrawal or puff challenge alone. These overlapping patterns of Fos-LI in specific regions of the brain, activated by both ethanol withdrawal and an anxiety provoking behavioral challenge, suggest that specific neuroanatomical sites in brain are associated with the symptom of anxiety observed during the "ethanol withdrawal syndrome."

Alcohol Withdrawal Delirium↗
PubMed1998

Effects of chronic ethanol exposure on oxidation and NMDA-stimulated neuronal death in primary cortical neuronal cultures.

Excessive oxidative radical formation has been implicated in a number of neurodegenerative processes, including glutamate-N-methyl-D-aspartate (NMDA)-mediated excitotoxicity. Previous studies have suggested that the formation of reactive oxygen species (ROS) during NMDA exposure is involved in triggering the excitotoxic cascade. Chronic exposure of primary neuronal cultures to ethanol has been shown to potentiate NMDA-mediated processes, such as nitric oxide formation and excitotoxicity. It was the objective of this study to investigate the role of NMDA and ROS formation in the development of NMDA supersensitivity after chronic ethanol exposure. The fluorescent dyes dichlorofluorescein diacetate (H2DCF) and propidium iodide, which have been shown to be reliable markers for the detection of cellular oxidation and cell death, respectively, in neuronal culture preparations, were used to examine the relationship between ROS production and NMDA-mediated neuronal death after chronic ethanol administration. Cultures treated chronically with ethanol (100 mM) for 96 hr displayed significantly less H2DCF oxidation when exposed to various concentrations of FeSO4 for 25 min. However, this decrease in intracellular oxidation did not have any apparent inhibitory effects on the amount of cell death observed 12 hr after the 25-min exposure to FeSO4. When NMDA-FeSO4-mediated oxidation was examined in cultures treated chronically with ethanol, dose-dependent increases in H2DCF oxidation were observed, but only in control-treated cultures. This blunting of intracellular H2DCF oxidation did not attenuate the potentiation of NMDA-mediated excitotoxicity observed after chronic ethanol exposure. These results suggest that the observed supersensitivity to NMDA is not due to increases in intracellular ROS formation and that chronic ethanol may induce neuronal factors that reduce ROS formation, but do not protect against normal death.

Animals↗
PubMed1997

Inferior collicular seizure generalization produces site-selective cortical induction of cyclooxygenase 2 (COX-2).

Given the potential role of mitogen-inducible cyclooxygenase (COX-2) in CNS damage, patterns of COX-2 induction were determined both before and after seizure generalization from the inferior collicular cortex into the forebrain. With midbrain seizures, no change was found in COX-2-like immunoreactivity, even at the site of seizure genesis. However, upon forebrain seizure generalization, dramatic, ipsilateral increases in COX-2-like immunoreactivity were found in layers II and II of perirhinal, entorhinal and temporal cortex, just dorsal to the perirhinal fissure, coursing from the level of the medial geniculate to the level of the inferior colliculus. No changes in COX-2-like immunoreactivity were found in contralateral cortical regions, retrosplenial cortex, dentate gyrus, subiculum, tenia tectum or inferior colliculus. Thus, initial seizure generalization into the forebrain induces COX-2 expression in a highly specific area of the cerebral cortex.

Animals↗
PubMed1997

Use of a multiwell fluorescence scanner with propidium iodide to assess NMDA mediated excitotoxicity in rat cortical neuronal cultures.

Glutamate mediated excitotoxicity is a major area of experimentation due to the potential for prevention of morbidity and brain damage associated with stroke and brain trauma. We have developed a simple rapid method to study excitotoxicity in primary cortical neuronal cultures using propidium iodide (PI) fluorescence read by a multiwell fluorescence scanner. Transient (25 min) or continuous N-methyl-D-aspartate (NMDA) treatment led to progressive neuronal death over 24 h that was blocked by 1 microM MK-801, 10 microM ifenprodil, and 200 mM ethanol. Results with PI fluorescence were identical to those found using the lactate dehydrogenase (LDH) release and trypan blue staining assays of excitotoxicity. This method provides a simple rapid means to test the effects of drugs during glutamate excitotoxicity and to do accurate time course experiments of delayed neuronal death.

Animals↗
PubMed1997

Chronic ethanol increases N-methyl-D-aspartate-stimulated nitric oxide formation but not receptor density in cultured cortical neurons.

The effects of prolonged ethanol exposure on excitatory amino acid receptor stimulated nitric oxide (NO) formation were examined in primary rat cortical neuronal cultures. Chronic ethanol (4 days, 100 mM) potentiated N-methyl-D-aspartate (NMDA)-stimulated NO formation as determined by measuring the conversion of [3H]arginine to [3H]citrulline. In contrast, chronic ethanol had no effect on NO formation stimulated by kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxalonepropionic acid, or the calcium ionophore ionomycin. Potassium chloride-stimulated NO formation was also enhanced by chronic ethanol treatment, but this effect was not seen in the presence of the ionotropic glutamate receptor antagonists MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione. Immunoblot analysis of expression of NR1, NR2A, and NR2B receptor subunits showed no difference between control and chronic ethanol-treated cultures. In support of this apparent lack of change in receptor density, there was no difference in the specific binding of 125I-MK-801 between control and chronic ethanol-treated groups. These results demonstrate that prolonged ethanol exposure selectively enhanced NMDA receptor-stimulated NO formation, which may play an important role in alcohol dependence, withdrawal, and alcohol-associated brain damage. These results also suggest that chronic ethanol-induced increases in NMDA receptor function may not be due to a simple increase in the number of NMDA receptors or change in NMDA receptor subunit composition but may instead reflect more complicated and subtle changes.

Animals↗
PubMed1997

NMDA receptor binding in adult rat brain after several chronic ethanol treatment protocols.

The amino acid L-glutamate is a major excitatory neurotransmitter that is involved in many CNS functions, including learning, memory, long-term potentiation, and synaptic plasticity. Acute exposures to ethanol (50 to 200 mM) have been shown to inhibit NMDA receptor responses, whereas chronic exposure to ethanol leads to adaptive supersensitivity thought to be involved in ethanol dependence and tolerance. To investigate the effects of chronic ethanol exposure on glutamate receptor density, we examined the binding of both NMDA and non-NMDA ligands in rat brain after several chronic ethanol treatment protocols using a number of different rat strains. No increases in the binding of [3H]MK-801, [3H]CGP 39653, or the polyamine specific competitive antagonist, [3H]ifenprodil, were seen after two well-used chronic ethanol treatments. These included the 2-week liquid diet developed by Frye et al. (J. Pharmacol. Exp. Ther. 216:306-314, 1981) and the 4-day binge treatment developed by Majchrowicz (Psychopharmacologia 43:245-254, 1975). However, small increases in the binding of both the NMDA noncompetitive antagonist [3H]MK-801, as well as the competitive NMDA antagonist [3H]CGP 39653, were seen in select frontal brain regions after 3 weeks of the Walker-Freund chronic ethanol liquid diet. When this chronic liquid diet treatment was extended to a period of 6 weeks, these increases in receptor binding were diminished to nonsignificant levels. The binding of the non-NMDA ligands [3H]AMPA and [3H]kainate were not significantly affected by either length of Walker-Freund liquid diet exposure. When rats were treated chronically with ethanol for 30 days using the paradigm developed by Tsukamoto et al. (Hepatology 5:224-232, 1985), small, but significant, increases in the binding of [3H]MK-801 were seen in the CA1 and dentate gyrus regions of the hippocampus. These studies indicate that robust increases in NMDA receptor binding do not occur with several chronic ethanol treatment protocols, and suggests that NMDA receptor supersensitivity during the development of tolerance and dependence to ethanol may not simply be due to changes in the density of NMDA receptors, but may involve other mechanisms.

2-Amino-5-phosphonovalerate↗
PubMed1996

Age-related loss of cholinergic-muscarinic coupling to PLC: comparison with changes in brain regional PLC subtypes mRNA distribution.

Activation of phospholipase C (PLC) coupled to phosphoinositide (PtdIns) hydrolysis occurs through one of the two pathways. One of the major pathways for the neurotransmitter signaling involves phosphoinositide (PtdIns) specific and G-protein dependent PLC-beta, which stimulates the formation of inositol triphosphate (IP3) and inositol tetraphosphate (IP4). Another pathway through the stimulation of calcium influx can directly activate all of the PLC isozymes. At least three isozymes of PLC have been characterized in the brain; PLC-A (alpha), PLC-I (beta) and PLC-II (gamma), which are shown to be localized differentially in brain regions. Muscarinic-cholinergic signals are mediated in large part through the hydrolysis of PtdIns by PLC. To investigate changes in muscarinic coupling to PLC during aging, we examined carbachol stimulated and calcium stimulated PtdIns hydrolysis in cerebral cortical membranes in young, middle aged and old rats. In order to determine whether PtdIns hydrolysis changes correspond to PLC isozyme expression in these animals, we examined three subtypes of PLC mRNA expression in brain sections of young and old rats using in situ hybridization technique. Our study indicated decreased carbachol-induced PLC activity in the cerebral cortex and, in contrast, increased PLC-beta mRNA in the frontal cortex and superficial cortical layer of aged rats. PLC-alpha mRNA was decreased in hippocampal regions of older rats. These studies suggest that during aging there is an uncoupling of muscarinic stimulated PtdIns hydrolysis, which is accompanied by an increased PLC-beta mRNA and decreased PLC-alpha mRNA that may represent compensatory changes in PLC expression.

Aging↗
PubMed1996

Effects of ethanol on ion channels.

Ion channels play critical roles in nervous system function, from initiating rapid synaptic activity to propagation of action potentials. Studies have indicated that many of the effects of ethanol on the nervous system are likely caused by the actions of ethanol on ion channels. Ion channels are multimeric structures that gate ions through subtle changes in tertiary structure. Ethanol readily enters molecular sites within multimeric ion channels, modifying intermolecular forces and bonds that are important for the open-close-inactivation kinetic properties of channels. The diversity of channel composition caused by the multimeric structure results in subtypes of channels that have a spectrum of sensitivity to ethanol that translates into brain regional differences in ethanol sensitivity, in part caused by differences in ion channel subunit composition. Ethanol has been shown to affect both receptor-activated ion channels and voltage-gated ion channels. The acute intoxicating and incoordinating effects of ethanol are probably related to inhibition of subtypes of NMDA-glutamate receptor ion channels and potentiation of certain subtypes of GABAA receptor ion channels. Effects on these channels, as well as glycine, nicotinic cholinergic, serotonergic, and other ion channels, likely contribute to the euphoric, sedative, and other acute actions of ethanol. Changes in ion channel subunit composition, density, and properties probably also contribute to ethanol tolerance, dependence, withdrawal hyperexcitability, and neurotoxicity. A substantial number of studies have implicated glutamate NMDA receptor, GABAA, and L-type voltage-gated calcium channels in the adaptive changes in the brain during chronic ethanol exposure. The diversity of ion channels subunits, their prominent role in brain function, and ethanol action are likely to make them important contributors to alcoholism and alcohol abuse.

Animals↗
PubMed1996

Further selection of rat lines differing in 5-HT-1A receptor sensitivity: behavioral and functional correlates.

It was previously reported that selection for differences in the hypothermic effects to the selective 5-HT-1A agonist, 8-OH-DPAT, occurred rapidly, with very substantial differences present by the fourth generation. The present communication summarizes the findings from the next five generations of selection and from behavioral and other functional studies on these rats. The rats which were more sensitive to 8-OH-DPAT (High DPAT Sensitive-HDS) exhibited decreases in temperature of 4 degrees C or more and the distribution did not overlap with that of the rats which were less sensitive to 8-OH-DPAT (Low DPAT Sensitive-LDS) which exhibited decreases in temperature of 1.5 degrees C or less. The randomly bred control group (Random DPAT Sensitive-RDS) exhibited intermediate temperature decreases (means of 1.6-1.8 degrees C), with time overlap with the distributions of the selected groups. Pretreatment with pindolol, a 5-HT-1A antagonist, reduced the hypothermic response to 8-OH-DPAT, but pretreatment with ritanserin, a 5-HT-7 and 5-HT-2A/C antagonist, had no effect, confirming that the hypothermic response to 8-OH-DPAT is mediated predominantly by 5-HT-1A receptors. The HDS rats were less mobile in a forced swim test and drank more saccharin solution in a two-bottle choice paradigm than the LDS or RDS rats over several generations. In contrast, there were no consistent differences among the groups for open field activity or performance in an elevated plus maze. There were no differences among the groups for voluntary alcohol intake, but the HDS rats exhibited greater suppression of alcohol and saccharin intake after injection of 8-OH-DPAT (0.125 mg kg-1). The HDS rats were also found to have a higher number of 5-HT-1A binding sites in cortical regions than the LDS or RDS rats, but there were no 5-HT-1A binding site differences in the raphe nuclei. These findings clearly show that consistent behavioral differences do occur in the 8-OH-DPAT-selected lines of rats, but only for behaviors related to possible depression or reward, not anxiety. The pattern of binding results suggests that these behavioral correlates of 8-OH-DPAT selection may be related to changes in cortical 5-HT-1A receptors rather than raphe autoreceptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1995

Age does not alter Protein kinase C isozymes mRNA expression in rat brain.

Calcium and phospholipid dependent Protein kinase C (PKC) may play a role in memory function and pathogenesis of many neurodegenerative disorders such as Alzheimer's disease (AD). Abnormal phosphorylation by PKC as well as reduced levels of PKC has been implicated in the neurodegeneration associated with AD and aging. Recently, many subtypes of PKC isozymes have been identified by molecular biology techniques which are expressed differentially in various regions of the brain. The reduction and alterations in the activities and distribution of these subtypes of PKC isozymes may be accountable for the decline of selective neurons during aging. In order to investigate the role of PKC isozymes during aging, we examined the distribution of PKC-alpha, beta, and gamma mRNA expressions between young (4 months) and old (25 months) rat brains using in situ hybridization histochemistry. Our studies showed that signals of three isoforms of PKC mRNA vary in cortical and hippocampal regions. However, no change was detected in any of the PKC isoforms mRNA expression in aged animals.

Aging↗
PubMed1995

Angiotensin II decreases inducible nitric oxide synthase expression in rat astroglial cultures.

Consistent with stimulation of expression of an inducible form of nitric oxide synthase (iNOS), exposure of rat astroglial cultures to lipopolysaccharide (LPS) caused a time-dependent increase in the accumulation of nitrite in the culture media. Addition of the peptide angiotensin II (ANG II) with LPS decreased subsequent formation of nitrite in a concentration-dependent manner (concentration inhibiting 50% of maximal response approximately 1 nM). The ANG II effect could be blocked by the ANG II type 1 (AT1 receptor antagonist losartan but not by the ANG II type 2 (AT2) receptor antagonist PD-123177. ANG II had no effect on nitrite formation stimulated by a combination of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma). A brief 10-min exposure to ANG II was sufficient to cause an approximately 30% inhibition of the LPS response, with maximal inhibition of approximately 65% after 3 h, and occurred only when ANG II was added during the iNOS induction phase. Consistent with partial inhibition of LPS-stimulated expression of iNOS, ANG II reduced the levels of both iNOS mRNA and iNOS protein. These results demonstrate that ANG II can decrease LPS-stimulated NO production in astroglia by inhibiting induction of iNOS expression.

Amino Acid Oxidoreductases↗
PubMed1994

Cholinergic and serotonergic stimulation of phosphoinositide hydrolysis is decreased in Alzheimer's disease.

Agonist-stimulated phosphoinositide (PPI) hydrolysis is a major signal transduction pathway in brain. These studies investigated neurotransmitter stimulated PPI hydrolysis in postmortem human brain. Preliminary studies using rat brain suggested that moderate postmortem delay has little effect on PPI hydrolysis and that human tissue might be reliably studied for differences in receptor-PLC coupling. Studies in human brain membranes (frontal cortex) indicated that the time course for GTP gamma S and carbachol/GTP gamma S-stimulated PPI hydrolysis was linear for at least 20 min. GTP gamma S-stimulated [3H]inositol phosphate (InsP) formation was enhanced by carbachol (232%) and 5-Hydroxytryptamine (5HT-147%). SAX-HPLC separation of [3H]inositol polyphosphates indicated that the major isomer of inositol trisphosphate (InsP3) was Ins(1.4.5)P3, the expected product of PtdIns(4,5)P2 hydrolysis. Ca2+ increased PPI hydrolysis progressively from 100 nM through 50 microM and synergistically enhanced carbachol/GTP gamma S stimulation. Comparisons of age-matched controls with Alzheimer's patients indicated that GTP gamma S, carbachol/GTP gamma S, and 5HT/GTP gamma S-stimulation of PPI hydrolysis is reduced approximately 50% in membranes prepared from Alzheimer's patients. Ca2+ of PPI hydrolysis was not different between controls and Alzheimer's patients suggesting that muscarinic cholinergic and serotonergic receptors are uncoupled from PLC in Alzheimer's disease. These studies indicate that there are changes in cholinergic and serotonergic signal transduction in Alzheimer's disease. Further, this method can be used to study signal transduction events in postmortem human brain.

Alzheimer Disease↗
PubMed1994

Magnesium and zinc potentiate ethanol inhibition of N-methyl-D-aspartate-stimulated nitric oxide synthase in cortical neurons.

The coupling of calcium mobilizing receptors to nitric oxide (NO) formation was examined in cerebral cortical cultures. Of the various agents tested, only glutamate, depolarization with KCl and the calcium ionophore ionomycin stimulated nitric oxide synthase (NOS) activity. Characterization of the glutamate response revealed that the ionotropic glutamate receptor agonists N-methyl-D-aspartate (NMDA), kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic all stimulated NOS activity with a relative maximal efficacy of NMDA > kainate > alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic. Ethanol, Mg++ and Zn++ produced a concentration-dependent inhibition of NMDA stimulation of NOS. The Mg++ inhibition was reversed by increasing concentrations of NMDA, whereas Zn++ inhibition was not. Ethanol (100 mM) produced an apparent competitive type inhibition as seen by a parallel right-shift in the NMDA concentration-response curve. However, ethanol inhibition was dependent upon the presence of Mg++ and/or Zn++ in a concentration-related manner. Whereas 100 mM ethanol did not significantly inhibit NMDA stimulation of NOS activity in the absence of Mg++ and Zn++, inclusion of a combination of these cations increased the sensitivity to ethanol such that the NMDA response was completely blocked by 100 mM ethanol (IC50 approximately 30 mM). The potency for inhibition of NMDA stimulation of NOS by several short-chain alcohols followed their hydrophobicity profile and showed a similar dependency upon Mg++ for inhibition, alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic, but not kainate, stimulation of NOS was also inhibited by ethanol (100 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1993

Activation of phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase during rat parotid acinar cell proliferation.

We have recently shown that beta-adrenergic agonist, isoproterenol-induced parotid acinar cell proliferation is in part mediated by elevated levels of surface galactosyltransferase which undergoes interaction with the EGF-R. The receptor subsequently undergoes autophosphorylation on the tyrosine residues in a manner similar to its 'receptor-ligand' interaction (Purushotham et al. (1992) Biochem. J. 284, 767-776). In this study, we provide evidence for phosphatidylinositol 3-kinase and 4-kinase as cytoplasmic signalling proteins involved in both the isoproterenol and EGF-stimulated signal transduction upon in vitro and in-vivo stimulation of parotid acinar cells. Total cell lysate activity for the PtdIns 4-kinase was 2- and 3-fold higher than unstimulated control cells, while the PtdIns 3-kinase was 1.4- and 2.8-fold higher following stimulation by isoproterenol or EGF, respectively. Increases of 6- and 2-fold in phosphatidylinositol 3-kinase were observed in anti-phosphotyrosine-antibody-immunoprecipitated cell lysates upon in-vitro growth stimulation with isoproterenol or EGF, respectively. There was an increase in tyrosine phosphorylation of the holoenzyme and association of the p85 subunit of phosphatidylinositol 3-kinase with EGF-R in response to both isoproterenol and EGF treatments. This corresponded with the mobilization of p85 from the cytoplasm to the plasma membrane upon growth stimulation. These results further implicate the phosphoinositide metabolites in the second messenger signalling pathways of isoproterenol-induced rat parotid cell proliferation. The parallel utilization of EGF indicate that the post-transductional mechanisms of isoproterenol-induced acinar cell proliferation are similar to the growth-factor-mediated activation of intracellular signalling pathways for cell growth.

1-Phosphatidylinositol 4-Kinase↗