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Ethanol and glutamate receptor function
ABSTRACTS A235
A238 FACTORS INFLUENCING CONCENTRATIONS OF GABA IN Theodore A. Hare. Mark Ii. CEREBIOSPINAL FLUID. Grossumn, James H.,Wood. Bruce S. Glaeeer and N.V. Bala Ma" am, Thomas Jef erson Univ., P 1 a., PT., Univ. of Penn., Phila.. PA and Vet. Admin..Med. 6 Reg. Office Cent., Wilmington, DE Degradation of cerebtospinal fluid (CSF) constituenta occurring during collection, storage and analysis may add artifactual variations GASA levels in untreated CSF deto CSF data. termined by ion-exchange/fluoromtric assay remained stable when stored at -70% for 3.5 GABA concenyears and at -20% for 1.5 years. trations in untreated CSF doubled during 2 hours at room temperature but did-not significantly change during 10 minutes at room GASA levels in temperature or 2 hours at 4%. deproteinized CSF were stable at -70°C but doubled during 11 months of storaqe at -20%. GASA concentrations in deproteinized CSF were stable for 49 hours at room temperature but increased 1.3-fold and 2.0-fold after 3 weeks at 4°C and tc-zamtemperature, respectively. The GABA content of homocarnosine standards in 0.1 N SC1 similarly increased during storage and at -20°C suggesting that the GABA elevations in inadequately stored or prepared CSF may be secondary to GABA-containing peptide degradation.
at roomtemperature
A236 LOW CEREBROSPINAL FLUID GABA CONCENTRATIONS IN EPILEPTIC PATIENTS: VARIATIONS WITH RESPECT TG SEIZURE TYPE AND ChRONIC CEREBELIAR GTI""LATION. James H. Wood, Theodore A. Hare, Bruce S. Glaeaer, Benjamin R. Brooks, James C. Sallenqer and Robert M. Post. Univ. of Penn., Phila., PA, Thomas Jefferson Univ., Phila.. PA. Johns Houkins Univ., Baltimore, MD, l&H, Be&he&, MD. I GABA has been implicated in the neurochemistry of epilepsy. The mean lumbar CSF CAB* concentration determined by ion-exchange/fluoromtric assay among 21 medicated patients with intraztable seizures was significantly lower (p 0.001) than that of 20 unmedicated normal^wlunteers. Patients with generalized grand ma1 and psychomotor seizures had significantly lower (p 0.05) CSF GASA concentrations than those with focal sensoey/rwtor epilepsy. Lumbar CSF GABA levels did not significantly correlate with serum concentrations of phenytoin, phenobarbital or primidone. Lumbar CSF GABA levels in four seizure patients were significantly leer during bilateral, continuous cerebella stimulation than those determined after a 7-&y period without stimulation. Thus GABA-mediated nauronal transmission appears depressed in epileptic patients and further reductions evoked by cerebella surface stimulation may compromise seizure control.
A237 PRODUCTION OF AN ANTISERLR(TO OECARBOXYLASES OF GLU-TAPATE (GAD) AN0 CYSTEINE SULPHIRTE (CSD) FROM RAT BRAIN W.H.Oertel,I.J.Kopin.M.L.Tappaz,O.H.Ransom and O.E.Schnechel Lab.Clin.Science, NIMH, Bldg.10, 20-55.Bethesda,Md.20205 GAD, the rate-limiting enzyme in GABA synthesis, and CSD. which forms hypotaurine, the precursor of the putative transmitter taurine, were partially copurified from rat brain. Polyvalent antisera against this preparation were raised in sheep. Imnunoelectmphoresis with less purified GAOfCSD and a polyvalent antiserum yielded three predpitin lines. Antisera to each predpitin line were then obtained in individual sheep. One of these antisera. 1440, had anti-W\D/CSD activity which precipitated both enzyme activities to similar degrees. On crossed i~unoelectrophoresis with intermediate gel containing 1440, the nobility of only one antigen in rat brain hanogenate supernatant was altered. The physfcal properties of the imnunopredpitate from rat brain hanogenate supernatant and 1440 were examined by 2-dlmcnsional electrophoresls. Irmnunocytochemlstryusing 1440 (1:ZOW) and the, Indirect PAP-method of Sternberger yielded specific locbliz&ion in cerebellum and other brain areas. GAO and CSD activity appear to be represented by one enzyme moleculeand synthesis of GABA and taurine, two major putatlve transmitters, may be controlled by one enzyme. W.H.Oertel is supported by OAAD. West Germany
A239 ETHANOL AND GLUTADlATERECEPTOR FUNCTION -- E. K. ,"ichaelis, M. L. Oichaelis. R. 1. Belieu, and R. D. Grubbe. Dept. of ""ma" Developaent, ""iv. OP Kansas, Laurence, Kansas 66045 The effects OF acute and chronic ethanol trsatnent on the activity of putative receptors for L-glutamic acid (L-Glu) in synaptosona1 n!enbra"*shss been examined. Receptor function in these membrane prsparrkione has bseg measured primarily by mmons OF radioactive ligand. L-C n)glutawste, binding assays et ligand concentrations in the range of 0.0, - 1 @4. Acute administration of ethanol did not cause significant changes in (3H)-glutsmic acid binding activity. vhsreas chronic oral administration of this alcohol brought abcut 8 significsnt increase in the density of Glu binding sites in synaptosomal pre,,arstions. Withdrawal from athanol intake resulted in e gradual return (in 6 days) to bass1 levels of Glu binding activity. The in
* exposure of synaptic membranes to 5 - 50rnl sttlano1 has also bsen found to cause significant increases in Clu binding sctiyity. In rscsnt studies YB have &Fined the role of synaptic membrane gangliooides II*endagenous inhibitors of Glu receptor function. Ethanol yss round to disru,,t the Glu membrane level.
receptor
-
gangliaside
interaction
at
the
These observations will be discussed in terns of ethanol's activity as a modulator of membrane recsptor Plmctio".
Supported by grants from NIAAA (AA-01911). from NICm9 (cm-22357). and from N~CHHD (HD-07066).
A240 GLUTAMATE DECAYBOXYLASE IN THE '$ERTEBRATERETINA. Christopher Bran on , Y.Y. Thomas Su , Domini M.K. Lam2, and f Department of Cell BiologyF and Cullen Eye Jang-Yen '9" Institute Baylor College of Medicine, Houston, Tex. 77030 Substantial biochemical and physiological evidence exists that GABA is a neumtransmitter in the vertebrate retina. Glutamate decarboxylase (GRD), the synthetic enzyme for GABA, has been purified fmm mouse brain and catfish brain; we have used specific antisera to these proteins for the inmunohistochemical localization of GAD in the retinas of the goldfish, frog. and rabbit. In the goldfish, GAD-positive reaction product "as seen in a broad band in sublamiba b of the inner plexiform layer (IPL), representing amacrine cell terminals synapsing on red-sensitive bipolar cells. In addition, Hi (red-sensitive) horizontal cells were heavily labeled. In the rabbit, four discrete, evenly-spaced laminae of reaction product. representing the terminals of a family of stratified amacrine cells involved in direction selectivity, were observed in the IPL, while no horizontal staining "as found. In the frog, labeling of both stratified and diffuse amacrine cell processes resulted in a dense stain throughout the IPL. In addition, horizontal cell bodies were frequently seen. These results support the notion that specific retinal cell types may be subdivided into classes which employ different neumtransmitters. Supported in part by NS13224 and a grant from the Huntington's Chorea Foundation.
,
,
A238 FACTORS INFLUENCING CONCENTRATIONS OF GABA IN Theodore A. Hare. Mark Ii. CEREBIOSPINAL FLUID. Grossumn, James H.,Wood. Bruce S. Glaeeer and N.V. Bala Ma" am, Thomas Jef erson Univ., P 1 a., PT., Univ. of Penn., Phila.. PA and Vet. Admin..Med. 6 Reg. Office Cent., Wilmington, DE Degradation of cerebtospinal fluid (CSF) constituenta occurring during collection, storage and analysis may add artifactual variations GASA levels in untreated CSF deto CSF data. termined by ion-exchange/fluoromtric assay remained stable when stored at -70% for 3.5 GABA concenyears and at -20% for 1.5 years. trations in untreated CSF doubled during 2 hours at room temperature but did-not significantly change during 10 minutes at room GASA levels in temperature or 2 hours at 4%. deproteinized CSF were stable at -70°C but doubled during 11 months of storaqe at -20%. GASA concentrations in deproteinized CSF were stable for 49 hours at room temperature but increased 1.3-fold and 2.0-fold after 3 weeks at 4°C and tc-zamtemperature, respectively. The GABA content of homocarnosine standards in 0.1 N SC1 similarly increased during storage and at -20°C suggesting that the GABA elevations in inadequately stored or prepared CSF may be secondary to GABA-containing peptide degradation.
at roomtemperature
A236 LOW CEREBROSPINAL FLUID GABA CONCENTRATIONS IN EPILEPTIC PATIENTS: VARIATIONS WITH RESPECT TG SEIZURE TYPE AND ChRONIC CEREBELIAR GTI""LATION. James H. Wood, Theodore A. Hare, Bruce S. Glaeaer, Benjamin R. Brooks, James C. Sallenqer and Robert M. Post. Univ. of Penn., Phila., PA, Thomas Jefferson Univ., Phila.. PA. Johns Houkins Univ., Baltimore, MD, l&H, Be&he&, MD. I GABA has been implicated in the neurochemistry of epilepsy. The mean lumbar CSF CAB* concentration determined by ion-exchange/fluoromtric assay among 21 medicated patients with intraztable seizures was significantly lower (p 0.001) than that of 20 unmedicated normal^wlunteers. Patients with generalized grand ma1 and psychomotor seizures had significantly lower (p 0.05) CSF GASA concentrations than those with focal sensoey/rwtor epilepsy. Lumbar CSF GABA levels did not significantly correlate with serum concentrations of phenytoin, phenobarbital or primidone. Lumbar CSF GABA levels in four seizure patients were significantly leer during bilateral, continuous cerebella stimulation than those determined after a 7-&y period without stimulation. Thus GABA-mediated nauronal transmission appears depressed in epileptic patients and further reductions evoked by cerebella surface stimulation may compromise seizure control.
A237 PRODUCTION OF AN ANTISERLR(TO OECARBOXYLASES OF GLU-TAPATE (GAD) AN0 CYSTEINE SULPHIRTE (CSD) FROM RAT BRAIN W.H.Oertel,I.J.Kopin.M.L.Tappaz,O.H.Ransom and O.E.Schnechel Lab.Clin.Science, NIMH, Bldg.10, 20-55.Bethesda,Md.20205 GAD, the rate-limiting enzyme in GABA synthesis, and CSD. which forms hypotaurine, the precursor of the putative transmitter taurine, were partially copurified from rat brain. Polyvalent antisera against this preparation were raised in sheep. Imnunoelectmphoresis with less purified GAOfCSD and a polyvalent antiserum yielded three predpitin lines. Antisera to each predpitin line were then obtained in individual sheep. One of these antisera. 1440, had anti-W\D/CSD activity which precipitated both enzyme activities to similar degrees. On crossed i~unoelectrophoresis with intermediate gel containing 1440, the nobility of only one antigen in rat brain hanogenate supernatant was altered. The physfcal properties of the imnunopredpitate from rat brain hanogenate supernatant and 1440 were examined by 2-dlmcnsional electrophoresls. Irmnunocytochemlstryusing 1440 (1:ZOW) and the, Indirect PAP-method of Sternberger yielded specific locbliz&ion in cerebellum and other brain areas. GAO and CSD activity appear to be represented by one enzyme moleculeand synthesis of GABA and taurine, two major putatlve transmitters, may be controlled by one enzyme. W.H.Oertel is supported by OAAD. West Germany
A239 ETHANOL AND GLUTADlATERECEPTOR FUNCTION -- E. K. ,"ichaelis, M. L. Oichaelis. R. 1. Belieu, and R. D. Grubbe. Dept. of ""ma" Developaent, ""iv. OP Kansas, Laurence, Kansas 66045 The effects OF acute and chronic ethanol trsatnent on the activity of putative receptors for L-glutamic acid (L-Glu) in synaptosona1 n!enbra"*shss been examined. Receptor function in these membrane prsparrkione has bseg measured primarily by mmons OF radioactive ligand. L-C n)glutawste, binding assays et ligand concentrations in the range of 0.0, - 1 @4. Acute administration of ethanol did not cause significant changes in (3H)-glutsmic acid binding activity. vhsreas chronic oral administration of this alcohol brought abcut 8 significsnt increase in the density of Glu binding sites in synaptosomal pre,,arstions. Withdrawal from athanol intake resulted in e gradual return (in 6 days) to bass1 levels of Glu binding activity. The in
* exposure of synaptic membranes to 5 - 50rnl sttlano1 has also bsen found to cause significant increases in Clu binding sctiyity. In rscsnt studies YB have &Fined the role of synaptic membrane gangliooides II*endagenous inhibitors of Glu receptor function. Ethanol yss round to disru,,t the Glu membrane level.
receptor
-
gangliaside
interaction
at
the
These observations will be discussed in terns of ethanol's activity as a modulator of membrane recsptor Plmctio".
Supported by grants from NIAAA (AA-01911). from NICm9 (cm-22357). and from N~CHHD (HD-07066).
A240 GLUTAMATE DECAYBOXYLASE IN THE '$ERTEBRATERETINA. Christopher Bran on , Y.Y. Thomas Su , Domini M.K. Lam2, and f Department of Cell BiologyF and Cullen Eye Jang-Yen '9" Institute Baylor College of Medicine, Houston, Tex. 77030 Substantial biochemical and physiological evidence exists that GABA is a neumtransmitter in the vertebrate retina. Glutamate decarboxylase (GRD), the synthetic enzyme for GABA, has been purified fmm mouse brain and catfish brain; we have used specific antisera to these proteins for the inmunohistochemical localization of GAD in the retinas of the goldfish, frog. and rabbit. In the goldfish, GAD-positive reaction product "as seen in a broad band in sublamiba b of the inner plexiform layer (IPL), representing amacrine cell terminals synapsing on red-sensitive bipolar cells. In addition, Hi (red-sensitive) horizontal cells were heavily labeled. In the rabbit, four discrete, evenly-spaced laminae of reaction product. representing the terminals of a family of stratified amacrine cells involved in direction selectivity, were observed in the IPL, while no horizontal staining "as found. In the frog, labeling of both stratified and diffuse amacrine cell processes resulted in a dense stain throughout the IPL. In addition, horizontal cell bodies were frequently seen. These results support the notion that specific retinal cell types may be subdivided into classes which employ different neumtransmitters. Supported in part by NS13224 and a grant from the Huntington's Chorea Foundation.
,
,