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K E Smith

Publications and source records attributed to K E Smith.

At least 109 records · Page 6Linked to original sources
PubMed1995

Re-evaluation of GABA transport in neuronal and glial cell cultures: correlation of pharmacology and mRNA localization.

Molecular cloning has revealed the existence of four distinct transporters for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), termed GAT-1, GAT-2, GAT-3, and BGT-1. To determine which of the cloned transporters are in neurons and which are in glia, we have undertaken a combined pharmacological and molecular biological study using cell cultures derived from rat brain. In neuronal cultures approximately 70% of GABA transport is sensitive to the GAT-1-selective ligand NNC-711 and drug potencies at this site correlate well with their potencies at GAT-1; GAT-1 mRNA is abundant in these cultures as determined by northern blot analysis. Drug potencies at the NNC-711-resistant component correlate well with their potencies at GAT-2 and GAT-3, whose pharmacological profiles are similar to one another. Northern blots reveal the presence of mRNA for GAT-3 in neuronal cultures, whereas GAT-2 and BGT-1 mRNAs are not detected. Type 1 astrocyte cultures exhibit very low levels of GABA transport activity, which has very low potency for GABA but high potency for taurine. Such cultures have mRNA for a taurine transporter and BGT-1, but not for GAT-1, GAT-2, and GAT-3. In cultures containing O-2A progenitor cells and Type 2 astrocytes, approximately 75% of GABA uptake is sensitive to NNC-711 and drug potencies at this site correlate well with their potencies at GAT-1; GAT-1 mRNA is abundant. Drug potencies at the NNC-711-resistant component correlate well with their potencies at GAT-2 and GAT-3; mRNAs for both of these transporters are present (though GAT-2 mRNA is the more abundant), as is BGT-1 mRNA. In summary, these data demonstrate heterogeneity of both neuronal and glial GABA transport.

Animals↗
PubMed1994

Tiagabine, SK&F 89976-A, CI-966, and NNC-711 are selective for the cloned GABA transporter GAT-1.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The synaptic action of GABA is terminated by rapid uptake into presynaptic terminals and surrounding glial cells. Molecular cloning has revealed the existence of four distinct GABA transporters termed GAT-1, GAT-2, GAT-3, and BGT-1. Pharmacological inhibition of transport provides a mechanism for increasing GABA-ergic transmission, which may be useful in the treatment of various neuropsychiatric disorders. Recently, a number of lipophilic GABA transport inhibitors have been designed and synthesized, which are capable of crossing the blood brain barrier, and which display anticonvulsive activity. We have now determined the potency of four of these compounds, SK&F 89976-A (N-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid), tiagabine ((R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3- piperidencarboxylic acid), CI-966 ([1-[2-[bis 4-(trifluoromethyl)phenyl]methoxy]ethyl]-1,2,5,6-tetrahydro-3- pyridinecarboxylic acid), and NNC-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,4,6-tetrahydro-3- pyridinecarboxylic acid hydrochloride), at each of the four cloned GABA transporters, and find them to be highly selective for GAT-1. These data suggest that the anticonvulsant activity of these compounds is mediated via inhibition of uptake by GAT-1.

Animals↗
PubMed1994

Design, synthesis and evaluation of substituted triarylnipecotic acid derivatives as GABA uptake inhibitors: identification of a ligand with moderate affinity and selectivity for the cloned human GABA transporter GAT-3.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. Molecular biology has revealed the presence of four high-affinity GABA transporters in the brain, GAT-1, GAT-2, GAT-3, and BGT-1, the latter transporting both GABA and the osmolyte Betaine. We have shown that known GABA uptake inhibitors such as SK&F 89976-A, CI-966, and Tiagabine exhibit high affinity and selectivity for GAT-1. In the present paper we describe the design and synthesis of a novel series of triarylnipecotic acid derivatives for evaluation as GABA uptake inhibitors. The design lead for this series of compounds was the nonselective GABA uptake inhibitor EGYT-3886, [(-)-2-phenyl-2-[(dimethylamino)ethoxy]-(1R)- 1,7,7-trimethylbicyclo[2.2.1]heptane]. From this series of compounds (S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-3-piperidinecarboxylic+ ++ (S)-1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-3-piperidinecarboxylic+ ++ acid, 4(S) was identified as a novel ligand with selectivity for GAT-3. 4(S) displayed an IC50 of 5 microM at GAT-3, 21 microM at GAT-2, > 200 microM at GAT-1, and 140 microM at BGT-1. This compound will be an important tool for evaluating the role of GAT-3 in neural function.

Animals↗
PubMed1994

Microbial transformations of steroids--VIII. Transformation of progesterone by whole cells and microsomes of Aspergillus fumigatus.

The filamentous fungus, Aspergillus fumigatus, efficiently hydroxylated exogenous progesterone producing, after 3 h of incubation, 11 alpha- and 15 beta-hydroxyprogesterone as major products, 7 beta-hydroxyprogesterone as a minor product and trace amounts of 7 beta, 15 beta- and 11 alpha, 15 beta-dihydroxyprogesterone. After 72 h the dihydroxyprogesterones were the sole metabolites in the culture medium. Microsomes, prepared by Ca2+ precipitation, catalysed only monohydroxylation of progesterone at the same sites as whole cells. Hydroxylation was dependent on NADPH (but not NADH) which was replaceable by NaIO4. Hydroxylation was inhibited by carbon monoxide and by the azole fungicide, ketoconazole. Microsomes gave a dithionite-reduced, carbon monoxide difference absorbance spectrum with a peak at 448 nm and a Type-I progesterone-binding spectrum typical of cytochrome P450 interaction with substrate. Ketoconazole inhibition studies suggest the presence of two non-inducible cytochrome P450 progesterone hydroxylases, one possessing 7 beta site-selectivity, the other 11 alpha/15 beta site-selectivity.

Aspergillus fumigatus↗
PubMed1994

Delayed treatment with AMPA, but not NMDA, antagonists reduces neocortical infarction.

We tested the abilities of two potent non-N-methyl-D-aspartate (non-NMDA) glutamate antagonists [2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline (NBQX)] and [1-(4-aminophenyl)-4-methyl-7,8-methylene-dioxy-5H-2,3-benzodiazep ine hydrochloride (GYKI 52466)] to reduce neocortical infarction following 2 h of transient middle cerebral artery occlusion in a hypertensive stroke model in the rat and compared these effects against, and in combination with, a potent NMDA antagonist [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-amine maleate (MK-801)]. In Expt. 1, an already established cytoprotective dose of Na(+)-NBQX (30 mg/kg i.p. x 3) was compared with saline (1 ml), the NMDA antagonist MK-801 (1 mg/kg i.p. x 3), and a combination of the same doses of both NBQX and MK-801. Initial doses were delayed to 90 min following occlusion with subsequent injections at the time of reperfusion and 30 min following reperfusion. Saline-treated rats sustained 181 +/- 32 mm3 (n = 15) of neocortical infarction (mean +/- SD). This was significantly reduced by NBQX to 137 +/- 25 mm3 (n = 15, p < 0.05) of damage. Neither MK-801 (170 +/- 33 mm3; n = 11) nor the combination of MK-801 and NBQX (169 +/- 20 mm3; n = 6) proved to be cytoprotective when given with a 90-min delay. In Expt. 2, NBQX (30 mg/kg) was dissolved (6 mg/ml) in 5% dextrose and compared with both saline and dextrose (1.2 ml) i.v. infusions given over a 4-h period starting 1 h after occlusion. Saline-treated rats had a mean infarct of 183 +/- 27 mm3 (n = 6), dextrose-treated had 200 +/- 30 mm3 (n = 9), while for NBQX-treated rats it was reduced to 129 +/- 60 mm3 (n = 10, p < 0.05). Intravenous NBQX precipitated into the renal tubules, causing nephrotoxicity. In Expt. 3, rats were given either saline (1 ml i.p.) or GYKI 52466 (10 mg/kg i.p.) at 30 and 90 min following occlusion and at 30, 90, and 150 min following reperfusion. Saline-treated rats sustained 187 +/- 27 mm3 of neocortical infarction (n = 7), while those treated with GYKI 52466 were protected, with 139 +/- 38 mm3 of infarction (n = 7, p < 0.05). A clinically useful role for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate antagonists in embolic stroke is envisaged if nontoxic drugs can be developed, since cerebroprotection was achieved with delayed treatment with both of these lead compounds.

Animals↗
PubMed1994

Quantification of facial surface change using a structured light scanner.

Three-dimensional surface changes that accompany facial surgical procedures were measured noninvasively and evaluated quantitatively. An optical three-dimensional surface scanner with 360-degree surface coverage of a subject's head and a subsecond data acquisition was used. The scanner employs six pairs of "white light" pattern projectors and digital TV cameras. A noncontact optical method to quantify facial surface morphology and objectively assess change resulting from reconstructive or cosmetic plastic surgery has been developed. This quantification technique was implemented and tested with the three-dimensional range scanner. This technique defines the entire surface of the head and face, as opposed to the conventional manual method of measuring surface points or facial landmarks. The method allows facial volume change assessment. The method was tested by repeatedly scanning a volunteer who was injected subcutaneously with known volumes of anesthetic solution. The measured and injected volumes were compared and showed little difference.

Computer Graphics↗
PubMed1994

Cloning of the human homologue of the GABA transporter GAT-3 and identification of a novel inhibitor with selectivity for this site.

Molecular cloning has revealed the presence of four high-affinity GABA transporters in the brain. The existence of three of these sites, GAT-2, GAT-3, and BGT-1, was unknown prior to their cloning and almost nothing is known of the role they play in regulating GABAergic transmission. In large measure our paucity of knowledge is attributable to the lack of specific inhibitors for these sites. In the present communication we describe the cloning and expression of the human homologue of GAT-3, and the identification of an inhibitor, (S)-SNAP-5114, with selectivity for this site. (S)-SNAP-5114 displays an IC50 of 5 microM at GAT-3, 21 microM at GAT-2, and > or = 100 microM at GAT-1 and BGT-1. Due to its lipophilicity, (S)-SNAP-5114 is also expected to cross the blood-brain-barrier and therefore, should be an important tool for evaluating the role of GAT-3 in neural function.

Amino Acid Sequence↗
PubMed1994

An atypical eating disorder with Crohn's disease in a fifteen-year-old male: a case study.

Most of the time, school or counseling psychologists are not specifically trained to work with children who have physical disorders that cause or exacerbate psychological problems. This article describes a 15-year-old male with delayed physical development who was referred for psychological evaluation and treatment of a suspected eating disorder. Six months after psychological intervention, appropriate eating behaviors were established, yet he had not gained weight as expected. After further medical evaluation, he was diagnosed as having Crohn's disease. Criteria for a DSM-III-R diagnosis of Anorexia Nervosa or Eating Disorder, Not Otherwise Specified, are discussed and symptoms of Crohn's disease described. Also discussed are differential diagnoses and practical recommendations for professionals in the field.

Adolescent↗
PubMed1994

The rat homologue of the bovine alpha 1c-adrenergic receptor shows the pharmacological properties of the classical alpha 1A subtype.

The cDNA for the rat alpha 1c-adrenergic receptor (AR) has been cloned using a probe derived from the bovine alpha 1c-AR sequence. Clone rB7a has a 2.6-kilobase insert with a 1390-base pair open reading frame and encodes a receptor of 466 amino acids. The cloned receptor has 91% amino acid identity with the bovine alpha 1c-AR. The rat alpha 1c-AR mRNA was detected in tissues known to be enriched for the alpha 1A-AR subtype, including vas deferens, heart, kidney, and hippocampus. Rat alpha 1c-AR mRNA was absent from liver and spleen when assayed by Northern blot analyses and RNase protection assays. In COS-7 cells transfected with cDNAs encoding the three rat alpha 1-ARs, WB-4101 and benoxathian had similar binding affinities for the alpha 1a/d-AR and the alpha 1c-AR and 10-fold lower affinities for the alpha 1b-AR. The affinity of 5-methylurapidil was found to be 10- and 30-fold higher at the alpha 1c-AR than at the alpha 1a/d- and alpha 1b-ARs, respectively. (S)-(+)-Niguldipine was found to have high affinity for the rat alpha 1c-AR, with 42- and 22-fold lower affinity at the alpha 1a/d- and alpha 1b-ARs, respectively. Treatment of intact transfected COS-7 cells with chlorethylclonidine resulted in the inactivation of 19% of the alpha 1c-ARs, in contrast to 72% and 85% inactivation of the alpha 1a/d- and alpha 1b-ARs, respectively. Similarly to the other two alpha 1-ARs, the rat alpha 1c-AR is coupled to the activation of phospholipase C. Our data suggest that the rat alpha 1c-AR cDNA encodes an alpha 1-AR with the pharmacological properties previously defined for the alpha 1A subtype found in tissues.

Adrenergic alpha-Agonists↗
PubMed1993

Effects of alcohol-induced lipid interdigitation on proton permeability in L-alpha-dipalmitoylphosphatidylcholine vesicles.

6-Carboxyfluorescein was employed to examine the effect of alcohol-induced lipid interdigitation on proton permeability in L-alpha-dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles. Proton permeability was measured by monitoring the decrease of 6-carboxyfluorescein fluorescence after a pH gradient from 3.5 (outside the vesicle) to 8.0 (inside the vesicle) was established. At 20 degrees C and below 1.2 M ethanol, the fluorescence decrease is best described by a single exponential function. Above 1.2 M ethanol, the intensity decrease is better described by a two-exponential decay law. Using the fitted rate constants and the vesicle radii determined from light-scattering measurements, the proton permeability coefficient, P, in DPPC vesicles was calculated as a function of ethanol concentration. At 20 degrees C, P increases monotonically with increasing ethanol content up to 1.0 M, followed by an abrupt increase at 1.2 M. The vesicle size also exhibits a sudden increase at around 1.2 M ethanol, which has been shown to result from vesicle aggregation rather than vesicle fusion. The abrupt increases in P and in vesicle size occur at the concentration region close to the critical ethanol concentration for the formation of the fully interdigitated gel state of DPPC. At 14 degrees C, the abrupt change in P shifts to 1.9-2.0 M ethanol, completely in accordance with the ethanol-temperature phase diagram of interdigitated DPPC. Effects of methanol and benzyl alcohol on lipid interdigitation have also been examined. At 20 degrees C, DPPC large unilamellar vesicles exhibit a dramatic change in P at 3 M methanol and at 40 mM benzyl alcohol. These concentrations come close to the critical methanol and benzyl alcohol concentrations for the formation of fully interdigitated DPPC structures determined previously by others. It can be concluded that proton permeability increases dramatically as DPPC is transformed from the noninterdigitated gel to the fully interdigitated gel state by high concentrations of alcohol. This marked increase in proton permeability can be attributed to the combined effect of the changes in membrane thickness and surface charge density, due to the ethanol-induced lipid interdigitation. The possible effects of the increased proton permeability caused by ingested ethanol on gastric mucosal membranes are discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

Further studies on the susceptibility of raccoons (Procyon lotor) to a rabies virus of skunk origin and comparative susceptibility of striped skunks (Mephitis mephitis).

Two raccoons (Procyon lotor) were inoculated in the masseter muscles with 10(5.9) mouse intracerebral lethal dose50 (MICD50) of a rabies virus isolated from a naturally infected Iowa (USA) striped skunk (Mephitis mephitis). Five striped skunks were inoculated with either 10(0.7) or 10(2.1) MICLD50 of the same isolate. All five skunks died within 35 days following inoculation. Both raccoons survived 273 days without adverse effects, and virus was not isolated from saliva samples taken at between 25 and 273 days following inoculation.

Animals↗
PubMed1993

AMPA antagonists: do they hold more promise for clinical stroke trials than NMDA antagonists?

The cytoprotective effects of MK-801 and NBQX, selective N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonists, respectively, were compared both singularly and in combination in models of transient severe forebrain and transient focal cerebral ischemia. After 10 minutes of four-vessel occlusion ischemia, the sodium salt of NBQX (30 mg/kg IP) given at the time of reperfusion and, subsequently, 15 and 30 minutes later produced a dramatic reduction in CA1 hippocampal necrosis at 7 days. This effect was not obtained with the intraperitoneal administration of either MK-801 (1 mg/kg x 3) or the combination of both NBQX and MK-801 given at the same time intervals. This effect of intraperitoneal NBQX alone was reproduced in a two-vessel occlusion/hypotension model using this same drug administration. Delayed treatment with both NBQX and GYKI 52466, but neither MK-801 nor the combination of NBQX and MK-801 given after a delay, produced a significant reduction in the mean volume of neocortical infarction after transient focal ischemia. We conclude that the AMPA receptor may play a more important role than the NMDA receptor in both selective ischemic necrosis of hippocampal neurons and in neocortical infarction. AMPA antagonists should be subjected to clinical stroke trials.

Animals↗
PubMed1992

Molecular heterogeneity of the gamma-aminobutyric acid (GABA) transport system. Cloning of two novel high affinity GABA transporters from rat brain.

cDNA clones encoding two novel gamma-aminobutyric acid (GABA) transporters (designated GAT-2 and GAT-3) have been isolated from rat brain, and their functional properties have been examined in mammalian cells. The transporters display high affinity for GABA (Km approximately 10 microM) and exhibit pharmacological properties distinct from the previously cloned neuronal GABA transporter (GAT-1). Both transporters require sodium and chloride for transport activity. The nucleotide sequences of GAT-2 and GAT-3 predict proteins of 602 and 627 amino acids, respectively, which can be modeled with 12 transmembrane domains, similar to the topology proposed for other cloned neurotransmitter transporters. Localization studies indicate that both transporters are present in brain and retina, while GAT-2 is also present in peripheral tissues. The cloning of these transporter genes from rat brain reveals previously undescribed heterogeneity in GABA transporters.

Amino Acid Sequence↗
PubMed1992

Immediate or delayed mild hypothermia prevents focal cerebral infarction.

The protective effect of mild hypothermia was studied in rodent models of both permanent and transient focal cerebral ischemia. In Expt. 1, Wistar rats were exposed to 6 h permanent ischemia by bilateral occlusion of both common carotid arteries and right middle cerebral artery. In Expt. 2, animals were exposed to 3 h transient ischemia followed by 21 h reperfusion, and in Expt. 3, 3 h transient ischemia was followed by 69 h of reperfusion. Expt. 4 used 3 h transient ischemia followed by 3 h reperfusion. In Expt. 1, animals maintained at 37 degrees C rectal (normothermia) suffered a mean infarct volume (+/- S.D.) of 142 +/- 44 mm3 (n = 6), which was reduced for those exposed to permanent hypothermic (32 degrees C) ischemia to 56 +/- 64 mm3 (n = 10) (P less than 0.05). In Expt. 2, normothermic ischemia and reperfusion resulted in an infarction of 211 +/- 35 mm3 (n = 6). Intra-ischemic hypothermia (32 degrees C) followed by 21 h of normothermic reperfusion resulted in 17 +/- 12 mm3 of infarction (n = 9) (P less than 0.001). Hypothermia for either the first or second 1.5 h of the 3 h ischemic insult reduced the infarct volume to 116 +/- 76 mm3 (n = 6) (P less than 0.05) or 108 +/- 73 mm3 (n = 7) (P less than 0.01), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effect of age on autonomic and cardiac responses in a rat stroke model.

The cardiovascular system and its responses change with increasing age. This has seldom been considered in experimental models of stroke, although most strokes occur in the elderly. We studied 57 male Wistar rats in three age groups: 47 to 70 days old (juvenile), 110 to 152 days old (young adult), and 186 to 245 days old (mature adult), each group being subdivided into experimental and sham operation groups. All rats underwent occlusion or sham occlusion of the left middle cerebral artery and monitoring of the mean arterial blood pressure, heart rate, sympathetic nerve activity, plasma catecholamine levels, and electrocardiogram. Eight of the 12 rats in the oldest group died within 6 hours of the middle cerebral artery occlusion; of these, the youngest was 186 days old. The mature adult rats that died before completion of the experiment showed the highest level of sympathetic nerve activity and the only significant increase in the QT interval of the electrocardiogram. Following middle cerebral artery occlusion, sympathetic nerve activity increased in the young adult rats but most strikingly in the mature adult rats that died before the end of the 6-hour experiments. Plasma norepinephrine levels were significantly elevated at 4 and 6 hours after middle cerebral artery occlusion in the oldest group and only at 6 hours in the juvenile rats. The results of this study are consistent with impaired sympathetic and cardiovascular regulation in the mature adult rat. High sympathetic activity may represent one mechanism leading to fatal cardiac arrhythmias. Age-related impairment of sympathetic regulation may contribute to the higher mortality seen among elderly patients with stroke.

Aging↗
PubMed1992

Differential regulation of muscarinic and nicotinic cholinergic receptors and their mRNAs in cultured sympathetic neurons.

Mechanisms regulating expression of neuronal muscarinic and nicotinic receptors were examined in cultures of neonatal rat sympathetic neurons. Two factors known to stimulate cholinergic transmitter development in sympathetic neurons were examined for their effects on cholinergic receptor expression. A membrane associated factor (MANS46) and a diffusible factor produced by cultured rat fibroblasts (RFCM) each decreased muscarinic receptor number. By contrast, neither treatment altered levels of nicotinic receptors. Levels of muscarinic (m2) receptor mRNA were decreased by MANS but not by RFCM, indicating that effects of the two treatments were mediated by different mechanisms. Neither MANS nor RFCM altered levels of nicotinic alpha 3 or beta 2 mRNAs, consistent with the lack of change in numbers of nicotinic receptors. These observations indicate that receptor phenotype in developing neurons is subject to regulation by multiple epigenetic factors. Further, the same signals which regulate transmitter development may also regulate receptor expression in sympathetic neurons.

Animals↗