Biomedical subjects

J A Kemp

Publications and source records attributed to J A Kemp.

At least 73 records · Page 4Linked to original sources

PubMed1994

Depression of high-threshold calcium currents by activation of human D2 (short) dopamine receptors expressed in differentiated NG108-15 cells.

1. This study examined the regulation of calcium currents in differentiated NG108-15 cells that had been stably transfected with cDNA encoding the short isoform of the human D2 dopamine receptor. Whole cell calcium currents were recorded by nystatin-perforated patch clamp recording. 2. Transient low-threshold calcium currents elicited by depolarizations from -100 mV to -20 mV were reversibly depressed by NiCl2 (84 +/- 8% at 30 microM; n = 3) and by omega-agatoxin IVA (15 +/- 5%; 100 nM, n = 7). These currents were unaffected by hD2 receptor activation. 3. High-threshold calcium currents elicited by depolarizations from -80 mV to 0 mV were partly blocked by omega-conotoxin GVIA (67 +/- 6% at 100 nM, n = 4) and by the subsequent addition of the dihydropyridine, nisoldipine (94 +/- 3% at 1 microM). Consistent with the presence of at least two distinct types of high-threshold calcium channels, nisoldipine alone (38 +/- 15% at 1 microM, n = 6) did not preclude the inhibition caused by omega-conotoxin GVIA (69 +/- 13% at 100 nM, n = 4). The residual current was completely blocked by 100 microM CdCl2 (98.8 +/- 0.4%, n = 7). 4. In hD2-transfected cells, but not untransfected cells, high-threshold currents were depressed by quinpirole (30 +/- 4% at 100 nM; n = 15) with a pEC50 of 8.61 +/- 0.22 (n = 5), as well as by (-)-noradrenaline (28 +/- 5% at 1 microM, n = 9). Responses to both agonists were selectively antagonized by S-(-)sulpiride (100 nM) but not by the alpha-adrenoceptor antagonist, phentolamine (1O microM). The depression caused by (-)-noradrenaline was positively correlated with that of quinpirole for each cell(r2 = 0.91, slope = 0.99).5. hD2-receptor-mediated inhibition of high-threshold calcium currents was abolished by pretreatment of cells with omega-conotoxin GVIA (100 nM; n = 4). However, a component of the high-threshold current was reversibly depressed by omega-conotoxin GVIA (67% to 45% depression after 10 min wash). This current was also depressed by hD2 receptor activation (59 +/- 9% depression in 100 nM quinpirole, n = 3),and was completely blocked by nisoldipine (95 +/- 2% at 1 MicroM).6. These data demonstrate that activation of hD2(short) dopamine receptors can regulate both wconotoxinGVIA, and dihydropyridine-sensitive high-threshold calcium currents in neuroblastoma cells.Morever, the ability of human D2 dopamine receptors to regulate more than one type of calcium current supports the notion that these receptors have a diverse functional role in the central nervous system.

Animals↗

PubMed1994

Block of human voltage-sensitive Na+ currents in differentiated SH-SY5Y cells by lifarizine.

1. The ability of lifarizine (RS-87476) to block human voltage-sensitive Na+ channel currents was studied by use of whole cell patch clamp recording from differentiated neuroblastoma cells (SH-SY5Y). 2. The Na+ conductance in differentiated SH-SY5Y cells (24.0 +/- 2.4 nS, n = 11) was half-maximally activated by 10 ms depolarizations to -37 +/- 2 mV and was half-maximally inactivated by predepolarizing pulses of 200 ms duration to -86 +/- 3 mV (n = 11). 3. At low stimulus frequencies (0.1 to 0.33 Hz) voltage-dependent sodium currents were completely blocked, in a concentration-dependent manner, by extracellular application of either tetrodotoxin (EC50 = 4 +/- 1 nM, n = 12) or by lifarizine (EC50 = 783 +/- 67 nM, n = 9). The onset of block by lifarizine (tau = 91 +/- 14 s at 10 microM) was considerably slower than that of tetrodotoxin (tau = 16 +/- 3 s at 100 nM). 4. Lifarizine (1 microM) reduced the peak sodium conductance in each cell (from 26.4 +/- 2.0 nS to 15.1 +/- 2.7 nS, n = 4) without changing the macroscopic kinetics of sodium current activation or inactivation (V1/2 = -35 1 mV and -87 +/- 4 mV respectively, n = 4). Similarly, lifarizine (1 microM) did not affect the reversal potential of the macroscopic sodium current (+14 +/- 5 mV in control and +16 +/- 2 mV in 1 microM lifarizine; n = 4) or reactivation time-constant (tau = 14.0 +/- 4.4 ms). 5. Block of the sodium channel open state by tetrodotoxin (30 nM) did not prevent the inhibition caused by a subsequent application of lifarizine (3 micro M). In contrast the depression caused by lifarizinewas readily reversible after pretreatment of cells with the local anaesthetic, lignocaine (1O mM).6. These data demonstrate that lifarizine is a use- and voltage-dependent antagonist of human voltage sensitive sodium currents. The slow kinetics and pharmacology of the block by lifarizine indicate that access of this drug to the channel is more restricted than that of tetrodotoxin and may involve an allosteric site or state of the channel that is also regulated by local anaesthetics.

Cell Differentiation↗

PubMed1994

Kinetic study of the interactions between the glutamate and glycine recognition sites on the N-methyl-D-aspartic acid receptor complex.

The N-methyl-D-aspartate (NMDA) receptor is unique among the ligand-gated ion channels, in that the gating process requires the binding of two independent coagonists, glutamate and glycine. Receptor binding experiments have suggested that the coagonist recognition sites interact with one another in an allosteric manner, and previous work in this laboratory has provided additional functional support in favor of an allosteric coupling; the affinity of glutamate for its recognition site was reduced when a partial agonist, (+)-HA-966, occupied the glycine site, compared with the affinity when glycine itself was bound to the receptor. The present experiments have taken these observations a step further and compare the effects of several glycine site ligands with different affinities and intrinsic activities (determined from equilibrium concentration-response curves) on glutamate off-rate. Thus, the dissociation rate for the decay of glutamate-activated membrane currents in voltage-clamped rat cortical neurons was fastest (160 +/- 28 msec) in the presence of saturating concentrations of (+)-HA-966 and progressively slower in the presence of D-cycloserine (258 +/- 27 msec), aminocyclopropanecarboxylic acid (330 +/- 21 msec), L-alanine (375 +/- 28 msec), and glycine (502 +/- 42 msec). We have also measured the affinities and intrinsic activities of several NMDA receptor ligands and report that a reciprocal interaction exists, such that the off-rate of glycine is influenced by the properties of the agonist occupying the glutamate coagonist site. Thus, the time constant for current decay after a brief exposure to glycine was fastest in the presence of a saturating concentration of cis-2,3-piperidinedicarboxylic acid (449 +/- 26 msec) and progressively slower in the presence of quinolinate (689 +/- 73 msec), NMDA (721 +/- 36 msec), and L-glutamate (1260 +/- 36 msec). The data suggested that the extent of the modulation of one site by the other is related to the intrinsic activity of the agonist, rather than its affinity. Specifically, we suggest that a partial agonist occupying one of the agonist recognition sites produces a conformational change that results in an accelerated off-rate for coagonist dissociation from the receptor; the lower the intrinsic activity, the greater is the effect on coagonist off-rate.

Animals↗

PubMed1994

Biological properties of the benzodiazepine amidine derivative L-740,093, a cholecystokinin-B/gastrin receptor antagonist with high affinity in vitro and high potency in vivo.

A novel series of 5-amino-1,4-benzodiazepin-2-one derivatives (amidines), which contain a cationic solubilizing group and which are antagonists for the cholecystokinin (CCK)-B receptor, have been identified. Optimization of this series led to the identification of an azabicyclononane amidine, L-740,093 [N-[(3R)-5-(3-azabicyclo[3.2.2]nonan-3-yl)-2,3-dihydro-1-methyl-2- oxo- 1H-1,4-benzodiazepin-3-yl]-N'-(3-methylphenyl)urea], that bound with high affinity of CCK-B receptors from guinea pig cerebral cortex (IC50 of 0.1 nM) and had a CCK-B/CCK-A receptor selectivity of 16,000. In comparison, L-365,260 had 85-fold lower affinity (8.5 nM) and was only 87-fold selective for CCK-B over CCK-A receptors. L-740,093 bound with high affinity to guinea pig gastrin receptors in vitro (IC50 of 0.04 nM). Electrophysiological studies on slices of rat ventromedial hypothalamic nucleus showed that L-740,093 produced rightward shifts of the concentration-response curve for the CCK-B receptor agonist pentagastrin (Kb of 0.06 nM). L-740,093 blocked pentagastrin-induced gastric acid secretion in anesthetized rats with a 50% inhibitory dose of 0.01 mg/kg, intraperitoneally, showing 100-fold greater activity, compared with L-365,260 (50% inhibitory dose of 1 mg/kg, intraperitoneally). An ex vivo binding assay in mice was used to investigate the interaction of L-740,093 with central CCK binding sites. After intravenous administration, L-740,093 inhibited ex vivo binding dose dependently, with a 50% effective dose of 0.2 mg/kg. These studies demonstrate that L-740,093 is the most potent and selective CCK-B antagonist yet described and that it has excellent central nervous system penetration.

Animals↗

PubMed1994

Molecular pharmacology of gamma-aminobutyric acid type A receptor agonists and partial agonists in oocytes injected with different alpha, beta, and gamma receptor subunit combinations.

Using systematic combination of alpha 1, alpha 3, and alpha 5 with beta 1, beta 2, and beta 3, together with gamma 1, gamma 2, and gamma 3, we have investigated the contributions of the various alpha, beta, and gamma subunits to the pharmacology of gamma-aminobutyric acid (GABA)A agonists. We have characterized GABA, (RS)-dihydromuscimol, piperidine-4-sulfonic acid, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol with recombinant human GABAA receptors expressed in Xenopus oocytes. Our observations indicate that the alpha subunit is the major determinant of efficacy for partial GABAA agonists. When alpha 1 and alpha 3 or alpha 1 and alpha 5 are coexpressed, the alpha 1 subunit determines the maximum efficacy, whereas the affinity is determined by the entire combination of subunits. Thus, the results of the present study demonstrate that the pharmacology of GABAA agonists is dependent on the subunit composition of the GABAA receptor complex. Functional GABAA receptors containing two different alpha subunits show pharmacological profiles distinctly different from those of receptors containing a single alpha subtype, indicating that two different alpha subunits can be coexpressed in one functional GABAA receptor complex.

Animals↗

PubMed1994

A guide for surveillance of patients with Barrett's esophagus.

OBJECTIVE: Barrett's esophagus (columnar metaplasia of the distal esophagus due to chronic gastroesophageal reflux) affects nearly 700,000 people in the United States, and carries a risk of esophageal adenocarcinoma that is 30-125 times that of an age-matched population. Patients who develop high grade dysplasia are at greatest risk. Current recommendations are for endoscopic surveillance to detect dysplasia and to diagnose carcinoma while it is in an early and possibly treatable stage. In addition, some authorities recommend esophagectomy for high grade dysplasia, whereas others reserve esophagectomy only for those with cancer. There are no controlled trials demonstrating that surveillance increases life expectancy in patients with Barrett's esophagus. Furthermore, endoscopic surveillance of this large group with Barrett's esophagus may be costly, and associated with considerable morbidity. Therefore, our objective was to assess the effectiveness and cost-effectiveness of endoscopic surveillance in patients with Barrett's esophagus. METHODS: Design--Decision analysis using a computer cohort simulation (Markov). We examined 12 strategies: (A) no endoscopic surveillance. Esophagectomy is performed only if cancer is detected by biopsy. (B) no surveillance. Esophagectomy is performed if high grade dysplasia is detected by biopsy: (C1-C5) surveillance at intervals from 1 to 5 yr, with esophagectomy if cancer is diagnosed, and (D1-D5) surveillance at intervals from 1 to 5 yr with esophagectomy if high grade dysplasia is diagnosed. We measured life expectancy, quality-adjusted life expectancy, and incremental cost-effectiveness ratios for each strategy. Data Sources--Medline Search and bibliographies of retrieved articles; expert opinion when published data were not available. RESULTS AND CONCLUSIONS: Annual surveillance with esophagectomy for high grade dysplasia prevents cancer and is the preferred strategy, if only length of life (life expectancy) is considered. For those who consider both length and quality of life, endoscopy every 2-3 yr will provide the greatest quality-adjusted life expectancy. When costs are considered, endoscopy every 5 yr also increases life expectancy and has an incremental cost-effectiveness ratio similar to common medical practices. The cumulative incidence of cancer and the quality of life with an esophagectomy had the greatest impact on the decision for surveillance and the optimal surveillance strategy.

Adenocarcinoma↗

PubMed1993

3-Acyl-4-hydroxyquinolin-2(1H)-ones. Systemically active anticonvulsants acting by antagonism at the glycine site of the N-methyl-D-aspartate receptor complex.

Most full antagonists at the glycine site of the NMDA receptor contain a carboxylic acid, which we believe to be detrimental to penetration of the blood-brain barrier. By consideration of a pharmacophore, novel antagonists at this site have been designed in which the anionic functionality is a vinylogous acid, in the form of a 4-hydroxyquinolin-2(1H)-one. In this series, a 3-substituent is necessary for binding, and correct manipulation of this group leads to compounds such as the 3-(3-hydroxyphenyl)propargyl ester 24 (L-701,273), with an IC50 for displacement of [3H]-L-689,560 binding of 0.17 microM and Kb against NMDA in the cortical slice of 1.39 microM. Compounds were tested for their ability to prevent audiogenic seizure in DBA/2 mice; the most potent compound in this series is the cyclopropyl ketone 42 (L-701,252), with an ED50 of 4.1 mg/kg ip. A model is proposed for binding to the glycine site, in which an important interaction is of a putative receptor cation with the pi-system of the 3-substituent.

Acylation↗

PubMed1993

Preferential co-assembly of recombinant NMDA receptors composed of three different subunits.

cDNAs ENCODING NMDA receptor subunits have recently been cloned and been shown to have different distributions in the CNS. However, no studies on the possible in vivo combinations or subunit stoichiometry have yet been carried out. By combining human NR1 with rat NR2A and NR2C we have studied the pharmacological properties of three possible NMDA receptor subtypes; NR1 + NR2A, NR1 + NR2C and NR1 + NR2A + NR2C. By performing glycine concentration-response curves and comparing EC50s, it was possible to show that the NR1 + NR2A + NR2C receptor preferentially co-assembled when all three subunit cDNAs were present. This receptor had an affinity for glycine intermediate between that of NR1 + NR2A and NR1 + NR2C, but a similar Hill coefficient. Thus, two different NR2 subunits can combine in the same receptor, conferring unique pharmacological properties, suggesting that it is likely that two or more NR2 subunits co-assemble together in the same NMDA receptor complex.

Animals↗

PubMed1993

Subtypes of metabotropic excitatory amino acid receptor distinguished by stereoisomers of the rigid glutamate analogue, 1-aminocyclopentane-1,3-dicarboxylate.

The 1R,3R- and 1R,3S-isomers of 1-aminocyclopentane-1,3-dicarboxylate (ACPD) failed to stimulate phosphoinositide turnover or modify A2b adenosine receptor-stimulated cyclic AMP accumulation in guinea-pig cerebral cortical slices. In contrast, both 1S,3R- and 1S,3S-ACPD elicited concentration-dependent stimulations of phosphoinositide turnover (EC50 values 35 and 97 microM, respectively) and potentiated A2b-stimulated cAMP formation (17 and 58 microM, respectively). When forskolin was used to elevate cyclic AMP levels, however, all four isomers elicited concentration-dependent inhibitions of cyclic AMP formation to the same extent (approximately 90% inhibition). For this response the rank order of potencies were (IC50 values): 1S,3S-(0.9 microM) > 1S,3R-(2.1 microM) > 1R,3R-(237 microM) > 1R,3S-ACPD (approximately 1 mM). These data suggest the presence in guinea-pig cerebral cortex of two distinct subtypes of ACPD receptor coupled to phosphoinositide hydrolysis (and the potentiation of A2b receptor-stimulated cAMP formation) and the inhibition of forskolin-stimulated cAMP accumulation. Furthermore, our results indicate the usefulness of 1S,3S-ACPD as a tool to selectively activate one of these subtypes.

Adenosine↗

PubMed1993

The glycine site of the NMDA receptor--five years on.

The glycine site on the NMDA receptor complex has generated an enormous amount of interest since it was first described five years ago. In this review by John Kemp and Paul Leeson the structure-activity relationships of agonists, partial agonists and antagonists acting at the glycine site are reviewed, along with what has been learned from studies with these compounds about the role of this site in physiological and pathological processes. Studies with prototype antagonists, and low-efficacy partial agonists that penetrate the brain, show that these compounds possess anticonvulsant and neuroprotective properties but lack some of the side-effects of other types of NMDA antagonists.

Animals↗

PubMed1993

Endogenous adenosine regulates the apparent efficacy of 1-aminocyclopentyl-1S,3R-dicarboxylate inhibition of forskolin-stimulated cyclic AMP accumulation in rat cerebral cortical slices.

In rat cerebral cortical slices, the 1-aminocyclopentyl-1S,3R-dicarboxylate (1S,3R-ACPD) isomer of the selective metabotropic excitatory amino acid agonist ACPD inhibited forskolin-stimulated cyclic AMP (cAMP) accumulation in a concentration-dependent manner with a maximal inhibition of 51 +/- 3% and a half-maximally effective concentration of 8.8 +/- 3.4 microM. Similarly, 1R,3S-ACPD inhibited the forskolin response in a concentration-dependent manner, but with an inhibition of 80 +/- 5% at 3 mM. In addition to inhibiting forskolin-stimulated cAMP levels, 1S,3R-ACPD, but not 1R,3S-ACPD, enhanced the cAMP response to A2b adenosine receptor activation. In the presence of 1.2 U/ml of adenosine deaminase (included to reduce the contribution of endogenous adenosine), the efficacy of 1S,3R-ACPD was increased (88 +/- 3% inhibition), but the potency was unchanged. The adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine also increased the inhibitory effect of 100 microM 1S,3R-ACPD, from 57 +/- 1 to 78 +/- 5%. These results indicate that endogenous adenosine plays an important role in regulating the apparent efficacy of 1S,3R-ACPD inhibition of forskolin-stimulated cAMP accumulation in rat cerebral cortical slices and that previous studies in rat hippocampus and hypothalamus in the absence of added adenosine deaminase may have underestimated the efficacy of this compound.

Adenosine↗

PubMed1993

The influence of the gamma 2L subunit on the modulation of responses to GABAA receptor activation.

1. Whole-cell patch clamp recordings were made from L-cells transfected with 2 combinations of subunits of the GABAA receptor. Log concentration-response curves were constructed to gamma-aminobutyric acid (GABA) on alpha 1,beta 1,gamma 2L containing cells and compared to those from alpha 1,beta 1 containing cells. The effects of flunitrazepam, pentobarbitone and alphaxalone on the concentration-response relationships were also examined. 2. From the log concentration-response curves, GABA had a mean (+/- s.e. mean) pEC50 = 5.2 +/- 0.09 and slope factor = 1.7 +/- 0.02 on alpha 1,beta 1,gamma 2L cells which were significantly different from the values obtained from alpha 1,beta 1 cells where the pEC50 = 5.6 +/- 0.02 and the slope = 1.5 +/- 0.02. 3. Flunitrazepam produced a parallel leftward shift of GABA concentration-response curves on alpha 1,beta 1,gamma 2L cells. The EC50 for flunitrazepam = 6.3 +/- 2.7 nM. No increase in the maxima of the GABA concentration-response curves was found in the presence of flunitrazepam. Flunitrazepam did not potentiate responses from alpha 1,beta 1 cells. 4. The log concentration-response curves from both populations of cells were shifted to the left by equal amounts by pentobarbitone. A significant increase in the maximal response to GABA was also produced by pentobarbitone. This occurred at lower concentrations of pentobarbitone on alpha 1,beta 1 cells. 5. Alphaxalone produced leftward shifts of GABA log concentration-response curves of similar magnitudes in both populations of cells. Significant increases in the maxima were found at 100 nM in alpha 1, beta 1 cells but not up to 1 microM in alpha 1,beta 1,gamma 2L cells.6. These results provide further evidence of the modulatory role of the gamma 2L subunit of the GABAA receptor containing alpha 1 and beta 2 subunits. As well as influencing the apparent affinity of GABA and conferring benzodiazepine modulation, it also appeared to regulate the increase in maximal response produced in the presence of barbiturates and steroids. This latter effect may imply that barbiturates and steroids increase the channel open-state probability in the presence of GABA and that this effect is diminished by the presence of the gamma 2L subunit.

Anesthetics↗

PubMed1993

Block of voltage-dependent sodium currents by the substance P receptor antagonist (+/-)-CP-96,345 in neurones cultured from rat cortex.

1. Whole cell patch clamp recordings of voltage- and tetrodotoxin-sensitive Na+ currents were made from cultured rat neocortical neurones (E18). The effects of the non-peptide NK1 receptor antagonist, (+/-)-CP-96,345 on Na+ currents was examined, relative to the effect of the local anaesthetic lignocaine and tetrodotoxin. 2. Sodium currents were reversibly depressed by bath application of (+/-)-CP-96,345 with a half-maximally effective concentration of 18 +/- 2 microM at a stimulation frequency of 0.1 Hz. Likewise the concentrations required to half-maximally inhibit sodium currents by tetrodotoxin and lignocaine were 10 +/- 2 nM and 1.3 +/- 0.2 mM respectively. 3. The depression of sodium currents by (+/-)-CP-96,345 (10 microM) was use-dependent in that raising the stimulus frequency from 0.1 Hz to 10 Hz further decreased the magnitude of sodium currents from 60 +/- 5% to 37 +/- 5% of control values respectively. Similarly, the depression of sodium currents by lignocaine (500 microM) and tetrodotoxin (30 nM) was also accentuated by raising the stimulus frequency from 0.1 Hz to 10 Hz. 4. The effect of (+/-)-CP-96,345 was not associated with a change in either the activation or steady-state inactivation characteristics of these currents, suggesting that its mechanism of action was via open channel blockade. 5. These data demonstrate that in addition to antagonizing NK1 receptors, (+/-)-CP-96,345 also acts as a channel blocker on sodium channels at micromolar concentrations, an effect which should be taken into consideration when examining the antinociceptive or anti-inflammatory action of this compound.

Animals↗

PubMed1993

Agonist response kinetics of N-methyl-D-aspartate receptors in neurons cultured from rat cerebral cortex and cerebellum: evidence for receptor heterogeneity.

Accumulating evidence from both radioligand binding and molecular cloning experiments has led to the suggestion that there are heterogeneous populations of N-methyl-D-aspartate (NMDA) receptors. In particular, the NMDA receptor associated with cerebellar neurons has been suggested to be different from that in higher brain regions. With these observations in mind, we considered the possibility that the NMDA receptors on cultured neurons from rat cortex and cerebellum may show differences in their affinity for the coagonists, glutamate and glycine, and the ion channel blocker ketamine. A kinetic approach was used to derive the association and dissociation constants for each of the ligands from their respective macroscopic time constants of binding and unbinding. The constants were assessed electrophysiologically by measuring the onset and decay of whole-cell currents in response to drug applications to voltage-clamped neurons. In addition, differences in coagonist affinity were examined using conventional equilibrium concentration-response curve analysis. These experiments revealed that current relaxations after fast applications of either glutamate or glycine decayed more rapidly in cerebellar neurons, compared with cortical neurons. Thus, assuming two independent binding sites per receptor, the microscopic decay time constants (tau off) for glutamate were 341 +/- 47 (n = 12) and 934 +/- 76 msec (n = 11, p < 0.0001) for granule cells and cortical neurons, respectively. The resulting apparent microscopic dissociation constant (mKd) for glutamate at cerebellar granule cells, calculated from the forward and reverse rate constants, was > 2-fold lower than that for cerebral cortex receptors (496 nM, compared with 251 nM). The difference between the two cell types in the tau off for glycine was more substantial, i.e., 558 +/- 53 (n = 15) and 2214 +/- 125 msec (n = 19, p < 0.0001) for cerebellum and cortex, respectively. Corresponding apparent mKd values for glycine differed by > 4-fold, i.e., 189 nM and 45 nM for cerebellar granule and cortical neurons, respectively. Analysis of data obtained from equilibrium concentration-response curves also revealed differences in coagonist affinity between the two cell populations. The mean mKd values for glutamate at cerebellum and cortical neurons were 1260 nM and 630 nM, respectively, and those for glycine were 316 nM and 63 nM, respectively. No obvious differences were found between the two cell types with respect to the ion channel-blocking kinetics of the dissociative anesthetic ketamine.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Functional comparison of the role of gamma subunits in recombinant human gamma-aminobutyric acidA/benzodiazepine receptors.

The effect of benzodiazepines on the activity of gamma-aminobutyric acid (GABA)A receptors has been shown to be influenced by different alpha subunits and can also be affected by the presence of different gamma subunits. Previous studies have shown that receptors without a gamma subunit or those containing gamma 1 are modulated to a lesser degree by benzodiazepines. Using the Xenopus oocyte expression system to express different subunit combinations, a detailed analysis of the pharmacological modulation of GABAA receptors by various benzodiazepine site ligands has been carried out. We analyzed 14 compounds, varying through full agonist, partial agonist, antagonist, and inverse agonist, with receptors consisting of alpha 2 beta 1, alpha 2 beta 1 gamma 2S, and alpha 2 beta 1 gamma 1 and we demonstrate differences in their extent of potentiation by different benzodiazepine-type ligands. Most compounds showed negligible effects on alpha 2 beta 1 and most agonists, particularly the imidazopyridines zolpidem, alpidem, and AHR14,749, exhibited less potentiation with alpha 2 beta 1 gamma 1 than with alpha 2 beta 1 gamma 2S. The inverse agonists dimethoxy-4-ethyl-beta-carboline-3-carboxylate and Ro15-4513 did not act as inverse agonists and produced slight potentiation of alpha 2 beta 1 gamma 1 receptors. Concentration-response curves were constructed for five selected agonists to evaluate both affinity and efficacy differences between alpha 2 beta 1 gamma 2 and alpha 2 beta 1 gamma 1 receptors. Most compounds showed lower efficacy and up to 10-fold lower affinity with alpha 2 beta 1 gamma 1. Zolpidem showed slightly higher affinity but an extremely low efficacy; FG8205 also showed a markedly lower efficacy and was the most selective compound for alpha 2 beta 1 gamma 2S versus alpha 2 beta 1 gamma 1 receptors. CL218,872 showed high efficacy with alpha 2 beta 1 gamma 1 and affinity similar to that with alpha 2 beta 1 gamma 2 (being the least selective compound), suggesting that some low efficacy partial agonists with gamma 2-containing receptors may be more efficacious with gamma 1-containing receptors. The antagonists Ro15-1788 and CGS8216, although they blocked flunitrazepam potentiation of alpha 2 beta 1 gamma 2, could not block potentiation of alpha 2 beta 1 gamma 1. This study demonstrates that unique pharmacological profiles can be conferred by receptors containing different gamma subunits.

Animals↗

PubMed1993

Role of the beta subunit in determining the pharmacology of human gamma-aminobutyric acid type A receptors.

A cDNA encoding the human gamma-aminobutyric acid (GABA)A receptor beta 2 subunit has been cloned and sequenced. The deduced amino acid sequence of this cDNA shows only a single amino acid change from the rat sequence (Asn-347 in rat, serine in human). Using polymerase chain reaction amplification of human-specific products from human x rodent somatic cell hybrid DNAs, the gene has been assigned to human chromosome 6. By expressing recombinant human GABAA receptors containing different beta subunits (beta 1, beta 2 or beta 3) in both transfected cells and Xenopus oocytes, we have been able to determine the influence of the beta subunit on the pharmacology of the receptor. For a number of benzodiazepine binding site compounds, a barbiturate, and several neurosteroids, neither the affinity nor the efficacy of the compounds is influenced by the type of beta subunit present in the receptor molecule. These data suggest that the beta subunit does not significantly influence the benzodiazepine, barbiturate, or steriod site pharmacologies of human GABAA receptor subtypes.

Amino Acid Sequence↗

PubMed1993

Differences in affinity and efficacy of benzodiazepine receptor ligands at recombinant gamma-aminobutyric acidA receptor subtypes.

Two forms of the multisubunit gamma-aminobutyric acid (GABA)A receptor were expressed in Xenopus oocytes by injecting mRNA encoding the bovine GABAA receptor subunit cDNAs for alpha 1 beta 1 gamma 2L and alpha 3 beta 1 gamma 2L. The properties of these two combinations were examined by electrophysiological recording of GABA currents using the two-electrode voltage-clamp method. The actions of several benzodiazepine site ligands were compared in terms of their affinity for and efficacy at these two subunit combinations. Flunitrazepam potentiated control GABA responses to a maximum of 77% with alpha 1 beta 1 gamma 2L and 105% with alpha 3 beta 1 gamma 2L, with EC50 values of 29 +/- 11 nM and 23 +/- 10 nM, respectively. Flunitrazepam also produced a greater shift to the left of the GABA concentration-response curve with alpha 3 beta 1 gamma 2L than with alpha 1 beta 1 gamma 2L. Concentration-response curves for the type I benzodiazepine receptor-preferring compounds zolpidem and CL218,872 showed a selectivity for the alpha 1 beta 1 gamma 2L receptor, with respective affinity ratios 7-fold and 17-fold higher, compared with alpha 3 beta 1 gamma 2L. The inverse agonist methyl-6,7-dimethoxy-4-ethyl-beta- carboline-3-carboxylate produced a maximum inhibition of 30% with both receptor combinations and also had a higher affinity for alpha 1 beta 1 gamma 2L than alpha 3 beta 1 gamma 2L. For the first time CL218,872 and FG8205 were shown to be partial agonists of individual receptor combinations, compared with a full agonist such as flunitrazepam. The FG8205 concentration-response curve reached a maximum approximately 60% that of a full agonist with both alpha 1 beta 1 gamma 2L and alpha 3 beta 1 gamma 2L. CL218,872 reached a lower maximum efficacy with alpha 3 beta 1 gamma 2L (30%) than with alpha 1 beta 1 gamma 2L (51%), demonstrating not only that different compounds can have varying levels of partial agonist activity but also that the same compound can have differing degrees of efficacy at different receptor combinations.

Animals↗

PubMed1992

Stable expression of mammalian type A gamma-aminobutyric acid receptors in mouse cells: demonstration of functional assembly of benzodiazepine-responsive sites.

The differential sensitivity of type A gamma-aminobutyric acid (GABAA) receptors to benzodiazepine ligands seen in the mammalian nervous system is thought to be generated by the existence of a number of different receptor subtypes, assembled from a range of closely related subunits (alpha 1-6, beta 1-3, gamma 1-3, and delta) encoded by discrete genes. The characteristics of a given subtype can be determined by the coexpression of cloned cDNAs encoding the subunits of interest. Two transient expression systems have so far been employed in the study of the ligand-binding characteristics and chloride channel properties of such GABAA receptors--Xenopus oocytes and transfected mammalian cells. Here we report on the use of a steroid-inducible promoter expression system for the production of a permanently transfected clonal cell line expressing the alpha 1 beta 1 gamma 2L GABAA receptor subtype. Using both immunoprecipitation by subunit-specific antisera and gel-exclusion chromatography, we have shown that the alpha 1, beta 1, and gamma 2L subunits coassemble to form receptor macromolecules that are of the same size as native GABAA receptors. Additionally, the recombinant receptors have the same benzodiazepine pharmacology as native alpha 1-containing GABAA receptors and function as GABA-gated chloride channels. Such cell lines expressing individual GABAA receptor subtypes will prove important tools in the study of the structure, function, and pharmacology of GABAA receptors and in the development of subtype-specific drugs.

Allosteric Regulation↗