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Biomedical subjects

M L Mayer

Publications and source records attributed to M L Mayer.

At least 55 records · Page 3Linked to original sources
PubMed1992

CDC43 and RAM2 encode the polypeptide subunits of a yeast type I protein geranylgeranyltransferase.

The question regarding the identity of the alpha and beta subunits of the yeast type I protein geranylgeranyltransferase was explored using prokaryotic expression of candidate genes. The Saccharomyces cerevisiae CDC43 and RAM2 genes were expressed in Escherichia coli and cell extracts examined for the ability to transfer [3H]geranylgeranyl diphosphate to an appropriate CaaX protein substrate. Individual expression of each gene yielded no activity; however, co-expression of the two genes resulted in high levels of [3H] geranylgeranyl incorporation into the substrate protein Ras-Cys-Val-Val-Leu. The activity was partially purified yielding approximately 12,600 units/liter. The partially purified enzyme geranylgeranylated the Ras-Cys-Val-Val-Leu, Ras-Cys-Ala-Ile-Leu, Ras-Cys-Ile-Ile-Leu, and Ras-Cys-Thr-Ile-Leu substrates but not the Ras-Cys-Val-Leu-Ser or Ras-Ser-Val-Leu-Ser substrates. The protein geranylgeranyltransferase was highly specific for geranylgeranyl diphosphate and poorly transferred farnesyl. The recombinant enzyme was indistinguishable from the native type I geranylgeranyltransferase in yeast extracts. As has been reported for the protein farnesyltransferase, the yeast type I protein geranylgeranyltransferase is also a magnesium-requiring, zinc metalloenzyme. Interestingly, the recombinant enzyme functioned with calcium as the only divalent cation, although addition of zinc increased calcium-dependent activity 2-fold.

Alkyl and Aryl Transferases↗
PubMed1992

Activation and desensitization of AMPA/kainate receptors by novel derivatives of willardiine.

Willardiine [(S)-1-(2-amino-2-carboxyethyl)pyrimidine-2,4-dione] is a naturally occurring heterocyclic excitatory amino acid present in the seeds of Acacia and Mimosa. A series of 5-substituted willardiines were synthesized in single enantiomeric forms and tested for activity at AMPA/kainate receptors, using whole-cell recording from mouse embryonic hippocampal neurons. The (S)- but not (R)-isomers of willardiine and 5-bromowillardiine were potent agonists, producing rapidly but incompletely desensitizing responses. At equilibrium, (S)-5-fluorowillardiine (EC50, 1.5 microM) was seven times more potent than (R,S)-AMPA (EC50, 11 microM) and 30 times more potent than willardiine (EC50, 45 microM); the potency sequence was fluoro greater than nitro greater than chloro approximately bromo greater than iodo greater than willardiine. Willardiines produce strikingly different degrees of desensitization: at saturating doses the equilibrium response to the weakly desensitizing agonist (S)-5-iodowillardiine was similar in amplitude to the response to kainate and 10 times larger than the response to the strongly desensitizing agonist (S)-willardiine. The desensitization sequence was fluoro greater than willardiine greater than nitro approximately chloro greater than bromo greater than iodo greater than kainate. Cross-desensitization experiments confirm that willardiines bind to the same receptors activated by kainate and AMPA, and show that both the rapidly desensitizing and equilibrium responses to willardiines are mediated by the same receptor: (S)-5-iodowillardiine blocked activation of the rapidly desensitizing response evoked by (S)-willardiine and (S)-5-fluorowillardiine, while the latter agonists blocked the equilibrium response to (S)-5-iodowillardiine. A slowly decaying inward tail current was recorded after a brief application of (S)-5-fluorowillardiine but not (S)-willardiine, consistent with a model in which willardiines bind with different affinity to desensitized receptors, such that following removal of agonist, receptors trapped in the desensitized state can return to the open state before dissociation of agonist terminates receptor activation. Willardiines are the first compounds characterized in which simple changes in molecular structure are associated with marked differences in the ability of agonists to produce desensitization of AMPA/kainate receptors.

Alanine↗
PubMed1992

Effect of extracellular pH on the potency of N-methyl-D-aspartic acid receptor competitive antagonists.

Structure-activity analysis reveals that acidic alpha-amino acids containing an omega-PO3H2 group are more potent antagonists at N-methyl-D-aspartate (NMDA) receptors than are analogs with omega-COOH or omega-tetrazole groups. At physiological values of extra-cellular pH the omega-PO3H2 group is only partially deprotonated and the corresponding antagonists exist as ions with one or two negative charges. In contrast, competitive antagonists with omega-COOH and omega-tetrazole groups are fully ionized at physiological pH but carry only a single negative charge. Dose-inhibition analysis was performed with (2R)-AP7 and its piperidine derivative LY 257883 to determine whether ionization of the omega-PO3H2 group influences NMDA receptor antagonist potency; these experiments revealed a > 3-fold increase in potency on raising of the extracellular pH from 7.3 to pH 8.2, consistent with the increase in the relative concentration of the ionic form of the antagonist in which the omega-PO3H2 group contains two negative charges. Experiments with the omega-COOH-containing analog of LY 257883 and with SDZ EAB 515, an omega-PO3H2-containing antagonist of novel structure, revealed only 1.5- and 1.3-fold increases in potency, respectively, over the same pH range. Analysis of the kinetics of block of NMDA-activated currents resulting from rapid application of LY 257883 suggests that the increase in potency on raising of the extracellular pH results largely from an increase in the antagonist association rate constant but also from a small decrease in the dissociation rate constant. Together, these results suggest that the fully ionized forms of the R-enantiomers of AP7 and LY 257883 act as the active antagonist species at NMDA receptors.

2-Amino-5-phosphonovalerate↗
PubMed1991

Bioavailability and kinetics of cibenzoline in patients with normal and impaired renal function.

To test the hypothesis that renal failure alters the disposition of cibenzoline in humans, an absolute bioavailability and elimination kinetic study was performed. We used the simultaneous administration of a stable isotope variant (SASIV). Eight healthy volunteers and eight matched hemodialysis patients each received simultaneously an 80-mg intravenous infusion of 15N-2-cibenzoline and a single 80-mg cibenzoline capsule. Cibenzoline plasma concentrations were assayed by a gas chromatographic-mass spectrometric assay. A compartment-independent kinetic analysis showed a plasma clearance of 707 mL/min and an elimination half-life of 7.3 hours after the intravenous dose in healthy volunteers. In renal-failure patients, cibenzoline clearance decreased to 224 mL/min and half-life increased to 22.4 hours. Decreased plasma clearance was due to decreases in both renal and nonrenal clearance. Absolute bioavailability was 83% and 90% in healthy volunteers and renal-failure patients, respectively. Hemodialysis accounted for only 13% of drug clearance.

Administration, Oral↗
PubMed1991

Kinetic analysis of interactions between kainate and AMPA: evidence for activation of a single receptor in mouse hippocampal neurons.

AMPA but not kainate produces a rapidly desensitizing response in mouse hippocampal neurons. The characteristic action of these agonists appears to arise from activation of a single receptor with active and desensitized states, for which AMPA and kainate have different relative affinity. The equilibrium potency of a series of five agonists that produce rapidly desensitizing responses at non-NMDA receptors (EC50 1 microM to 4 mM) was similar to their equilibrium potency for block of kainate responses. Increasing the concentration of kainate overcame such block, but in the presence of AMPA the rate of activation of responses to kainate was slowed. Conversely, in the presence of kainate the amplitude of rapidly desensitizing responses evoked by AMPA was reduced, and the rate of onset of desensitization was slowed.

Animals↗
PubMed1991

Modulation of excitatory synaptic transmission by drugs that reduce desensitization at AMPA/kainate receptors.

Desensitization at AMPA/kainate receptors has been proposed to contribute to the decay of excitatory synaptic currents. We examined the action of aniracetam, wheat germ agglutinin (WGA), and concanavalin A (Con A), drugs that act via separate mechanisms to reduce desensitization evoked by L-glutamate in rat hippocampal neurons. The decay of excitatory synaptic currents, and sucrose-evoked miniature excitatory postsynaptic currents (EPSCs) was slowed 2- to 3-fold by aniracetam. In contrast, WGA increased the EPSC decay time constant only 1.3-fold and Con A had no effect. Aniracetam increased the magnitude of stimulus-evoked EPSCs 1.9-fold; variance analysis suggests a postsynaptic mechanism of action. WGA and Con A reduced EPSC amplitude via a presynaptic mechanism. Aniracetam increased the burst length of L-glutamate-activated single-channel responses. Simulations suggest that aniracetam either slows entry into a desensitized state or decreases the closing rate constant for ion channel gating.

Animals↗
PubMed1991

Kinetic analysis of antagonist action at N-methyl-D-aspartic acid receptors. Two binding sites each for glutamate and glycine.

Antagonism of glutamate-receptor responses activated by N-methyl-D-aspartic acid (NMDA) was studied using whole cell voltage clamp recording from mouse dissociated hippocampal neurons cultured for 10-15 d. The kinetics of onset of and recovery from NMDA receptor block during continuous application of NMDA together with either glycine, or L-alanine, were recorded in response to concentration jump application of NMDA- and glycine-binding site directed competitive antagonists, applied with a multibarrel flow pipe under conditions which allowed rapid solution changes around the cell less than 10 ms. Mathematical solutions for both one- and two-equivalent site models for competitive antagonism were determined according to the differential equations outlined by Colquhoun and Hawkes (1977. Proc. R. Soc. Lond. B. 199:231-262). The kinetics of action of D-CPP and D-AP5, NMDA binding site antagonists, and 7Cl-kynurenic acid, a glycine binding site antagonist, were examined for each model. For all these antagonists, the kinetics for the onset of and recovery from antagonism were better fit by the two-equivalent site model, which yielded antagonist microscopic kBoff/kBon values which closely approximated Ki values determined from analysis of equilibrium dose response curves. These results suggest that two molecules of NMDA and two molecules of glycine must bind to the NMDA receptor for activation of ion channel gating.

2-Amino-5-phosphonovalerate↗
PubMed1991

Structure-activity analysis of binding kinetics for NMDA receptor competitive antagonists: the influence of conformational restriction.

1. The kinetics of action of 17 structurally related NMDA receptor competitive antagonists were measured under voltage clamp in mouse hippocampal neurones. Analysis of the response to rapid changes in antagonist concentration during constant application of agonist was used to estimate microscopic association (kon) and dissociation (koff) rate constants for antagonist binding, assuming a two-equivalent site model for competitive antagonism. Dose-inhibition curves were analysed to estimate antagonist equilibrium dissociation constants. 2. For a series of 11 omega-phosphono, alpha-amino acids kon and koff varied 26 and 107 fold respectively. Rapid association and dissociation rate constants were obtained for flexible antagonist molecules such as D-2-amino-7-phosphonoheptanoic acid (D-AP7): kon 1.4 x 10(7) M-1 s-1; koff 20.3 s-1. For conformationally restrained molecules such as 3S,4aR,6S,8aR-6-phosphonomethyl-decahydroisoquinoline- 3-carboxylic acid (LY 235959), association and dissociation rate constants were much slower: kon 1.1 x 10(6) M-1 s-1; koff 0.2 s-1. For the D- and L-isomers of 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) estimates for kon were similar, but for the L-isomer koff was 10 fold faster than for the D-isomer. 3. For 2-amino-5-phosphonopentanoic acid (AP5) and its piperidine derivative cis-4-(phosphonomethyl)piperidine-2-carboxylic acid (CGS 19755), an increase in chain length of two methylene groups between the omega-phosphono and alpha-carboxylate moieties caused a 1.6 to 1.8 fold decrease in kon with little change in koff. In contrast, for AP5, CPP and its omega-carboxylate analogue, addition of a double bond close to the phosphonate moiety caused a 1.3 to 1.6 fold increase in kon. 4. For antagonists with an omega-tetrazole moiety, kon and koff were 2.8-4.6 times faster than for the parent omega-phosphono compounds. A similar, but smaller increase in kon and koff was observed for antagonists with an omega-carboxylate moiety. 5. The slow kinetics of action of potent NMDA receptor antagonists were not an artefact of buffered diffusion. In neurones equilibrated with 200 microM D-AP7, 2 microM LY 235959 and 10 microM NMDA, a transient agonist response was recorded following a rapid switch to D-AP7-free solution. This can only be explained by differences in the binding kinetics of AP7 and LY 235959, since at equilibrium, with these concentrations, either antagonist essentially eliminates the agonist response to 10 microM NMDA. 6. For all antagonists studied, the ratio koff/k0. was consistent with equilibrium Ki values obtained under similar experimental conditions, over a 40 fold range of potency. Comparison of these values with Ki estimates determined from both agonist ([3H]-glutamate), and antagonist ([3H]-CGS 19755 and [3H]- CPP) radioligand competition studies revealed good correlation between data from voltage clamp and binding experiments. However, Ki values obtained in antagonist binding assays showed on average 6.5 fold higher affinity than those obtained in voltage clamp experiments; in contrast Ki values obtained in agonist binding assays showed only 1.4 fold higher affinity. 7. The insights gained from our experiments may be of use for predicting the structural features required to generate more potent NMDA receptor antagonists, and suggest that novel acyclic compounds will have greater potential for high potency than derivatives of conformationally rigid compounds with piperazine, piperidine or bicyclic ring structures.

2-Amino-5-phosphonovalerate↗
PubMed1990

Excitatory amino acid receptors, second messengers and regulation of intracellular Ca2+ in mammalian neurons.

As we have learnt from earlier articles in our series on the pharmacology of excitatory amino acids, neurons and glia express several subtypes of excitatory amino acid receptor, the activation of which increases intracellular free calcium ion concentration. In this sixth article, Mark Mayer and Richard Miller detail the mechanisms and consequences of this activity. Ionotropic excitatory amino acid receptors, most notably those activated by NMDA, allow Ca2+ influx as a result of passive diffusion down its concentration gradient. Metabotropic receptors trigger an increase in polyphosphoinositide metabolism, and release of Ca2+ from intracellular stores. The increase in [Ca2+]i mediated by excitatory amino acids underlies a complex cellular physiology, including the activation of other second messenger systems, and contributes to the initiation of long-term potentiation.

Amino Acids↗
PubMed1990

Modulation of N-methyl-D-aspartic acid receptor desensitization by glycine in mouse cultured hippocampal neurones.

1. Responses to N-methyl-D-aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using the tight-seal, whole-cell, patch-clamp technique for voltage clamp. A rapid perfusion system, with an exchange time constant of less than 10 ms, was used to apply NMDA under conditions which minimized slow, calcium-sensitive desensitization. With no added glycine, responses to 100 microM-NMDA applied for 1.5 s declined by greater than 90%, due to an additional component of desensitization of time constant 250 ms. 2. Adding glycine to the extracellular solution, over the range 30 nM to 3 microM, both potentiated responses to NMDA and to L-glutamate, and reduced fast desensitization. In the presence of 3 microM-glycine responses to NMDA declined by only 10%. Similar potentiation and reduction of desensitization was obtained with 3 microM concentrations of the glycine analogues D-alanine and D-serine. 3. Analysis of dose-response curves for the action of glycine on responses to 100 microM-NMDA revealed a 3-fold higher potency of glycine for potentiation of peak versus steady-state responses, with concentrations for half-activation of 134 and 382 nM, respectively. The competitive glycine antagonist 7-chlorokynurenic acid produced a similar shift of both the peak and steady-state dose-response curves for glycine, consistent with an equilibrium dissociation constant of 280 nM for interaction of 7-chlorokynurenic acid with the glycine binding site on NMDA receptors. 4. In the presence of 100 nM-glycine, 10 microM-7-chlorokynurenic acid produced nearly complete block of the response to 3 nM-NMDA, suggesting that glycine is absolutely required for activation of the NMDA receptor channel complex. 5. In some neurones responses to NMDA showed essentially no desensitization in the presence of 3 microM-glycine. Under these conditions, 7-chlorokynurenic acid produced a concentration-dependent block of both the initial and equilibrium response to NMDA, with a 4-fold greater sensitivity for block of the steady-state current (IC50 = 2.25 microM) than for block of the peak current (IC50 = 8.96 microM). As a result, in the presence of 7-chlorokynurenic acid, responses to NMDA showed strong desensitization, even in the presence of 3 microM-glycine. 6. Our results show that glycine-evoked potentiation of NMDA receptor activity is accompanied by reduced desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

A kinetic analysis of the modulation of N-methyl-D-aspartic acid receptors by glycine in mouse cultured hippocampal neurones.

1. Responses to N-methyl-D-aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using whole-cell patch-clamp recording. A fast perfusion system, with an exchange time constant of less than 10 ms, was used to study modulation of NMDA receptor desensitization by glycine. 2. The onset of NMDA receptor desensitization was well fitted by a single-exponential function; with 30 nM-glycine the time constant was 250 ms, corresponding to a rate of 4 s-1. The rate of onset of desensitization became faster with increasing glycine concentration, with a slope of 0.87 x 10(7) M-1 s-1. Recovery from desensitization, studied with a twin-pulse technique, was also well fitted by a single-exponential function; with 30 nM-glycine the time constant of recovery was 1.95 s-1. The rate of recovery from desensitization became faster with increasing glycine concentration, with a slope of 0.76 x 10(7) M-1 s-1. These results are consistent with a model in which the effect of glycine occurs via an increase in the rate constant for recovery from desensitization, with little effect on the rate constant for onset of desensitization. Over the range 30-300 nM-glycine, the ratio of the rate constants calculated for recovery and onset of desensitization was a good predictor of the degree of desensitization recorded at equilibrium. 3. Concentration jump experiments with glycine were performed with 100 microM-NMDA present continuously, and for a single binding site model gave estimates of the association (1.1 x 10(7) M-1 s-1) and dissociation (3.1 s-1) rate constants for interaction of glycine with the NMDA receptor. In the presence of NMDA, concentration jumps from 3 microM-glycine to lower concentrations gave relaxations which became slower with decreasing glycine concentration over the range 1 microM-30 nM. A similar slowing of desensitization occurred when the glycine concentration was altered over the same range. 4. Glycine analogues of lower affinity produced desensitization with faster kinetics. D-Alanine, 150 nM, produced desensitization with a time constant of 175 ms, faster than recorded with an equipotent concentration of glycine (50 nM, time constant 259 ms). Responses of similar peak amplitude, recorded with 60 microM-L-alanine, and 500 microM-D,L-homoserine, did not produce strong desensitization, consistent with desensitization too rapid to resolve in our experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗
PubMed1990

Concentration-jump experiments with NMDA antagonists in mouse cultured hippocampal neurons.

1. Voltage-clamp experiments were used to study N-methyl-D-aspartic acid (NMDA) receptor antagonists applied by fast perfusion to mouse hippocampal neurons in dissociated culture. 2. Preincubation with the NMDA antagonists zinc (3-30 microM) and magnesium (30-300 microM) reduced subsequent responses to 100 microM NMDA applied together with these antagonists. No time dependence of antagonism was observed when responses were measured at the start and at the end of NMDA pulses 1.25-1.5 s in duration. 3. Two competitive antagonists of similar affinity in equilibrium experiments, D-2-amino-5-phosphonopentanoic acid (D-AP5) and 3-((+-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), had different profiles of action when applied as described above. With D-AP5, pulses of NMDA produced fast-on, fast-off responses, of reduced amplitude, similar to the effect of Zn and Mg. Responses to NMDA in the presence of CPP were also of reduced amplitude but, in addition, showed slow activation, such that the antagonist action of CPP decreased with time after the application of NMDA. 4. In the presence of 3 microM glycine and NMDA receptor antagonists with activity at the glycine modulatory site, either kynurenic acid (Kyn), 7-chlorokynurenic acid (7Cl-Kyn), or 5-chloro-indole-2-carboxylic acid (5Cl-I2CA), NMDA-evoked responses showed apparent use-dependent antagonism, such that the peak response to NMDA was much greater than the equilibrium response. A similar effect was produced by preincubation with low concentrations of glycine (less than 300 nM), which enhances desensitization of responses to NMDA. The apparent use-dependent action of glycine antagonists could be reversed on raising the glycine concentration and did not vary appreciably with changes in membrane potential over the range -60 to +50 mV. 5. Concentration-jump application of NMDA antagonists, in the presence of 100 microM NMDA and 3 microM glycine, were used to study antagonist association and dissociation kinetics directly. For D-AP5 and CPP, the dissociation rate was independent of antagonist concentration, and approximately 15 times faster for D-AP5 (19.6 s-1) than for CPP (1.36 s-1). The association rate for D-AP5 and CPP increased with antagonist concentration in a linear manner over the range 3-30 microM and was slower for CPP than for D-AP5, consistent with their similar potency at equilibrium. 6. In contrast to results obtained with CPP and D-AP5, the association rate for 7Cl-Kyn was approximately 3 times slower than the dissociation rate and did not change with concentration of antagonist.(ABSTRACT TRUNCATED AT 400 WORDS)

2-Amino-5-phosphonovalerate↗
PubMed1990

Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-D-aspartate and quisqualate receptors.

Dose-response curves for activation of excitatory amino acid receptors on mouse embryonic hippocampal neurons in culture were recorded for 15 excitatory amino acids, including the L-isomers of glutamate, aspartate, and a family of endogenous sulfur amino acids. In the presence of 3 microM glycine, with no extracellular Mg, micromolar concentrations of 11 of these amino acids produced selective activation of N-methyl-D-aspartate (NMDA) receptors. L-Glutamate was the most potent NMDA agonist (EC50 2.3 microM) and quinolinic acid the least potent (EC50 2.3 mM). Dose-response curves were well fit by the logistic equation, or by a model with 2 independent agonist binding sites. The mean limiting slope of log-log plots of NMDA receptor current versus agonist concentration (1.93) suggests that a 2-site model is appropriate. There was excellent correlation between agonist EC50S determined in voltage clamp experiments and KdS determined for NMDA receptor binding (Olverman et al., 1988). With no added glycine, and 1 mM extracellular Mg, responses to NMDA were completely blocked; responses to kainate and quisqualate were unchanged. Under these conditions, glutamate and the sulfur amino acids activated a rapidly desensitizing response, similar to that evoked by micromolar concentrations of quisqualate and AMPA, but mM concentrations of L-aspartate, homoquinolinic acid, and quinolinic acid failed to elicit a non-NMDA receptor-mediated response. Except for L-glutamate (EC50 480 microM), the low potency of the sulfur amino acids prevented the study of complete dose-response curves for the rapidly desensitizing response at quisqualate receptors. Small-amplitude nondesensitizing quisqualate receptor responses were activated by much lower concentrations of all quisqualate receptor agonists. Full dose-response curves for the nondesensitizing response were obtained for 9 amino acids; L-glutamate was the most potent endogenous agonist (EC50 19 microM). Domoate (EC50 13 microM) and kainate (EC50 143 microM) activated large-amplitude, nondesensitizing responses.

Amino Acids↗
PubMed1989

Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine.

Responses to the excitatory amino acid N-methyl-D-aspartate (NMDA) are markedly potentiated by nanomolar concentrations of glycine. This is due to the action of glycine at a novel strychnine-resistant binding site with an anatomical distribution identical to that for NMDA receptors, suggesting that the NMDA receptor channel complex contains at least two classes of amino-acid recognition site. Antagonists at the glycine-binding site associated with NMDA receptors act as potent non-competitive antagonists, but do not alter the mean open time or conductance, as estimated by fluctuation analysis. The mechanisms by which glycine acts on NMDA receptors are unknown, but single-channel recording experiments show an increase in opening frequency with no change in mean open time or conductance, suggesting that glycine could regulate transitions to states that are intermediate between binding of NMDA receptor agonists and ion-channel gating. It has been suggested that glycine acts as a co-agonist at the NMDA receptor, and that responses to NMDA cannot be obtained in the complete absence of glycine, but in these experiments the response to NMDA was measured at equilibrium, and it is unlikely that sufficient temporal resolution was achieved to detect rapid alterations in receptor gating. Using a fast perfusion system we find that glycine regulates desensitization at NMDA receptors; this has a major effect on the response to NMDA measured at equilibrium, as would occur with slower applications of agonist. Reduction of NMDA receptor desensitization by glycine provides an example of a novel mechanism for regulation of ion-channel activity.

Animals↗