Biomedical subjects

L E Chavez-Noriega

Publications and source records attributed to L E Chavez-Noriega.

15 recordsLinked to original sources

PubMed2001

Dual action of n-alcohols on neuronal nicotinic acetylcholine receptors.

Alcohol is known to modulate the activity of a variety of neuroreceptors and ion channels. Recently, neuronal nicotinic acetylcholine receptors (nnAChRs) have become a specific focus of study because not only are they potently modulated by alcohol but also they regulate the release of various transmitters, including gamma-aminobutyric acid (GABA) and dopamine, which play an important role in the behavioral effects of ethanol. Whereas the potency of normal alcohols (n-alcohols) to potentiate GABA(A) receptors and to inhibit N-methyl-D-aspartate receptors increases with carbon chain length, we have found that n-alcohols, depending on the carbon chain length, exert a dual action, potentiation and inhibition, on nnAChRs in primary cultured rat cortical neurons. The mechanism of dual action of n-alcohols on nnAChRs was further analyzed using human embryonic kidney cells expressing the alpha 4 beta 2 subunits. Shorter chain alcohols from methanol to n-propanol potentiated acetylcholine (ACh)-induced currents, whereas longer chain alcohols from n-pentanol to n-dodecanol inhibited the currents. n-Butanol either potentiated or inhibited the currents depending on the concentrations of ACh and butanol. The parameters for both potentiation (log EC(200)) and inhibition (log IC(50)) were linearly related to carbon number, albeit with different slopes. The slope for potentiation was -0.299, indicating a change in free energy change (Delta Delta G) of 405 cal/mol/methylene group, whereas the slope for inhibition was -0.584, indicating a Delta Delta G of 792 cal/mol. These results suggest that potentiating and inhibitory actions are exerted through two different binding sites. Ethanol decreased the potency of n-octanol to inhibit ACh currents, possibly resulting from an allosteric mechanism.

1-Octanol↗

PubMed2000

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors alpha3beta2 and alpha4beta2 stably expressed in HEK293 cells.

HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations alpha3beta2 or alpha4beta2. [(3)H]-(+/-)Epibatidine ([(3)H]-(+/-)EPI) bound to membranes from A3B2 (alpha3beta2) and A4B2.2 (alpha4beta2) cells with K(d) values of 7.5 and 33.4 pM and B(max) values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca(2+) concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-beta-erythroidine (DHbetaE) in A3B2 cells and MEC=DHbetaE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in alpha3beta2 and alpha4beta2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca(2+), respectively. Our results indicate that stably expressed alpha3beta2 and alpha4beta2 human nAChRs are pharmacologically and functionally distinct.

Blotting, Northern↗

PubMed2000

Subunit-dependent inhibition of human neuronal nicotinic acetylcholine receptors and other ligand-gated ion channels by dissociative anesthetics ketamine and dizocilpine.

UNLABELLED: Background The neuronal mechanisms responsible for dissociative anesthesia remain controversial. N-methyl-D-aspartate (NMDA) receptors are inhibited by ketamine and related drugs at concentrations lower than those required for anesthetic effects. Thus, the authors studied whether ligand-gated ion channels other than NMDA receptors might display a sensitivity to ketamine and dizocilpine that is consistent with concentrations required for anesthesia. METHODS: Heteromeric human neuronal nicotinic acetylcholine receptors (hnAChR channels alpha2beta2, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2 and alpha4beta4), 5-hydroxytryptamine3 (5-HT3), alpha1beta2gamma2S gamma-aminobutyric acid type A (GABAA) and alpha1 glycine receptors were expressed in Xenopus oocytes, and effects of ketamine and dizocilpine were studied using the two-electrode voltage-clamp technique. RESULTS: Both ketamine and dizocilpine inhibited hnAChRs in a noncompetitive and voltage-dependent manner. Receptors containing beta1 subunits were more sensitive to ketamine and dizocilpine than those containing beta2 subunits. The inhibitor concentration for half-maximal response (IC50) values for ketamine of hnAChRs composed of beta4 subunits were 9.5-29 microM, whereas those of beta2 subunits were 50-92 microM. Conversely, 5-HT3 receptors were inhibited only by concentrations of ketamine and dizocilpine higher than the anesthetic concentrations. This inhibition was mixed (competitive/noncompetitive). GABAA and glycine receptors were very resistant to dissociative anesthetics. CONCLUSIONS: Human nAChRs are inhibited by ketamine and dizocilpine at concentrations possibly achieved in vivo during anesthesia in a subunit-dependent manner, with beta subunits being more critical than alpha subunits. Conversely, 5-HT3, GABAA, and glycine receptors were relatively insensitive to dissociative anesthetics.

Anesthetics, Dissociative↗

PubMed1999

Human neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes predict efficacy of halogenated compounds that disobey the Meyer-Overton rule.

BACKGROUND: According to the Meyer-Overton rule, anesthetic potency of a substance can be predicted by its lipid solubility, but a group of halogenated volatile compounds predicted to induce anesthesia does not obey this rule. Thus, these compounds are useful tools for studies of molecular targets of anesthetics. Human neuronal nicotinic acetylcholine receptor (hnAChR) subunits have been recently cloned, which allowed the authors to assess whether these receptors could differentiate among volatile anesthetic and nonimmobilizer compounds. This study provides the first data regarding anesthetic sensitivity of hnAChRs. METHODS: alpha2beta4, alpha3beta4, and alphaabeta2 hnAChRs were expressed in Xenopus oocytes, and effects of volatile anesthetics isoflurane and F3 (1-chioro-1,2,2-triflurocyclobutane, 1A) and nonimmobilizers F6 (1,2-dichlorohexafluorocyclobutane, 2N) and F8 (2,3-dichlorooctafluorobutane) on the peak acetylcholine-gated currents were studied using the two-electrode voltage-clamp technique. RESULTS: Isoflurane and F3 inhibited all the hnAChRs tested in a concentration-dependent manner. Isoflurane at a concentration corresponding to 1 minimum alveolar concentration (MAC) inhibited 83, 69, and 71% of ACh-induced currents in alpha2beta4, alpha3beta4, and alpha4beta2 hnAChRs, respectively, and 1 MAC of F3 inhibited 64, 44, and 61% of currents gated in those receptors. F6 (8-34 microM) did not cause any changes in currents gated by any of the receptors tested. F8 (4-18 microM) did not alter the currents gated in either alpha3beta4 or alpha4beta2 receptors, but caused a small potentiation of alpha2beta4 hnAChRs without a concentration-response relation. CONCLUSION: The in vivo potency and effectiveness of volatile anesthetic and nonimmobilizer compounds were consistent with their actions on hnAChRs expressed in a recombinant expression system, suggesting a potential participation of these receptors in the mechanisms of anesthesia.

Acetylcholine↗

PubMed1999

Effects of ethanol on recombinant human neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

Alcohol and tobacco use is highly correlated in humans, and studies with animal models suggest an interaction of alcohol with neuronal nicotinic acetylcholine receptors (nAChRs). The aim of the present study was to characterize the effect of acute ethanol treatment on different combinations of human nAChR (hnAChR) subunits expressed in Xenopus oocytes. Ethanol (75 mM) potentiated ACh-induced currents in alpha2beta4, alpha4beta4, alpha2beta2, and alpha4beta2 receptors. This effect was due to an increase in Emax, without a change in the EC50 or Hill coefficient. hnAChR alpha2beta4 did not develop tolerance to repeated applications of ethanol or continuous exposure (10 min). The alpha3beta2 and alpha3beta4 combinations were insensitive to ethanol. Low concentrations of ethanol (25 and 50 mM) significantly inhibited homomeric alpha7 receptor function, but these receptors showed highly variable responses to ethanol. These results indicate that ethanol effects on hnAChRs depend on the receptor subunit composition. In light of recent evidence indicating that nAChRs mediate and modulate synaptic transmission in the central nervous system, we postulate that acute intoxication might involve ethanol-induced alterations in the function of these receptors.

Acetylcholine↗

PubMed1998

Conformationally restricted analogues of nicotine and anabasine.

A series of conformationally restricted analogues of nicotine has been synthesized and evaluated as agonists of neuronal acetylcholine receptors. Compound 2 (SIB-1663), which selectively activated human recombinant alpha 2 beta 4 and alpha 4 beta 4 nAChRs, was shown to be active in animal models of Parkinson's disease and pain.

Anabasine↗

PubMed1998

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents.

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than nonspecific agents such as nicotine.

Animals↗

PubMed1998

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells.

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.

Bridged Bicyclo Compounds, Heterocyclic↗

PubMed1997

Pharmacological characterization of recombinant human neuronal nicotinic acetylcholine receptors h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7 expressed in Xenopus oocytes.

Human neuronal nicotinic acetylcholine receptors (nAChRs) h alpha 2 beta 2, h alpha 2 beta 4, h alpha 3 beta 2, h alpha 3 beta 4, h alpha 4 beta 2, h alpha 4 beta 4 and h alpha 7 were expressed in Xenopus oocytes and tested for their sensitivities to the nicotinic agonists acetylcholine (ACh), nicotine, cytisine (CYT) and 1,1-dimethyl-4-phenylpiperazinium (DMPP) and the nAChR. antagonists mecamylamine (MEC), d-tubocurarine and dihydro-beta-erythroidine. CYT was the least efficacious agonist at hnAChRs containing beta 2 subunits, but it displayed significant activity at h alpha 2 beta 4, h alpha 3 beta 4, h alpha 4 beta 4 and h alpha 7 nAChRs. ACh was one of the most efficacious agonists at all hnAChRs, except at h alpha 3 beta 2, where DMPP was markedly more efficacious than ACh. ACh was among the least potent agonists at all hnAChRs. The rank order of potency displayed by h alpha 3 beta 2 and h alpha 3 beta 4 nAChRs (DMPP approximately CYT approximately nicotine > ACh and DMPP > CYT approximately nicotine > ACh, respectively), differs from that reported for their rat homologs (Luetje and Patrick, 1991; Covernton et al., 1994). The agonist profile observed in h alpha 7 also differs from that reported for its rat homolog (Seguela et al., 1993). Human alpha 4 beta 2 and h alpha 4 beta 4 nAChRs were more sensitive to dihydro-beta-erythroidine than d-tubocurarine, whereas h alpha 7 and h alpha 3 beta 4 were more sensitive to d-tubocurarine than dihydro-beta-erythroidine. These antagonists were equipotent at h alpha 2 beta 2, h alpha 3 beta 2 and h alpha 2 beta 4 nAChRs. MEC (3 microM) inhibited h alpha 2 beta 4 and h alpha 4 beta 4 nAChRs by > 80%, whereas h alpha 2 beta 2, h alpha 4 beta 2 and h alpha 7 nAChRs were inhibited by approximately 50%. Taken together, the differential sensitivities observed at various recombinant hnAChR subtypes indicate that both alpha and beta subunits contribute to the pharmacology of these ligand-gated channels. The unique selectivity profiles displayed by human nAChRs constitute a valuable tool for the development of selective nicotinic analogs as potential therapeutic drugs.

Animals↗

PubMed1996

Comparative structure of human neuronal alpha 2-alpha 7 and beta 2-beta 4 nicotinic acetylcholine receptor subunits and functional expression of the alpha 2, alpha 3, alpha 4, alpha 7, beta 2, and beta 4 subunits.

cDNA clones encoding human neuronal nicotinic acetylcholine receptor alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha 7, beta 2, beta 3, and beta 4 subunits were isolated from brainstem, hippocampus, prefrontal cortex, substantia nigra, thalamus, and IMR32 libraries. Human alpha 2 and alpha 6 and full-length beta 3 and beta 4 clones have not been previously reported. Deduced amino acid sequences of the alpha 2, alpha 6, beta 3, and beta 4 predicted mature peptides are 503 residues (56.9 kDa), 464 residues (53.7 kDa), 440 residues (50.8 kDa), and 477 residues (54.1 kDa), respectively. These sequences show 84 (alpha 2), 87 (alpha 6), 89 (beta 3), and 84% (beta 4) identity to the corresponding rat sequences. The amino termini of the human alpha 2 and beta 3 mature peptides contain 23 and six additional residues, respectively, compared to those of rat alpha 2 and beta 3. Recombinant receptors were expressed in Xenopus laevis oocytes injected with in vitro transcripts encoding either alpha 7 alone or alpha 2, alpha 3, or alpha 4 in pairwise combination with beta 2 or beta 4. Inward currents were elicited by the application of acetylcholine (1-100 microM) and other agonists; these responses were blocked 65-97% by application of 10 microM d-tubocurare, confirming functional expression of human nicotinic receptors.

Acetylcholine↗

PubMed1994

Increased transmitter release at excitatory synapses produced by direct activation of adenylate cyclase in rat hippocampal slices.

The field EPSP recorded in the CA1 region of rat hippocampal slices is potentiated by bath application of the direct adenylate cyclase activator forskolin (Chavez-Noriega and Stevens, 1992a). We have now used the whole-cell patch-clamp technique to analyze the effect of forskolin on evoked synaptic currents and on spontaneous and miniature excitatory postsynaptic currents (sEPSCs and mEPSCs) recorded in rat hippocampal slices in order to determine the relative contributions of pre- and postsynaptic mechanisms to this increased synaptic strength. Application of 50 microM forskolin in the presence of 3-isobutyl-1-methylxanthine (IBMX; a phosphodiesterase inhibitor) enhanced the evoked EPSC (eEPSC) peak amplitude to 230 +/- 43% of control (n = 13). No significant change in sEPSC or in mEPSC amplitude was detected after forskolin addition (106 +/- 7%, n = 9), indicating that postsynaptic receptor sensitivity at synaptic junctions is not greatly affected. In contrast, a large increase in sEPSC and mEPSC frequency was noted in all cells (299 +/- 81%). Following forskolin application, the amplitude distribution of evoked synaptic currents shifted to larger values, but more significantly, a sharp decrease in failure rate was produced in all cells tested. Also, a significant correlation was found between the potentiation produced by forskolin in IBMX on the eEPSC and the ratio of the squared coefficient of variation (CV = SD/mean). Finally, a quantal analysis of four cells was consistent with the hypothesis that transmitter release was increased by forskolin/IBMX with, if anything, a concomitant decrease in quantal size. Together, these observations indicate that presynaptic mechanisms significantly contribute to the enhancement produced by this diterpene.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1992

Modulation of synaptic efficacy in field CA1 of the rat hippocampus by forskolin.

Activation of cAMP-dependent protein kinase (kinase A) has recently been shown to enhance responses evoked by stimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in cultured hippocampal pyramidal neurons. Here we report results of experiments designed to determine if activation of the cAMP cascade potentiates synaptic strength in field CA1 of rat hippocampal slices. We find that bath application of the direct adenylate cyclase activator forskolin (50 microM) enhances the field excitatory postsynaptic potential (EPSP) slope and population spike amplitude evoked by stimulation of Schaffer/commissural afferents. This effect is potentiated by the phosphodiesterase inhibitor and adenosine receptor antagonist 3-isobutyl-1-methylxanthine (IBMX). The enhancement produced by forskolin is suppressed in the presence of adenylate cyclase inhibitors and is not mimicked by the inactive forskolin analogue 1,9-dideoxyforskolin, indicating that, indeed, activation of adenylate cyclase mediates the effects of forskolin in field CA1. Our observations support the idea that changes in intracellular cAMP levels can modulate synaptic efficacy of excitatory glutamatergic synapses in the mammalian hippocampus.

1-Methyl-3-isobutylxanthine↗

PubMed1990

A decrease in firing threshold observed after induction of the EPSP-spike (E-S) component of long-term potentiation in rat hippocampal slices.

Two components of long-term potentiation (LTP) are distinguished with extracellular recording electrodes: a synaptic and an EPSP-Spike (E-S) component. The latter consists of the enhancement produced in the population spike amplitude in excess of that predicted by EPSP potentiation alone. The experiments carried out in this study were designed to investigate intracellular correlates of E-S potentiation and to examine the hypothesis that an increased postsynaptic excitability underlies E-S potentiation. CA1 pyramidal neurons were synaptically activated from stratum radiatum. LTP, defined as a stable increase in the probability of firing to afferent stimulation, was found to be related to a decrease in the intracellular PSP peak amplitude and slope required to fire the cells at a probability of 0.5. These changes were accompanied by a decrease in threshold to direct activation. No significant changes in input resistance or resting potential were recorded. These excitability changes were only observed in cells displaying LTP; they were not related to the potentiation of the synaptic component (PSP amplitude). Our results support the hypothesis that different mechanisms underlie the two components of LTP, and that a reduction in threshold for neuronal discharge accompanies tetanus-induced E-S potentiation. It is suggested that an increase in the ratio of synaptically evoked excitation/inhibition and a reduction in tonic synaptic inhibition through GABAA channels contribute to E-S potentiation.

Action Potentials↗

PubMed1990

Failure to reverse long-term potentiation by coupling sustained presynaptic activity and N-methyl-D-aspartate receptor blockade.

The proposal that long-term potentiation (LTP) is a mechanism underlying memory in the mammalian brain rests on a number of properties of LTP that parallel characteristics of memory defined by behavior. A prominent feature of behaviorally defined memory is its reversibility. LTP is induced at synapses that correlate in their activity, and the signal for induction is calcium influx through N-methyl-D-aspartate (NMDA) receptor channels. By analogy to the reversibility of behaviorially defined memory, uncorrelated synaptic activity might be expected to reverse LTP, an anti-Hebbian effect called long-term depression, which has only recently been described in the hippocampus [Stanton, P. K. & Sejnowski, T. J. (1989) Nature (London) 339, 215-218]. Because the extent to which synaptic activity is correlated is represented by postsynaptic calcium concentrations, it seemed likely to us that long-term depression is related to the failure of calcium to pass through the NMDA channel. One way to block the calcium influx that signals correlated synaptic activity is with the NMDA receptor antagonist D-(-)-2-amino-5-phosphonovalerate. We performed a series of experiments in rat hippocampal slices designed to estimate the amount of synaptic depression per afferent test pulse under these conditions. Schaffer collateral-commissural afferents to field CA1 were repetitively stimulated in the presence of 2-amino-5-phosphonovalerate. No enduring synaptic depression nor reversal of LTP could be detected. We conclude that some other mechanism underlies long-term depression in the hippocampus.

2-Amino-5-phosphonovalerate↗

PubMed1989

The EPSP-spike (E-S) component of long-term potentiation in the rat hippocampal slice is modulated by GABAergic but not cholinergic mechanisms.

Long-term potentiation of synaptic efficacy (LTP) can be shown to consist of two components: a synaptic and an excitatory postsynaptic potential (EPSP)-spike (E-S) component. The E-S component is expressed as a leftward shift in the curve relating population spike amplitude as a function of EPSP slope. The participation of cholinergic and GABAergic processes in E-S potentiation was studied in field CA1 of rat hippocampal slices. Atropine, a muscarinic antagonist, did not prevent tetanus-induced E-S potentiation. The cholinergic agonist carbachol and the GABAA antagonist picrotoxin produced a leftward shift in the E-S relation; picrotoxin, but not carbachol, prevented the expression of tetanus-induced E-S potentiation. These observations indicate that an increase in the ratio of evoked excitation to inhibition and/or a reduction in tonic inhibition mediated by the activation of GABAA receptors contribute to E-S potentiation produced by high-frequency stimulation.

Action Potentials↗