Biomedical subjects

C Chavkin

Publications and source records attributed to C Chavkin.

At least 55 records · Page 3Linked to original sources

PubMed1995

Activation of inwardly rectifying potassium channels (GIRK1) by co-expressed rat brain cannabinoid receptors in Xenopus oocytes.

The neuronal cannabinoid receptor clone was expressed of saturable [3H]WIN 55,212-2 binding sites. Co-expression of the cannabinoid receptor with cRNA coding for the G-protein-gated inwardly rectifying K+ channel (GIRK1) resulted in oocytes exhibiting large inward K+ currents in response to the cannabinoid agonist WIN 55,212-2. The activation of the potassium current by WIN 55,212-2 was dose-dependent with an EC50 of 630 nM. These results suggest that activation of inwardly rectifying K+ channels may be an additional effector mechanism for brain cannabinoid receptors.

Analgesics↗

PubMed1995

Agonist-induced desensitization of the mu opioid receptor-coupled potassium channel (GIRK1).

In Xenopus oocytes expressing the rat mu receptor and the G protein-gated, inwardly rectifying K+ channel (known as KGA or GIRK1), application of [D-Ala2,MePhe4,Glyol5]enkephalin) a mu opioid agonist, evoked a dose-dependent increase in K+ conductance. With sustained agonist exposure, the amplitude of the response decayed with a t1/2 of 8 +/- 2 min. In oocytes coexpressing the mu and 5HT1A receptors with GIRK1, stimulation of either receptor resulted in heterologous desensitization of the subsequent response to the other. Injection of guanosine 5'-O-(thiotriphosphate) (1 mM) increased the basal GIRK1 activity and the total response to the application of agonist, but did not affect the rate of desensitization. Basal channel activity in the absence of agonist also desensitized at the same rate when the oocytes were exposed to high K+ (96 mM) solution. The above results indicate that the desensitization of the response occurred at a site downstream of the receptor, possibly at the channel. The rate of desensitization was not significantly altered by any of the following treatments: removal of external Ca2+, preloading the oocytes with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-tetra-(acetoxymethyl)-ester (0.5-1 mM), elevation of cAMP levels, treatment with phorbol esters (1 microM), staurosporine (0.5 microM), okadaic acid (1 microM), or cytochalasin B (0.5 microM). These results suggest that desensitization may not involve a calcium or phosphorylation-dependent mechanism.

Animals↗

PubMed1995

Altered behavior and long-term potentiation in type I adenylyl cyclase mutant mice.

The murine Ca(2+)-stimulated adenylyl cyclase (type I) (EC 4.6.1.1), which is expressed predominantly in brain, was inactivated by targeted mutagenesis. Ca(2+)-stimulated adenylyl cyclase activity was reduced 40-60% in the hippocampus, neocortex, and cerebellum. Long-term potentiation in the CA1 region of the hippocampus from mutants was perturbed relative to controls. Both the initial slope and maximum extent of changes in synaptic response were reduced. Although mutant mice learned to find a hidden platform in the Morris water task normally, they did not display a preference for the region where the platform had been when it was removed. These results indicate that disruption of the gene for the type I adenylyl cyclase produces changes in behavior and that the cAMP signal transduction pathway may play an important role in synaptic plasticity.

Adenylyl Cyclases↗

PubMed1995

Immunocytochemical localization of delta opioid receptors in mouse brain.

An affinity-purified anti-peptide antibody generated against the carboxy-terminal region of the delta opioid receptor was used to localize delta opioid receptors in mouse brain. delta Opioid receptor immunoreactivity was found in axons and nerve terminals in regions of the olfactory bulb, hippocampal formation, cerebral and cerebellar cortex, midbrain and hindbrain. The immunocytochemical distribution correlated well, though not completely with autoradiographic distribution of delta opioid receptors in mouse brain using either [3H][2-D-penicillamine, 5-D-penicillamine]-enkephalin (DPDPE) or [3H]naltrindole. Confocal microscopy of double-labeled tissue provided direct evidence that delta opioid receptors are principally expressed on GABAergic terminals in the hippocampus. These anatomical findings complement extensive physiological studies to provide a more detailed description of endogenous opioid circuitry.

Animals↗

PubMed1995

L-type calcium channels mediate dynorphin neuropeptide release from dendrites but not axons of hippocampal granule cells.

Granule cells in the guinea pig dentate gyrus release kappa opioid neuropeptides, dynorphins, from dendrites as well as from axon terminals. We have found that both L- and N-type calcium channel antagonists inhibited dendritic dynorphin release. In contrast, N-type but not L-type calcium channel antagonists inhibited axonal dynorphin release. Neither L- nor N-type channel antagonists directly altered the effects of kappa opioid receptor activation. By inhibiting dynorphin release, L-type channel antagonists also facilitated the induction of long-term potentiation of the perforant path-granule cell synapse. These studies establish that a single cell type can release a transmitter from two different cellular domains and provide new distinction between axonal and dendritic transmitter release mechanisms.

Animals↗

PubMed1995

Kappa-opioid receptors couple to inwardly rectifying potassium channels when coexpressed by Xenopus oocytes.

Xenopus oocytes expressed kappa-opioid specific binding sites after injection of cRNA prepared from a clone of the rat kappa-opioid receptor. Coinjection of kappa receptor cRNA with cRNA coding for a G protein-linked, inwardly rectifying, K+ channel (GIRK1, or KGA) resulted in oocytes that responded to the kappa agonist U-69593 by activating a large (1.0-1.5-microA) K+ current. U-69593 exhibited an EC50 of 260 +/- 50 nM and was blocked by the opioid antagonists norbinaltorphimine and naloxone. The kappa agonist bremazocine was 200-fold more potent than U-69593 in eliciting K+ current but exhibited a partial agonist profile in this expression system. The present results indicate that stimulation of inwardly rectifying K+ channels may be a potential effector mechanism for kappa-opioid receptors.

Amino Acid Sequence↗

PubMed1995

Colocalization of mu opioid receptors with GIRK1 potassium channels in the rat brain: an immunocytochemical study.

Affinity-purified anti-peptide antibodies generated against the carboxy-terminal region of the mu opioid receptor and the GIRK1 (Kir 3.1) ion channel were used to localize these two proteins in the rat brain. Mu opioid receptor immunoreactivity was detected in brain regions that were previously found to contain mu opioid binding sites using autoradiography. The distribution of GIRK1 immunoreactivity in the brain correlated well with a previous in situ hybridization study. Confocal microscopy of rat brain sections double-labelled with anti-mu opioid receptor and anti-GIRK1 antibodies revealed colocalization of GIRK1 and mu opioid receptor immunoreactivities in somata of subpopulations of neurons in the cerebral cortex, anterior olfactory nucleus, nucleus accumbens, globus pallidus, substantia nigra, peripeduncular nucleus, hippocampal formation, diagonal band, thalamus, locus coeruleus, dorsal raphe, red nucleus, nucleus of the trapezoid body, reticular nucleus, vestibular nucleus, inferior colliculus and the mesencephalic trigeminal nucleus. These anatomical findings suggest that the mu opioid receptor may couple to GIRK1 in some but not all regions of the rat brain.

Amino Acid Sequence↗

PubMed1994

Oocytes from Xenopus laevis contain an intrinsic sigma 2-like binding site.

In preparation for expression studies for rat brain sigma-binding sites, Xenopus oocytes were tested for the presence of [3H]di-o-tolylguanidine (DTG)-binding sites. Native oocytes were found to contain two intrinsic [3H]DTG-binding sites, a high-affinity site (Kd = 32 +/- 6 nM, Bmax of 45.7 +/- 19 pmol/mg protein) and a low-affinity binding site (Kd = 1.3 +/- 0.7 microM, Bmax of 3.2 +/- 0.7 nmol/mg protein). In a series of radioligand-binding-displacement studies, the high-affinity binding sites were found to have a binding profile which has a similar Kd to that of the mammalian sigma 2-binding site (32 vs. 38 nM). Comparison of the IC50 values for inhibition of [3H]DTG binding in rat liver and oocytes for DTG, haloperidol (HAL), (-)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ((+)-3-PPP), (+)-pentazocine and Zn2+, showed similarity in rank (r2 = 0.913) but a 7-fold lower potency in oocytes. These results suggest that the high-affinity [3H]DTG-binding site in oocytes represents a sigma 2-like binding site.

Animals↗

PubMed1994

Conducting states of a mammalian serotonin transporter.

We have studied permeation at a cloned rat 5-HT transporter expressed in Xenopus oocytes. [3H]5-HT uptake and [125I]RTI-55 binding yield a turnover rate of approximately 1/s that does not depend on membrane potential. However, in voltage-clamp experiments, three distinct currents results from 5-HT transporter expression. First, a steady-state, voltage-dependent transport-associated current is induced by 5-HT application. Second, a transient inward current is activated by voltage jumps to high negative potentials in the absence of 5-HT and is blocked by 5-HT itself. Third, a small leakage current is observed in the absence of 5-HT. All the observed currents are blocked by inhibitors of 5-HT uptake but are differentially affected by Na+, Li+, K+, Ba2+, Cs+, Cl-, and amiloride. The conducting states of the 5-HT transporter may reflect the existence of a permeation pathway similar to that of ionic channels.

Amiloride↗

PubMed1994

Inhibition of glutamate release by presynaptic kappa 1-opioid receptors in the guinea pig dentate gyrus.

1. Activation of kappa 1-opioid receptors inhibits excitatory transmission in the hippocampal dentate gyrus of the guinea pig. The present studies used both anatomic and physiological approaches to distinguish between a pre- and postsynaptic localization of these receptors. 2. The entorhinal cortex was lesioned unilaterally to cause degeneration of perforant path afferents to the dentate molecular layer, and kappa 1-opioid binding sites were measured by labeling with the selective agonist, [3H]-U69593. Binding density was reduced significantly in the dentate gyrus molecular layer ipsilateral to the lesion compared with the contralateral molecular layer and with sham-lesioned controls. 3. Paired-pulse facilitation is a neurophysiologic paradigm that has been used to differentiate pre- and postsynaptic sites of action for agents that inhibit excitatory neurotransmission. U69593 reduced the amplitude of single population spikes and increased the degree of paired pulse facilitation. The potentiation of paired-pulse facilitation was maintained when the stimulation intensity was increased to compensate for the inhibition of excitatory transmission. These effects of kappa 1-receptor activation were similar to those seen after presynaptic inhibition of excitatory neurotransmitter release and support the hypothesis that U69593 presynaptically inhibits excitatory amino acid release in the dentate gyrus. 4. Local application of glutamate by pressure ejection in the dentate molecular layer evoked field excitatory postsynaptic potentials that mimicked those evoked by electrical stimulation of the perforant path. Both responses were sensitive to the non-N-methyl-D-aspartate glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. U69593 inhibited responses evoked by perforant path stimulation but had no effect on responses evoked by glutamate application.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗

PubMed1994

Dynorphin opioids present in dentate granule cells may function as retrograde inhibitory neurotransmitters.

The granule cell population response to perforant path stimulation decreased significantly within seconds following release of endogenous dynorphin from dentate granule cells. The depression was blocked by the opioid receptor antagonists naloxone and norbinaltorphimine, suggesting that the effect was mediated by dynorphin activation of kappa 1 type opioid receptors. Pharmacological application of dynorphin B in the molecular layer was effective at reducing excitatory synaptic transmission from the perforant path, but application in the hilus had no significant effect. These results suggest that endogenous dynorphin peptides may be released from a local source within the dentate molecular layer. By light microscopy, dynorphin-like immunoreactivity (dynorphin-LI) was primarily found in granule cell axons in the hilus and stratum lucidum with only a few scattered fibers evident in the molecular layer. At the extreme ventral pole of the hippocampus, a diffuse band of varicose processes was also seen in the molecular layer, but this band was not present in more dorsal sections similar to those used for the electrophysiological studies. Electron microscopic analysis of the molecular layer midway along the septotemporal axis revealed that dynorphin-LI was present in dense-core vesicles in both spiny dendrites and unmyelinated axons with the majority (74%) of the dynorphin-LI-containing dense-core vesicles found in dendrites. Neuronal processes containing dynorphin-LI were observed throughout the molecular layer. The results suggest that dynorphin release from granule cell processes in the molecular layer regulates excitatory inputs entering the hippocampus from cerebral cortex, thus potentially counteracting such excitation-induced phenomena as epileptogenesis or long-term potentiation.

Animals↗

PubMed1994

Kappa opioids inhibit induction of long-term potentiation in the dentate gyrus of the guinea pig hippocampus.

NMDA receptor-mediated long-term potentiation (LTP) of dentate granule cell responses to perforant path stimulation was inhibited by the kappa 1 opioid receptor agonist U69,593. This inhibition was reversed stereospecifically by naloxone and blocked by the selective kappa 1 antagonist norbinaltorphimine (NBNI). NBNI, by itself, had no effect on LTP induced by threshold stimulation but significantly enhanced LTP from more prolonged stimulation. This effect of NBNI suggests that endogenous opioids can regulate LTP in the dentate gyrus. In support of this hypothesis, stimulation of dynorphin-containing fibers also blocked LTP production in an NBNI-sensitive manner. Finally, dynorphin-mediated inhibition of LTP acts primarily on mechanisms of induction rather than maintenance or expression, since dynorphin released immediately before, but not immediately after, perforant path stimulation blocked LTP. Thus, exogenous and endogenous kappa opioids can inhibit induction of long-term potentiation at the perforant path-granule cell synapse and may therefore regulate plastic changes in synaptic transmission in a brain region thought to play an important role in processes of both learning and memory and epileptogenesis.

Animals↗

PubMed1994

Kappa 2 opioid receptors inhibit NMDA receptor-mediated synaptic currents in guinea pig CA3 pyramidal cells.

The role of the endogenous opioid peptide dynorphin (1-17) in regulating NMDA receptor-mediated synaptic currents was examined in guinea pig hippocampus. Schaffer collateral/commissural fiber-evoked NMDA synaptic currents were recorded using whole-cell patch-clamp techniques in CA3 pyramidal cells. Dynorphin was found to have dual effects on NMDA synaptic currents, increasing currents at low concentrations and decreasing currents at high concentrations. Only the inhibitory action of dynorphin was sensitive to naloxone, indicating that this effect was mediated by an opioid receptor. The inhibitory effect was mimicked by bremazocine, but not by U69,593, U50,488, [D-Ala2, N-Me-Phe4, Gly-ol]-enkephalin, or [D-Pen2,5]-enkephalin. Bremazocine's effect was blocked by naloxone, but not by nor-binaltorphimine, cyprodime, or naltrindole. These findings suggest that bremazocine's effect was mediated by the kappa 2 subtype of opioid receptor. In addition, 1 microM naloxone and antisera to dynorphin (1-17) were found to increase NMDA-mediated synaptic currents. Nor-binaltorphimine, cyprodime, naltrindole, and antisera to met-enkephalin did not increase the NMDA synaptic current. These findings suggest that endogenous dynorphin was acting at kappa 2 receptors to inhibit NMDA receptor-mediated synaptic currents. Overall, these findings indicate that dynorphin is an endogenous agonist for kappa 2 receptors in the CA3 region of the guinea pig hippocampus and that these receptors regulate NMDA receptor function.

Animals↗

PubMed1993

Atrial G protein-activated K+ channel: expression cloning and molecular properties.

Activity of several ion channels is controlled by heterotrimeric GTP-binding proteins (G proteins) via a membrane-delimited pathway that does not involve cytoplasmic intermediates. The best studied example is the K+ channel activated by muscarinic agonists in the atrium, which plays a crucial role in regulating the heartbeat. To enable studies of the molecular mechanisms of activation, this channel, denoted KGA, was cloned from a rat atrium cDNA library by functional coupling to coexpressed serotonin type 1A receptors in Xenopus oocytes. KGA displays regions of sequence homology to other inwardly rectifying channels as well as unique regions that may govern G-protein interaction. The expressed KGA channel is activated by serotonin 1A, muscarinic m2, and delta-opioid receptors via G proteins. KGA is activated by guanosine 5'-[gamma-thio]triphosphate in excised patches, confirming activation by a membrane-delimited pathway, and displays a conductance equal to that of the endogenous channel in atrial cells. The hypothesis that similar channels play a role in neuronal inhibition is supported by the cloning of a nearly identical channel (KGB1) from a rat brain cDNA library.

Amino Acid Sequence↗

PubMed1993

Endogenous dynorphins inhibit excitatory neurotransmission and block LTP induction in the hippocampus.

Although anatomical and neurochemical studies suggest that endogenous opioids act as neurotransmitters, their roles in normal and pathophysiological regulation of synaptic transmission are not defined. Here we examine the actions of prodynorphin-derived opioid peptides in the guinea-pig hippocampus and show that physiological stimulation of the dynorphin-containing dentate granule cells can release endogenous dynorphins, which then activate kappa 1 opioid receptors present in the molecular layer of the dentate gyrus. Activation of kappa 1 receptors by either pharmacologically applied agonist or endogenously released peptide reduces excitatory transmission in the dentate gyrus, as shown by a reduction in the excitatory postsynaptic currents evoked by stimulation of the perforant path, a principal excitatory afferent. In addition, released dynorphin peptides were found to block the induction of long-term potentiation (LTP) at the granule cell-perforant path synapse. The results indicate that endogenous dynorphins function in this hippocampal circuit as retrograde, inhibitory neurotransmitters.

Action Potentials↗

PubMed1993

Receptors that couple to 2 classes of G proteins increase cAMP and activate CFTR expressed in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- channel activated by phosphorylation, was expressed in Xenopus oocytes along with various combinations of several other components of the cAMP signalling pathway. Activation of the coexpressed beta 2 adrenergic receptor increased cAMP and led to CFTR activation. The activation of CFTR (1) requires only short (15 s) exposure to isoproterenol, (2) occurs for agonist concentrations 100-1000 fold lower than those that produce cAMP increases detectable by a radioimmunoassay, (3) requires injection of only 5 pg of receptor cRNA per oocyte, and (4) can be increased further by coexpression of cRNA for adenylyl cyclase type II or III or for Gs alpha. In addition, CFTR activation and cAMP increases by beta 2 activation were enhanced by activation of the coexpressed 5HT1A receptor, which is thought to couple to Gi. The additional activation by the 5HT1A receptor was enhanced by coexpression of adenylyl cyclase type II but not with type III and may proceed via the beta gamma subunits of a G protein. The sensitivity of the assay system is also demonstrated by responses to vasoactive intestinal peptide and to pituitary adenylate cyclase-activating polypeptide in oocytes injected with cerebral cortex mRNA.

Adenylyl Cyclases↗

PubMed1992

Endogenous opioid regulation of norepinephrine release in guinea pig hippocampus.

Release of endogenous norepinephrine was detected in guinea pig hippocampal slices using a radioligand displacement assay. Focal electrical stimulation released endogenous norepinephrine and caused a calcium-dependent reduction in specific [3H]propranolol binding at beta-adrenergic receptors in the brain slice. The mu-opioid agonist PL017 decreased norepinephrine release, and the inhibition by PL017 could be blocked by the opioid antagonist naloxone. Endogenous opioid peptides concomitantly released by tissue stimulation also decreased norepinephrine release in a naloxone-sensitive manner. These results support the hypothesis that endogenous opioids can regulate excitability in the hippocampus by presynaptic modulation of norepinephrine release.

Animals↗

PubMed1992

8-Bromo-cAMP blocks opioid activation of a voltage-gated potassium current in isolated hippocampal neurons.

We have previously shown that mu-selective opioid agonists activate both an inward rectifying and a voltage-gated potassium conductance in acutely dissociated non-pyramidal neurons from rat hippocampus. We now report that the opioid-activated voltage-gated potassium conductance was blocked by the membrane permeable cAMP analogue 8-bromo-cAMP. In contrast, 8-bromo-cGMP failed to inhibit opioid activation of the voltage-gated potassium current. These results suggest that the opioid-activated potassium channel is regulated by cAMP-dependent phosphorylation.

8-Bromo Cyclic Adenosine Monophosphate↗