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1.
EMBO J ; 20(4): 767-76, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11179221

ABSTRACT

To understand the requirements for binding to G protein betagamma subunits, phage-displayed random peptide libraries were screened using immobilized biotinylated betagamma as the target. Selected peptides were grouped into four different families based on their sequence characteristics. One group (group I) had a clear conserved motif that has significant homology to peptides derived from phospholipase C beta (PLC beta) and to a short motif in phosducin that binds to G protein beta subunits. The other groups had weaker sequence homologies or no homology to the group I sequences. A synthetic peptide from the strongest consensus group blocked activation of PLC by G protein betagamma subunits. The peptide did not block betagamma-mediated inhibition of voltage-gated calcium channels and had little effect on betagamma-mediated inhibition of Gs-stimulated type I adenylate cyclase. Competition experiments indicated that peptides from all four families bound to a single site on betagamma. These peptides may bind to a protein-protein interaction 'hot spot' on the surface of betagamma subunits that is used by a subclass of effectors.


Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Amino Acid Sequence , Coliphages/chemistry , Heterotrimeric GTP-Binding Proteins/chemistry , Molecular Sequence Data , Peptides/metabolism , Potassium Channels/metabolism , Protein Binding , Sequence Homology, Amino Acid
2.
J Pharmacol Exp Ther ; 294(2): 500-9, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10900225

ABSTRACT

The cellular correlates of desensitization or tolerance are poorly understood. To address this, we studied acute and long-term mu-opioid desensitization, with respect to Ca(2+) currents, in cultured rat dorsal root ganglion (DRG) neurons. Exposure of DRG neurons to the mu-agonist [D-Ala(2),N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO; 3 microM) reduced whole-cell currents approximately 35%, but with continued agonist application, 52% of the response was lost over 10 to 12 min. In contrast, exposure of DRG neurons to DAMGO for 24 h resulted in a nearly complete loss of Ca(2+) channel regulation after washing and re-exposure to DAMGO. Responses to the gamma-aminobutyric acid(B) agonist baclofen were not affected in these neurons. Acute desensitization preferentially affected the voltage-sensitive component of mu-opioid and gamma-aminobutyric acid(B) responses. Facilitation of both the DAMGO- and baclofen-inhibited current by a strong depolarizing prepulse was significantly attenuated in acutely desensitized neurons. Because G(betagamma)-subunits mediate neurotransmitter-induced changes in channel voltage-dependent properties, these data suggest an altered interaction of the G(betagamma)-subunit with the Ca(2+) channel. Block of N-type Ca(2+) channels with omega-conotoxin GVIA revealed a component of the opioid response that did not desensitize over 10 min. We conclude that acute and long-term mu-opioid desensitization in DRG neurons occurs by different mechanisms. Acute desensitization is heterologous and functionally compartmentalized: the pathway targeting non-N-type channels is relatively resistant to the early effects of continuous agonist exposure; the pathway targeting N-type channels in a largely voltage-insensitive manner is partially desensitized; and the pathway targeting N-type channels in a largely voltage-sensitive manner is completely desensitized.


Subject(s)
Analgesics, Opioid/pharmacology , Calcium Channels/physiology , Neurons, Afferent/drug effects , Receptors, Opioid, mu/physiology , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels/classification , Calcium Channels, N-Type/physiology , Cells, Cultured , Drug Tolerance , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , GTP-Binding Proteins/physiology , Ganglia, Spinal/cytology , Ganglia, Spinal/drug effects , Ganglia, Spinal/physiology , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Kinetics , Neurons, Afferent/physiology , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Receptors, GABA-B/physiology , Receptors, Opioid, mu/agonists , omega-Conotoxin GVIA/pharmacology
3.
Proc Natl Acad Sci U S A ; 96(18): 10385-90, 1999 Aug 31.
Article in English | MEDLINE | ID: mdl-10468617

ABSTRACT

Morphine and other micro opioids regulate a number of intracellular signaling pathways, including the one mediated by phospholipase C (PLC). By studying PLC beta3-deficient mice, we have established a strong link between PLC and mu opioid-mediated responses at both the behavioral and cellular levels. Mice lacking PLC beta3, when compared with the wild type, exhibited up to a 10-fold decrease in the ED(50) value for morphine in producing antinociception. The reduced ED(50) value was unlikely a result of changes in opioid receptor number or affinity because no differences were found in whole-brain B(max) and K(d) values for mu, kappa, and delta opioid receptors between wild-type and PLC beta3-null mice. We also found that opioid regulation of voltage-sensitive Ca(2+) channels in primary sensory neurons (dorsal root ganglion) was different between the two genotypes. Consistent with the behavioral findings, the specific mu agonist [D-Ala(2),(Me)Phe(4),Gly(ol)(5)]enkephalin (DAMGO) induced a greater whole-cell current reduction in a greater proportion of neurons isolated from the PLC beta3-null mice than from the wild type. In addition, reconstitution of recombinant PLC protein back into PLC beta3-deficient dorsal root ganglion neurons reduced DAMGO responses to those of wild-type neurons. In neurons of both genotypes, activation of protein kinase C with phorbol esters markedly reduced DAMGO-mediated Ca(2+) current reduction. These data demonstrate that PLC beta3 constitutes a significant pathway involved in negative modulation of mu opioid responses, perhaps via protein kinase C, and suggests the possibility that differences in opioid sensitivity among individuals could be, in part, because of genetic factors.


Subject(s)
Brain/metabolism , Enkephalins/pharmacology , Gene Expression Regulation, Enzymologic , Isoenzymes/genetics , Isoenzymes/metabolism , Morphine/pharmacology , Neurons, Afferent/physiology , Pain/genetics , Receptors, Opioid, mu/metabolism , Type C Phospholipases/genetics , Type C Phospholipases/metabolism , Animals , Calcium Channels/genetics , Cell Membrane/metabolism , Enkephalin, Ala(2)-MePhe(4)-Gly(5)- , Ganglia, Spinal/physiology , Gene Expression Regulation , Isoenzymes/deficiency , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Mice, Knockout , Neurons, Afferent/drug effects , Pain/physiopathology , Phospholipase C beta , Receptors, Opioid, delta/metabolism , Receptors, Opioid, kappa/metabolism , Type C Phospholipases/deficiency
4.
Neuroscience ; 84(4): 1209-22, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9578407

ABSTRACT

We have recently demonstrated that eight, daily flurothyl-induced generalized clonic seizures, followed by a four week stimulus-free interval, results in a long-lasting reduction in generalized seizure threshold and a change in the type of seizure expressed in response to flurothyl from clonic to tonic. There is a progressive increase in the probability that a mouse will express a tonic seizure during the four week interval, suggesting that prior flurothyl seizures initiate a proepileptogenic process that requires time to develop. In this study, the immunohistochemical detection of the c-fos protein (Fos) was used to evaluate whether seizure-induced epileptogenesis resulted in regional differences in the degree of neuronal activation. Fos immunoreactivity was examined 1.5 h following either a single generalized seizure, the last of eight consecutive daily seizures or a retest seizure evoked two weeks after the last of eight seizures. In each condition, generalized seizure behaviours were elicited in C57BL/6 mice using flurothyl and classified as either "forebrain" (face and forelimb clonus) or "brainstem" (running/bouncing, treading, tonic extension). The spatial distribution of Fos induction was compared on the basis of the seizure phenotype and the seizure history. The predominant differences in Fos distribution were found to be related to the type of seizure expressed regardless of the seizure history. Furthermore, the different motor components that make up a "brainstem" seizure could not be distinguished by the pattern of Fos labelling suggesting that multiple convulsive behaviours are mediated by one anatomical system. Finally, Fos induction in the ventromedial hypothalamic nucleus preceded and predicted the change in seizure type from "forebrain" to "brainstem". These data support the concept that separate anatomical systems mediate the expression of the two generalized seizure phenotypes. In addition, the ventromedial nucleus of the hypothalamus may be a point of interaction between the systems and may play a role in seizure-induced neural reorganization.


Subject(s)
Brain Chemistry/physiology , Central Nervous System Stimulants/pharmacology , Convulsants/pharmacology , Flurothyl/pharmacology , Kindling, Neurologic/metabolism , Proto-Oncogene Proteins c-fos/biosynthesis , Animals , Behavior, Animal/drug effects , Brain/anatomy & histology , Brain Chemistry/drug effects , Central Nervous System Depressants/pharmacology , Ethanol/pharmacology , Kindling, Neurologic/drug effects , Male , Mice , Mice, Inbred C57BL , Phenotype , Seizures/physiopathology
5.
J Neurosci ; 17(14): 5581-90, 1997 Jul 15.
Article in English | MEDLINE | ID: mdl-9204939

ABSTRACT

This study examined both the acute and long-lasting changes in seizure susceptibility that occur in response to the repeated induction of generalized seizure activity. Daily flurothyl-induced generalized clonic seizures resulted in a progressive decrease in both the generalized seizure threshold and the latency to the first myoclonic jerk. The threshold reduction was significant as early as the second trial and was maximal by trial 5. However, a minimum of eight seizures was necessary for the maximal reduction to be long-lasting. The present study also examined the effects of the number of seizures and the duration of the stimulation-free interval on the type of generalized seizure expressed. During the induction phase of the experiment, only generalized clonic seizures ("forebrain seizures") were expressed. If, however, the animal was retested after a 1, 2, 3, or 4 week stimulation-free interval, a progressive increase in both the proportion of animals expressing "brainstem seizure" behaviors and the median seizure score was observed. The progression of flurothyl-induced generalized seizure behaviors was significantly altered if (1) a minimum of eight generalized clonic seizures had been expressed, and (2) a minimum of a 2 week stimulation-free interval followed. Fewer generalized clonic seizures failed to reliably produce changes in seizure phenotype, even after extended stimulus-free intervals. These data indicate that specific kindling processes are initiated during the interval of repeated seizure induction and evolve in the absence of continued seizure induction. Furthermore, these mechanisms of epileptogenesis were found to be manifest predominantly as a change in the seizure phenotype expressed and to proceed independent of changes in the generalized seizure threshold.


Subject(s)
Behavior, Animal/physiology , Epilepsy, Generalized/physiopathology , Animals , Disease Models, Animal , Male , Mice , Mice, Inbred C57BL , Time Factors
6.
Epilepsia ; 38(6): 631-6, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9186244

ABSTRACT

PURPOSE: We have developed and characterized a novel model of epileptogenesis based on the convulsive actions of flurothyl in mice. The hallmark feature of this model is a reliable change in the type of seizure expressed in response to flurothyl from generalized clonic to generalized tonic seizures. The purpose of our study was to evaluate the effects of chronic administration of valproate (VPA), phenytoin (PHT), and MK-801 on the change in seizure phenotype observed in our model system. METHODS: Male C57BL/6J mice received flurothyl seizures on 8 consecutive days. Two hours after the last generalized seizure, chronic drug or vehicle was administered twice daily at 12-h intervals for 28 days. The drugs evaluated were VPA (250 mg/kg), PHT (30 mg/kg), and MK-801 (0.5 mg/kg). After a 7-day drug washout period, mice were retested with flurothyl. RESULTS: Among uninjected or vehicle-injected control mice, there was a significant increase in the proportion of animals expressing tonic seizures after the 28-day stimulation-free interval. Chronic administration of VPA or MK-801, but not PHT, blocked the characteristic change in seizure type from clonic to tonic. CONCLUSIONS: The change in seizure phenotype observed after exposure to our paradigm indicates a fundamental reorganization in the propagation of flurothyl-initiated seizures. As in electrical kindling, VPA and MK-801 are effective at blocking or retarding the reorganization, whereas PHT is not. The concordance in pharmacologic profiles between kindling and our model suggests that the processes underlying changes in seizure susceptibility in these two models share mechanisms in common.


Subject(s)
Anticonvulsants/pharmacology , Disease Models, Animal , Dizocilpine Maleate/pharmacology , Epilepsy/chemically induced , Epilepsy/prevention & control , Flurothyl , Phenytoin/pharmacology , Valproic Acid/pharmacology , Animals , Behavior, Animal/drug effects , Brain Stem/drug effects , Dose-Response Relationship, Drug , Epilepsy/classification , Epilepsy, Generalized/chemically induced , Epilepsy, Generalized/prevention & control , Epilepsy, Tonic-Clonic/chemically induced , Epilepsy, Tonic-Clonic/prevention & control , Flurothyl/pharmacology , Kindling, Neurologic/drug effects , Kindling, Neurologic/physiology , Male , Mice , Mice, Inbred C57BL , Motor Activity/drug effects , Neural Pathways/drug effects
7.
Exp Neurol ; 143(2): 255-68, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9056388

ABSTRACT

The expression of generalized clonic and generalized tonic seizures has been suggested to result from the activation of different and independent neuronal circuits. Using the induction of the c-fos protein (Fos) as a marker of neuronal activity, we identified brain structures that are differentially associated with the expression of electroconvulsive shock-induced generalized clonic and generalized tonic seizures. Expression of either seizure phenotype resulted in a similar bilaterally symmetrical increase in Fos immunoreactivity in many forebrain structures, including the bed nucleus of the stria terminalis, hippocampal dentate gyrus, amygdala, and piriform cortex, compared to controls. However, following tonic hindlimb extension (THE), the degree of labeling in specific thalamic, hypothalamic, and brain stem areas was significantly greater than that of either controls or animals exhibiting clonic seizures. While a greater number of neurons in the hypothalamus (e.g., ventromedial nucleus), subparafascicular thalamic nucleus, peripeduncular area, deep medial superior colliculus, dorsal and lateral central gray, and paralemniscal nuclei were robustly labeled following THE, noticeably fewer cells were immunoreactive following face and forelimb clonic seizure behaviors. These differences were also found to be independent of the stimulus magnitude. In animals stimulated with the same current intensity but expressing either of the two seizure phenotypes, the pattern of Fos induction was consistent with the seizure phenotype expressed. These results demonstrate that specific subsets of neurons are differentially activated following the expression of different generalized seizure behaviors and that activity in discrete mesencephalic and diencephalic structures is more frequently associated with the expression of generalized tonic seizures than with the expression of generalized clonic seizures.


Subject(s)
Epilepsy, Generalized/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Animals , Disease Models, Animal , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL
8.
Epilepsy Res ; 15(3): 201-5, 1993 Jul.
Article in English | MEDLINE | ID: mdl-8223416

ABSTRACT

Investigations into the neurochemical or molecular biological mechanisms underlying the kindled state require a seizure induction procedure for eliciting generalized tonic-clonic seizures in naive animals. Such seizure controls are necessary for dissociating the influence of ictal motor events on measures of interest from the influence of the kindling process on these same measures. In this study three procedures for inducing seizures in naive animals were evaluated against a set of criteria considered ideal.


Subject(s)
Epilepsy, Tonic-Clonic/physiopathology , Kindling, Neurologic/physiology , Seizures/physiopathology , Amygdala/physiology , Animals , Behavior, Animal/physiology , Electric Stimulation , Electrodes, Implanted , Electroshock , Male , Prefrontal Cortex/physiology , Rats , Rats, Sprague-Dawley
9.
Epilepsy Res ; 10(2-3): 142-7, 1991.
Article in English | MEDLINE | ID: mdl-1817954

ABSTRACT

Amygdala kindling was observed to increase significantly the proportion of rats that exhibited tonic hindlimb extension in response to corneal electroshock stimulation. Mechanical brainstem lesions which abolished electroshock-induced tonic hindlimb extension failed to alter either the expression of fully generalized kindled seizures or the development of amygdala kindled seizures. Results suggest that while kindling can alter the sensitivity of brainstem systems involved in the expression of tonic hindlimb extension, these same systems are not necessary for either the development or expression of amygdala kindled seizures.


Subject(s)
Brain Stem/physiopathology , Electroshock , Kindling, Neurologic/physiology , Seizures/physiopathology , Animals , Disease Models, Animal , Male , Rats , Rats, Inbred Strains
10.
J Pharmacol Exp Ther ; 252(3): 1125-33, 1990 Mar.
Article in English | MEDLINE | ID: mdl-2319464

ABSTRACT

Midazolam is a new ultra short-acting benzodiazepine whose physical dependence properties have not been well characterized. Our laboratory has demonstrated previously that physical dependence to the long-acting chlordiazepoxide in the rat is inducible by a single intoxicating dose, whereas maximal dependence required chronically equivalent maximally tolerable dosing b.i.d. for 5 weeks. Based on the methods developed in our laboratory to quantify benzodiazepine intoxication and withdrawal, Trs were designed to evaluate midazolam's capacity to induce dependence in the rat after definable acute (120 mg/kg p.o.), sub-acute (120 mg/kg q.i.d. x 3 days) and chronic (120-180 mg/kg bid. x 5 weeks) dosing that was near maximally tolerable. A single dose of midazolam failed to produce withdrawal signs. Tolerance and dependence increased as a function of midazolam dose and duration of Tr.


Subject(s)
Midazolam/adverse effects , Substance Withdrawal Syndrome/physiopathology , Substance-Related Disorders/etiology , Animals , Behavior, Animal/drug effects , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Tolerance , Male , Midazolam/administration & dosage , Rats , Rats, Inbred Strains
13.
J Pharmacol Exp Ther ; 239(3): 775-83, 1986 Dec.
Article in English | MEDLINE | ID: mdl-3098961

ABSTRACT

A single intoxicating dose of chlordiazepoxide HCl (p.o.) in the rat can induce quantifiable manifestations of physical dependence. Dependence was revealed by antagonist precipitation (Ro 15-1788, CGS-8216) as well as spontaneous emergence of neurobehavioral signs of withdrawal observed by multiple raters blind to treatments. Ro 15-1788 was 45% more effective than CGS-8216 in both reversing chlordiazepoxide intoxication and expressing withdrawal signs. The severity of Ro 15-1788-precipitated withdrawal varied with chlordiazepoxide dose, Ro 15-1788 dose and the agonist-antagonist dose interval. Maximal precipitated dependence was evoked 3 days after chlordiazepoxide HCl (450 mg/kg) by Ro 15-1788 (25 mg/kg i.p.). The precipitated syndrome consisted of tail erection, reduced motor activity, high step, curled claw, arched back, muscle hypertonus and piloerection. Ro 15-1788-precipitated dependence emerged between 28 and 52 hr, peaked at 76 hr and disappeared by 124 hr. Spontaneous withdrawal had emerged from 100 to 124 hr and then faded gradually. The neurobehavioral expression of central nervous system depression and its reversal were necessary but not sufficient conditions for the induction and expression of acute chlordiazepoxide dependence. These results suggest caution in reviving acute benzodiazepine-overdosed patients to avoid iatrogenic withdrawal analogous to naloxone for opiates.


Subject(s)
Chlordiazepoxide , Substance-Related Disorders/physiopathology , Animals , Behavior, Animal/drug effects , Dose-Response Relationship, Drug , Drug Interactions , Drug Tolerance , Flumazenil/pharmacology , Male , Pyrazoles/pharmacology , Rats , Rats, Inbred Strains , Substance Withdrawal Syndrome/physiopathology , Time Factors
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