Biomedical subjects

B Attali

Publications and source records attributed to B Attali.

50 records · Page 3Linked to original sources

PubMed1991

Cloning, expression, pharmacology and regulation of a delayed rectifier K+ channel in mouse heart.

Neonatal mouse cardiac poly(A)+ mRNA microinjection into Xenopus oocytes directed the expression of a delayed rectifier K+ current. A cDNA encoding this channel, called mIsK, was cloned from a neonatal mouse heart cDNA library whose properties were studied after expression of the complementary RNA in Xenopus oocytes. Among the different known K+ channel blockers, only the class III antiarrhythmic clofilium inhibited mIsK in the 10-100 microM range. The channel was completely insensitive to other antiarrhythmics such as quinine, quinidine, sotalol or amiodarone. mIsK was enhanced by increasing intracellular Ca2+ and by microinjected Ca(2+)-calmodulin dependent protein kinase II. These stimulations were reversed by the calmodulin antagonist W7. Conversely, the phorbol ester PMA, the diacylglycerol analog OAG and microinjected purified protein kinase C inhibited mIsK. This inhibitory effect could be prevented by the protein kinase C inhibitor staurosporine. These results were consistent with the presence of consensus sequences for kinase II and kinase C in the mIsK structure. Cultured newborn mouse ventricular cardiac cells exhibited a delayed rectifier K+ current which had biophysical properties similar to those of cloned mIsK and which was inhibited by clofilium and protein kinase C activators. In situ hybridization experiments revealed that mIsK mRNA was homogeneously distributed in the cardiac tissue. Neonatal mouse heart expressed the most mIsK mRNA compared with various other rat and mouse tissues. Since this K+ channel generates a current which appears to be involved in the control of both the action potential duration and the beating rate, these results suggest an important role for the mIsK channel in cardiac cell physiology and cardiac pathology.

Amino Acid Sequence↗

PubMed1991

Phorbol ester pretreatment desensitizes the inhibition of Ca2+ channels induced by kappa-opiate, alpha 2-adrenergic, and muscarinic receptor agonists.

Acute treatment of rat spinal cord-dorsal root ganglion cocultured neurons with 12-O-tetradecanoylphorbol 13-acetate (TPA), a known activator of protein kinase C, inhibited the dihydropyridine-sensitive voltage-dependent 45Ca2+ influx measured in these cells (IC50 of approximately 100 nM, 66% inhibition at 1 microM TPA). However, prolonged preincubation (24 h) of the cells with 100 nM TPA followed by extensive washing completely abolished, i.e., desensitized, the capacity of a second application of TPA to inhibit the activity of the voltage-dependent Ca2+ channels. Moreover, this treatment also abolished the inhibition of Ca2+ influx produced by kappa-opiate as well as by alpha 2-adrenergic and muscarinic receptor agonists. Substantial desensitization was already observed following a 1-h pretreatment with 100 nM TPA. In contrast to TPA, an inactive phorbol ester (4 beta-phorbol 13-acetate) did not affect the inhibition of the voltage-dependent Ca2+ influx by these receptor agonists. These results suggest that protein kinase C may have a role in the modulation of Ca2+ channels by kappa-opiate, alpha 2-adrenergic, and muscarinic receptor agonists.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1990

Characterization of kappa opiate receptors in rat spinal cord-dorsal root ganglion cocultures and their regulation by chronic opiate treatment.

We have investigated the expression and regulation of kappa opiate receptors in rat spinal cord-dorsal root ganglion primary cocultures. The density of opiate receptors increased markedly during the differentiation of the cultures; after 10 days in vitro the number of [3H]diprenorphine binding sites reached 244 +/- 47 fmol/mg protein. Most of the binding sites were of the kappa type, representing about 65-80% of total opiate receptors, while mu sites were expressed at a lower density (ca. 20% of total opiate sites). Following this period of development, the number of kappa and mu receptors did not change significantly. No detectable delta sites were observed at any time of culture (up to 4 weeks in vitro). Chronic opiate agonist treatment (24 h) of the cultured cells with either 10 microM U50488 (a selective kappa agonist), or 1 microM etorphine (a nonselective opiate agonist), did not change the number of kappa receptors and their binding affinity to [3H]diprenorphine. On the other hand, 50% of the mu receptor sites down-regulated following 24 h treatment with 1 microM etorphine. Chronic antagonist exposure (5 days) with 10 microM naloxone, markedly up-regulated the mu receptors (261% of control), whereas kappa sites exhibited a much weaker upregulation (164% of control). These data demonstrate that kappa opiate receptors are expressed at high concentration in spinal cord-dorsal root ganglion cocultures and that contrary to mu sites, kappa receptor density is less susceptible to modulation by chronic opiate treatment. The results also suggest that postreceptor components are important in regulating the kappa receptor function following prolonged opiate exposure.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1990

Pre- and postnatal development of opiate receptor subtypes in rat spinal cord.

We have studied the developmental expression of opiate binding sites in the rat spinal cord at various prenatal and postnatal stages. For each developmental stage, we have compared the expression pattern of kappa receptors with that of mu and delta receptor subtypes. Both mu and kappa receptors appear relatively early during spinal cord ontogeny (from the 15th prenatal day), while delta sites are expressed later at the postnatal period (starting at the 1st postnatal day). The number of kappa sites predominates throughout the development (55-80% of total opiate sites) with two peaks of binding activity: one at the 20th gestational day, and the other around the 7th postnatal day. mu sites represent 20-38% of the total opiate receptor population with one peak of binding activity appearing at the 1st postnatal day. The densities of mu and kappa receptors at the adult stage are lower by 40-50% than the peak values observed at the early postnatal periods. The relative amounts of delta sites remain low throughout the ontogeny (4-8% of the total opiate sites). The binding properties of neonatal (1 day after birth) kappa sites (ligand binding affinities, regulation of agonist binding by guanosine triphosphate and various cations) are similar to those displayed by kappa receptors in adult spinal cord.

Aging↗

PubMed1990

Differential effect of mu, delta, and kappa ligands on G protein alpha subunits in cultured brain cells.

Rat and guinea pig fetal brain cell cultures and immunoblotting techniques were used to study the effect of receptor selective opioids on the level of the membrane-bound alpha i and alpha o GTP binding protein subunits. Incubation of rat hindbrain cultures with the mu selective peptide DAGO decreased the amount of both alpha proteins. The reduction observed was equivalent to 36% in alpha o and 41% in alpha i. On the other hand, incubation of rat forebrain cultures with this peptide had an opposite effect, increasing the alpha o and alpha i levels by 66% and 68%, respectively. This differential effect of the peptide on the G proteins at the two brain areas may reflect the selective interaction at the receptor level; DAGO induced a fast and effective receptor down-regulation (50% decrease in Bmax) in hindbrain but not in forebrain cultures. Moreover, delta and mu selective ligands differed in their effect, as indicated by the finding that the delta selective peptide DPDPE increased the amount of both alpha proteins in hindbrain cultures by 40%. Similar experiments conducted with guinea pig brain aggregate cultures indicated that the kappa selective agonist U50,488 decreased the amount of the membrane bound alpha i protein subunit by 56%. The results thus indicate that opioid agonists, interacting selectively with the three types of opioid receptors, induce a complex repertoire of changes in the immunoreactive levels of the membrane-bound alpha GTP binding protein subunits in various CNS structures.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1989

Kappa opiate agonists inhibit Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. Involvement of a GTP-binding protein.

The aim of the present study has been to characterize the regulation by opiates of 45Ca2+ influx in rat spinal cord-dorsal root ganglion cocultures. We have demonstrated that K+-induced depolarization, in the presence of the Ca2+ channel agonist Bay K8644, stimulated Ca2+ influx (3-4-fold) via the dihydropyridine class of voltage-dependent Ca2+ channels. While mu and delta opiates had no effect, kappa opiate agonists (e.g. U50488, dynorphin) profoundly depressed the stimulated Ca2+ influx (86% inhibition at 100 microM U50488). The kappa agonist action was stereospecific and could be reversed by the opiate antagonist naloxone. The inhibition produced by kappa agonists was greatly diminished following pertussis toxin treatment, and this effect was accompanied by toxin-induced ADP-ribosylation of a 40-41-kDa protein. This suggests that kappa opiate receptors are negatively coupled to voltage-dependent Ca2+ channels, via a pertussis toxin-sensitive GTP-binding protein. Basal 45Ca2+ uptake, stimulated by adenylate cyclase activators (forskolin and cholera toxin), was potently inhibited by kappa opiates suggesting that, under conditions of neurohormonal stimulation of adenylate cyclase, kappa receptors are coupled to Ca2+ channels indirectly via the adenylate cyclase complex. In addition, cAMP-independent coupling pathways may also be involved.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1989

Long-term opiate exposure leads to reduction of the alpha i-1 subunit of GTP-binding proteins.

Desensitization or tolerance is a major consequence of long-term opiate exposure. The mechanism of opiate desensitization is only poorly understood. We report that exposure of rat spinal cord-dorsal root ganglion cocultured neurons to kappa-opiate agonist is accompanied by a 60-70% reduction in the level of the alpha i subunit of GTP-binding proteins. Using selective antibodies, which discriminate among the various alpha i subunit forms, it was found that the opiate treatment leads to a reduction in the amount of the alpha i-1 subunit. The levels of alpha s, alpha o, and beta subunits remain unchanged. This molecular event could underlie the development of tolerance and cross-tolerance to opiates.

Blotting, Western↗

PubMed1989

Kappa-opiate agonists inhibit adenylate cyclase and produce heterologous desensitization in rat spinal cord.

The nature of the opiate modulation of adenylate cyclase following acute and chronic agonist exposure has been investigated in rat spinal cord. Using membranes of both adult rat spinal cord and spinal cord-dorsal root ganglion cocultures, we found that kappa-opiate receptors are negatively coupled to adenylate cyclase. The kappa-opiate agonists (e.g., U50488) inhibit significantly and dose-dependently the basal and the forskolin-stimulated cyclase activities, whereas mu and delta agonists are ineffective. The regulatory action is stereospecific and requires the presence of GTP. EGTA treatment of the plasma membranes abolished the effect of kappa-opiate agonists on the basal cyclase activity, and this inhibitory effect could not be restored by subsequent addition of Ca2+. The EGTA treatment did not affect the kappa agonist inhibition of the forskolin-stimulated cyclase. The results also show that following chronic exposure of cultured cells to etorphine or U50488, there is a loss of kappa agonist inhibition of the cyclase. Moreover, this desensitization process appears to be heterologous, because alpha 2-adrenergic agonists (e.g., clonidine or norepinephrine) and the muscarinic agonist (carbachol) exhibited significantly lower potency for inhibiting cyclase activity when compared to untreated cultures. This pattern of heterologous desensitization suggests that chronic exposure to kappa opiates leads to alterations in postreceptor regulatory components, possibly GTP-binding proteins.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1986

Inhibition of adenylate cyclase and induction of heterologous desensitization by kappa agonists in rat spinal cord.

Using crude P2 membranes of adult rat spinal cord we were able to show that the K opiate agonist U50488 significantly and dose-dependently inhibited the basal cyclase activity, while mu (DAGO) and delta (DADL) agonists were ineffective. The regulatory action was stereospecific and required the presence of GTP plus Na+ as well as Ca2+ ions. This inhibitory effect of K agonists was also observed when the cyclase activity was stimulated by forskolin. Similar inhibition was observed in spinal cord-dorsal root ganglion cocultures. Following chronic exposure of cultured cells to etorphine or U50488, the K agonists lost their ability to inhibit the cyclase. Furthermore, the desensitization process appeared to be heterologous, since the alpha 2 adrenergic agonist, norepinephrine and the muscarinic agonist, carbachol exhibited significant lower potency for inhibiting cyclase activity when compared to control cultures. These data suggest that in spinal cord, opiate receptors of the K type are negatively coupled to adenylate cyclase and the induction of tolerance produced by K agonists is related to alterations of post-receptor regulatory components.

Adenylyl Cyclase Inhibitors↗

PubMed1986

Expression and regulation of kappa opiate receptors in rat spinal cord-dorsal root ganglion cocultures.

We have been using rat spinal cord-dorsal root ganglion primary cocultures (SC-DG) as a model system for exploring K receptor regulation. During the first 10 days in culture, the total number of opiate receptors increased markedly, reaching a Bmax of 180 fmoles/mg protein for K sites and a Bmax of 50 fmoles/mg protein for mu sites. Following this period of development, the K and mu receptor number did not change significantly. No detectable delta sites were observed at any time of culture. The binding of [3H]diprenorphine to K sites was found to be saturable, of high affinity and stereospecific. After chronic agonist treatment of cultured cells, K receptors did not down-regulate, whereas more than 50% of the mu receptor sites did. Following chronic antagonist treatment, mu receptors were markedly up-regulated (260% of control), while K sites exhibited a weaker up-regulation response (160% of control). These data demonstrate that K opiate receptors are expressed at high concentrations in SC-DG cultures and that contrary to mu receptors in spinal cord and delta receptors in NG10815 cells, K binding sites are less susceptible to modulation following chronic agonist or antagonist treatment. This suggests that K receptors may be regulated by different control mechanisms.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1983

Interaction of selective mu and delta ligands with the kappa 2 subtype of opiate binding sites.

In the lumbo-sacral spinal cord of rat and guinea-pig 3H-etorphine selectively interacts with the kappa 2 subtype whose binding properties are distinct from those of mu, delta and kappa sites. The affinity of some mu (DAGO, morphiceptin, morphine) and delta (DADLE, DSTLE) ligands for this site are ranging from 40 to 2000nM, suggesting their significant cross-reactivity with this kappa 2 subtype. The saturation curves of 3H-etorphine in the presence of morphiceptin (2 or 10 microM) or DAGO (0.1 or 0.2 microM) or DSTLE (0.1 microM) reveal an affinity decrease without any change of the binding capacity. The KI values extrapolated from the double-reciprocal plots are similar to those calculated from the monophasic displacement curves. These results can be interpreted only in terms of a competitive-type interaction with a single class of kappa 2 sites and confirms the absence of mu and delta sites in the lumbo-sacral spinal cord of both species.

Animals↗

PubMed1982

Evidence for multiple "Kappa" binding sites by use of opioid peptides in the guinea-pig lumbo-sacral spinal cord.

Binding properties of [3H]-etorphine and [3H]-ethylketocyclazocine have been studied in the lumbo-sacral spinal cord of guinea-pig which does not contain mu or delta binding sites. [3H]-etorphine binds to a single class of high affinity sites, whereas [3H]-ethylketocyclazocine interacts with a high and a low affinity component. Using a discriminative procedure, 5 microM (D-Ala2, D-Leu5) enkephalin (DAL), the high affinity component of [3H]-ethylketocyclazocine can be resolved in two classes of sites, (D-Ala2, D-Leu5) enkephalin sensitive sites (DALS sites) and (D-Ala2, D-Leu5) enkephalin insensitive sites (DALI sites). In these conditions, there is a total loss of [3H]-etorphine sites, whose binding capacity and properties strictly correspond to the DALS sites labelled by [3H]-ethylketocyclazocine. Pharmacological investigations indicate that DALI sites for which dynorphin (1 leads to 17) is the best ligand, can be related to kappa sites previously described in guinea-pig brain, whereas DALS sites for which (Arg6, Phe7) Met-enkephalin possesses a good affinity, closely correspond to benzomorphan sites recently characterized in rat brain and spinal cord. [3H]-ethylketocyclazocine interacts additionally with "non opiate" low affinity sites, for which only benzomorphan drugs exhibit a good affinity, whereas morphine, naloxone, phencyclidine or endogenous opioid peptides do not present any affinity for them. On the basis of these data, a new subdivision of "kappa" sites is discussed.

Animals↗

PubMedDate not supplied

Characterization of [3H]-etorphine binding in guinea-pig striatum after blockade of mu and delta sites.

The guinea-pig striatum contains an apparent homogenous population of [3H]-etorphine high affinity sites (KD = 0.56 +/- 0.12 nM; Bmax = 267 +/- 47 fmoles/mg protein). The specific binding is completely abolished by 5 microM (D-Ala2, D-Leu5) enkephalin whereas an important residual binding is still present after the blockade of mu and delta sites. The binding properties of these residual sites are very similar to those of the benzomorphan sites characterized in rat brain and spinal cord. From the different binding properties of kappa and benzomorphan sites, the subdivision into kappa1 (kappa sites) and kappa2 (benzomorphan sites) is discussed.

Animals↗

PubMedDate not supplied

Differential interaction of opiates to multiple "kappa" binding sites in the guinea-pig lumbo-sacral spinal cord.

Binding characteristics of [3H]-etorphine and [3H]-ethylketocyclazocine are different in the lumbo-sacral spinal cord of guinea-pig. [3H]-etorphine binds to a single class of high affinity sites whereas [3H]-ethylketocyclazocine interacts with two components, a high affinity and a low affinity components. In the presence of 5 microM (D-Ala2, D-Leu5) enkephalin (DAL), the total high affinity sites can be resolved in two classes of sites, DAL sensitive sites (DALs sites) and DAL insensitive sites (DALI sites). In these conditions, [3H]-etorphine binding is completely abolished, and the binding capacity and properties of [3H]-etorphine correspond to the DALs sites. Pharmacological investigations indicated that DALI sites represent the kappa sites, whereas DALs sites closely correspond to benzomorphan sites described in rat brain and spinal cord. From these results, a new subclassification of "kappa" sites is proposed.

Analgesics, Opioid↗