Inhibition of protein kinase C (PKC) response of voltage-gated calcium (Cav)2.2 channels expressed in Xenopus oocytes by Cavß subunits.

Cav2.2 channels are a substrate for phosphorylation by protein kinase C (PKC) isozymes. The contribution of Cavß, an auxiliary subunit of these channels, in the PKC modulation was studied. Cav2.2 channels were expressed in Xenopus oocytes in various subunit combinations with or without Cavß subunits. Currents were recorded using a two-electrode voltage clamp with barium as the charge carrier (IBa). Acetyl-ß-methylcholine (MCh), an activator of PKCα, potentiated Cav2.2 currents expressed with Cav2.2α1 alone or Cav2.2α1α2/δ. Similarly PKC isozymes α, ßII or ɛ potentiated IBa through Cav2.2α1 subunit channels. In contrast, MCh failed to potentiate currents expressed with Cav2.2α1 and Cavß1b, ß2a, ß3 or ß4 subunits. Similarly, in the presence of Cavß1b subunits, PKC isozymes failed to potentiate these currents; contrarily, PKCs α or ßII decreased the IBa. MCh failed to potentiate Cav2.2α1 subunit currents in the serine/threonine (Ser/Thr)→alanine mutants, T422A, S1757A or S2132A of Cav2.2α1 subunits. Hence Thr-422, Ser-1757 and Ser-2132 may be PKCα isozyme target sites. The action of PKC on these sites was further substantiated by the increased basal IBa along with the loss of MCh potentiation when Ser/Thr was mutated to aspartate. The observation that MCh or PKC isozymes failed to affect Cav2.2 currents in the presence of Cavß subunits suggests that these subunits may have interfered with the interaction between PKC and Ser/Thr sites of Cav2.2α1 subunits. In addition to affecting channel expression and current kinetics, Cavß subunits may also modulate the response of these channels to neurochemicals.

Effects of isoflurane on the expressed Cav2.2 currents in Xenopus oocytes depend on the activation of protein kinase Cδ and its phosphorylation sites in the Cav2.2α1 subunits.

The effects of isoflurane on the modulation of two neuronal voltage-gated calcium channels (Ca(v); Ca(v)2.1 and 2.2) by protein kinase C (PKC) isozymes ßII, ε or δ and their combination were examined. Ca(v)2.1α1 or Ca(v)2.2α1 with ß1b and α2δ subunits were expressed in Xenopus oocytes and the currents (I(Ba)) were recorded by two-electrode voltage clamp. Isoflurane (0.70 mM) decreased both Ca(v)2.1 and 2.2 currents by 20-35% and also caused translocation of PKCδ to the membrane. Compared to the wild type (WT), isoflurane caused greater inhibition of Ca(v)2.2 currents in the absence of stimulatory PKC sites (Thr-422, Ser-1757, Ser-2108, Ser-2132) and in the presence of inhibitory PKC site (Ser-425). In contrast, isoflurane caused less inhibition of I(Ba) in the oocytes expressing S425A, the inhibitory site mutant, compared to WT. PKCδ by itself did not modulate Ca(v)2.2 currents, but potentiated these currents in the presence of isoflurane. PKCε increased Ca(v)2.2 currents either alone or in combination with isoflurane. Ca(v)2.1 currents were not modulated by phorbol-12-myristate, 13-acetate (PMA) or acetyl-ß-methylcholine (MCh), activators of PKC. Yet the presence of isoflurane caused PMA (but not MCh) to enhance Ca(v)2.1 currents. PKCßII and PKCε isozymes activated by PMA, did not alter Ca(v)2.1 currents. However, in the presence of isoflurane, these two isozymes together potentiated Ca(v)2.1 currents. The variable responses of Ca(v)2.1 currents to PKCßII and PKCε and Ca(v)2.2 currents to PKCδ in the presence of isoflurane may be due to increased affinity or accessibility of these isozymes to their Ser/Thr PKC sites of Ca(v)α1 subunits.

Site-specific regulation of CA(V)2.2 channels by protein kinase C isozymes betaII and epsilon.

Ca(v)2.2 high voltage-gated calcium channels are regulated by phorbol-12-myristae, 13-acetate (PMA) via Ser/Thr protein kinase C (PKC) phosphorylation sites in the I-II linker and C-terminus of the alpha(1) 2.2 subunit. Here we show that PMA enhancement of Ca(v)2.2 currents expressed in Xenopus oocytes can be blocked by inhibitors of PKC betaII or PKC epsilon isozymes, as shown previously for Ca(v)2.3 currents, and that microinjection of PKC betaII or PKC epsilon isozymes in the oocytes expressing the WT Ca(v)2.2 channels increases the basal barium current (I(Ba)). The I-V plot shows a large increase in current amplitude with PKC betaII and PKC epsilon isozymes with only a small shift in the peak I(Ba) in the hyperpolarizing direction. The potentiation of Ca(v)2.2 currents by microinjection of PKC betaII and PKC epsilon isozymes was not altered by the inhibition of G proteins with GDPbetaS. The combination of isozyme specific inhibitors with previously generated Ser/Thr to Ala mutants of alpha(1) 2.2 subunit revealed that PKC betaII or PKC epsilon isozymes (but not PKC alpha or delta) can provide full enhancement through the stimulatory site (Thr-422) in the I-II linker but that PKC epsilon is better at decreasing channel activity through the inhibitory site Ser-425. The enhancing effect of PKC betaII or epsilon at Thr-422 is dominant over the inhibitory effect at Ser-425. Injected PKC betaII also enhances Ca(v)2.2 current when any of the potential stimulatory sites (Ser-1757, Ser-2108 and Ser-2132) are available in the C-terminus. PKC epsilon provides lesser enhancement with C-terminal sites and only with Ser-2108 and Ser-2132. Sites Ser-1757 and Ser-2132, but not Ser-2108, are dominant over the inhibitory site Ser-425. Collectively, these results reveal a hierarchy of regulatory sites in Ca(v)2.2 channels. Site-specific regulation by different PKC isozymes may allow graded levels of channel activation and susceptibility or resistance to subsequent stimulatory events.

The effects of isoflurane on native and chimeric muscarinic acetylcholine receptors: the role of protein kinase C.

UNLABELLED: By using two electrode voltage clamps, we investigated the effects of isoflurane on m3 and chimeric m1/m3 muscarinic receptors and the role of protein kinase C (PKC) in the effects. Muscarinic receptors were expressed by injection of mRNA into Xenopus oocytes, and Ca(2+)-activated Cl(-) currents were measured after the application of acetyl-beta-methylcholine. We constructed chimeric m1/m3 receptor DNA encoding the third intracellular loop of m1 and the remainder from the m3 receptor. Chimeric and m3 receptors were inhibited by isoflurane, but the m1 receptor was not. PKC activation with phorbol-12-myrisate-13-acetate (50 nM) decreased signaling of both chimeric and m3 receptors significantly. Chelerythrine (20 microM, PKC inhibitor) abolished the effect of isoflurane on chimeric and m3 signaling. Whereas isoflurane inhibition of chimeric and m3 receptors was completely reversible after washout with Tyrode's solution for 3 min, treatment with okadaic acid (500 nM, protein phosphatase inhibitor) rendered the inhibition irreversible. Taken together, our results suggest that isoflurane inhibits m3 and chimeric m1/m3 muscarinic signaling by enhancing PKC activity and that the site of action is located outside of the third intracellular loop. IMPLICATIONS: By use of the Xenopus oocyte expression system, we investigated the effects of isoflurane on muscarinic signaling and the role of protein kinase C in these effects. Our findings suggest that isoflurane inhibits muscarinic receptors through activation of protein kinase C and that the relevant phosphorylation sites are located outside the third intracellular loop.

Differential sensitivity of expressed L-type calcium channels and muscarinic M(1) receptors to volatile anesthetics in Xenopus oocytes.

Since volatile anesthetics inhibited high voltage-gated calcium channels and G-protein-coupled M(1) muscarinic signaling, their effects upon M(1) receptor-induced modulation of L-type (alpha1C) calcium channel was investigated. Voltage-clamped Ba(2+) currents (I(Ba)) were measured in Xenopus oocytes coexpressed with L-type channels and M(1) muscarinic receptors. M(1) receptor agonist, acetyl-beta-methylcholine (MCh) inhibited the peak and late components of I(Ba) in a dose-dependent manner. Analysis of I(Ba) after the treatment with MCh or volatile anesthetics revealed that the inactivating component, its time constant, and the noninactivating current were all decreased by these agents. MCh-induced inhibition followed a second messenger pathway that included G-proteins, phospholipase C, inositol-1,4,5-trisphosphate, and intracellular calcium [Ca(2+)](i). Although halothane or isoflurane inhibited I(Ba,) their effect was not mediated through these intracellular second messengers. By using volatile anesthetics and MCh sequentially, and in various combinations, the susceptibility of L-type currents and their modulation by M(1) receptors to volatile anesthetics were investigated. When MCh and volatile anesthetics were administered together simultaneously, a pronounced inhibition that was approximately equal to the sum of their individual effects was seen. Halothane or isoflurane further inhibited the I(Ba) when either volatile anesthetic was administered following the inhibition produced by prior administration of MCh. However, when MCh was administered following either volatile anesthetic, its effect was significantly reduced. Thus, whereas volatile anesthetics appear to directly inhibit L-type channels, they also interfere with channel modulation by G-protein-coupled receptors, which may have functional implications for both neuronal and cardiovascular tissues.

Inhibition of m3 muscarinic acetylcholine receptors by local anaesthetics.

1. Muscarinic m1 receptors are inhibited by local anaesthetics (LA) at nM concentrations. To elucidate in more detail the site(s) of LA interaction, we compared these findings with LA effects on m3 muscarinic receptors. 2. We expressed receptors in Xenopus oocytes. Using two-electrode voltage clamp, we measured the effects of lidocaine, QX314 (permanently charged) and benzocaine (permanently uncharged) on Ca(2+)-activated Cl(-)-currents (I(Cl(Ca))), elicited by acetyl-beta-methylcholine bromide (MCh). We also characterized the interaction of lidocaine with [(3)H]-quinuclydinyl benzylate ([(3)H]-QNB) binding to m3 receptors. Antisense-injection was used to determine the role of specific G-protein alpha subunits in mediating the inhibitory effects of LA. Using chimeric receptor constructs we investigated which domains of the muscarinic receptors contribute to the binding site for LA. 3. Lidocaine inhibited m3-signalling in a concentration-dependent, reversible, non-competitive manner with an IC(50) of 370 nM, approximately 21 fold higher than the IC(50) (18 nM) reported for m1 receptors. Intracellular inhibition of both signalling pathways by LA was similar, and dependent on the G(q)- protein alpha subunit. In contrast to results reported for the m1 receptor, the m3 receptor lacks the major extracellular binding site for charged LA. The N-terminus and third extracellular loop of the m1 muscarinic receptor molecule were identified as requirements to obtain extracellular inhibition by charged LA.

Effects of volatile anesthetics on the direct and indirect protein kinase C-mediated enhancement of alpha1E-type Ca(2+) current in Xenopus oocytes.

The effect of the volatile anesthetics (VAs) halothane (0.59 mM) and isoflurane (0.70 mM) on protein kinase C (PKC)-mediated modulation of alpha1E type of high-voltage-gated Ca(2+) channels was examined in Xenopus oocytes coexpressing m1 muscarinic acetylcholine receptors. Phorbol-12-myristate-13-acetate (PMA) or 1, 2-dioctanoyl-sn-glycerol (DOG) was used to activate PKC directly, whereas indirect activation was induced with acetyl-beta-methylcholine (MCh). The interaction between PKC activators and VAs was examined by perfusing either VA before, during, or after the administration of PMA, DOG, or MCh. In addition, the effect of VAs was studied after the down-regulation of PKC. The application of VAs inhibited Ba(2+) current (I(Ba)), whereas PMA (500 nM), DOG (100 microM), or MCh (1 and 10 microM) markedly potentiated I(Ba). VAs inhibited PMA- or DOG-enhanced I(Ba) to the same extent as seen in controls. The inhibition of I(Ba) induced by VAs was not reversed by PMA or DOG. The administration of VAs in combination with PMA, DOG, or MCh (1 microM) led to the inhibition of I(Ba). MCh (10 microM) counteracted the inhibitory effect of VAs when administered together and reversed the inhibition of I(Ba) produced by VAs. These differences in the responses between PMA and MCh (10 microM) may be based on the involvement of various pools of PKC. It is suggested that VAs act directly at the membrane, because they blocked the membrane-based action of PMA, whereas the receptor-based action of MCh was only partially blocked. It is possible that some PKC isoforms may not be a direct target of VAs.

Volatile anesthetic inhibition of neuronal Ca channel currents expressed in Xenopus oocytes.

The genes encoding the alpha(1A), alpha(1B), alpha(1C) and alpha(1E) subunits of neuronal high voltage-gated Ca channels (HVGCCs) were separately expressed with beta(1B) and alpha(2)/delta subunits in Xenopus oocytes to determine the effects of volatile anesthetics (VAs) on currents through each specific channel. VA effects were determined on currents carried by Ba(2+) (I(Ba)) using the two electrode voltage clamp technique. Although time to peak was unaffected, both halothane (0.59 mM) and isoflurane (0.70 mM) reversibly inhibited peak I(Ba) by 25-35% and late current (at 830 ms) by 50-60%. A hyperpolarizing shift in steady-state inactivation of alpha(1E)-current was found which could contribute up to one third of observed decrease in the peak current. The rate of inactivation of I(Ba) seen with alpha(1A), alpha(1B) and alpha(1E)-type Ca channels was consistently increased by halothane and isoflurane. To more clearly quantify these effects, I(Ba) inactivation was fit by a single exponential function. The anesthetics depressed both the inactivating and non-inactivating residual components of I(Ba) and decreased the time constant of inactivation. In the case of I(Ba) through alpha(1C)-type channels, inactivation was minimal; however, the average current was inhibited by VAs. Similar inhibition of all these HVGCCs by halothane and isoflurane suggests that a common structural component may be involved. Furthermore, the inhibition of such neuronal HVGCCs in situ could alter synaptic neurotransmitter release and contribute to the anesthetic state.

The alpha5 subunit of the murine type A GABA receptor.

GABA[A] receptors in the brain convert binding of GABA (gamma-aminobutyric acid) to inhibition by chloride currents. Several important classes of drugs, including benzodiazepines and alcohol, modulate these receptors, which have also been implicated in epilepsy. We describe the alpha5 subunit of GABAA receptors in mice, comparing inbred DBA/2J mice, prone to juvenile audiogenic seizures, with seizure resistant C57BL/6J mice. We find no sequence differences between the strains, although there are several interesting amino acid differences from the rat. We also compare the expression of the alpha5 subunit in whole brains of DBA/2J mice to that in C57BL/6J mice at 21 days, the peak of the former's seizure susceptibility, again finding no significant difference. We further describe the pattern of expression of alpha5 mRNA during mouse brain development, with a peak at 3 days after birth, and among five brain regions in the adult mouse, with the highest levels in the hippocampus. Finally, we present preliminary evidence for rare alternative splicing of this subunit's message, in the N-terminal extracellular domain, to give a form not translatable into a functional protein.

Inhibition of neuronal Na+ channels by antidepressant drugs.

Although tricyclic antidepressant (TCA) blockade of cardiac Na+ channels is appreciated, actions on neuronal Na+ channels are less clear. Therefore, the effects of TCAs (amitriptyline, doxepin and desipramine) as well as trazdone and fluoxetine on voltage-gated Na+ current (INa) were examined in bovine adrenal chromaffin cells using the whole-cell patch-clamp method. Amitriptyline produced concentration-dependent depression of peak INa evoked from a holding potential of -80 mV with KD value of 20.2 microM and a Hill coefficient of 1.2. Although 20 microM amitriptyline induced no change in the rate or voltage dependence of INa activation, steady-state inactivation demonstrated a 15-mV hyperpolarizing shift. Similar results were observed for doxepin and desipramine. This shift in steady-state inactivation was associated with a slowed rate of recovery from the inactivated state. Contrasting results were observed with the atypical antidepressants: while 20 microM fluoxetine depressed peak INa by 61% and caused a 7-mV hyperpolarizing shift in steady-state inactivation, 100 microM trazodone decreased peak INa by only 19% and caused only a 3-mV shift. Although the magnitude of fluoxetine effects was similar to those of the TCAs, the onset of fluoxetine effects was substantially slower than for amitriptyline. In voltage-clamp and current-clamp measurements from neonatal rat dorsal root ganglion neurons, 20 microM amitriptyline decreased INa by 52% and depressed action potential dynamics consistent with enhanced Na+ channel inactivation. The effects of the TCAs on INa are similar to local anesthetic behavior and could contribute to certain analgesic actions.

Volatile anaesthetics have differential effects on recombinant m1 and m3 muscarinic acetylcholine receptor function.

Muscarinic acetylcholine signalling plays major roles in regulation of consciousness, cognitive functioning, pain perception and circulatory homeostasis. Halothane has been shown to inhibit m1 muscarinic signalling. However, no comparative data are available for desflurane, sevoflurane or isoflurane, nor have the anaesthetic effects on the m3 subtype (which is also prominent in the brain) been studied. Therefore, we have investigated the effects of these compounds on isolated m1 and m3 muscarinic receptor function. Defolliculated Xenopus oocytes expressing recombinant m1 or m3 muscarinic or (for comparison) AT1A angiotensin II receptors were voltage clamped, and Ca(2+)-activated Cl- currents (ICl(Ca)) induced by acetyl-beta-methylcholine (Mch) or angiotensin II were measured in the presence of clinically relevant concentrations of halothane, sevoflurane, desflurane or isoflurane. To determine the site of action of the volatile anaesthetics we compared anaesthetic effects on m1, m3 and AT1A receptor function and studied the effects of volatile anaesthetics on signalling induced by intracellular injection of the second messenger IP3. Desflurane had a biphasic effect on m1 signalling, enhancing at a concentration of 0.46 mmol litre-1 but depressing at 0.92 mmol litre-1. A similar, although not significant, trend was observed with m3 signalling. Isoflurane had no effect on m1 signalling, but profoundly inhibited m3 signalling. Sevoflurane depressed the function of m1 and m3 signalling in a dose-dependent manner. Halothane, similar to its known effect on m1 signalling, dose-dependently depressed m3 function. ICl(Ca) induced by intracellular injections of IP3 were unaffected by all four anaesthetics. Similarly, none of the anaesthetics tested affected AT1A signalling. Absence of interference with AT1A signalling and intracellular pathways suggest that the effects of anaesthetics on muscarinic signalling most likely result from interactions with the m1 or m3 receptor molecule. Multiple interaction sites with different affinities may explain the biphasic response to desflurane. Anaesthetic-specific effects on closely related receptor subtypes suggest defined sites of action for volatile anaesthetics on the receptor protein.

GABAA receptor beta 1, beta 2, and beta 3 subunits: comparisons in DBA/2J and C57BL/6J mice.

GABAA receptors link binding of GABA (gamma-aminobutyric acid) to inhibitory chloride flux in the brain. They are the site of action of several important classes of drugs, and have been implicated in animal models of epilepsy and in the actions of alcohol. We compare the sequence and expression of the beta 1, beta 2 and beta 3 subunits of GABAA receptors in two inbred strains of mice, DBA/2J and C57BL/6J, which differ markedly in seizure susceptibility and in a variety of behaviors related to alcohol. Only the beta 3 subunit had strain differences in cDNA nucleotide sequence, which did not affect amino acid sequence but which did create restriction fragment length polymorphisms (RFLPs) potentially useful in gene mapping. We have also tested mouse beta 1 and beta 2 subunits for internal alternative splicing, detecting none.

GABAA receptor subtypes: from pharmacology to molecular biology.

GABAA receptors are GABA (gamma-aminobutyric acid)-gated chloride channels, which are major mediators of neuronal inhibition in the brain and are modulated by benzodiazepines, barbiturates, alcohol, and other important centrally acting drugs. Although previous pharmacological and biochemical data had suggested a degree of heterogeneity, recent cloning of at least 15 different receptor subunits, thought to be combined in groups of five, indicates that the brain may contain a truly astonishing variety of GABAA receptor subtypes. This review describes the little that is known about these subtypes, emphasizing possible molecular bases of receptor heterogeneity. We also discuss approaches to establishing the subunit composition of subtypes.

Effect of anticonvulsant felbamate on GABAA receptor system.

Felbamate (FBM, 2-phenyl-1,3-propanediol dicarbamate), a potential antiepileptic drug (AED), has an unknown mechanism of action. We examined possible interaction of FBM with GABAA ergic transmission. FBM did not alter specific binding of ligands to GABA. benzodiazepine, and picrotoxin sites of the oligomeric GABAA receptor complex to rat brain membranes, nor did it enhance the effect of GABA on 36Cl-influx in well-characterized cultured spinal cord neurons. These results suggest that the anticonvulsant effect of FBM does not involve GABAA ergic transmission.

Tricyclic antidepressants inhibit Ca(2+)-activated K(+)-efflux in cultured spinal cord neurons.

The effect of tricyclic antidepressants and monoamine oxidase inhibotors on the Ca(2+)-activated K(+)-efflux was studied using 86Rb-efflux assay in primary cultured mouse spinal cord neurons. Depolarization of the cultured cells with 100 mM KCl increased the 86Rb-efflux significantly in Ca(2+)-containing buffer, but not in Ca(2+)-free buffer. All the antidepressants examined, except the monoamine oxidase inhibitors, inhibited the 86Rb-efflux. Desipramine exhibited additivity with tetraethyl ammonium (TEA) and quinine sulfate (QSO4), but not with GABAB receptor agonist baclofen. The inhibitory action of antidepressants was not mediated through the GABAB receptors, since GABAB receptor antagonist, phaclofen, was unable to antagonize this effect. The ability of tricyclic antidepressants to inhibit calcium ionophore (A 23187)-induced 86Rb-efflux suggests that these drugs do not act at the level of voltage-gated Ca(2+)-channels. Furthermore, this effect does not seem to involve the G-proteins, adenylate cyclase, or protein kinase C systems, since pertussis toxin (PTX) and the activators of adenylate cyclase and protein kinase C did not reverse the effect of tricyclics on 86Rb-efflux. Taken together, these results suggest that antidepressants inhibit Ca(2+)-activated K(+)-channels at a stage subsequent to the voltage-gated Ca(2+)-channels.

A functional assay to measure postsynaptic gamma-aminobutyric acidB responses in cultured spinal cord neurons: heterologous regulation of the same K+ channel.

The stimulation of postsynaptic gamma-aminobutyric acid (GABA)B receptors leads to slow inhibitory postsynaptic potentials due to the influx of K(+)-ions. This was studied biochemically, in vitro in mammalian cultured spinal cord neurons by using 86Rb as a substitute for K+. (-)-Baclofen, a GABAB receptor agonist, produced a concentration-dependent increase in the 86Rb-influx. This effect was stereospecific and blocked by GABAB receptor antagonists like CGP 35 348 (3-aminopropyl-diethoxymethyl-phosphonic acid) and phaclofen. Apart from the GABAB receptors, both adenosine via adenosine1 receptors and 5-hydroxytryptamine (5-HT) via 5-HT1 alpha agonists also increased the 86Rb-influx. These agonists failed to show any additivity between them when they were combined in their maximal concentration. In addition, their effect was antagonized specifically by their respective antagonists without influencing the others. These findings suggest the presence of GABAB, adenosine1 and 5-HT1 alpha receptors in the cultured spinal cord neurons, which exhibit a heterologous regulation of the same K(+)-channel. The effect of these agonists were antagonized by phorbol 12,13-didecanoate, an activator of protein kinase C, and pretreatment with pertussis toxin. This suggests that these agonists by acting on their own receptors converge on the same K(+)-channel through the Gi/Go proteins. In summary, we have developed a biochemical functional assay for studying and characterizing GABAB synaptic pharmacology in vitro, using spinal cord neurons.

Functional coupling of presynaptic GABAB receptors with voltage-gated Ca2+ channel: regulation by protein kinases A and C in cultured spinal cord neurons.

Depolarization-induced 86Rb efflux, an index of K+ efflux, was developed by using mammalian cultured spinal cord neurons to study the effect of gamma aminobutyric acid (GABAB) receptor activation on Ca2(+)-activated K(+)-channels. The Ca2(+)-activated 86Rb efflux was obtained by using two methods. The first method utilized depolarizing concentrations of KCl (100 mM) to study the voltage-gated Ca2+ channel activation, whereas in the second method, calcium ionophore A23187 was used to get the voltage-independent Ca2(+)-activated 86Rb efflux. The GABAB receptor agonist baclofen inhibited the efflux induced by depolarization but not by A23187, whereas tricyclic antidepressant desipramine inhibited the efflux induced by both depolarization and A23187. These results suggest that the GABAB receptor activation inhibits 86Rb efflux by inhibiting the voltage-gated Ca2+ channels. Moreover, forskolin and the analogs of cAMP antagonized the action of baclofen, suggesting that the GABAB receptors are negatively coupled to adenylate cyclase. Furthermore, protein kinase C activators antagonized this action of baclofen, while the antagonists of protein kinase C reversed their action on baclofen. In addition, the inactive forskolin, 1,9-dideoxyl forskolin, and the inactive phorbol analog, phorbol 12,13-didecanoate, did not influence the action of baclofen. Thus, it is suggested that the GABAB receptor activation inhibited the voltage-gated Ca2+ influx and that this action is under modulatory control by kinases A and C.

GABAB receptor activation inhibits Ca2(+)-activated 86Rb-efflux in cultured spinal cord neurons via G-protein mechanism.

86Rb-efflux assay in primary cultured spinal cord neurons was developed to study the effect of GABAB receptor activation on Ca2(+)-activated K(+)-channels. Depolarization of the cultured cells with 100 mM KCl increased the 86Rb-efflux significantly. This efflux was blocked partly by quinine sulfate, tetraethylammonium, and La3+, indicating the involvement of Ca2(+)-activated K(+)-channels. Both (-)-baclofen and GABA inhibited the Ca2(+)-activated 86Rb-efflux. This inhibition seems to be mediated through GABAB receptor activation as it was blocked by GABAB antagonist phaclofen, but not by bicuculline. Moreover, pertussis toxin blocked the ability of (-)-baclofen to inhibit the Ca2(+)-activated 86Rb-efflux, showing that GABAB receptor activation involves G-protein mechanism. Further, forskolin and phorbol ester also attenuated the action of (-)-baclofen. This suggests that the GABAB receptors are negatively coupled to adenylate cyclase. These results show that the action of GABAB receptors involved G-proteins and adenylate cyclase. This assay may provide an ideal model to study GABAB receptor pharmacology.

Effect of short course chemotherapy on salivary paracetamol elimination.

1. Salivary elimination of paracetamol was studied in six male pulmonary tuberculosis patients on short course chemotherapy. 2. Peak paracetamol concentration showed a steady decrease at the end of first week and continued until the end of the study. 3. The elimination half-life of paracetamol decreased 5 weeks after the therapy with a corresponding increase in the clearance rate. 4. Adjustment of dosage of drugs that are metabolized by the microsomal enzymes may be required in patients on anti-tubercular drug regimen in which rifampicin is included.