Spinal morphine/clonidine antinociceptive synergism: involvement of G proteins and N-type voltage-dependent calcium channels

When morphine and clonidine are coadministered into the spinal cord (intrathecally) the resulting antinociception is greater than would be expected if the drug responses were additive; thus, a synergistic interaction. The mechanism for this synergistic interaction was investigated using agents which alter calcium channel function and G protein function. Drugs were administered intrathecally to mice and antinociception was measured using the tail flick test. The L-type calcium channel antagonists nifedipine (15 micrograms) and verapamil (15 micrograms) and the N-type antagonist omega-conotoxin GVIA (3 and 30 ng) decreased ED50 values for both morphine and clonidine three-to five-fold. The L-type calcium channel activator Bay K 8644 had a biphasic effect; 1.7 ng increased, although 170 ng decreased, morphine and clonidine ED50 values. None of the calcium channel modifiers affected the morphine/clonidine synergism. In mice pretreated with pertussis toxin (PTX, one, 10-ng dose 21 days previously), the morphine ED50 value increased two-fold, although the clonidine ED50 value was not changed. PTX pretreatment did not alter the morphine/clonidine synergism. Also, in PTX-pretreated mice, nifedipine and 1.7 ng Bay K 8644 did not alter the morphine/clonidine synergism. However, in PTX-pretreated animals omega-conotoxin GVIA (3 ng) changed the morphine/clonidine synergism to an additive interaction. Thus, both N-type calcium channels and PTX-sensitive G proteins are likely involved in spinal morphine/clonidine synergism.     

The importance of calmodulin in the accessory olfactory bulb in the formation of an olfactory memory in mice

Female mice form an olfactory memory to the pheromones of the mating male, during a critical period after mating. Failure to form this memory results in the male being treated as strange, and hence, his pheromones block pregnancy. Previous studies have shown that formation of this memory is dependent on synaptic mechanisms in the accessory olfactory bulb. A number of studies have pointed to calmodulin as a critical mediator of synaptic plasticity. In this study we have examined the effects of local infusions of drugs which block calmodulin-regulated processes, into the accessory olfactory bulb on the formation of this memory. Infusions of the calmodulin antagonist calmidazolium during the critical period prevented memory formation. However, the specific inhibitor of calcium/calmodulin-dependent protein kinase II, KN-62, or the selective inhibitor of calcium/calmodulin-dependent protein phosphatase 2B (calcineurin), FK506, was without effect on memory formation at any of the doses used. Instead of preventing memory formation, FK506 permitted the formation of a non-selective memory to strange male pheromones in the presence of mating, although FK506 alone could not induce a memory without the occurrence of mating. These results suggest that calmodulin in the accessory olfactory bulb is important in the formation of the olfactory memory to male pheromones. However, memory formation may be independent of calmodulin-kinase II. Calcineurin may play a role in processes antagonizing memory formation.     

High-voltage-activated calcium currents in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus

We studied the high-voltage-activated (HVA) calcium currents in cells isolated from the ventrobasal nucleus of the rat thalamus with the use of the whole cell patch-clamp technique. Low-voltage-activated current was inactivated by the use of long voltage steps or 100-ms prepulses to -20 mV. We used channel blocking agents to characterize the currents that make up the HVA current. The dihydropyridine (DHP) antagonist nimodipine (5 microM) reversibly blocked 33 +/- 1% (mean +/- SE), and omega-conotoxin GVIA (1 microM) irreversibly blocked 25 +/- 5%. The current resistant to DHPs and omega-conotoxin GVIA was inhibited almost completely by omega-conotoxin MVIIC (90 +/- 5% at 3-5 microM) and was partially inhibited by omega-agatoxin IVA (54 +/- 4% block at 1 microM). We conclude that there are at least four main HVA currents in thalamic neurons: N current, L current, and two omega-conotoxin MVIIC-sensitive currents that differ in their sensitivity to omega-agatoxin IVA. We also examined modulation of HVA currents by strong depolarization and by G protein activation. Long (approximately 1 s), strong depolarizations elicited large, slowly deactivating tail currents, which were sensitive to DHP antagonists. With guanosine 5'-0-(3-thiotriphosphate) (GTP-gamma-S) in the intracellular solution, brief (approximately 20 ms), strong depolarization produced a voltage-dependent facilitation of the current (44 +/- 5%), compared with cells with GTP (22 +/- 7%) or guanosine 5'-O-(2-thiodiphosphate) (7 +/- 4%). However, the HVA current was inhibited only weakly by 100 microM acetylcholine (8 +/- 4%). Effects of the gamma-aminobutyric acid-B agonist baclofen were variable (3-39% inhibition, n = 12, at 10-50 microM).     

Met]enkephalin in the spinal cord is involved in the antinociception induced by intracerebroventricularly-administered etorphine in the mouse

We have recently reported that the antinociception induced by etorphine given i.c.v. is mediated in part by the stimulation of both mu- and epsilon-opioid receptors and the activation of both monoaminergic and opioidergic descending pain control systems. [Xu J. Y. et al. (1992) J. Pharmac. exp. Ther. 263, 246-252]. Since the opioid epsilon-receptor-mediated antinociception induced by beta-endorphin is mediated by the release of [Met]enkephalin and subsequent stimulation of delta-opioid receptors in the spinal cord, the present studies were designed to determine if beta-endorphin-like action is also involved in etorphine-induced antinociception. The tail-flick test was used to assess the antinociceptive response performed in male ICR mice. Etorphine at doses from 5 to 20 ng given i.c.v. produced a dose-dependent inhibition of the tail-flick response. The inhibition of the tail-flick response induced by etorphine given i.c.v. was antagonized by intrathecal pretreatment for 60 min with antiserum against [Met]enkephalin (10 microg), but not with antiserum against [Leu]enkephalin (10 microg) or dynorphin A (1-13) (10 microg). Desensitization of delta-opioid receptors in the spinal cord by intrathecal pretreatment with [Met]enkephalin (5 microg) for 60 min attenuated i.c.v. administered etorphine-induced tail-flick inhibition. However, intrathecal pretreatment with [Leu]enkephalin (5 microg) or dynorphin A (1-17) (0.1 microg) for 60 min did not attenuate i.c.v. administered etorphine-induced tail-flick inhibition. The results indicate that antinociception induced by etorphine given i.c.v. is mediated in part by the stimulation of the epsilon-opioid receptor at the supraspinal sites and by the release of [Met]enkephalin, which subsequently stimulates delta-opioid receptors in the spinal cord.     

Energy levels in resting and mitogen-stimulated human lymphocytes during treatment with FK506 or cyclosporin A in vitro

The effects of the adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA), on both the increase in intracellular free Ca2+ concentration ([Ca2+]i) and on the release of endogenous glutamate in rat hippocampal synaptosomes were studied. The inhibitory effect of CPA on the increase in [Ca2+]i stimulated with 4-aminopyridine was neutralized by the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The inhibitory effect of CPA was greater in synaptosomes from the CA1 subregion than in whole hippocampal synaptosomes. The inhibitory effects of both CPA and of the Ca2+ channel blockers, omega-conotoxin GVIA, omega-conotoxin MVIIC or omega-conotoxin GVIA plus omega-conotoxin MVIIC, were greater than those caused by the Ca2+ channel blockers. The release of endogenous glutamate was inhibited by 41% by CPA. The inhibition observed when CPA and omega-conotoxin GVIA or CPA and omega-conotoxin MVIIC were present was also greater than the inhibition by the Ca2+ channel blockers alone. The presence of both omega-conotoxin GVIA and omega-conotoxin MVIIC did not completely inhibit the release of glutamate, and CPA significantly enhanced this inhibition. The membrane potential and the accumulation of [3H]tetraphenylphosphonium of polarized or depolarized synaptosomes was not affected by CPA, suggesting that adenosine did not increase potassium conductances. The present results suggest that, in hippocampal glutamatergic nerve terminals, adenosine A1 receptor activation partly inhibits P/Q- and other non-identified types of Ca2+ channels.     

Calcium oxalate crystal attachment to cultured kidney epithelial cell lines

Lamprey spinal neurons exhibit a fast afterhyperpolarization and a late afterhyperpolarization (AHP) which is due to the activation of apamin-sensitive SK Ca2+-dependent K+ channels (KCa) activated by calcium influx through voltage-dependent channels during the action potential (Hill et al. 1992, Neuroreport, 3, 943-945). In this study we have investigated which calcium channel subtypes are responsible for the activation of the KCa channels underlying the AHP. The effects of applying specific calcium channel blockers and agonists were analysed with regard to their effects on the AHP. Blockade of N-type calcium channels by omega-conotoxin GVIA resulted in a significant decrease in the amplitude of the AHP by 76.2+/-14.9% (mean +/- SD). Application of the P/Q-type calcium channel blocker omega-agatoxin IVA reduced the amplitude of the AHP by 20.3+/-10.4%. The amplitude of the AHP was unchanged during application of the L-type calcium channel antagonist nimodipine or the agonist (+/-)-BAY K 8644, as was the compound afterhyperpolarization after a train of 10 spikes at 100 Hz. The effects of calcium channel blockers were also tested on the spike frequency adaptation during a train of action potentials induced by a 100-200 ms depolarizing pulse. The N- and P/Q-type calcium channel antagonists decreased the spike frequency adaptation, whereas blockade of L-type channels had no effect. Thus in lamprey spinal cord motor- and interneurons, apamin-sensitive KCa channels underlying the AHP are activated primarily by calcium entering through N-type channels, and to a lesser extent through P/Q-type channels.     

Involvement of calcium and L-type channels in nicotine-induced antinociception

The nature of the signaling process activated by neuronal nicotinic receptors has not been fully defined; however, several recent studies have implicated the involvement of calcium ion fluxes in the response to nicotine on a cellular level. Alteration of nicotine-induced antinociception in mice after systemic administration was therefore investigated in the presence of several drugs that increase intracellular calcium. Calcium, (+/-)-BAYK 8644, thapsigargin, glyburide and A23187 administered intrathecally (i.t.) were found to enhance nicotine-induced antinociception by shifting its dose-response curve to the left. Conversely, i.t. administration of agents which decrease intracellular calcium, such as EGTA and alpha-calcitonin gene-related peptide, blocked nicotine-induced antinociception. These findings support a role for spinal intracellular calcium in the pharmacological effects of nicotine. Additionally, blockade of antinociception by nimodipine and nifedipine indicates that a L-type calcium channel is involved in nicotine's effect. However, nicotine did not compete for [3H] nitrendipine binding. Intrathecal administration of mecamylamine, a nicotinic antagonist, resulted in a blockade of antinociception produced by the i.t. injection of thapsigargin, A23187, calcium and (+/-)-BAYK 8644. The mechanism of mecamylamine's antagonism of nicotine is uncertain. However, these results suggest that mecamylamine blocks the effects of drugs which increase intracellular calcium by either a modulation of intracellular calcium-dependent mechanisms or a blockade of calcium channels. Thus, mecamylamine could modulate a calcium signaling process secondary to receptor activation resulting in blockade of antinociception produced by diverse agents.     

The effect of dark and equiluminant occlusion on the interocular transfer of visual aftereffects

Because changes in intracellular Ca2+ affect progression through the mitotic cell cycle, we investigated the role of Ca2+-binding proteins in regulating cell cycle progression. Evidence was found demonstrating that the activation of Ca2+/calmodulin-dependent protein kinase (CaM kinase) inhibits cell cycle progression in small cell lung carcinoma (SCLC) cells. We also demonstrated that SCLC cells express both CaM kinase type II (CaMKII) and CaM kinase type IV (CaMKIV). Five independent SCLC cell lines expressed proteins reactive with antibody to the CaMKII beta subunit, but none expressed detectable proteins reactive with antibody to the CaMKII alpha subunit. All SCLC cell lines tested expressed both the alpha and beta isoforms of CaMKIV. Immunoprecipitation of CaMKII from SCLC cells yielded multiple proteins that autophosphorylated in the presence of Ca2+ / calmodulin. Autophosphorylation was inhibited by the CaMKII(281-302) peptide, which corresponds to the CaMKII autoinhibitory domain, and by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine (KN-62), a specific CaM kinase antagonist. Influx of Ca2+ through voltage-gated Ca2+ channels stimulated phosphorylation of CaMKII in SCLC cells, and this was inhibited by KN-62. Incubation of SCLC cells of KN-62 potently inhibited DNA synthesis, and slowed progression through S phase. Similar anti-proliferative effects of KN-62 occurred in SK-N-SH human neuroblastoma cells, which express both CaMKII and CaMKIV, and in K562 human chronic myelogenous leukemia cells, which express CaMKII but not CaMKIV. The expression of both CaMKII and CaMKIV by SCLC cells, and the sensitivity of these cells to the anti-proliferative effects of KN-62, suggest a role for CaM kinase in regulating SCLC proliferation.     

Differences in the influence of AIN-purified and non-purified diets on the development of hypertension in SHR and DOCA-salt hypertensive rats

The effect of nicotine administered supraspinally on antinociception induced by supraspinally administered opioids was examined in ICR mice. The intracerebroventricular (i.c.v.) injection of nicotine alone at doses from 1 to 12 micrograms produced only a minimal inhibition of the tail-flick response. Morphine (0.2 micrograms), beta-endorphin (0.1 microgram), D-Pen2.5-enkephalin (DPDPE; 0.5 microgram), trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzeocetamide (U50, 488H; 6 micrograms) caused only slight inhibition of the tail-flick response. Nicotine dose dependently enhanced inhibition of the tail-flick response induced by i.c.v. administered morphine (0.2 microgram) or beta-endorphin (0.1 microgram). The degree of enhancing effect of nicotine toward beta-endorphin-induced inhibition of the tail-flick response was greater than toward morphine-induced inhibition of the tail-flick response. However, i.c.v. administered nicotine at the same doses was not effective in enhancing the inhibition of the tail-flick response induced by DPDPE (0.5 microgram) or U50, 488H (6 micrograms) administered i.c.v. Our results suggest that stimulation of supraspinal nicotinic receptors may enhance antinociception induced by morphine (a mu-opioid receptor agonist) and beta-endorphin (an epsilon-opioid receptor agonist) administered supraspinally. However, the activation of nicotinic receptors at supraspinal sites may not be involved in enhancing the antinociception induced by DPDPE (a delta-opioid receptor agonist) or U50, 488H (a kappa-opioid receptor agonist) administered supraspinally.     

Inhibition of acetylcholine release from presynaptic terminals of skate electric organ by calcium channel antagonists: a detailed pharmacological study

Release of acetylcholine (ACh) from the presynaptic terminals in skate electric organ was tested for its sensitivity to calcium channel antagonists. A pharmacological profile was established by measuring inhibition of K(+)-stimulated release of [3H]ACh from prelabelled tissue slices. Peptide antagonists of N-type (omega-conotoxins GVIA and MVIIA) and P-type (omega-agatoxin-IVA) channels had no effect, whereas both omega-conotoxins MVIIC and SVIB produced concentration-dependent inhibition and could completely block ACh release. omega-Conotoxin GVIA and omega-agatoxin IVA did not attenuate the block by omega-conotoxin MVIIC. The inorganic ions, Cd2+ and Ni2+, also produced a full inhibition of release (Cd2+ > > Ni2+) and Gd3+ a partial one. Drugs targeting L-type channels (diltiazem, nifedipine and verapamil) at low microM concentrations and a synthetic analogue of the polyamine toxin from funnel web spider venom (sFTX) at 1 mM were all non-inhibitory. Inhibition by omega-conotoxins MVIIC (IC50 25 nM) and SVIB (IC50 500 nM) was reversible and modulated by external concentrations of Ca2+. Inhibitory potency was increased by lowering and decreased by elevating external Ca2+. This "antagonistic" effect of Ca2+ was also seen with Cd2+ inhibition. The inhibitory potency of omega-conotoxin MVIIC was unaffected by predepolarisation. End plate potentials generated by release of endogenous ACh in electrically-stimulated slices were also reversibly blocked by Cd2+ and omega-conotoxins MVIIC and SVIB but were unaffected by omega-conotoxin GVIA and omega-agatoxin IVA. It is concluded that ACh release in skate electric organ depends on presynaptic calcium channels which have different pharmacological properties from established sub-types.     

Competitive inhibitors of free and chitosan-immobilized urease

Renal proximal tubules isolated from the rat possess nitric oxide synthase (NOS) activity that is calcium/calmodulin dependent and stereoselectively inhibited by NG-monomethyl-arginine (NMMA). In the absence of added Ca2+ and calmodulin, activity was reduced 84 +/- 13% compared with the activity in the presence of 2 mM Ca2+ and 25 micrograms/mL calmodulin. Inhibition by EGTA (10 mM) was 95 +/- 5% and by calmidazolium (R24571, 250 microM) was 99 +/- 1%. Inhibition by L-NMMA (100 microM) was 78 +/- 13% and by D-NMMA (100 microM) was 7 +/- 7%. The majority of NOS activity was found in the soluble fraction. NOS activity in isolated proximal tubules was also examined 6 hr after a single i.v. injection of lipopolysaccharide. Activity was increased significantly (P < 0.05) in the soluble fraction by 2-fold [from 0.320 +/- 0.052 to 0.648 +/- 0.046 (nmol/mg protein/30 min)] and in the particulate fraction by 3-fold [from 0.081 +/- 0.030 to 0.256 +/- 0.034 (nmol/mg protein/30 min)]. All activities were inhibited by EGTA. These data demonstrate that proximal tubules express a calcium/calmodulin-dependent NOS activity that is increased in vivo by lipopolysaccharide.     

(R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors mediate a calcium-dependent inhibition of the metabotropic glutamate receptor-stimulated formation of inositol 1,4,5-trisphosphate

L-glutamate (3-1,000 microM) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 10-1,000 microM), a selective agonist for the metabotropic glutamate receptor, stimulated the formation of inositol 1,4,5-trisphosphate in a concentration-dependent manner. L-Glutamate was half as efficacious as 1S,3R-ACPD. N-methyl-D-aspartate (NMDA; 1 nM to 1 mM) did not significantly influence the response to a maximally effective concentration of 1S,3R-ACPD (100 microM). On the other hand, coapplication of (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 1-300 nM) produced a concentration- and time-dependent inhibition of the 1S,3R-ACPD effect, with a maximal inhibition (97%) at 100 nM. Ten micromolar 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of the AMPA receptor, blocked the inhibitory effect of AMPA. Reduced extracellular calcium concentration, as well as 10 microM nimodipine, an L-type calcium channel antagonist, inhibited the AMPA influence on the 1S,3R-ACPD response. W-7, a calcium/calmodulin antagonist, prevented the inhibition by AMPA, whereas H-7, an inhibitor of protein kinase C, had no effect. These data suggest that activation of AMPA receptors has an inhibitory influence on inositol 1,4,5-trisphosphate formation mediated by stimulation of the metabotropic glutamate receptor. The mechanism of action involves calcium influx through L-type type calcium channels and possible activation of calcium/calmodulin-dependent enzymes.     

The distribution of omega-conotoxin MVIICnle-binding sites in rat brain measured by autoradiography

An analogue of omega-conotoxin MVIIC, [125I]omega-MVIICnle, has been employed in an autoradiographic assay to define the distribution of binding sites in rat brain of this neuronal calcium channel antagonist. In comparison with N-type channels (labeled by [125I]omega conotoxin GVIA), omega-MVIICnle sites are much denser in cerebellum (molecular layer) than in forebrain. Binding in thalamus is also comparatively high for omega-MVIICnle. Under these conditions, [125I]omega-MVIICnle binding to rat brain sections is not displaceable by the N-channel antagonist, omega-conotoxin GVIA. The calcium channel blocker [125I]omega-conotoxin MVIICnle labels a unique set of binding sites in mammalian brain.     

Solution and gel rheology of a new polysaccharide excreted by the bacterium Alteromonas sp. strain 1644

The present series of experiments were designed to examine a potential role for central descending pain facilitatory systems in mediating secondary hyperalgesia produced by topical application of mustard oil and measuring the nociceptive tail-flick reflex in awake rats. Topical application of mustard oil (100%) to the lateral surface of the hind leg produced a facilitation of the tail-flick reflex that was significantly reduced in spinal transected animals. Mustard oil hyperalgesia was also inhibited in animals that had received electrolytic lesions in the rostral ventromedial medulla (RVM). Intrathecal (i.t.) administration of the non-selective cholecystokinin (CCK) receptor antagonist proglumide (10 micrograms) prior to mustard oil application completely blocked both the lesser and greater hyperalgesic responses observed in spinal transected and normal animals, respectively, and produced an inhibition of the tail-flick reflex in normal animals. Administration of the selective CCKB receptor antagonist L-365260 i.t. dose-dependently inhibited mustard oil hyperalgesia (ID50 = 364 ng) at doses approximately 5-fold less than the CCKA receptor antagonist devazepide (ID50 = 1760 ng). Similar to spinal proglumide, microinjection of the neurotensin antagonist SR48692 (3.5 micrograms) into the RVM blocked mustard oil hyperalgesia and inhibited the tail-flick reflex. These data suggest that secondary hyperalgesia produced by mustard oil is mediated largely by a central, centrifugal descending pain facilitatory system which involves neurotensin in the RVM and spinal CCK (via CCKB receptors). The inhibition of the tail-flick reflex produced by mustard oil following spinal or supraspinal administration of receptor antagonists suggests concurrent activation of central descending facilitatory and inhibitory systems.     

Graded compression ultrasonography in the assessment of the "tough decision" acute abdomen in childhood

The three-dimensional structure of omega-conotoxin MVIID has been determined in aqueous solution by two-dimensional 1H NMR techniques. A total of 267 relevant upper-bound distance restraints were used to obtain a family of convergent structures using molecular dynamics methods. A standard simulated annealing protocol using the XPLOR program included in ARIA provided a total of 18 final structures. The averaged RMSD between these structures and the mean atomic coordinates was 0.8 +/- 0.3 A for the backbone atoms. The highest mobility was observed in the segments between residues 10 to 13, comprising Tyr 13, one of the residues shown to be important for binding of omega-conotoxin GVIA and MVIIA to N-type calcium channels. The three-dimensional structure is stabilised by the three disulfide bonds and includes a short antiparallel beta-strand between residues 5-8, 23-25 and 19-21. The folding for this non-N-type calcium channel blocker is similar to that previously calculated for omega-conotoxins GVIA, MVIIA and MVIIC. This suggests the disulfide bond pattern fixes the structure. The reported three-dimensional information can be used to advantage in order to highlight the structural parameters involved in discrimination among calcium channel subtypes.     

ATP-activated chloride permeability in biliary epithelial cells is regulated by calmodulin-dependent protein kinase II

Previous studies in freshly isolated rat biliary epithelial cells and in the human cholangiocarcinoma cell line Mz-ChA-1 have demonstrated that ATP activates a calcium-dependent chloride conductance. The coupling between the rise in intracellular calcium and activation of chloride channels has not previously been investigated. In the present study, we evaluated the potential role of calmodulin-dependent protein kinase II (CaMKII) in ATP-activated chloride permeability in Mz-ChA-1 cells. ATP stimulated [125I] efflux, a marker for Cl- movement. Peak efflux rates were inhibited by approximately 60% in cells pretreated with the calmodulin antagonist, W-7. In whole-cell patch clamp recordings, ATP and ionomycin activated calcium-dependent Cl- currents. Pretreatment of cells with the CaMKII inhibitor KN-62 blocked activation by either agent. It is concluded that calcium-dependent activation of chloride currents in Mz-ChA-1 cells is coupled to a CaMKII-dependent process.     

Differential antagonism by MK-801 against antinociception induced by opioid receptor agonists administered supraspinally in mice

Various doses of MK-801 ((+/-)-5-methyl-10,11-dihydro-5H-dibenzo(a,d) cyclohepten-5, 10-imine maleate), a non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist (0.001-1 microgram) injected intracerebroventricularly (i.c.v.) alone did not show any antinociceptive effect. MK-801 (0.001-1 microgram i.c.v.) dose dependently attenuated the inhibition of the tail-flick and hot plate responses induced by i.c.v. administered morphine (1 microgram), [D-Pen2, D-Pen5]enkephalin (DPDPE; 10 micrograms), and U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeoce tamide ) 60 micrograms). However, the inhibition of the tail-flick and hot plate responses induced by i.c.v. administered beta-endorphin (1 microgram) was not changed by i.c.v. administered MK-801. Our results indicate that, at the supraspinal level, NMDA receptors are involved in the production of antinociception induced by supraspinally administered morphine, DPDPE, and U50,488H but not beta-endorphin.     

Molecular genetics of long-QT syndrome

We have recently shown that spinal calmodulin inhibitors (W-7 and calmidazolium) dose-dependently inhibit the nociceptive reaction (biting, scratching, licking, BSL) evoked by intrathecal N-methyl-D-aspartate (NMDA) and septide, an agonist of the neurokinin (NK) NK1 receptor. To compare this effect with that induced by standard analgesics, we now report a study of the effects of calmidazolium (14420 nmol), bupivacaine (29-582 nmol) and morphine (26-260 nmol) when coadministered intrathecally with either NMDA (4 microg) or septide (0.5 microg). Calmidazolium had the highest potency for inhibiting septide-induced nociceptive behaviour, acting over a dose range of 34-130 nmol (dose eliciting a half-maximal response, ED50, 67 nmol) lower than that of bupivacaine [ED50 234 (115-475) nmol]. Only the highest dose of morphine (260 nmol) inhibited septide-evoked BSL [ED50=133 (69-255) nmol]. Higher doses of morphine could not be tested due to the appearance of an excitatory aversive reaction. Both calmidazolium [ED50=232 (138-388) nmol] and bupivacaine [ED50=123 (59-256) nmol] dose-dependently reduced NMDA-induced BSL reaching an almost maximal inhibition at the highest doses assayed (420 and 291 nmol, respectively). In contrast, morphine had less effect on NMDA-induced behaviour, inducing only a partial reduction of BSL even with the highest dose assayed (260 nmol). Overall, it can be concluded that the calmodulin inhibitor calmidazolium inhibits septide- and NMDA-evoked nociceptive behaviour with a potency and efficacy at least as high as those of morphine and bupivacaine.     

Activation of brain nitric oxide synthase in depolarized human temporal cortex slices: differential role of voltage-sensitive calcium channels

1. Nitric oxide (NO) synthase activity was studied in slices of human temporal cortex samples obtained in neurosurgery by measuring the conversion of L-[3H]-arginine to L-[3H]-citrulline. 2. Elevation of extracellular K+ to 20, 35 or 60 mM concentration-dependently augmented L-[3H]-citrulline production. The response to 35 mM KCl was abolished by N(G)-nitro-L-arginine (100 microM) demonstrating NO synthase specific conversion of L-arginine to L-citrulline. Increasing extracellular MgCl2 concentration up to 10 mM also prevented the K+ (35 mM)-induced NO synthase activation, suggesting the absolute requirement of external calcium ions for enzyme activity. 3. However, the effect of high K+ (35 mM) on citrulline synthesis was insensitive to the antagonists of ionotropic and metabotropic glutamate receptors dizocilpine (MK-801), 6-nitro-7-sulphamoylbenzo(f)-quinoxaline-2-3-dione (NBQX) or L-2-amino-3-phosphonopropionic acid (L-AP3) as well as to the nicotinic receptor antagonist, mecamylamine. 4. The 35 mM K+ response was insensitive to omega-conotoxin GVIA (1 microM) and nifedipine (100 microM), but could be prevented in part by omega-agatoxin IVA (0.1 and 1 microM). The inhibition caused by 0.1 microM omega-agatoxin IVA (approximately 30%) was enhanced by adding omega-conotoxin GVIA (1 microM) or nifedipine (100 microM). Further inhibition (up to above 70%) could be observed when the three Ca2+ channel blockers were added together. Similarly, synthetic FTX 3.3 arginine polyamine (sFTX) prevented (50% at 100 microM) the K+-evoked NO synthase activation. This effect of sFTX was further enhanced (up to 70%) by adding 1 microM omega-conotoxin GVIA plus 100 microM nifedipine. No further inhibition could be observed upon addition of MK-801 or/and NBQX. 5. It was concluded that elevation of extracellular [K+] causes NO synthase activation by external Ca2+ entering cells mainly through channels of the P/Q-type. Other Ca2+ channels (L- and N-type) appear to contribute when P/Q-channels are blocked.