Inositolhexakisphosphate is convulsant in mice and rats in the nanomolar range

The effect of i.c.v. administration of dodecasodium and dicalcium inositolhexakisphosphate (Na12IP6 and Ca2IP6, respectively) to mice and rats was studied. In mice, Na12IP6 (1-300 nmol) or Ca2IP6 (10-500 nmol) induced: ataxia, ground-hugging, tremor (often continuous), scratching, hyperlocomotion, wild running, myoclonic jerks, jumping, clonic muscle spasms, tonic seizure, followed by death or full recovery. The CD50 values for clonic seizures for Na12IP6 and Ca2IP6 were 16 and 49 nmol, respectively. The convulsant effect of Na12IP6 (15 nmol i.c.v.) was not blocked by pretreatment with D(-)-4-(3-phosphonoprop-2-enyl)-piperazine-2-carboxylate, but was dose dependently reduced by pretreatment with CaCl2 (30-60 nmol i.c.v.) and abolished by coadministration of CaCl2 (30 nmol) with Na12IP6 (i.c.v.). In rats, Na12IP6 (50 nmol i.c.v.) induced severe electroencephalographic seizures in the hippocampus and cortex. The potent convulsant effect of IP6 (administered i.c.v.) depends at least in part on a calcium-chelating action.     

Anticonvulsant activity of a mGlu(4alpha) receptor selective agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid

The metabotropic Group III agonist, (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-1), selective for the mGlu(4alpha) receptor, suppresses sound-induced seizures in DBA/2 mice following its intracerebroventricular (i.c.v.) administration (ED(50) 5.6 [2.9-10.7], nmol i.c.v., 15 min, clonic phase) and in genetically epilepsy-prone (GEP) rats following focal administration into the inferior colliculus (ED(50) 0.08 [0.01-0.50], nmol, 60 min, clonic phase). ACPT-1 also protects against clonic seizures induced in DBA/2 mice by the Group I agonist, (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG) (ED(50) 0.60 [0.29-1.2], nmol i.c.v.) and by the Group III antagonist, (RS)-alpha-methylserine-O-phosphate (MSOP) (ED(50) 49.3 [37.9-64.1], nmol i.c.v.). Another Group III agonist, (RS)-4-phosphonophenyl-glycine (PPG), preferentially activating the mGlu(8) receptor, previously shown to protect against sound-induced seizures in DBA/2 mice and GEP rats, also protects against seizures induced in DBA/2 by 3,5-DHPG (ED(50) 3.7 [2.4-5.7], nmol i.c.v.) and by the Group III antagonist, MSOP (ED(50) 40.2 [21.0-77.0], nmol i.c.v.). At very high doses (500 nmol i.c.v. and above), Group III antagonists have pro-convulsant and convulsant activity. The anticonvulsant protection against sound-induced seizures in DBA/2 mice provided by a fully protective dose (20 nmol, i.c.v.) of the mGlu(4) receptor agonist ACPT-1, is partially reversed by the co-administration of the Group III antagonists, MSOP, (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG) or (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4), in the 20-50 nmol dose range. At doses of 50-200 nmol, MPPG and MAP4 cause further reversal of the ACPT-1 anticonvulsant protection, while the MSOP effect on ACPT-1 protection is abolished at higher doses. In contrast, the anticonvulsant protection against sound-induced seizures in DBA/2 mice provided by a fully protective dose (20 nmol, i.c.v.) of the mGlu(8) receptor agonist PPG, is not significantly affected by the co-administration of the same Group III antagonists, MSOP, MPPG or MAP4. We conclude that activation of either mGlu(4alpha) or mGlu(8) receptors confer anticonvulsant protection in DBA/2 mice. Furthermore, the metabotropic Group III receptor antagonists, MSOP, MPPG, and MAP4 appear to be functionally selective for the mGlu(4) receptor in this system.     

Effects of phorbol ester on carbachol-induced contraction in bovine ciliary muscle: possible involvement of protein kinase C

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M₃ subtype. Acute exposure to phorbol esters (1 μM phorbol 12,13-dibutyrate, PDB, or 0.1 μM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 μM) and H₇ [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 μM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [³H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Anticonvulsant actions of LY 367385 ((+)-2-methyl-4-carboxyphenylglycine) and AIDA ((RS)-1-aminoindan-1,5-dicarboxylic acid)

We have studied the effects in three rodent models of generalised convulsive or absence epilepsy of two antagonists of group I metabotropic glutamate receptors that are selective for the mGlu1 receptor. LY 367385 ((+)-2-methyl-4-carboxyphenylglycine) and AIDA ((RS)-1-aminoindan-1,5-dicarboxylic acid) have been administered intracerebroventricularly (i.c.v.) to DBA/2 mice and lethargic mice (lh/lh), and focally into the inferior colliculus of genetically epilepsy prone rats (GEPR). In DBA/2 mice both compounds produce a rapid, transient suppression of sound-induced clonic seizures (LY 367385: ED50 = 12 nmol, i.c.v., 5 min; AIDA: ED50 = 79 nmol, i.c.v., 15 min). In lethargic mice both compounds significantly reduce the incidence of spontaneous spike and wave discharges on the electroencephalogram, from <30 to >150 min after the administration of AIDA, 500 nmol, i.c.v., and from 30 to >150 min after the administration of LY 367385, 250 nmol, i.c.v. LY 367385, 50 nmol, suppresses spontaneous spike and wave discharges from 30 to 60 min. In genetically epilepsy prone rats both compounds reduce sound-induced clonic seizures. LY 367385, 160 nmol bilaterally, fully suppresses clonic seizures after 2-4 h. AIDA is fully effective 30 min after 100 nmol bilaterally. It is concluded that antagonists of mGlu1 receptors are potential anticonvulsant agents and that activation of mGlu1 receptors probably contributes to a variety of epileptic syndromes.     

The structural requirements for phorbol esters to enhance noradrenaline and dopamine release from rat brain cortex.

1. The effects of various protein kinase C (PKC) activators on the stimulation-induced (S-I) release of noradrenaline and dopamine was studied in rat cortical slices pre-incubated with [3H]-noradrenaline or [3H]-dopamine. The aim was to investigate a possible structure-activity relationship for these agents on transmitter release. 2. 4 beta-Phorbol 12,13-dibutyrate (4 beta PDB, 0.1-3.0 microM), enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner whereas the structurally related inactive isomer 4 alpha-phorbol 12, 13-dibutyrate (4 alpha PDB, 0.1-3.0 microM) and phorbol 13-acetate (PA, 0.1-3.0microM) were without effect on noradrednaline release. Another group of phorbol 12, 13-diesters containing a common 13-ester substituent (phorbol 12, 13-diacetate, PDA, 0.1-3.0 microM; phorbol 12-myristate 13-acetate, PMA, 0.1-3.0 microM; phorbol 12-methylaminobenzoate 13-acetate, PMBA, 0.03-3.0 microM) also enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner with PMA being the least potent. 3. The 12-deoxyphorbol 13-substituted monoesters, 12-deoxyphorbol 13-acetate (dPA, 0.1-3.0 microM), 12-deoxyphorbol 13-angelate (dPAng, 0.1-3.0 microM), 12-deoxyphorbol 13-isobutyrate (dPiB, 0.03-3.0 microM) and 12-deoxyphorbol 13-phenylacetate (dPPhen, 0.1-3.0 microM) enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner. In contrast, 12-deoxyphorbol 13-tetradecanoate (dPT, 0.1-3.0 microM) was without effect. 4. The involvement of PKC in mediating the effects of the various phorbol esters was further investigated. PKC was down-regulated by 20 h exposure of the cortical slices to 4 beta-phorbol 12,13-dibutyrate (1 microM). In this case the facilitatory effect of 4 beta PDB and dPA was abolished whilst that of dPAng was significantly attenuated. This indicates that these agents were acting selectively at PKC. In support of this the PKC inhibitors, polymyxin B (21 microM) and bisindolylmaleimide I (3 microM), attenuated the facilitatory effect of 4 beta PDB and dPAng although that of dPA was not significantly altered. 5. The effects of these agents on transmitter release were not correlated with their in vitro affinity and isozyme selectivity for PKC. Short chain substituted mono- and diesters of phorbol were more potent enhancers of action-potential evoked noradrenaline and dopamine release than the long chain esters. Interestingly, these former agents are the least potent or non effective (e.g. dPA, PDA) tumour promoters. We suggest that the reason for the poor effects of lipophilic long chain phorbol esters (PMA, dPT) on transmitter release is that they are sequestered in the plasmalemma and do not access the cell cytoplasm where the PKC may be located.     

A phorbol ester inhibits the release of endothelium-derived relaxing factor

The effect of the phorbol ester phorbol-12,13-dibutyrate (PDB), an activator of protein kinase C, on endothelium-dependent relaxation was studied in noradrenaline-constricted isolated aortic ring preparations of the rabbit. Endothelium-dependent relaxation induced by acetylcholine or substance P was inhibited by PDB (greater than or equal to 10(-7) M). Endothelium-dependent relaxation induced by the calcium ionophore A23187 (7.5 X 10(-8) and 10(-7) M) was unaffected by PDB (to 10(-6) M). The mechanical responses to acetylcholine or sodium nitroprusside in endothelium-denuded rings were not altered by PDB (to 10(-6) M). The results suggest a role for protein kinase C in receptor-mediated EDRF release mechanisms.     

Differential abilities of phorbol esters in inducing protein kinase C (PKC) down-regulation in noradrenergic neurones

1. The ability of several phorbol ester protein kinase C (PKC) activators (phorbol 12, 13-dibutyrate, PDB; phorbol 12, 13-diacetate, PDA; and 12-deoxyphorbol 13-acetate, dPA) to down-regulate PKC was studied by assessing their effects on electrical stimulation-induced (S-I) noradrenaline release from rat brain cortical slices and phosphorylation of the PKC neural substrate B-50 in rat cortical synaptosomal membranes. 2. In cortical slices which were incubated for 20 h with vehicle, acute application of PDB, PDA and dPA (0.1 - 3.0 microM) enhanced the S-I noradrenaline release in a concentration-dependent manner to between 200 - 250% of control in each case. In slices incubated with PDB (1 microM for 20 h), subsequent acute application of PDB (0.1 - 3.0 microM) failed to enhance S-I release, indicating PKC down-regulation. However, in tissues incubated with PDA or dPA (3 microM) for 20 h, there was no reduction in the facilitatory effect of their respective phorbol esters or PDB (0.1 - 3.0 microM) when acutely applied, indicating that PKC was not down-regulated. This was confirmed using Western blot analysis which showed that PDB (1 microM for 20 h) but not PDA (3 microM for 20 h) caused a significant reduction in PKCalpha. 3. Incubation with PDB for 20 h, followed by acute application of PDB (3 microM) failed to increase phosphorylation of B-50 in synaptosomal membranes, indicating down-regulation. In contrast, tissues incubated with PDA or dPA for 20 h, acute application of their respective phorbol ester (10 microM) or PDB (3 microM) induced a significant increase in B-50 phosphorylation. 4. Acutely all three phorbol esters elevate noradrenaline release to about the same extent, yet PDA and dPA have lower affinities for PKC compared to PDB, suggesting unique neural effects for these agents. This inability to cause functional down-regulation of PKC extends their unusual neural properties. Their neural potency and lack of down-regulation may be related to their decreased lipophilicity compared to other phorbol esters. 5. We suggest that PKC down-regulation appears to be related to binding affinity, where agents with high affinity, irreversibly insert PKC into artificial membrane lipid and generate Ca(2+)-independent kinase activity which degrades and deplete PKC. We suggest that this mechanism may also underlie the ability of PDB to down-regulate PKC in nerve terminals, in contrast to PDA and dPA.     

The responses to phorbol esters which stimulated protein kinase C in canine Purkinje fibers.

1. The effects of phorbol esters on canine Purkinje fibers were examined using conventional microelectrode techniques. 2. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and 4-beta-phorbol-12,13-dibutyrate (PDB), which are specific activators of protein kinase C (PKC), decreased the action potential amplitude and the maximum rate of depolarization (Vmax) at 3 x 10(-7) M or higher. These phorbol esters had little effect on the resting potential. 3. PDB (1-3 x 10(-7) M) also reduced the contractile force, accompanied with initial increase (in 5 out of 8 experiments), whereas TPA did not decrease it to any significant extent. 4. An inactive analog of phorbol esters, 4-alpha-phorbol-12,13-didecanoate (PDD), decreased the action potential amplitude and Vmax, and slightly increased the action potential duration. However, PDD failed to produce any inotropic effect. 5. Post-rest potentiation of the contractile force after a rest from stimulation for 30 sec was inhibited in the presence of 3-10 x 10(-7) M TPA or 3 x 10(-7) M PDB. 6. Isoproterenol 10(-7) M augmented the action of PDB 3 x 10(-7) M. 7. These results suggest that activation of PKC may modulate myocardial Ca2+ homeostasis and influence the excitation-contraction process.     

Anticonvulsant activity of two metabotropic glutamate group I antagonists selective for the mGlu5 receptor: 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and (E)-6-methyl-2-styryl-pyridine (SIB 1893)

The selective mGlu5 antagonists, MPEP, 2-methyl-6-phenylethynyl-pyridine, and SIB1893, (E)-6-methyl-2-styryl-pyridine, have been evaluated as antiepileptic drugs in DBA/2 mice and lethargic mice. Clonic seizures induced by the selective mGlu5 agonist, (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG), 3 micromol intracerebroventricularly (i.c.v.), are potently suppressed by both compounds (MPEP, ED(50)=0.42 [0.28-0.62] mg/kg intraperitoneally (i.p.); SIB 1893 ED(50)=0.19 [0.11-0.33] mg/kg i.p. ). Clonic seizures induced by the mGlu1,5 agonist, 3, 5-dihydroxyphenylglycine (DHPG), 1.5 micromol i.c.v., are less potently suppressed by both compounds (MPEP, ED(50)=22 [13-38] mg/kg i.p., 110 [67-180] nmol i.c.v.; SIB1893, ED(50)=31 [18-54] mg/kg i.p. , 95 [82-110] nmol i.c.v.). Sound-induced seizures in DBA/2 mice are suppressed at 15 min by MPEP and SIB 1893 (MPEP ED(50) clonic seizures=18 [10-32] mg/kg i.p., 93 [69-125] nmol i.c.v.; tonic seizures=6.1 [4.5-8.3] mg/kg i.p., 46 [26-80] nmol i.c.v.; SIB 1893 ED(50) clonic seizures=27 [17-44] mg/kg i.p., 825 [615-1108] nmol i. c.v., tonic seizures=5.4 [3.4-8.6] mg/kg i.p., 194 [113-332] nmol i. c.v.). The ED(50) for MPEP for impaired rotarod performance is 128 [83-193] mg/kg i.p., at 15 min, i.e. a therapeutic index for sound-induced seizures of 5-20. In lethargic mice (lh/lh), a genetic absence model, MPEP, 50 mg/kg i.p., caused a marked reduction in the incidence of spontaneous spike-and-wave discharges. These selective antagonists of mGlu5 block seizures due to activation of mGlu5 at very low systemic doses. At rather higher doses they block convulsive and non-convulsive primary generalised seizures.     

Non-competitive N-methyl-D-aspartate antagonists protect against sound-induced seizures in DBA/2 mice

Non-competitive antagonists of the N-methyl-D-aspartate (NMDA) receptor have been evaluated as anticonvulsants against sound-induced seizures in DBA/2 mice. The ED50 values for protection against sound-induced clonic seizures 15 min following the intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration are: MK-801, ED50 = 0.5 nmol (i.c.v.); 0.14 mumol/kg (i.p.); phencyclidine, ED50 = 14 nmol (i.c.v.); 1.9 mumol/kg (i.p.); dextrorphan, ED50 = 35 nmol (i.c.v.); 18.5 mumol/kg (i.p.); tiletamine, ED50 = 40 nmol (i.c.v.); 5.6 mumol/kg (i.p.); SKF-10047, ED50 = 50 nmol (i.c.v.); 23.5 mumol/kg (i.p.); dextromethorphan, ED50 = 70 nmol (i.c.v.); 28.0 mumol/kg (i.p.); ketamine, ED50 = 110 nmol (i.c.v.); 15.5 mumol/kg (i.p.). The anticonvulsant effects of ketamine and tiletamine are of short duration (10-30 min), whereas the anticonvulsant effects of MK-801 and dextromethorphan last for 45 min or longer. The effects of phencyclidine, SKF-10047 and dextrorphan are of intermediate duration. Mild to moderate behavioural excitation is associated with the anticonvulsant activity of all the non-competitive NMDA antagonists. For MK-801, phencyclidine, dextrorphan, SKF-10047 and ketamine there is a close correlation between their relative anticonvulsant potencies and their potencies for displacing [3H]MK-801. The anticonvulsant effect is likely to be primarily mediated via NMDA antagonism at the PCP/MK-801 site.     

The effects of phorbol esters on choline phospholipid hydrolysis in heart and brain

The efflux of choline was determined in rat striatal slices, incubated chicken atria and perfused chicken hearts. 4 beta-Phorbol-12 beta,13 alpha-dibutyrate (PDB) and 4 beta-phorbol-12 beta-myristate, 13 alpha-acetate (PMA) were used to stimulate protein kinase C. The other phorbol esters, 4 beta-phorbol-13 alpha-acetate (PAc) and 4 alpha-phorbol-12 beta,13 alpha-didecanoate (4 alpha PDD), known to be inactive, were tested to evaluate the specificity of the responses. PDB markedly enhanced the efflux of choline in all of the three preparations. The PDB-evoked efflux of choline in incubated chicken atria was equal to the net production of choline and, therefore, was not caused by translocation of intracellular free choline. After inhibition of the cholinesterase activity, PDB linearly increased the efflux of choline in rat striatal slices, but failed to alter the spontaneous efflux of acetylcholine. Thus acetylcholine did not serve as the source of the PDB-evoked efflux of choline. PMA was as effective as PDB, whereas PAc and 4 alpha PDD failed to alter the choline efflux in the perfused heart. Both infusion of a Ca2(+)-free EGTA-containing Tyrode solution and mepacrine reduced the spontaneous efflux of choline by about 40% and blocked the PDB-evoked efflux of choline. In contrast, a Ca2(+)-free solution without EGTA failed to alter the spontaneous and the PDB-evoked choline efflux. It is concluded that phorbol esters stimulate the hydrolysis of choline-containing phospholipids in heart and brain via activation of protein kinase C.     

Receptor site specificity for the acute effects of beta-N-methylamino-alanine in mice

DL-beta-N-methylamino-alanine (DL-BMAA; 1-10 mumol i.c.v.) in mice induced a syndrome of: ataxia, ptosis, scratching, jumping, myoclonic jerks, clonic muscle spasms and tonic seizure, which was unaffected by pretreatment with D(-)-4-(3-phosphonoprop-2-enyl)-piperazine-2-carboxylate (D(-)-CPPene; i.p.), or by co-administration of gamma-D-glutamylamino-methylsulphonate (gamma-D-GAMS with DL-BMAA; i.c.v.). Pretreatment with 1-(aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (GYKI 52466; i.v.) decreased the incidence of clonic seizures for DL-BMAA, kainic acid and RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (RS-AMPA; i.c.v.). These results suggest an involvement of the AMPA/quisqualate subtype of excitatory amino acid receptors in acute BMAA toxicity.     

A comparison of the priming effect of phorbol myristate acetate and phorbol dibutyrate on fMet-Leu-Phe-induced oxidative burst in human neutrophils

Phorbol esters such as phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBU) are generally considered to have similar effects through a similar mechanism, i.e. protein kinase C (PKC) activation. We recently suggested that this was not the case in human neutrophils. To identify further differences between the two phorbol esters, we compared their priming effects on fMet-Leu-Phe-induced superoxide anion (O2-) production, cytosolic PKC activity and binding of fMet-Leu-Phe. Priming could be initiated with a low (0.2 nM) concentration of both PDBU and PMA. Their effects on the pattern of fMet-Leu-Phe-induced superoxide production were similar in both Ca2(+)-containing and Ca2(+)-free medium. PDBU, like PMA, abolished the Ca2+ dependency of fMet-Leu-Phe-induced O2- production in a dose-dependent manner. In cytochalasin B-treated cells and in the presence of Ca2+, priming with PDBU or PMA did not alter the enhancing effect of cytochalasin B on fMet-Leu-Phe-induced O2- production. In Ca2(+)-free medium, priming abolished the Ca2+ dependency of fMet-Leu-Phe stimulation in cytochalasin B-treated cells. Cytochalasin B, however, enhanced the effect of PMA but not that of PDBU. Priming with PDBU was not associated under any experimental conditions with a decrease in cytosolic PKC activity, or an increase in PKM activity before or after fMet-Leu-Phe stimulation. Furthermore, priming effects were abolished by cell washing but not by H-7 or staurosporine, which are potent PKC inhibitors. PDBU, in contrast to PMA, increased fMet-Leu-Phe binding to PMNs through a decrease in the dissociation constant and induced degranulation of specific granules as measured by the release of vitamin B12 binding protein. These findings show that the priming effects of PDBU differ in certain respects from those of PMA, namely with regard to its synergism with cytochalasin B and the expression of fMet-Leu-Phe receptors. In addition, priming concentrations of PDBU, like PMA, did not alter cytosolic PKC activity in fMet-Leu-Phe-stimulated neutrophils.     

Anti-epileptic activity of group II metabotropic glutamate receptor agonists (--)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268) and (--)-2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY389795).

The selective group II metabotropic glutamate receptor (mGlu(2/3)) agonists (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268) and (-)-2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY389795) have been evaluated as anti-epileptic drugs in dilute brown agouti (DBA/2) mice, lethargic (lh/lh) mice, genetically epilepsy-prone-9 (GEP) rats and amygdala-kindled rats. Sound-induced clonic seizures in DBA/2 mice were transiently inhibited by both agonists intracerebroventricularly (i.c.v.), LY379268 ED(50)=0.08 [0.02-0.33]nmol and LY389795 ED(50)=0.82 [0.27-3.24]nmol or intraperitoneally (i.p.), LY379268 ED(50)=2.9 [0.9-9.6]mg/kg and LY389795 ED(50)=3.4 [1.0-11.7]mg/kg. Both mGlu(2/3) agonists inhibited seizures induced by the group I mGlu receptor agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG), where LY379268, i.c.v. ED(50)=0.3 [0.02-5.0]pmol and LY389795, i.c.v. ED(50)=0.03 [0.05-0.19]nmol. The spike and wave discharge (SWD) duration of absence seizures in lh/lh mice was significantly reduced by both agonists at 1 and 10nmol (i.c.v.) up to 90min following infusion. The electrically induced seizure score and afterdischarge duration of amygdala-kindled rats was partially inhibited by the agonists 30min after i.p. injection of 10mg/kg. The agonists did not inhibit sound-induced seizures in GEP rats (0.1-1mg/kg, 30min 1h, i.p.), but were proconvulsant following sound stimulus (> or =0.1mg/kg). These findings identify a potential role for mGlu(2/3) agonists in the amelioration of generalised and partial epileptic seizures.     

Mechanisms for cardiac depression induced by phorbol myristate acetate in working rat hearts.

1. The effects of the phorbol ester, phorbol myristate acetate (PMA) were examined on function and energy metabolism in the isolated working heart of the rat. 2. At a concentration of 10(-9) M PMA produced a rapid loss in cardiac function in terms of aortic flow rate (AFR) and coronary flow rates (CFR) whereas a similar concentration of 4 alpha-phorbol 12,13-didecanoate was ineffective. At a concentration of 10(-10) M, the PMA-induced depression was more gradual but nevertheless very pronounced with an almost total loss in AFR after 30 min perfusion. The reduction in CFR was more moderate than that observed with respect to AFR. 3. The protein kinase C (PKC) inhibitor (+/-)-1-O-hexadecyl-2-O-acylglycerol significantly attenuated the loss in AFR and CFR following addition of PMA. 4. Two inhibitors of Na+/H+ exchange, amiloride and quinacrine, totally prevented the reduction in AFR. Although the PMA-induced depression in CFR was also attenuated by both amiloride and quinacrine, these effects were not significant, probably reflecting the less pronounced effect of PMA on this parameter. 5. Nifedipine, a dihydropyridine calcium channel blocker reduced PMA toxicity to a similar degree as Na+/N+ exchange inhibition whereas the calcium channel agonist Bay K 8644 was without effect. 6. Tissue content of energy metabolites including high energy phosphates, total adenine nucleotides or lactate were not significantly affected by PMA perfusion. 7. We conclude that PKC activation is necessary for phorbol ester-induced cardiac dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Discrimination of histamine H1 and muscarinic receptor-mediated signalling pathways by phorbol ester in human astrocytoma cells

1. Histamine H1 receptor-mediated signalling was compared with muscarinic receptor-mediated signalling in 1321N1 human astrocytoma cells. 2. Short-term (2 min) treatment of cells with phorbol 12-myristate 13-acetate (PMA) resulted in a reduction of increases in intracellular Ca2+ ([Ca2+]i) induced by carbachol or histamine. 3. Carbachol-induced increases in [Ca2+]i were 10-fold more sensitive to PMA than the histamine-induced increases. 4. When cells were treated with PMA for 48 or 72 h (long-term treatment), protein kinase C (PKC) was down-regulated and PMA did not inhibit carbachol-induced increases in [Ca2+]i. 5. Histamine-induced increases in [Ca2+]i were significantly reduced by long-term treatment with PMA. 6. These findings suggest that the signalling pathways mediated by histamine H1 and muscarinic receptors can be distinguished by using PKC in 1321N1 human astrocytoma cells.     

Genital grooming and emesis induced by vanilloids in Suncus murinus, the house musk shrew.

The potential of resiniferatoxin and capsaicin to modulate emesis and genital grooming was investigated in Suncus murinus. Resinifertoxin (3-30 nmol, i.c.v.), E-capsaicin (10-100 nmol, i.c.v.) and Z-capsaicin (100 nmol, i.c.v.) induced emesis (P<0.05) and subsequently antagonised the emetic response induced by intragastric copper sulphate (480.6 micromol/kg; P<0.05). However, resiniferatoxin failed to affect nicotine-induced (30.7 mol/kg, s.c.) emesis (P>0.05). Only resiniferatoxin induced genital grooming that was antagonised (P<0.05) by capsazepine (300-600 nmol, i.c.v.) and ruthenium red (3 nmol, i.c.v.). E-capsaicin-induced emesis was antagonised by capsazepine (300-600 nmol, i.c.v.; P<0.05) and ruthenium red (3 nmol, i.c.v.; P<0.05) but resiniferatoxin-induced emesis was resistant to capsazepine (30-600 nmol, i.c.v.; P>0.05). The emetic action of resiniferatoxin but not E-capsaicin was subject to tachyphylaxis. In cross-tachyphylaxis experiments, E-capsaicin reduced the genital grooming induced by resiniferatoxin (P<0.05). The data are discussed in relation to the classification of vanilloid receptors and mechanisms involved in emesis and genital grooming.     

Characterization of phorbol esters activity on individual mammalian protein kinase C isoforms, using the yeast phenotypic assay

An alternative in vivo assay, based on growth inhibition of yeast expressing an individual mammalian protein kinase C (PKC) isoform (proportional to the degree of PKC activation), was used to characterize the activities of phorbol-12-myristate-13-acetate (PMA) and its analogues on classical (alpha and betaI), novel (delta and eta) and atypical (zeta) PKC isoforms. Effects of PMA, 4alpha-PMA, phorbol-12-myristate-13-acetate-4-O-methyl-ether (MPMA), phorbol-12-monomyristate (PMM), phorbol-12,13-diacetate (PDA), phorbol-13-monoacetate (PA), phorbol-12,13-dibutyrate (PDB), phorbol-12,13-didecanoate (PDD) and 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA), on growth of yeast expressing individual PKC isoforms was determined. PMA-induced growth inhibition on all isoforms tested (except on PKC-zeta). PDD and PDB presented an efficacy similar to PMA; the other PMA-analogues presented lower efficacies. MPMA and 4alpha-PMA stimulated growth of yeast expressing classical PKCs and reduced the PMA-induced growth inhibition, effects similar to those exhibited by the PKC inhibitors chelerythrine and R-2,6-diamino-N-[[1-(1-oxotridecyl)-2-piperidinyl]methyl]-hexanamide dihydrochloride (NPC 15437). This study reveals that phorbol esters differ on their potency to activate a given PKC isoform, and presents their isoform-selectivity. Furthermore, MPMA and 4alpha-PMA caused effects similar to those expected from PKC inhibition.     

Release of endothelium-derived relaxing factor from pig cultured aortic endothelial cells, as assessed by changes in endothelial cell cyclic GMP content, is inhibited by a phorbol ester.

1. Cultured aortic endothelial cells of the pig respond to the endothelium-derived relaxing factor (EDRF) they release with an increase in cyclic GMP content. This response is inhibited by haemoglobin or by L-NG-monomethyl-arginine (L-NMMA), and has been used to investigate the effects of phorbol esters on EDRF release. 2. Pretreatment with phorbol-12,13-dibutyrate (PDB) but not the inactive 4 alpha-phorbol-12,13,-didecanoate (PDD), inhibited increases in cyclic GMP induced by substance P (10(-8) M) in a time and concentration-dependent manner. PDB did not affect basal cyclic GMP levels. 3. PDB (3 x 10(-7) M), but not PDD (3 x 10(-7) M), also inhibited ATP (10(-5) M)-induced increases in cyclic GMP, but did not affect those induced by bradykinin (10(-7) M). 4. Increases in cyclic GMP induced by low (10(-7) M) but not high (10(-6) M) concentrations of the calcium ionophore A23187 were inhibited by PDB (3 x 10(-7) M). This inhibitory effect was due to enhanced destruction of EDRF by superoxide anions rather than inhibition of EDRF release, as the inhibition was abolished in the presence of superoxide dismutase (SOD, 30 mu ml-1) and catalase (CAT, 100 mu ml-1). 5. SOD and CAT did not affect the inhibitory action of PDB on substance P or ATP-induced increases in cyclic GMP. 6. Increases in endothelial cell cyclic GMP content induced by sodium nitroprusside (10(-5) M) were unaffected by PDB pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of systemic, spinal and supraspinal L-arginine-nitric oxide-cGMP pathway in thermal hyperalgesia caused by intrathecal injection of glutamate in mice.

The intrathecal (i.t.) administration of glutamate (10-100 nmol) caused dose-related hyperalgesia (mean ED50 of 35 nmol) when assessed in the thermal behaviour model of nociception, the hot-plate test maintained at 50 degrees C. The i.p., i.t. or intracerebroventricular (i.c.v.) injection of the nitric oxide synthase inhibitors, L-NOARG and L-NAME, did not induce any detectable effect per se, but instead, produced dose-related inhibition of glutamate-induced hyperalgesia. D-NAME, the inactive enantiomer of L-NAME, had no effect. The i.c.v. or i.t. administration of L-NIO caused graded attenuation of glutamate-induced hyperalgesia. L-arginine (3.4 mmol kg(-1), i.p.), but not D-arginine (3.4 mmol kg(-1), i.p.) significantly potentiated glutamate (10 nmol)-induced hyperalgesia, an action that was prevented by L-NOARG (137 nmol kg(-1)). The co-injection of S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (0.22 micromol) or 8-bromo-cGMP (22.5 nmol) with glutamate (10 nmol), via either i.t. or i.c.v. routes, also significantly enhanced glutamate-induced hyperalgesia. The guanylate cyclase inhibitors LY 83583 (0.1-1.0 nmol) or ODQ (30-300 pmol) co-administered with glutamate, dose-dependently antagonised the glutamate-induced hyperalgesia. Collectively, these results demonstrate that the i.t. injection of glutamate into the spinal cord of mice produces dose-related hyperalgesia an effect that was largely mediated by the L-arginine-nitric oxide-cGMP pathway from both spinal and supraspinal sites.