Determination of effects of antiepileptic drugs on SNAREs-mediated hippocampal monoamine release using in vivo microdialysis

1. To elucidate possible mechanisms underlying the effects of carbamazepine (CBZ), valproate (VPA) and zonisamide (ZNS) on neurotransmitter exocytosis, the interaction between these three antiepileptic drugs (AEDs) and botulinum toxins (BoNTs) on basal, Ca(2+)- and K(+)-evoked release of dopamine (DA) and serotonin (5-HT) were determined by microdialysis in the hippocampus of freely moving rats. 2. Basal release of monoamine was decreased by pre-microinjection of the syntaxin inhibitor, BoNT/C, but only weakly affected by the synaptobrevin inhibitor, BoNT/B. Ca(2+)-evoked release was inhibited by BoNT/C selectively. K(+)-evoked release was reduced by BoNT/B predominantly and BoNT/C weakly. 3. Perfusion with low and high concentrations of CBZ and ZNS increased and decreased basal monoamine release, respectively. Perfusion with VPA increased basal 5-HT release concentration-dependently, whereas basal DA release was affected by VPA biphasic concentration-dependently, similar to CBZ and ZNS. This stimulatory action of AEDs on basal release was inhibited by BoNT/C predominantly. 4. Ca(2+)-evoked monoamine release was increased by low concentrations of CBZ, ZNS and VPA, but decreased by high concentrations. These effects of the AEDs on Ca(2+)-evoked release were inhibited by BoNT/C, but not by BoNT/B. 5. K(+)-evoked monoamine release was reduced by AEDs concentration-dependently. The inhibitory effect of these three AEDs on K(+)-evoked release was inhibited by BoNT/B, but not by BoNT/C. 6. These findings suggest that the therapeutic-relevant concentration of CBZ, VPA and ZNS affects exocytosis of DA and 5-HT, the enhancement of syntaxin-mediated monoamine release during resting stage, and the inhibition of synaptobrevin-mediated release during depolarizing stage.     

Adenosine receptors in rat basophilic leukaemia cells: transductional mechanisms and effects on 5-hydroxytryptamine release.

1. The presence of adenosine receptors linked to adenylate cyclase activity and their functional role in calcium-evoked 5-hydroxytryptamine (5-HT) release was investigated in rat basophilic leukaemia (RBL) cells, a widely used model for studying the molecular mechanisms responsible for stimulus-secretion coupling. 2. In [3H]-5-HT-loaded cells triggered to release by the calcium ionophore A23187, a biphasic modulation of 5-HT secretion was induced by adenosine analogues, with inhibition of stimulated release at nM and potentiation at microM concentrations, suggesting the presence of adenosine receptor subtypes mediating opposite effects on calcium-dependent release. This was also confirmed by results obtained with other agents interfering with adenosine pharmacology, such as adenosine deaminase and the non-selective A1/A2 antagonist 8-phenyl-theophylline. 3. Similar biphasic dose-response curves were obtained with a variety of adenosine analogues on basal adenylate cyclase activity in RBL cells, with inhibition and stimulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) production at nM and microM concentrations, respectively. The rank order of potency of adenosine analogues for inhibition and stimulation of adenylate cyclase activity and the involvement of G-proteins in modulation of cyclic AMP levels suggested the presence of cyclase-linked A1 high-affinity and A2-like low-affinity adenosine receptor subtypes. However, the atypical antagonism profile displayed by adenosine receptor xanthine antagonists on cyclase stimulation suggested that the A2-like receptor expressed by RBL cells might represent a novel cyclase-coupled A2 receptor subtype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dopamine and ethanol cause translocation of epsilonPKC associated with epsilonRACK: cross-talk between cAMP-dependent protein kinase A and protein kinase C signaling pathways

We found previously that neural responses to ethanol and the dopamine D2 receptor (D2) agonist 2,10,11-trihydroxy-N-propylnorapomorphine hydrobromide (NPA) involve both epsilon protein kinase C (epsilonPKC) and cAMP-dependent protein kinase A (PKA). However, little is known about the mechanism underlying ethanol- and D2-mediated activation of epsilonPKC and the relationship to PKA activation. In the present study, we used a new epsilonPKC antibody, 14E6, that selectively recognized active epsilonPKC when not bound to its anchoring protein epsilonRACK (receptor for activated C-kinase), and PKC isozyme-selective inhibitors and activators to measure PKC translocation and catalytic activity. We show here that ethanol and NPA activated epsilonPKC and induced translocation of both epsilonPKC and its anchoring protein, epsilonRACK to a new cytosolic site. The selective epsilonPKC agonist, pseudo-epsilonRACK, activated epsilonPKC but did not cause translocation of the epsilonPKC/epsilonRACK complex to the cytosol. These data suggest a step-wise activation and translocation of epsilonPKC after NPA or ethanol treatment, where epsilonPKC first translocates and binds to its RACK and subsequently the epsilonPKC/epsilonRACK complex translocates to a new subcellular site. Direct activation of PKA by adenosine-3',5'-cyclic monophosphorothioate, Sp-isomer (Sp-cAMPS), prostaglandin E1, or the adenosine A2A receptor is sufficient to cause epsilonPKC translocation to the cytosolic compartment in a process that is dependent on PLC activation and requires PKA activity. These data demonstrate a novel cross-talk mechanism between epsilonPKC and PKA signaling systems. PKA and PKC signaling have been implicated in alcohol rewarding properties in the mesolimbic dopamine system. Cross-talk between PKA and PKC may underlie some of the behaviors associated with alcoholism.     

ATP- and adenosine-mediated signaling in the central nervous system: adenosine stimulates glutamate release from astrocytes via A2a adenosine receptors

Adenosine enhanced intracellular Ca(2+) concentrations in astrocytes via A(2a) adenosine receptors involving protein kinase A (PKA) activation. The Ca(2+) rise is inhibited by brefeldin A, an inhibitor of vesicular transport; but not by neomycin and U73122, phospholipase C inhibitors; xestospongin, an IP(3)-receptor inhibitor; ryanodine, a ryanodine-receptor inhibitor; TMB-8, an endoplasmic reticulum calcium-release blocker; octanol, a gap-junction inhibitor; or cadmium, a non-selective, calcium-channel blocker. Adenosine stimulates astrocytic glutamate release via an A(2a) adenosine receptors/PKA pathway, and the release is inhibited by the vesicular transport inhibitors brefeldin A and bafilomycin A1. A(2a) adenosine receptors and the ensuing PKA events, thus, are endowed with vesicular Ca(2+) release from an unknown intracellular calcium store and vesicular glutamate release from astrocytes.     

Biochemical mechanisms in 5-hydroxytryptamine-induced human platelet aggregation

The activation of human platelets by 5-hydroxytryptamine (5-HT) is not accompanied by detectable release of ATP or TXB2. The process is unaffected by cyclooxygenase, thromboxane synthetase or combined cyclooxygenase/lipoxygenase inhibition (suprofen, indomethacin, R 19091, dazoxiben, N.D.G.A, BW755C, esculetin), indicating the absence of involvement of arachidonic acid metabolites. Transmembrane Ca2+-entry blockers (flunarizine, nifedipine, nimodipine) have no effect either, indicating that the activator calcium released by 5-HT comes from intracellular stores. The 5-HT-induced platelet activation is inhibited by stimulators of adenylate cyclase (PGE1, PGE2, isoprenaline, adenosine) and inhibitors of cAMP phosphodiesterase (papaverine, anagrelide, RA233), indicating that also for this type of platelet activation cAMP behaves as a unidirectional, inhibitory regulator.     

Rapid non-genomic activation of cytosolic cyclic AMP-dependent protein kinase activity and [Ca(2+)](i) by 17beta-oestradiol in female rat distal colon

1. In this study, the effect of 17beta-oestradiol on adenosine 3' : 5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase (PKA) activity was investigated. 2. Rapid (within 15 min) activation of basal PKA activity was observed in cytosolic fractions by 17beta-oestradiol but not by 17alpha-oestradiol, progesterone or testosterone. This stimulation was abolished by the specific PKA inhibitor PKI but not by the classical oestrogen receptor antagonist tamoxifen. 3. 17beta-Oestradiol did not stimulate basal PKA activity in membrane fractions or in cytosolic fractions from male rats. 4. The increase in cytosolic PKA activity was indirect as (i) it was inhibited by the adenylyl cyclase inhibitor SQ22536, (ii) it was mimicked by forskolin and (iii) 17beta-oestradiol did not cause a stimulation of basal PKA activity in either type I or type II commercially available PKA holoenzymes. 5. Protein kinase Cdelta (PKCdelta) was directly activated by 17beta-oestradiol. The specific PKC inhibitor, bisindolylmaleimide I (GF 109203X), abolished the 6. 17beta-oestradiol-induced PKA activation. 17beta-Oestradiol stimulate an increase in free intracellular calcium ion concentration ([Ca(2+)](i)) in isolated female but not male rat colonic crypts. This was inhibited by verapamil, nifedipine and zero extracellular [Ca(2+)] but unaffected by tamoxifen. 17alpha-Oestradiol, testosterone and progesterone failed to increase [Ca(2+)](i). 7. PKC and PKA inhibitors abolished the 17beta-oestradiol-induced increase in [Ca(2+)](i). 8. These results demonstrate the existence of a novel 17beta-oestradiol-specific PKA and Ca(2+) signalling pathway, which is both sex steroid- and gender-specific, in rat distal colonic epithelium.     

Noradrenaline synthesis after sympathetic nerve activation in rat atria and its dependence on calcium but not CAM kinase II and protein kinases A or C.

1. The biosynthesis of noradrenaline following sympathetic nerve activation was investigated in rat atria. In particular the time course of noradrenaline synthesis changes, the relationship of changes in synthesis to transmitter release and the possible roles of second messengers and protein kinases were examined. 2. Rat atria incubated with the precursor [3H]-tyrosine synthesized [3H]-noradrenaline. Synthesis was enhanced following pulsatile electrical field stimulation (3 Hz for 5 min) with the bulk of the increase occurring in the first 45 min after the commencement of electrical stimulation. In separate experiments rat atria were pre-incubated with [3H]-noradrenaline and the radioactive outflow in response to electrical field stimulation (3 Hz for 5 min) was taken as an index of noradrenaline release. 3. Stimulation-induced (S-I) noradrenaline synthesis was significantly correlated to S-I noradrenaline release for a variety of procedures which modulate noradrenaline release by mechanisms altering Ca2+ entry into the neurone (r2 = 0.99): those which decreased release: tetrodotoxin (0.3 microM), Ca(2+)-free medium, lowering the frequency of nerve activation to 1 Hz, and those which increased release, tetraethylammonium (0.3 mM), phentolamine (1 microM) and the combination of phentolamine (1 microM) and adenosine (10 microM). On the strength of this relationship we suggest that Ca2+ entry is a determining factor in S-I synthesis changes rather than the amount of noradrenaline released. Indeed the reduction in noradrenaline release with the calmodulin-dependent protein (CAM) kinase II inhibitor KN-62 (10 microM) which acts subsequent to Ca2+ entry, did not affect S-I synthesis. 4. The cell permeable cyclic AMP analogue, 8-bromoadenosine 3'',5''-monophosphate (BrcAMP, 90 and 270 microM), dose-dependently increased basal [3H]-noradrenaline synthesis in unstimulated rat atria. This effect was antagonized by the selective protein kinase A (PKA) antagonist, Rp-8-chloroadenosine 3'',5''-cyclic monophosphorothioate (RClcAMPS, 300 microM), suggesting that PKA activation enhances basal noradrenaline biosynthesis in sympathetic nerve terminals. 5. The protein kinase inhibitors, KN-62 (CAM kinase II, 10 microM), RClcAMPS (PKA, 300 microM), polymyxin B (protein kinase C (PKC), 21 microM) and staurosporine (PKC, PKA and CAM kinase II, (0.1 microM) did not affect S-I synthesis, although KN-62, polymyxin B and staurosporine decreased S-I release. We conclude that S-I synthesis is triggered by Ca2+ entering the neurone but that the signalling pathway does not involve classical protein kinases and appears distinct from the steps involved in transmitter release.     

Characterization of ERK1/2 signalling pathways induced by adenosine receptor subtypes in newborn rat cardiomyocytes

1. Adenosine A(1), A(2A), and A(3) receptors (ARs) and extracellular signal-regulated kinase 1/2 (ERK1/2) play a major role in myocardium protection from ischaemic injury. In this study, we have characterized the adenosine receptor subtypes involved in ERK1/2 activation in newborn rat cardiomyocytes. 2. Adenosine (nonselective agonist), CPA (A(1)), CGS 21680 (A(2A)) or Cl-IB-MECA (A(3)), all increased ERK1/2 phosphorylation in a time- and dose-dependent manner. The combined maximal response of the selective agonists was similar to adenosine alone. Theophylline (nonselective antagonist) inhibited completely adenosine-mediated ERK1/2 activation, whereas a partial inhibition was obtained with DPCPX (A(1)), ZM 241385 (A(2A)), and MRS 1220 (A(3)). 3. PD 98059 (MEK1; ERK kinase inhibitor) abolished all agonist-mediated ERK1/2 phosphorylation. Pertussis toxin (PTX, G(i/o) blocker) inhibited completely CPA- and partially adenosine- and Cl-IB-MECA-induced ERK1/2 activation. Genistein (tyrosine kinase inhibitor) and Ro 318220 (protein kinase C, PKC inhibitor) partially reduced adenosine, CPA and Cl-IB-MECA responses, without any effect on CGS 21680-induced ERK1/2 phosphorylation. H89 (protein kinase A, PKA inhibitor) abolished completely CGS 21680 and partially adenosine and Cl-IB-MECA responses, without any effect on CPA response. 4. Cl-IB-MECA-mediated increases in cAMP accumulation suggest that A(3)AR-induced ERK1/2 phosphorylation involves adenylyl cyclase activation via phospholipase C (PLC) and PKC stimulation. 5. In summary, we have shown that ERK1/2 activation by adenosine in cardiomyocytes results from an additive stimulation of A(1), A(2A), and A(3)ARs, which involves G(i/o) proteins, PKC, and tyrosine kinase for A(1) and A(3)ARs, and Gs and PKA for A(2A)ARs. Moreover, the A(3)AR response also involves a cAMP/PKA pathway via PKC activation.     

Opposing facilitatory and inhibitory modulation of glutamate release elicited by cAMP production in cerebrocortical nerve terminals (synaptosomes)

Activation of cAMP-protein kinase A (PKA) is widely reported to facilitate synaptic transmission. Here, we examined the presynaptic loci of PKA action using isolated nerve terminals (synaptosoms). The adenylyl cyclase (AC) activator, forskolin, failed to have any effect on 4-aminopyridine (4-AP)-evoked glutamate release, when added alone. However, in the presence of the alkylxanthine, IBMX, forskolin strongly facilitated glutamate release. This potentiation of release was blocked by the PKA inhibitors Rp-cAMPS and H7. Given that IBMX has dual activity, antagonizing adenosine receptors as well as inhibiting cAMP phosphodiesterase, we examined the effect of a selective adenosine A(1) receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and RO20-1724, a specific phosphodiesterase inhibitor. Both unmasked the forskolin-mediated modulation of glutamate release. Conversely, the adenosine analogue, N(6)-cyclohexyladenosine (CHA), reversed the facilitation induced by forskolin+RO20-1724. Adenosine A(1) receptor activation, therefore, appears to curtail cAMP/PKA-induced potentiation of glutamate release. Looking at the targets for cAMP/PKA-mediated potentiation of glutamate release, while synaptosomal excitability was only marginally increased, basal and 4-AP-evoked-increases in [Ca(2+)](c) were substantially enhanced by forskolin+IBMX. Moreover, glutamate release elicited by Ca(2+)-ionophore (ionomycin)-induced Ca(2+)-entry was facilitated by forskolin+IBMX. cAMP/PKA-mediated facilitation of glutamate release may therefore involve modulation of Ca(2+)-entry, as well as downstream events controlling synaptic vesicle recruitment and exocytosis.     

Modulation of lysophosphatidic acid-induced Cl- currents by protein kinases A and C in the Xenopus oocyte

The roles of protein kinase C (PKC) and protein kinase A (PKA) in the regulation of lysophosphatidic acid (LPA)-induced Cl- currents in Xenopus oocytes were examined. PKC activation by phorbol 12-myristate 13-acetate (PMA) treatment completely blocked LPA-induced Cl- currents by inhibiting inositol 1,4,5-trisphosphate (IP3) elevation. This inhibitory effect of PMA on the LPA response was blocked by pretreatment of oocytes with staurosporine and 3-[N-(dimethylamino)propyl-3-indiolyll-4-[3-indolyl]maleimide (GF109203X), PKC inhibitors. In addition, treatment of oocytes with GF109203X enhanced the LPA response by increasing IP3 production. Elevation of the intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration by treating oocytes with either forskolin (FK) plus isobutylmethylxanthine (IBMX) or 2'-O-dibutyryl-cAMP (dB-cAMP) reduced LPA-induced Cl- currents. The effect of activation of the cAMP pathway appears to be mediated by PKA, since treatment of oocytes with FK plus IBMX or dB-cAMP enhanced PKA activity. Furthermore, the inhibitory effect of dB-cAMP on the LPA response was blocked by treatment of oocytes with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulframide-2 HCl (H-89), a selective inhibitor of PKA. Both FK plus IBMX and dB-cAMP treatment reduced IP3 generation in response to LPA stimulation. Inhibition of PKA activity with H-89 or Rp-cyclic 3',5'-hydrogen phosphorothioate adenosine triethylammonium had no effect on LPA-induced Cl- currents. Finally, inhibition of the LPA response by activation of PKA was independent of extracellular Ca2+. These results demonstrate that both PKC and PKA play active roles in modulating the LPA-induced signaling pathway.     

Activation of mGluRII induces LTD via activation of protein kinase A and protein kinase C in the dentate gyrus of the hippocampus in vitro

The involvement of metabotropic glutamate receptor group II (mGluRII) in the induction of long-term depression (LTD) was investigated in the medial perforant path of the rat dentate gyrus, a region with a very high density of mGluRII. Perfusion of either of two potent mGluRII agonists, (2S,1R,2R,3R)-2-(2S, 1'R, 2'R, 3'R)-2 (2' 3'-dicarboxycyclopropyl)glycine (DCG-IV) or (+)-2- aminobicyclo[3.1.0]hexane-2-6-dicarboxylic acid (LY354740) induced a reversible inhibition of the field EPSP followed, upon washout of the agonist, by LTD. The reversible inhibition was associated with a change in paired pulse depression, indicating an underlying presynaptic reduction in the probability of transmitter release, whereas the LTD was not associated with a change in paired pulse depression, indicating either a presynaptic reduction in the number of active release sites, or a postsynaptic change. Further evidence that the DCG-IV-induced LTD was generated by activation of mGluRII was the finding that the mGluRII antagonist (RS)-alpha-methylserine-O-phosphate monophenylphosphoryl ester (MSOPPE) prevented the induction of the LTD induced by DCG-IV. The DCG-IV-induced LTD showed mutual occlusion with LFS-induced LTD. The generation of the agonist-induced LTD required, in part, activation of N-methyl-D-aspartate receptors (NMDAR), as LTD induction was partially blocked in the presence of the NMDAR antagonist D-2-amino-5-phosphonopentanoate (AP5). Evidence for involvement of protein kinase C (PKC) and protein kinase (PKA) in the induction of LTD by activation of mGluRII was obtained by showing an inhibition of the DCG-IV-induced LTD by the PKC inhibitors Ro-31-8220 and bisindolylmaleimide I, and also by the PKA inhibitor H-89. The study demonstrates that activation of mGluRII induces LTD via activation the PKA and PKC pathways in the medial perforant path of the dentate gyrus.     

Cyclic AMP and protein kinase A stimulate Cdc42: role of A(2) adenosine receptors in human mast cells

The functional activity of Cdc42 is known to be regulated by proteins that control its GDP/GTP-bound state. However, there is still limited information on how Cdc42 is controlled by G-protein-coupled receptors. Adenosine receptors belong to the G-protein-coupled receptor family of cell surface receptors. Human HMC-1 mast cells express the high-affinity A(2A) and the low-affinity A(2B) subtypes of adenosine receptors known to increase intracellular cAMP levels. We found that both subtypes of A(2) adenosine receptors activate Cdc42 in HMC-1 cells. Furthermore, stimulation of adenylate cyclase with forskolin, or loading of HMC-1 with the cell-permeable cAMP analog 8-Br-cAMP, activated Cdc42. Stimulation of Cdc42 by cAMP was also observed in CHO-K1 and COS-7 cells. Protein kinase A (PKA)-mediated phosphorylation is likely involved in cAMP-dependent Cdc42 activation, because transient expression of the PKA catalytic subunit in COS-7 cells activated Cdc42. Inhibition of protein phosphatases 1 and 2A with calyculin A potentiated the effects of 5'-N-ethylcarboxamidoadenosine and 8-Br-cAMP, whereas the selective PKA inhibitor H-89 reversed the activation of Cdc42. We demonstrated that Cdc42 is a poor substrate for PKA phosphorylation in vitro and in intact cells. Our data suggest that PKA does not phosphorylate Cdc42 directly. Instead, the proteins that modulate the GDP/GTP-bound state of Cdc42 may be the primary targets of PKA phosphorylation.     

Effect of 5-hydroxy-L-tryptophan on the release of 5-HT in rat hypothalamus in vivo as measured by microdialysis.

The effect of systemic administration of the 5-HT precursor, 5-hydroxy-L-tryptophan (5-HTP) on the release of 5-HT in the lateral hypothalamus of the chloral hydrate-anaesthetized rat in vivo was examined using brain microdialysis. Administration of 5-HTP caused an immediate increase of 5-HT in dialysates, which was long lasting (greater than or equal to 140 min) and dose-dependent (30-100 mg/kg i.p.). When calcium was omitted from the perfusion medium, thereby limiting exocytosis, levels of basal 5-HT were significantly decreased and the 5-HTP-induced response of 5-HT was markedly attenuated. Administration of the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (0.25 mg/kg i.p.), which selectively inhibits serotoninergic neuronal activity by activation of the somatodendritic 5-HT autoreceptor, significantly decreased basal levels of 5-HT and markedly attenuated the 5-HTP-induced increase in 5-HT. The data demonstrate that systemic administration of 5-HTP caused an increase in the release of 5-HT in the hypothalamus. Furthermore, this release occurred by a calcium-dependent mechanism (probably exocytosis), was dependent on serotoninergic neuronal activity and predominantly derived from 5-HT neurones. The findings are discussed in relation to the behavioral and neuroendocrine effects of increasing availability of the 5-HT precursor.     

Involvement of protein kinase C in 5-HT-evoked thermal hyperalgesia and spinal fos protein expression in the rat

The present study was designed to characterize nociceptive response induced by 5-hydroxytryptamine (5-HT) and to investigate effects of inhibition of protein kinase C (PKC) in the periphery on noxious stimulus-evoked activity of the secondary neurons in the spinal cord. Subcutaneous injection of 5-HT (50 microg) and alpha-methylserotonin (alpha-m-5-HT, 5-HT2A receptor agonist, 50 microg) into the unilateral hindpaw evoked significant decreases in paw withdrawal latency (PWL). The 5-HT-induced hyperalgesia was abolished by ketanserin (5-HT2A antagonist, 10 microg, intraplantarly or i.pl.), but not by WAY100635 (5-HT1A antagonist, 100 microg, i.pl.). 5-HT and alpha-m-5-HT also evoked numerous expressions of c-Fos-like immunoreactivity (c-fos-LI) in the ipsilateral dorsal horn (predominantly laminae I-II) of the lumbar spinal cord. However, treatment with 8-OH-DPAT (5-HT1A receptor agonist, 100 microg, i.pl.) elicited only moderate thermal hyperalgesia and very limited expression of spinal c-fos-LI. Intraplantar chelerythrine (2, 6 or 10 microg), a PKC inhibitor, dose-dependently attenuated the hyperalgesia evoked by alpha-m-5-HT. Chelerythrine (10 microg, i.pl.) also completely prevented the development of hyperalgesia evoked by 5-HT but not by 8-OH-DPAT. Furthermore, pretreatment with chelerythrine significantly inhibited the expressions of c-fos-LI evoked by alpha-m-5-HT in laminae I-VI and by 5-HT in laminae I-II. These results demonstrate that PKC activation was involved in the development of nociceptive responses elicited by 5-HT and activation of peripheral 5-HT2A, but not 5-HT1A, receptors. The study also provides evidence at a cellular level that inhibition of PKC in the periphery suppresses the 5-HT-evoked neuronal activity in the central nervous system.     

Separate purinoceptors mediate enhancement by adenosine of concanavalin A-induced mediator release and the cyclic AMP response in rat mast cells

Adenosine caused a concentration-related enhancement of concanavalin A (Con A)-induced 5-hydroxytryptamine (5-HT) release from rat purified peritoneal mast cells. This was accompanied by an enhancement and prolongation of the cyclic AMP response to Con A. The cyclic AMP response but not enhancement of 5-HT release was blocked by 8-phenyltheophylline suggesting the two events to be unrelated. The effects of AMP and ADP, adenosine analogues and adenosine uptake inhibitors suggest the enhancement of 5-HT release to be mediated by a P1-cell surface purinoceptor which does not show the characteristics of either A1 or A2 subtypes.     

Protein kinase associated with gating and closing transmission mechanisms in temporoammonic pathway

The entorhinal cortex (EC) is a major source of afferent input to the hippocampus via the perforant and temporoammonic pathways; however, the detailed transmission mechanism in the temporoammonic pathway remains to be clarified. Thus, we determined interaction among GABA(A), AMPA/glutamate receptors and protein kinases (PKA and PKC) in the exocytosis of GABA and glutamate using multiprobe microdialysis, as well as propagation of neuronal excitability using optical recording in the EC-Hippocampal formation. Multiprobe microdialysis demonstrated that EC-evoked GABA release in ventral CA1 was predominantly regulated by the PKC-related rather than PKA-related exocytosis mechanism and was augmented by the activation of glutamatergic transmission. Contrary to GABA release, EC-evoked glutamate release was predominantly regulated by PKA-related rather than PKC-related mechanisms and was suppressed by activation of GABAergic transmission. Optical recording demonstrated that there are two sub-pathways in the temporoammonic pathway; direct projects from EC layers (II-IV) to dendrites on pyramidal cells and GABAergic interneurons in ventral hippocampal CA1. PKC activation enhanced trisynaptic transmission, whether the GABA(A) receptor was functional or blocked, whereas PKC activation enhanced and inhibited temporoammonic transmission when the GABA(A) receptor was functional and blocked, respectively. Thus, GABAergic inhibition, which is regulated by PKC activity, in the temporoammonic pathway is more significant than that in the trisynaptic pathway.     

Second messenger-dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

1. The present study investigated the role of second messenger-dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108-15 mouse neuroblastomaxrat glioma hybrid cells. 2. In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12-myristate 13-acetate (PMA) or by purinoceptor stimulation using uridine 5'-triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin-stimulated cyclic AMP accumulation but did not affect cyclic AMP responses mediated by the prostanoid-IP receptor agonist iloprost, or the A(2) adenosine receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA). 3. In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of G(s)-coupling components in NG108-15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation. 4. Cells exposed to the PKA inhibitor H-89, exhibited a time-dependent increase in secretin receptor responsiveness compared to control cells. This effect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not affected by H-89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time-dependent increase in secretin receptor responsiveness. 5. Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108-15 cells.     

Xaliproden (SR57746A) induces 5-HT1A receptor-mediated MAP kinase activation in PC12 cells

Neurotrophic growth factors are involved in cell survival. However, natural growth factors have a very limited therapeutic use because of their short half-life. In the present study, we investigated the mechanism of action of a non-peptidic neurotrophic drug, Xaliproden, a potential molecule for the treatment of motoneuron diseases, since the transduction pathways of this synthetic 5-HT1A agonist are very poorly understood. Xaliproden does not activate the Trk receptor but causes a rapid increase in the activities of the ERK1 and ERK2 isoforms of MAP kinase, which then rapidly decrease to the basal level. We demonstrate that isoforms of the SHC adapter protein are phosphorylated independently of each other and are probably not the source of the Xaliproden-induced MAP kinases activation. The inhibitor of Ras farnesylation, FPT-1, and the protein kinase C inhibitors, GF 109203X and chelerythrine, inhibited the Xaliproden-induced MAP kinase activation, suggesting p21Ras and PKC involvement. Moreover, the observations that the 5-HT1A antagonist, pindobind, and pertussis toxin abolished the Xaliproden-induced ERK stimulation suggested that Xaliproden activates the MAP kinase pathways by stimulating the G protein-coupled receptor, 5-HT1A. These results demonstrate clearly that the non-peptidic compound, Xaliproden, exerts its neurotrophic effects through a mechanism of action differing from that of neurotrophins. These findings suggest that this compound does not involve MAPK activation by TrkA receptor stimulation but acts by MAP kinase pathway by a pertussis toxin-sensitive mechanism involving 5-HT1A receptors, p21 Ras and MEK-1 and by PKC and Akt pathways.     

Additive effect of ADP and CGRP in modulation of the acetylcholine receptor channel in Xenopus embryonic myocytes.

1. We have previously shown that the activation of either protein kinase A (PKA) or protein kinase C (PKC) enhanced the responses of muscle membrane to acetylcholine (ACh) by increasing the mean open time of embryonic-type ACh channels in Xenopus cultured myocytes. In the present study, we further investigated the interaction between these two kinases in the modulation of ACh channels by using the receptor ligands, adenosine diphosphate (ADP) and calcitonin gene-related peptide (CGRP) which selectively activate PKC and PKA, respectively. 2. ADP concentration-dependently increased the mean open time of embryonic-type ACh channels and 0.3 mM ADP is sufficient to achieve the maximal potentiating effect. alpha, beta-Methylene ATP and PMA (phorbol 12-myristate 13-acetate) but not adenosine, AMP, dibutyryl cyclic GMP have similar potentiating action. 3. Suramin (0.3 mM) pretreatment abolished the potentiating effect of ADP but left that of PMA unchanged. 4. CGRP increased the mean open time of embryonic-type ACh channels in a concentration-dependent manner and 1 microM CGRP produced the maximal effect. 5. The maximal effects of both ADP (0.3 mM) and CGRP (1 microM) in the prolongation of mean open time of ACh channels were additive. 6. These results suggest that the modulation of embryonic-type ACh channels by the endogenously released ligands via the activation of PKA and PKC is additive and possibly different sites of ACh channels may be involved in the potentiation effect of either PKC or PKA.     

Towards a quantitative representation of the cell signaling mechanisms of hallucinogens: measurement and mathematical modeling of 5-HT1A and 5-HT2A receptor-mediated ERK1/2 activation

Through a multidisciplinary approach involving experimental and computational studies, we address quantitative aspects of signaling mechanisms triggered in the cell by the receptor targets of hallucinogenic drugs, the serotonin 5-HT2A receptors. To reveal the properties of the signaling pathways, and the way in which responses elicited through these receptors alone and in combination with other serotonin receptors' subtypes (the 5-HT1AR), we developed a detailed mathematical model of receptor-mediated ERK1/2 activation in cells expressing the 5-HT1A and 5-HT2A subtypes individually, and together. In parallel, we measured experimentally the activation of ERK1/2 by the action of selective agonists on these receptors expressed in HEK293 cells. We show here that the 5-HT1AR agonist Xaliproden HCl elicited transient activation of ERK1/2 by phosphorylation, whereas 5-HT2AR activation by TCB-2 led to higher, and more sustained responses. The 5-HT2AR response dominated the MAPK signaling pathway when co-expressed with 5-HT1AR, and diminution of the response by the 5-HT2AR antagonist Ketanserin could not be rescued by the 5-HT1AR agonist. Computational simulations produced qualitative results in good agreement with these experimental data, and parameter optimization made this agreement quantitative. In silico simulation experiments suggest that the deletion of the positive regulators PKC in the 5-HT2AR pathway, or PLA2 in the combined 5-HT1A/2AR model greatly decreased the basal level of active ERK1/2. Deletion of negative regulators of MKP and PP2A in 5-HT1AR and 5-HT2AR models was found to have even stronger effects. Under various parameter sets, simulation results implied that the extent of constitutive activity in a particular tissue and the specific drug efficacy properties may determine the distinct dynamics of the 5-HT receptor-mediated ERK1/2 activation pathways. Thus, the mathematical models are useful exploratory tools in the ongoing efforts to establish a mechanistic understanding and an experimentally testable representation of hallucinogen-specific signaling in the cellular machinery, and can be refined with quantitative, function-related information.