Cyclic Monophosphothioate Triethylamine Research Articles

GDPbS Activates Excitatory Synapses in CA1 Pyramidal Cells by Disinhibiting the PKA Activating Pathway

Objective: During the early postnatal brain developmental stages, excitatory synapses mediated by AMPA receptors are weak or silent. Activity-dependent insertion of AMPA receptors into synapses depends on the activation of protein kinase A. In this work, we investigated the effect of Guanosine 5’-[βthiol] diphosphate (GDPsS) on excitatory and inhibitory synaptic currents in CA1 pyramidal cells at postnatal days 9-12. Methods: Whole-cell patch-clamp recordings from identified hippocampal CA1 pyramidal cells were used. GDPβS Was applied through the recording electrode. Results: GDPβS induces an increase in excitatory synaptic current amplitude, but not in the inhibitory synaptic current amplitude. An analysis of the change in excitatory synaptic current amplitude in the presence of GDPβS revealed a progressive increase, which is blocked by the protein kinase A inhibitor Rp-3’,5’-cyclic monophosphothioatetriethylamine (Rp-cAMP), suggesting that GDPβS inhibits G-protein with a tonic negative control on a protein kinase A activating pathway. In addition, GDPβS has no effect on paired-pulse facilitation, suggesting that the glutamate release machinery is not affected. Moreover, as GDPβS was applied to postsynaptic neurons, the increase in excitatory postsynaptic current amplitude is related to changes at the postsynaptic side. Conclusion: Those results suggest that in developing hippocampal CA1 pyramidal cells, the tonic inhibition of a protein kinase A activating pathway by a G-protein prevents the activation of excitatory synapses

Ethanol Activation of Protein Kinase A Regulates GABAAα1 Receptor Function and Trafficking in Cultured Cerebral Cortical Neurons

Ethanol exposure produces alterations in GABAergic signaling that are associated with dependence and withdrawal. Previously, we demonstrated that ethanol-induced protein kinase C (PKC) γ signaling selectively contributes to changes in GABAA α1 synaptic receptor activity and surface expression. Here, we demonstrate that protein kinase A (PKA) exerts opposing effects on GABAA receptor adaptations during brief ethanol exposure. Cerebral cortical neurons from day 0-1 rat pups were tested after 18 days in culture. Receptor trafficking was assessed by Western blot analysis, and functional changes were measured using whole-cell patch-clamp recordings of evoked and miniature inhibitory postsynaptic current (mIPSC) responses. One-hour ethanol exposure increased membrane-associated PKC and PKA, but steady-state GABAA α1 subunit levels were maintained. Activation of PKA by Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine alone increased GABAA α1 subunit surface expression and zolpidem potentiation of GABA responses, whereas coexposure of ethanol with the PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine decreased α1 subunit expression and zolpidem responses. Exposure to the PKC inhibitor calphostin-C with ethanol mimicked the effect of direct PKA activation. The effects of PKA modulation on mIPSC decay τ were consistent with its effects on GABA currents evoked in the presence of zolpidem. Overall, the results suggest that PKA acts in opposition to PKC on α1-containing GABAA receptors, mediating the GABAergic effects of ethanol exposure, and may provide an important target for the treatment of alcohol dependence/withdrawal.

Obestatin as contractile mediator of excised frog heart

AbstractThe aim of this study is to investigate the mechanism of positive inotropic effect of obestatin on in vitro heart preparations of Rana ridibunda frog. The application of increasing amounts of obestatin in the concentration range from 1 μmol/l to 1 μmol/l significantly enhances the force of contraction of excised and cannulated frog hearts. This effect was partially reduced in the presence of prazosin (3 μmol/l). Propranolol (30 μmol/l), pertussis toxin (2 ng/ml) and the specific inhibitor of cAMP-dependent protein kinase (PKA) Rp-adenosine 3′,5′-cyclic monophosphothioate triethylamine (30 μmol/l) completely blocked the obestatin-induced increase of the force of frog heart contractions. It is concluded that, via its receptor molecule, obestatin activates neuronal pertussis toxin sensitive G-protein(s) that further enhance the secretion of epinephrine from sympathetic neurons. This epinephrine activates mainly the myocardial β-adrenoreceptors and PKA downstream targets, and is responsible for the observed positive inotropic effect of obestatin. An alternative explanation of our data is that obestatin directly enhances the effect of myocardial β-adrenergic signaling.

Activation of Pedunculopontine Tegmental PKA Prevents GABAB Receptor Activation–Mediated Rapid Eye Movement Sleep Suppression in the Freely Moving Rat

The pedunculopontine tegmental (PPT) GABAergic system plays a crucial role in the regulation of rapid eye movement (REM) sleep. I recently reported that the activation of PPT GABA(B) receptors suppressed REM sleep by inhibiting REM-on cells. One of the important mechanisms for GABA(B) receptor activation-mediated physiological action is the inhibition of the intracellular cAMP-dependent protein kinase A (cAMP-PKA) signaling pathway. Accordingly, I hypothesized that the PPT GABA(B) receptor activation-mediated REM sleep suppression effect could be mediated through inhibition of cAMP-PKA activation. To test this hypothesis, a GABA(B) receptor selective agonist, baclofen hydrochloride (baclofen), cAMP-PKA activator, Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine (SpCAMPS), and vehicle control were microinjected into the PPT in selected combinations to determine effects on sleep-waking states of chronically instrumented, freely moving rats. Microinjection of SpCAMPS (1.5 nmol) induced REM sleep within a short latency (12.1 +/- 3.6 min) compared with vehicle control microinjection (60.0 +/- 6.5 min). On the contrary, microinjection of baclofen (1.5 nmol) suppressed REM sleep by delaying its appearance for approximately 183 min; however, the suppression of REM sleep by baclofen was prevented by a subsequent microinjection of SpCAMPS. These results provide evidence that the activation of cAMP-PKA within the PPT can successfully block the GABA(B) receptor activation-mediated REM sleep suppression effect. These findings suggest that the PPT GABA(B) receptor activation-mediated REM sleep regulating mechanism involves inactivation of cAMP-PKA signaling in the freely moving rat.

Pituitary adenylate cyclase-activating polypeptide promotes differentiation of mouse neural stem cells into astrocytes

We have found that pituitary adenylate cyclase-activating polypeptide (PACAP) employed at the physiological concentrations induces the differentiation of mouse neural stem cells into astrocytes. The differentiation process was not affected by cAMP analogues such as dibutylic cAMP (db-cAMP) or 8Br-cAMP or by the specific competitive inhibitor of protein kinase A, Rp-adenosine-3′,5′-cyclic monophosphothioate triethylamine salt (Rp-cAMP). Expression of the PACAP receptor (PAC1) in neural stem cells was detected by both RT–PCR and immunoblot using an affinity-purified antibody. The PACAP selective antagonist, PACAP 6-38, had an inhibitory effect on the PACAP-induced differentiation of neural stem cells into astrocytes. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, with the signal then transmitted intracellularly via a PAC1-coupled G protein, does not involve Gs. This signaling mechanism may thus play a crucial role in the differentiation of neural stem cells into astrocytes.

Central nNOS is involved in restraint stress-induced fever: evidence for a cGMP pathway

It has been shown that the NO pathway plays a major role in restraint stress-induced fever, and that the neuronal nitric oxide synthase (nNOS) seems to be the NOS isoform that accounts for the pyretic effect of NO in psychological stress-induced fever. However, no information exists as to localization of the nNOS, i.e., in the peripheral or in the central nervous system (CNS). Thus, in the present study, we tested the hypothesis that NO arising from nNOS in the CNS participates in restraint stress-induced fever. Moreover, we also assessed the involvement of cyclic guanosine monophosphate (cGMP) in the mediation of the NO effects. To this end, intracerebroventricular S-methyl- l-thiocitrulline (SMTC; a selective nNOS inhibitor), sodium nitroprusside (an NO donor) or Rp-guanosine 3′,5′-cyclic monophosphothioate triethylamine (Rp-cGMPS; a specific membrane-permeable inhibitor of the activation by cGMP of cGMP-dependent protein kinase) were injected, and the colonic temperature ( T c) of restrained or unrestrained rats was recorded. Both SMTC (0.5 mg/μl) and Rp-cGMPS (10 μg/μl) intracerebroventricular injections enhanced restraint fever, whereas intracerebroventricular injections of sodium nitroprusside (100 μg/μl) reduced this response. These data indicate that NO produced in the CNS, arising from nNOS and acting via cGMP, plays an antipyretic role in the restraint stress-induced fever.

β 1-Adrenoceptor antibodies induce apoptosis in adult isolated cardiomyocytes

β 1-Adrenoceptor autoantibodies are present in about 30% of patients suffering from dilated cardiomyopathy. The apoptotic effects mediated by β 1-adrenoceptor antibodies remain to be studied. Monoclonal antibodies were raised against a synthetic peptide corresponding to the second extracellular loop of the human β 1-adrenoceptor in balb/C mouse, and were characterized by enzyme immunoassay. Purified immunoglobulin G from nonimmunized animals (controls) did not influence the rate of apoptosis. β 1-Adrenoceptor antibodies caused a dose-related increase in apoptotic cells: annexin test (dilution 1:2: 21±1.1% apoptotic cells vs. 4±0.4% apoptotic cells in controls; p<0.01); TdT-mediated dUTP nick end labeling (TUNEL) test (dilution 1:2: 26±2% apoptotic cells vs. 10±2% apoptotic cells in controls; p<0.01). The effect of the β 1-adrenoceptor antibodies was blocked by the antigenic peptide and by the antagonist metoprolol (10 μmol/l). The apoptotic effect induced by isoproterenol was attenuated by the β 1-adrenoceptor antibody. After pre-incubation of cardiomyocytes with the protein kinase A inhibitor Rp-Adenosine-3′,5′-cyclic monophosphothioate triethylamine (RpcAMPS), β 1-adrenoceptor antibody was not capable of inducing an increase of the rate of apoptosis. β 1-Adrenoceptor antibodies induced apoptosis in adult rat cardiomyocytes via the protein kinase A cascade.

Presynaptic H3 autoreceptors modulate histamine synthesis through cAMP pathway.

Histamine H3 receptors modulate histamine synthesis, although little is known about the transduction mechanisms involved. To investigate this issue, we have used a preparation of rat brain cortical miniprisms in which histamine synthesis can be modulated by depolarization and by H3 receptor ligands. When the miniprisms were incubated in presence of forskolin, dibutyryl-cAMP, or 3-isobutyl-1-methylxanthine (IBMX), histamine synthesis was stimulated in 34, 29, and 47%, respectively. These stimulations could be prevented by the selective cAMP protein kinase blocker Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPs). Preincubation with the H3 receptor agonist imetit prevented IBMX- (100% blockade) and forskolin- (70% blockade) induced stimulation of histamine synthesis. The H3 inverse agonist thioperamide enhanced histamine synthesis in the presence of 1 mM IBMX or 30 mM potassium (+47 and +45%, respectively). Similarly, the H3 antagonist clobenpropit enhanced histamine synthesis in the presence of 30 mM potassium (+ 59%). The cAMP-dependent protein kinase blockers Rp-cAMPs and PKI14-22 could impair the effects of thioperamide and clobenpropit, respectively. These results indicate that the adenylate cyclase-protein kinase A pathway is involved in the modulation of histamine synthesis by H3 autoreceptors present in histaminergic nerve terminals.

Inhibition of protein kinase A activity interferes with long-term, but not short-term, memory of conditioned taste aversions.

The present experiments examined whether inhibition of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activity interferes with conditioned taste aversion (CTA) memories. Rats were centrally infused with the selective PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) before conditioning. Direct infusions of Rp-cAMPS into the amygdala showed no interference with short-term memory but did show significant attenuation of long-term memory and more rapid extinction. Results suggest that PKA activity is involved in the consolidation of long-term memory of CTAs, and that the amygdala may be 1 site that is important for this activity.

Adenosine exerts multiple effects in dorsal horn neurones of the adult rat spinal cord

In the present study, we have examined the effects of adenosine and its analogues on the electrophysiological properties of dorsal horn neurones in the rat adult spinal cord. Adenosine and the A 1 receptor agonist R-phenylisopropyl adenosine (RPIA) reversibly hyperpolarised these neurones via the generation of an outward current at −60 mV that was inhibited by pre-application of barium or Rp-adenosine 3′, 5′-cyclic monophosphothioate triethylamine. In contrast, the A 2a receptor agonist 2-[ p-(2-carboxyethyl)phenylethylamino]-5′- N-ethylcarboxamidoadenosine (CGS21680) had no effect on the resting membrane properties of these neurones. Stimulation of the dorsal root evoked non-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) at −60 mV that were completely abolished by 2,3-dihydroxy-6-nitro-7-sulophamoyl-benzo(F)quinoxalone (NBQX). Bath application of adenosine or RPIA reversibly inhibited these EPSCs in a concentration-dependent manner via a presynaptic action. In contrast, CGS21680 increased the amplitude of the EPSC in 20% of neurones tested and decreased the EPSC amplitude in 30% of neurones tested. It is concluded that adenosine exerts multiple effects upon the electrophysiological properties of dorsal horn neurones in the adult spinal cord via interaction with multiple receptors. These findings have important implications in the understanding of adenosine action in preclinical models of pain.

Ionotropic Histamine Receptors and H2Receptors Modulate Supraoptic Oxytocin Neuronal Excitability and Dye Coupling

Histaminergic neurons of the tuberomammillary nucleus (TM) project monosynaptically to the supraoptic nucleus (SON). This projection remains intact in our hypothalamic slices and permits investigation of both brief synaptic responses and the effects of repetitively activating this pathway. SON oxytocin (OX) neurons respond to single TM stimuli with fast IPSPs, whose kinetics resemble those of GABA(A) or glycine receptors. IPSPs were blocked by the Cl(-) channel blocker picrotoxin, but not by bicuculline or strychnine, and by histamine H(2), but not by H(1) or H(3) receptor antagonists, suggesting the presence of an ionotropic histamine receptor and the possible nonspecificity of currently used H(2) antagonists. G-protein mediation of the IPSPs was ruled out using guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS), pertussis toxin, and Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPs), none of which blocked evoked IPSPs. We also investigated the effects of synaptically released histamine on dye coupling and neuronal excitability. One hundred seventy-three OX neurons were Lucifer yellow-injected in horizontal slices. Repetitive TM stimulation (10 Hz, 5-10 min) reduced coupling, an effect blocked by H(2), but not by H(1) or H(3), receptor antagonists. Because H(2) receptors are linked to activation of adenylyl cyclase, TM-stimulated reduction in coupling was blocked by GDP-betaS, pertussis toxin, and Rp-cAMPs and was mimicked by 8-bromo-cAMP, 3-isobutyl-1-methylxanthine, and Sp-cAMP. Membrane potentials of OX and vasopressin neurons were hyperpolarized, accompanied by decreased conductances, in response to bath application of 8-bromo-cAMP but not the membrane-impermeable cAMP. These results suggest that synaptically released histamine, in addition to evoking fast IPSPs in OX cells, mediates a prolonged decrease in excitability and uncoupling of the neurons.

PDGF-BB REGULATES IGF-MEDIATED IGFBP-4 PROTEOLYSIS IN FETAL LUNG FIBROBLASTS

Insulin-like growth factor (IGF)-stimulated lung fibroblast proliferation may be regulated by locally produced IGF-binding proteins (IGFBPs) during lung development. Recent evidence has shown that many growth factors participate in the regulation of cell proliferation by regulating IGFBPs. Because platelet-derived growth factor-BB (PDGF-BB)is highly expressed during lung development and is known to regulate IGFBP-4 production bylung cells, we examined the mechanisms by which PDGF-BB regulates IGFBP-4 production using primary cultures of 19-day gestation rat lung fibroblasts. Exposure of fetal rat lung fibroblasts to PDGF-BB increased IGFBP-4 mRNA transcript abundance by 3.6- and 2.4-fold at 18 and 40 hours, respectively. Addition of Rp-adenosine 3'-5'-cyclic monophosphothioa tetriethylamine (rp-cAMPS),a competitive inhibitor of protein kinase A, blunted the PDGF-BB-stimulated increase in conditioned medium (CM)IGFBP-4 and the increase in IGFBP-4 mRNA. Proteolysis of IGFBP-4 was detected in aliquots of cell-free CM from cells exposed to SFMfor 48 hours. IGFBP-4 proteolysis was inhibited by EDTA and 1,10-phenanthroline and was accentuated by the addition of IGF-I and IGF-IIand, to a lesser extent, by des(1-3)IGF-I. Exposure of cells to PDGF-BB for 48 hours resulted in an inhibition of IGFBP-4 proteolysis that was associated with a decrease in the concentration of IGF-I in CM. These studies demonstrate that PDGF-BB increases the accumulation of IGFBP-4 in fetal rat lung fibroblasts CM through increased production and by inhibiting IGF-mediated IGFBP-4 proteolysis.

Conditioned locomotion in rats following amphetamine infusion into the nucleus accumbens: blockade by coincident inhibition of protein kinase A.

Recent studies demonstrate a role for cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in the nucleus accumbens (NAc) in reward-related learning. To clarify this role, we assessed the effect of PKA inhibition on the unconditioned and conditioned locomotor activating properties of intra-NAc amphetamine. Rats underwent three 60 min conditioning sessions, pairing a test environment with bilateral co-infusions of amphetamine (25 microg/side) and the PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) (0, 2.5, 250, 500 ng, 1, 10 or 20 microg/side). Two additional groups - receiving amphetamine explicitly unpaired with the environment or saline/environment pairings - served as controls. In a subsequent drug-free 60 min session, animals that received amphetamine/environment pairings demonstrated conditioned locomotion relative to controls. Rp-cAMPS co-treatment during pairing sessions differentially affected conditioned and unconditioned locomotor activation. Amphetamine-induced unconditioned activity was significantly enhanced by 500 ng and 1 microg Rp-cAMPS, locomotor sensitization was enhanced by 250 ng-1 microg Rp-cAMPS, and conditioned activity was attenuated by 1 microg Rp-cAMPS and blocked by 10 and 20 microg Rp-cAMPS. Thus, unconditioned activity and locomotor sensitization were enhanced at doses (250 ng-1 microg) that did not affect or attenuated conditioned activity, while conditioned activity was reduced or blocked at doses (1-20 microg) that enhanced or did not affect overall unconditioned activity. These results demonstrate that the activation of PKA plays a critical role in the process by which properties of drugs become associated with environmental stimuli.

Stimulant action of pituitary adenylate cyclase-activating peptide on normal and drug-compromised peristalsis in the guinea-pig intestine.

1. Pituitary adenylate cyclase-activating peptide (PACAP) is known to influence the activity of intestinal smooth muscle. This study set out to examine the action of PACAP on normal and drug-inhibited peristalsis and to shed light on its site and mode of action. 2. Peristalsis in isolated segments of the guinea-pig small intestine was elicited by distension through a rise of the intraluminal pressure. Drug-induced motility changes were quantified by alterations of the peristaltic pressure threshold at which aborally moving peristaltic contractions were triggered. 3. PACAP (1-30 nM) stimulated normal peristalsis as deduced from a concentration-related decrease in the peristaltic pressure threshold (maximum decrease by 55%). The peptide's stimulant effect remained intact in segments pre-exposed to apamin (0.5 microM), N-nitro-L-arginine methyl ester (300 microM), naloxone (0.5 microM), atropine (1 microM) plus naloxone (0.5 microM) or hexamethonium (100 microM) plus naloxone (0.5 microM). 4. PACAP (10 nM) restored peristalsis blocked by morphine (10 microM), noradrenaline (1 microM) or N6-cyclopentyladenosine (0.3 microM) and partially reinstated peristalsis blocked by Rp-adenosine-3',5'-cyclic monophosphothioate triethylamine (100 microM) but failed to revive peristalsis blocked by hexamethonium (100 microM) or atropine (1 microM). The peptide's spectrum of properistaltic activity differed from that of naloxone (0.5 microM) and forskolin (0.3 microM). 5. The distension-induced ascending reflex contraction of the circular muscle was facilitated by PACAP (1-30 nM) which itself evoked transient nerve-mediated contractions of intestinal segment preparations. 6. These data show that PACAP stimulates normal peristalsis and counteracts drug-induced peristaltic arrest by a stimulant action on excitatory enteric motor pathways, presumably at the intrinsic sensory neurone level. The action of PACAP seems to involve multiple signalling mechanisms including stimulation of adenylate cyclase.

Involvement of endothelium/nitric oxide in vasorelaxation induced by purified green tea (−)epicatechin

The present study investigated the involvement of endothelial nitric oxide in relaxation induced by purified green tea (−)epicatechin in rat isolated mesenteric arteries. (−)Epicatechin caused both endothelium-dependent and -independent relaxation. N G-Nitro- l-arginine methyl ester ( l-NAME, 100 μM) and methylene blue (10 μM) significantly attenuated (−)epicatechin-induced relaxation in endothelium-intact tissues. l-Arginine (1 mM) partially antagonized the effect of l-NAME. (−)Epicatechin-induced relaxation was inhibited by Rp-guanosine 3′,5′-cyclic monophosphothioate triethylamine. In contrast, indomethacin and glibenclamide had no effect. (−)Epicatechin (100 μM) significantly increased the tissue content of cyclic GMP and N G-nitro- l-arginine (100 μM) or removal of the endothelium abolished this increase. (−)Epicatechin (100 μM) induced an increase in intracellular Ca 2+ levels in cultured human umbilical vein endothelial cells. Iberiotoxin at 100 nM attenuated (−)epicatechin-induced relaxation in endothelium-intact arteries and this effect was absent in the presence of 100 μM l-NAME. In summary, (−)epicatechin-induced endothelium-dependent relaxation is primarily mediated by nitric oxide and partially through nitric oxide-dependent activation of iberiotoxin-sensitive K + channels. In addition, there may be a causal link between increased Ca 2+ levels and nitric oxide release in response to (−)epicatechin.

Interplay between the NO pathway and elevated [Ca2+]i enhances ciliary activity in rabbit trachea.

1. Average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were simultaneously measured in rabbit airway ciliated cells in order to elucidate the molecular events that lead to ciliary activation by purinergic stimulation. 2. Extracellular ATP and extracellular UTP caused a rapid increase in both [Ca2+]i and CBF. These effects were practically abolished by a phospholipase C inhibitor (U-73122) or by suramin. 3. The effects of extracellular ATP were not altered: when protein kinase C (PKC) was inhibited by either GF 109203X or chelerythrine chloride, or when protein kinase A (PKA) was inhibited by RP-adenosine 3', 5'-cyclic monophosphothioate triethylamine (Rp-cAMPS). 4. Activation of PKC by phorbol 12-myristate, 13-acetate (TPA) had little effect on CBF or on [Ca2+]i, while activation of PKA by forskolin or by dibutyryl-cAMP led to a small rise in CBF without affecting [Ca2+]i. 5. Direct activation of protein kinase G (PKG) with dibutyryl-cGMP had a negligible effect on CBF when [Ca2+]i was at basal level. However, dibutyryl-cGMP strongly elevated CBF when [Ca2+]i was elevated either by extracellular ATP or by ionomycin. 6. The findings suggest that the initial rise in [Ca2+]i induced by extracellular ATP activates the NO pathway, thus leading to PKG activation. In the continuous presence of elevated [Ca2+]i the stimulated PKG then induces a robust enhancement in CBF. In parallel, activated PKG plays a central role in Ca2+ influx via a still unidentified mechanism, and thus, through positive feedback, maintains CBF close to its maximal level in the continuous presence of ATP.

Selective blockade of a slowly inactivating potassium current in striatal neurons by (+/-) 6-chloro-APB hydrobromide (SKF82958).

The ion channels of rat striatal neurons are known to be modulated by stimulation of D1 dopamine receptors. The susceptibility of depolarization-activated K+ currents to be modulated by the D1 agonist, 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetra-hydro-1H-3-benzaze pine (APB) was investigated using whole-cell voltage-clamp recording techniques from acutely isolated neurons. APB (0.01-100 microM) produced a concentration-dependent reduction in the total K+ current. At intermediate concentrations (ca. 10 microM), APB selectively depressed the slowly inactivating A-current (I(As)). A similar effect was produced by application of the D1 agonist, 7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1-H-2-benzazepine (SKF38393, 10 microM). APB reduced I(As) rapidly, having onset and recovery time constants of 1.2 sec and 1.6 sec, respectively. Unexpectedly, the effect of APB could not be mimicked by application of Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Sp-cAMPS, 100-200 microM), a membrane-permeable analog of cyclic AMP (cAMP), or by pretreatment with forskolin (25 microM), an activator of adenylyl cyclase. The reduction in I(As) also was not blocked by pretreatment with the D1 receptor antagonist, R(+)-SCH23390 hydrochloride (SCH23390, 10-20 microM). In addition, intracellular dialysis with guanosine-5'-O-(2-thiodiphosphate (GDP-beta-S, 200 microM) did not preclude the APB-induced inhibition of I(As), nor did dialysis with guanosine-5'-O-(3-thiotriphosphate (GTP-gamma-S, 400 microM) prevent reversal of the effect. The effect of APB was produced by a reduction in the maximal conductance of I(As) without changing the voltage-dependence of the current. Collectively, these results argue that APB does not inhibit I(As) through D1 receptors coupled to stimulation of adenylyl cyclase, but rather by allosterically regulating or blocking the channels giving rise to this current.

Selective blockade of a slowly inactivating potassium current in striatal neurons by (±) 6‐chloro‐APB hydrobromide (SKF82958)

The ion channels of rat striatal neurons are known to be modulated by stimulation of D1 dopamine receptors. The susceptibility of depolarization-activated K+ currents to be modulated by the D1 agonist, 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetra-hydro-1H-3-benzazepine (APB) was investigated using whole-cell voltage-clamp recording techniques from acutely isolated neurons. APB (0.01–100 μM) produced a concentration-dependent reduction in the total K+ current. At intermediate concentrations (ca. 10 μM), APB selectively depressed the slowly inactivating A-current (IAs). A similar effect was produced by application of the D1 agonist, 7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1-H-2-benzazepine (SKF38393, 10 μM). APB reduced IAs rapidly, having onset and recovery time constants of 1.2 sec and 1.6 sec, respectively. Unexpectedly, the effect of APB could not be mimicked by application of Sp-adenosine 3′,5′-cyclic monophosphothioate triethylamine (Sp-cAMPS, 100–200 μM), a membrane-permeable analog of cyclic AMP (cAMP), or by pretreatment with forskolin (25 μM), an activator of adenylyl cyclase. The reduction in IAs also was not blocked by pretreatment with the D1 receptor antagonist, R(+)-SCH23390 hydrochloride (SCH23390, 10–20 μM). In addition, intracellular dialysis with guanosine-5′-O-(2-thiodiphosphate (GDP-β-S, 200 μM) did not preclude the APB-induced inhibition of IAs, nor did dialysis with guanosine-5′-O-(3-thiotriphosphate (GTP-γ-S, 400 μM) prevent reversal of the effect. The effect of APB was produced by a reduction in the maximal conductance of IAs without changing the voltage-dependence of the current. Collectively, these results argue that APB does not inhibit IAs through D1 receptors coupled to stimulation of adenylyl cyclase, but rather by allosterically regulating or blocking the channels giving rise to this current. Synapse 29:213–224, 1998. © 1998 Wiley-Liss, Inc.

Activity-dependent regulation of [Ca2+]i in avian cochlear nucleus neurons: roles of protein kinases A and C and relation to cell death.

Neurons of the cochlear nucleus, nucleus magnocellularis (NM), of young chicks require excitatory afferent input from the eighth nerve for maintenance and survival. One of the earliest changes seen in NM neurons after deafferentation is an increase in intracellular calcium concentration ([Ca2+]i). This increase in [Ca2+]i is due to loss of activation of metabotropic glutamate receptors (mGluR) that activate second-messenger cascades involved in [Ca2+]i regulation. Because mGluRs are known to act via the phospholipase C and adenylate cyclase signal transduction pathways, the goal of this study was to determine the roles of protein kinases A (PKA) and C (PKC) activities in the regulation of NM neuron [Ca2+]i by eighth nerve stimulation. Additionally, we sought to determine the relationship between increased [Ca2+]i and cell death as measured by propidium iodide incorporation. [Ca2+]i of individual NM neurons in brain stem slices was monitored using fura-2 ratiometric fluorescence imaging. NM field potentials were monitored in experiments in which the eighth nerve was stimulated. Five hertz orthodromic stimulation maintained NM neuron [Ca2+]i at approximately 110 nM for 180 min. In the absence of stimulation, NM neuron [Ca2+]i increased steadily to a mean of 265 nM by 120 min. This increase was attenuated by superfusion of PKC activators phorbol-12,13-myristate acetate (100 nM) or dioctanoylglycerol (50 microM) and by activators of PKA: 1 mM 8-bromoadenosine-3',5'-cyclophosphate sodium (8-Br-cAMP), 50 microM forskolin or 100 microM Sp-adenosine 3',5'-cyclic monophosphothioate triethylamine. Inhibition of PKA (100 microM Rp-cAMPS) or PKC (50 nM bisindolymaleimide or 10 microM U73122) during continuous orthodromic stimulation resulted in an increase in NM neuron [Ca2+]i that exceeded 170 and 180 nM, respectively, by 120 min. Nonspecific kinase inhibition with 1 microM staurosporine during stimulation resulted in an [Ca2+]i increase that was greater in magnitude than that seen with either PKA or PKC inhibition alone, equal to that seen in the absence of stimulation, but much smaller than that seen with inhibition of mGluRs. In addition, manipulations that resulted in a [Ca2+]i increase >/=250 nM resulted in an increase in number and percentage of propidium iodide-labeled NM neurons. These results suggest that eighth nerve activity maintains [Ca2+]i of NM neurons at physiological levels in part via mGluR-mediated activation of PKA and PKC and that increases in [Ca2+]i due to activity deprivation or interruption of the PKA and PKC [Ca2+]i regulatory mechanisms are predictive of subsequent cell death.

Contribution of cAMP to the inhibitory effect of non‐steroidal anti‐inflammatory drugs in rat uterine smooth muscle

1. The effect of the non-steroidal anti-inflammatory drugs naproxen, mefenamic acid, phenylbutazone, piroxicam and tolmetin on the vanadate (0.3 mM)-induced tonic contraction, as well as the modifications of these effects by the G-protein inhibitor pertussis toxin, and the inhibitors of protein kinase A, Rp-cAMPS (Rp-Adenosine 3',5'-cyclic monophosphothioate triethylamine salt) and protein kinase C, H-7 [1(5-isoquinolynilsulfonyl)-2-methyl-piperazine], have been assayed to study the possible nature of intracellular mediators contributing to the inhibitory effects of NSAIDs in rat uterine smooth muscle incubated in medium lacking calcium plus EDTA. The effect of phorbol 12,13-dibutyrate on vanadate contraction and its modification with H-7 has also been examined. 2. Naproxen (6-600 microM), mefenamic acid (6-300 microM), phenylbutazone (6-300 microM), piroxicam (6-600 microM) and tolmetin (6-600 microM) produced concentration-dependent relaxation of vanadate-induced tonic contraction. The potency order, in accordance with their respective IC50 values was: phenylbutazone > or = mefenamic acid > or = naproxen > tolmetin > or = piroxicam. 3. The relaxant effects of naproxen, phenylbutazone, piroxicam and tolmetin were significantly antagonized with pertussis toxin (50 ng ml-1), Rp-cAMPS (100 microM) and H-7 (1 microM). However, the effect of mefenamic acid was unmodified by the three drugs. This suggests that the effect of mefenamic acid and other NSAIDs occur by different mechanisms. 4. Phorbol 12,13-dibutyrate relaxed the vanadate contraction but the maximal relaxation achieved (54.8 +/- 8.3%, n = 4) was lower than those induced with the NSAIDs. On the other hand, H-7 (1 microM) did not modify the relaxant effect of phorbol 12,13-dibutyrate. This suggests that H-7 behaves as a PKA, but not a PKC inhibitor, under the present experimental conditions. 5. The relaxation by naproxen, phenylbutazone, piroxicam and tolmetin is presumably produced by increasing cAMP because the effects of these are antagonized with Rp-cAMPS and H-7, and by pertussis-toxin-sensitive mechanisms.