Protein kinase C inhibitors block generation of anoxia-induced long-term potentiation

The aim of this study was to study the possible intracellular mechanisms underlying the anoxia-induced long-term potentiation (anoxic LTP) in the CA1 neurons of rat hippocampal slices using extra- and intracellular recording techniques. Superfusion of the hippocampal slices with the protein kinase C (PKC) inhibitors NPC-15437 (20 microM) or H-7 (20 microM) specifically prevented the induction of anoxic LTP. Moreover, the anoxic LTP was completely abolished in neurons intracellularly recorded with the selective PKC inhibitor PKCI 19-36 (50 microM). The specific cAMP-dependent protein kinase (PKA) inhibitor Rp-cyclic adenosine 3',5'-monophosphate (Rp-cAMPS, 25 microM) had no effect on the anoxic LTP. It is concluded that induction of anoxic LTP requires the activation of postsynaptic PKC.     

Developmental dissociation of excitability and secretory ability in Aplysia bag cell neurons

1. Despite the considerable progress made in understanding the role of electrical activity in triggering secretion, the developmental relationships between excitability and secretion are not well understood. The well-characterized bag cell neurons of Aplysia provide an advantageous system in which to investigate developmental interactions of these two key properties of neurons. 2. A prolonged afterdischarge triggers egg laying hormone (ELH) secretion in mature bag cell neurons. To investigate secretion in the developmental framework of excitability, we first examined whether immature neurons, which are incapable of the mature form of excitability (afterdischarge), contain ELH and whether this hormone is packaged in vesicles. We used immunoelectron microscopy to compare vesicular localization of ELH and to compare the size and density of ELH-containing vesicles in neurons from adult and juvenile Aplysia. This comparison revealed that immature neurons contain ELH in vesicles in the size range of secretory vesicles. However, they lack a class of large vesicles (> 250 nm in diameter) that is characteristic of mature neurons. 3. To investigate whether the ELH contained in immature bag cell neurons could be secreted in response to electrical activity, we used the potassium channel blocker tetraethylammonium (TEA) combined with nerve stimulation to depolarize neurons from both juvenile animals (ovotestes do not contain eggs) and from adult Aplysia (ovotestes contain eggs). Using radioimmunoassay, we have found that the duration and amount of ELH secreted from bag cell neurons from juvenile Aplysia in response to TEA does not depend on whether or not the cells can be induced to afterdischarge, and the amount and duration of ELH secreted from bag cell neurons of juvenile Aplysia (whether or not they afterdischarged) differed from those secreted by adult neurons. However, by normalizing for body size, we found that the final estimated hemolymph concentration of ELH would be similar in juvenile and adult animals. 4. We investigated the potential functional significance of secretion of bag cell hormones in juvenile Aplysia by attempting to bypass the bag cell neurons and directly activate downstream elements with extract from adult bag cell neurons (BCE), known to contain ELH and other peptides. We found that juvenile Aplysia exhibit at least one component of egg-laying behavior, cessation of locomotion, in response to BCE during a developmental period (as measured by weight) in which they normally would possess neurons incapable of afterdischarge. Thus developmental regulation of excitability in the bag cell neurons may prevent inappropriate hormone release and subsequent premature expression of reproductive behaviors.     

Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A

Protein kinase A (PKA) stimulates Cl secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR), a tightly regulated Cl- channel in the apical membrane of many secretory epithelia. The CFTR channel is also modulated by protein kinase C (PKC), but the regulatory mechanisms are poorly understood. Here we present evidence that PKA-mediated phosphorylation alone is not a sufficient stimulus to open the CFTR chloride channel in the presence of MgATP; constitutive PKC phosphorylation is essential for acute activation of CFTR by PKA. When patches were excised from transfected Chinese hamster ovary cells, CFTR responses to PKA became progressively smaller with time and eventually disappeared. This decline in PKA responsiveness did not occur in the presence of exogenous PKC and was reversed by the addition of PKC to channels that had become refractory to PKA. PKC enhanced PKA stimulation of open probability without increasing the number of functional channels. Short-term pretreatment of cells with the PKC inhibitor chelerythrine (1 microM) reduced the channel activity that could be elicited by forskolin in cell-attached patches. Moreover, in whole cell patches, acute stimulation of CFTR currents by chlorophenylthio-cAMP was abolished by two chemically unrelated PKC inhibitors, although an abrupt, partial activation was observed after a delay of >15 min. Modulation by PKC was most pronounced when basal PKC phosphorylation was reduced by briefly preincubating cells with chelerythrine. Constitutive PKC phosphorylation in unstimulated cells permits the maximum elevation of open probability by PKA to reach a level that is approximately 60% of that attained during in vitro exposure to both kinases. Differences in basal PKC activity may contribute to the variable cAMP responsiveness of CFTR channels in different cell types.     

Modulation of protein kinase C and cAMP-dependent protein kinase by delta-opioid

Modulation of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities by delta-opioid receptor specific agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) was investigated in neuroblastoma x glioma hybrid NG 108-15 cells. DPDPE activated PKC in a dose-dependent manner, with the maximal response at 5 min. The DPDPE-stimulated PKC activation could be blocked by naltrindole. The activation of PKC by DPDPE was dependent on Ca2+ and was inhibited by chelerythrine chloride (10 microM), but not by H89 (1 microM). Pretreatment of NG 108-15 cells with pertussis toxin (100 ng/ml for 24 h) completely abolished DPDPE-stimulated PKC activation. In contrast to the result from the acute treatment with DPDPE, which had no significant effect on PKA activity, chronic treatment of DPDPE (1 microM for 24 h) increased PKA activity, but reduced the basal activity of PKC. These results demonstrated that DPDPE differentially modulated PKC and PKA activities via a receptor-mediated, PTX sensitive pathway.     

Potentiation of K252a, a protein kinase inhibitor-induced polyploidization by cAMP in cultured fibrosarcoma cell line

We found that K252a, a potent inhibitor of protein kinases (PK), induced DNA re-replication of Meth-A cells, i.e., DNA synthesis at a higher DNA ploidy without undergoing cytokinesis (polyploidization). The K252a-induced polyploidization was inhibited by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, suggesting that the polyploidization is caused through inhibition of PKC. By contrast, the polyploidization was potentiated by adenosine 3':5'-cyclic monophosphate (cAMP), a cAMP-dependent protein kinase (PKA) activator. These findings suggest that the cAMP-dependent signaling pathway and diacylglycerol (DAG)-dependent signaling pathway play an important role in regulating the induction of polyploidization in Meth-A cells, through a possible "cross-talk" between the two pathways.     

Involvement of cAMP-dependent protein kinase in the nucleus accumbens in cocaine self-administration and relapse of cocaine-seeking behavior

cAMP-dependent protein kinase (PKA) in the nucleus accumbens (NAc) has been implicated in cocaine addiction because (1) cocaine reinforcement is mediated by dopamine receptors that modulate cAMP formation, and (2) repeated exposure to cocaine upregulates the cAMP system in NAc neurons. This study tested PKA involvement in cocaine self-administration and relapse of cocaine-seeking behavior by infusing cAMP analogs that activate or inhibit PKA into the NAc of rats. Bilateral intra-NAc infusions of the PKA inhibitor Rp-cAMPS reduced baseline cocaine self-administration, shifted the dose-response curve for cocaine self-administration to the left, and induced relapse of cocaine-seeking behavior after extinction from cocaine self-administration, consistent with an enhancement of cocaine effects in each paradigm. In contrast, pretreatment with intra-NAc infusions of a PKA activator, Sp-cAMPS or dibutyryl cAMP, increased baseline cocaine self-administration during the second hour of testing and shifted the dose-response curve to the right, consistent with an antagonist-like action. After extinction from cocaine self-administration, similar infusions of Sp-cAMPS induced generalized responding at both drug-paired and inactive levers. As an index of PKA activity in vivo, NAc infusions of Rp-cAMPS reduced basal levels of dopamine-regulated phosphoprotein-32 phosphorylation and blocked amphetamine-induced increases in cAMP response element-binding protein (CREB) phosphorylation. Conversely, NAc infusions of Sp-cAMPS increased phosphorylation of CREB. Together, these results suggest that sustained upregulation of the cAMP system in the NAc after repeated cocaine exposure could underlie tolerance to cocaine reinforcement, whereas acute inhibition of this system may contribute to drug craving and relapse in addicted subjects.     

Prolonged hormone secretion from neuroendocrine cells of Aplysia is independent of extracellular calcium

The role of Ca2+ from extracellular and intracellular sources in stimulating neurosecretion was investigated in four experiments using neuroendocrine bag cells of the marine mollusk Aplysia. (i) Bag cells were treated with either an extracellular calcium chelator (BAPTA) or Co(2+)-substitution within 30 s after onset of an electrical afterdischarge to prevent influx of Ca2+ from extracellular fluid. These treatments shortened the duration of the afterdischarge, but did not significantly affect the overall pattern or total amount of egg laying hormone (ELH) secretion, suggesting that extracellular Ca2+ is not required for maintenance of ELH release. (ii) Substitution of Ba2+ for Ca2+ has previously been shown to support bag cell afterdischarges that trigger transient elevations in intracellular Ca2+. We showed that this treatment also stimulates ELH secretion, suggesting that Ca2+ release from intracellular stores can stimulate ELH secretion. (iii) To raise intracellular Ca2+ levels in the absence of an afterdischarge, the calcium ionophore X537A was used to transport Ca2+ across plasma and organelle membranes. When this treatment was combined with extracellular calcium chelators so that the only source of Ca2+ was from intracellular compartments, ELH secretion was stimulated. Taken together, these findings are consistent with the hypothesis that release of Ca2+ from intracellular stores is sufficient to stimulate ELH secretion.     

Stimulation of cell elongation in teleost rod photoreceptors by distinct protein kinase inhibitors

The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic-AMP-dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN-62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC.     

Transforming growth factor-beta1 stimulates protein kinase A in mesangial cells

We recently demonstrated that transforming growth factor-beta (TGF-beta) stimulates phosphorylation of the type I inositol 1,4, 5-trisphosphate receptor (Sharma, K., Wang, L., Zhu, Y., Bokkala, S., and Joseph, S. (1997) J. Biol. Chem. 272, 14617-14623), possibly via protein kinase A (PKA) activation in murine mesangial cells. In the present study, we evaluated whether TGF-beta stimulates PKA activation. Utilizing a specific PKA kinase assay, we found that TGF-beta increases PKA activity by 3-fold within 15 min of TGF-beta1 treatment, and the enhanced kinase activity was completely reversed by the inhibitory peptide for PKA (PKI; 1 microM). In mesangial cells transfected with a PKI expression vector, enhanced PKA activity could not be demonstrated with TGF-beta1 treatment. TGF-beta1 was also found to stimulate translocation of the alpha-catalytic subunit of PKA to the nucleus by Western analysis of nuclear protein as well as by confocal microscopy. TGF-beta1-mediated phosphorylation of cAMP response element-binding protein was completely reversed by H-89 (3 microM), a specific inhibitor of PKA. Stimulation of fibronectin mRNA by TGF-beta1 was also attenuated in cells overexpressing PKI. We thus conclude that TGF-beta stimulates the PKA signaling pathway in mesangial cells and that PKA activation contributes to TGF-beta stimulation of cAMP response element-binding protein phosphorylation and fibronectin expression.     

Opposite modulation of opiate withdrawal behaviors on microinfusion of a protein kinase A inhibitor versus activator into the locus coeruleus or periaqueductal gray

Chronic opiate administration upregulates the cAMP pathway in the locus coeruleus (LC). This adaptation is thought to increase the electrical excitability of LC neurons and contribute to the dramatic increase in LC firing induced by opioid receptor antagonists in opiate-dependent animals. The goal of the present study was to evaluate directly a role of the cAMP pathway in opiate withdrawal behaviors by studying, in vivo, whether withdrawal is influenced by intra-LC infusion of compounds known to activate or inhibit protein kinase A (PKA). Infusions into amygdala or periaqueductal gray (PAG) were studied for comparison. In one series of experiments the effect of intra-LC, intra-amygdala, or intra-PAG infusions of the PKA inhibitor Rp-cAMPS on naloxone-precipitated withdrawal from morphine was examined. Intra-LC infusions of Rp-cAMPS significantly attenuated several prominent behavioral signs of morphine withdrawal. Intra-PAG infusions of Rp-cAMPS also significantly attenuated opiate withdrawal behaviors, although different behaviors were affected. In contrast, intra-amygdala infusions of Rp-cAMPS were without significant effect. In a second series of experiments the effect of intra-LC or intra-PAG infusions of the PKA activator Sp-cAMPS on behavior in nondependent drug-naive animals was determined. Sp-cAMPS infusions into either brain region induced a quasi-withdrawal syndrome, but the observed behaviors differed between the two groups. Analysis of the phosphorylation state of tyrosine hydroxylase, a well characterized substrate for PKA, confirmed the ability of Rp-cAMPS and Sp-cAMPS to inhibit and activate, respectively, PKA activity in vivo. Together, these data provide direct evidence for involvement of the cAMP-PKA system in the LC, as well as in the PAG, in opiate withdrawal and withdrawal-related behaviors.     

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. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Opposing effects on modulation of angiogenesis by protein kinase C and cAMP-mediated pathways

The role of cAMP-mediated pathway in modulating angiogenesis was investigated. We have shown previously that activators of protein kinase C (PKC) caused a marked increase in angiogenesis, while the specific inhibitor of PKC, Ro 318220 suppressed angiogenesis. Here we show that forskolin, which activates adenylate cyclase and elevates the intracellular levels of cAMP, and the Sp-diastereomer of adenosine cyclic-3',5'-monophosphothioate (Sp-cAMPS), caused a dose-dependent suppression of collagenous protein biosynthesis and angiogenesis in the chick chorioallantoic membrane system (CAM). The opposite modulation of angiogenesis by activators of PKC and elevated cAMP levels was further confirmed by the suppression of 4 beta-phorbol-12-myristate-13-acetate (4 beta-PMA)-stimulated angiogenesis by either forskolin or Sp-cAMPS. On the contrary, the Rp-diastereomer of adenosine cyclic-3',5'-monophosphothioate (Rp-cAMPS), which antagonises endogenous cAMP biochemical actions, had no effect on angiogenesis alone and did not suppress 4 beta-PMA stimulated angiogenesis. However, Rp-cAMPS antagonised the effect of forskolin and Sp-cAMPS on 4 beta-PMA induced angiogenesis. Similar results were obtained in the human umbilical vein endothelial cell tube formation assay. In this system, the PKC inhibitor, Ro 318220, caused a dose-dependent inhibition of 4 beta-PMA reversed this effect. Also, forskolin and Sp-cAMPS caused an inhibition in tube formation. These results indicate that increased levels of intracellular cAMP have a negative effect in normal angiogenesis and cause a large reduction of the promotion of angiogenesis resulting from PKC activation.     

Activation of protein kinase C potentiates postsynaptic acetylcholine response at developing neuromuscular synapses

1. Phorbol 12-myristate 13-acetate (TPA, 1 microM) and phorbol 12,13-dibutyrate (PDBu, 2 microM), activators of protein kinase C (PKC), increased the mean amplitude and decay time of the spontaneous synaptic currents of Xenopus nerve-muscle coculture, whereas, 4 alpha-phorbol (2 microM) which is an inactive phorbol analogue had no effect. 2. Staurosporine (0.5 microM) and H-7 (10 microM), inhibitors of PKC, inhibited the potentiation effects of TPA on the spontaneous synaptic currents. 3. Effects of TPA on the postsynaptic acetylcholine (ACh) sensitivity were examined by iontophoresis of ACh to the surface of embryonic muscle cells of 1-day-old Xenopus cultures. TPA increased both the amplitude and decay time of ACh-induced whole-cell currents in isolated myocytes. 4. TPA concentration-dependently increased the mean open time of low-conductance ACh channels but did not affect those of high-conductance ACh channels. PDBu but not 4 alpha-phorbol exhibited similar effects to TPA. Staurosporine and H-7 inhibited the increasing effects of TPA. 5. These results suggest that activation of PKC might be involved in synaptogenesis at developing neuromuscular synapses by the postsynaptic potentiation of ACh sensitivity.     

Multiple second-messenger system modulation of voltage-activated calcium currents in teleost retinal horizontal cells

Two voltage-activated calcium currents, a transient T-type and a PL-sustained type, have been measured in isolated, cultured white bass horizontal cells. These two voltage-activated calcium currents were found to be modulated by two independent second-messenger systems. Furthermore, activation of either second-messenger system led to similar changes in calcium current activity. Activation of the cyclic AMP second-messenger pathway or the sn-1,2-diacylglycerol (DAG) second-messenger system resulted in a significant decrease in the amplitude of the transient current and a simultaneous large increase in the amplitude of the sustained current. Both second-messenger systems achieved their effects through protein phosphorylation. The cyclic AMP pathway resulted in the activation of protein kinase A (PKA) and the DAG pathway worked to activate protein kinase C (PKC). Two protein kinase inhibitors were analyzed in this study for their ability to inhibit second-messenger activated protein kinase activity and separate the two pathways. The peptide cyclic AMP-dependent protein kinase inhibitor and staurosporine were found to be nonspecific at high concentrations and inhibited both second-messenger pathways. At low concentrations however, staurosporine specifically inhibited only PKC, whereas adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase inhibitor was selective for PKA. Both second-messenger systems were activated by the neuromodulator, dopamine. Thus one agonist can initiate multiple second-messenger systems leading to similar changes in voltage-activated calcium current activity. The modulatory action on calcium currents produced by one second-messenger system added to the modulatory action resulting from activation of the other second-messenger system. The effect is to alter the magnitude of the horizontal cell calcium currents.     

Pressure-induced expression of heat shock protein 70 mRNA in adult rat heart is coupled both to protein kinase A-dependent and protein kinase C-dependent systems

BACKGROUND: Production of heat shock protein 70 (HSP70) in the heart is induced by hemodynamic stress, but its intracellular signal transduction system has not been elucidated well. OBJECTIVE: To investigate the hypothesis that protein kinase A (PKA)-dependent and protein kinase C (PKC)dependent systems are involved in the pressure-induced expression of HSP70 mRNA in perfused adult rat heart METHODS: Isolated tetrodotoxin-arrested Sprague-Dawley rat hearts were perfused as Langendorff preparations at a constant aortic pressure of 60 mmHg. Aortic pressure in rats of the pressure-overloaded group was elevated from 60 to 120 mmHg for 2-120 min. cAMP contents and rates of synthesis of protein were measured by radioimmunoassay and the incorporation of [14C]-phenylalanine into total heart protein, respectively. Expression of HSP70 mRNA was determined by Northern blot analysis. RESULTS: Elevation of aortic pressure significantly increased cAMP content after 2 min of perfusion (by 41%), significantly increased rates of synthesis of protein during the second hour of perfusion (by 41%), and induced expression of HSP70 mRNA maximally after 60 min of perfusion (2.7-fold the control value). Exposure to glucagon, forskolin or 1 -methyl-3-isobutylxanthine mimicked increases in these parameters caused by elevation of aortic pressure. Administration of a selective PKA inhibitor, H-89, significantly prevented induction of increases in expression of HSP70 mRNA and rates of synthesis of protein by a high pressure overload and exposure to agents that increase cAMP content. Furthermore, administration of phorbol ester induced expression of HSP70 mRNA. Administration of a PKC inhibitor, calphostin C, significantly prevented induction of increases in expression of HSP70 mRNA by a pressure overload and by exposure to phorbol ester. CONCLUSIONS: These results suggest that the pressure-induced induction of production of HSP70 is regulated both by PKA-dependent and by PKC-dependent systems during periods of active synthesis of protein in adult rat heart.     

Functional modulation of P2X2 receptors by cyclic AMP-dependent protein kinase

It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage- and ligand-gated channels are modulated. The intracellular carboxyl terminus of P2X2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P2X2 purinoceptor could be regulated by the protein phosphorylation. Whole-cell voltage-clamp recording was used to record ATP-evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P2X2 receptor. Dialyzing HEK 293 cells with phorbol 12-myristate 13-acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP-induced cationic current. The role of PKA phosphorylation in modulating the function of the P2X2 receptor was investigated by internally perfusing HEK 293 cells with 8-bromo-cAMP or the purified catalytic subunit of PKA. Both 8-bromo-cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP-activated current without affecting the inactivation kinetics and the value of reversal potential. Site-directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser431) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P2X2 receptors, intracellular perfusion of 8-bromo-cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP-evoked current. These results suggest that as with other ligand-gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P2X2 receptor and ATP-mediated physiological effects in the nervous system.     

Gonadotropin-releasing hormone and pituitary adenylate cyclase-activating polypeptide affect levels of cyclic adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) subunits in the clonal gonadotrope alphaT3-1 cells: evidence for cross-talk between PKA and protein kinase C pathways

We have shown previously that protein kinase A (PKA) subunit levels are regulated by activation of PKA or protein kinase C (PKC) in anterior pituitary cells. GnRH also influenced PKA subunit levels, suggesting that hormonal regulation occurs in gonadotrophs, and therefore, we have reexamined this question using the clonal gonadotrope-derived cell line (alphaT3-1 cells). Western blot analysis, using specific immunoaffinity purified immunoglobulins, revealed expression of catalytic (Cat) and regulatory type I (RI) and type II (RII) subunits of PKA in these cells. Activation of adenylyl cyclase (AC) with forskolin, or of PKC with tetradecanoyl phorbol acetate (TPA), caused a rapid (detectable at 0.5-1 h) and concentration-dependent loss of all PKA subunits. Forskolin (10-100 microM) reduced Cat and RI by 60% and RII by 30%, whereas TPA (0.1-1 microM) reduced Cat and RII by 50% and RI by 40%. Simultaneous activation of PKA and PKC caused the expected dose-dependent reductions in Cat, and the effects of forskolin or TPA were nearly additive. RI and RII were reduced similarly by 10 nM TPA, whereas 100 nM TPA tended to prevent the reduction of RI or RII caused by forskolin. GnRH, which activates phosphoinositidase C and not AC in these cells, caused a clear loss of Cat or RII at all concentrations tested and of RI at 0.1 nM. Pituitary adenylate cyclase-activating polypeptide 38, which acts via PVR-1 receptors to stimulate both phosphoinositidase C and AC in these cells, also caused a clear dose-dependent decrease in Cat, RI, and RII, although higher concentrations were needed for the latter effects. Together, the data demonstrate that catalytic and regulatory subunits of PKA are subject to both hormonal and receptor-independent regulation in alphaT3-1 cells, reinforcing the possibility that such effects occur in nonimmortalized gonadotropes. Whereas the effects of PKA activators very likely involve proteolytic degradation of the dissociated PKA holoenzyme, the effects of TPA and GnRH occur in the absence of cAMP elevation by unknown mechanisms. Whatever the mechanisms involved, the data reveal a mechanism for cross-talk between phosphoinositidase C and AC-mediated hormonal signals, in which PKC activation seems to play a pivotal role.     

Cyclic AMP-dependent protein kinase enhances hippocampal dentate granule cell GABAA receptor currents

1. The effects of activation of endogenous adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), intracellular application of PKA and inhibition of endogenous PKA by protein kinase inhibitory peptide (PKIP) on hippocampal dentate granule cell gamma-aminobuturic acid A (GABAA) receptor (GABAR) currents were characterized. 2. GABAR currents evoked by repeated application of GABA (30 or 100 microM) were enhanced by application of 1 mM norepinephrine (52 +/- 26%; mean +/- SE; n = 11) and of 500 mM 8-bromo cAMP (15 +/- 2%, n = 7). 3. GABA concentration response curves were obtained from six dentate granule cells before and after application of 500 microM 8-bromo cAMP. The maximal current was increased significantly by 89 +/- 36%, but the mean EC50 was not significantly changed (68 +/- 42 microM vs. 25 +/- 10 microM). 4. The GABA concentration response relationship was studied in a group of 7 granule cells recorded with pipettes containing PKIP and 2 mM ATP and compared with another group of 12 cells recorded with 2 mM ATP in the pipette. When currents were recorded with intracellular PKIP, the mean EC50 for GABA was no different (43 +/- 9 microM vs. 45 +/- 16 microM); however, the maximal current obtained was smaller, (961 +/- 102 pA vs. 658 +/- 104 pA). 5. Concentration response data were obtained from four granule cells using recording pipettes containing the cPKA and an ATP regeneration system and compared with seven cells recorded with the ATP regeneration system. With cPKA, the maximal GABAR current was significantly larger (1,224 +/- 132 pA vs. 718 +/- 56 pA), but the EC50 for GABA was not significantly altered (21 +/- 2.0 microM vs. 79 +/- 25 microM).     

Estradiol-induced enhancement of object memory consolidation involves NMDA receptors and protein kinase A in the dorsal hippocampus of female C57BL/6 mice.

This study examined the role of dorsal hippocampal NMDA receptors and PKA activation in 17β-estradiol (E?)-induced enhancement of object memory consolidation. Mice explored two identical objects during training, after which they immediately received intraperitoneal injections of 0.2 mg/kg E?, and bilateral dorsal hippocampal infusions of Vehicle, the NMDA receptor antagonist APV (2.5 μg/side), or the cAMP inhibitor Rp-cAMPS (18.0 μg/side). Retention was tested 48 hours later. The enhanced object memory and increased ERK phosphorylation observed with E? alone was reduced by APV and Rp-cAMPS, suggesting that estrogenic enhancement of object memory involves NMDA receptors and PKA activation within the dorsal hippocampus. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Parathyroid hormone-related protein increases DNA synthesis in proximal tubule cells by cyclic AMP- and protein kinase C-dependent pathways

The present study was performed to characterize the possible involvement of cAMP synthesis and protein kinase C (PKC) activation in the DNA synthesis-stimulating effect of parathyroid hormone-related protein (PTHrP) in proximal tubule cells. We found that DNA synthesis was stimulated by 10 microM 8BrcAMP, and 1 microM Sp-cDBIMPS, two cAMP analogs, and also by 1 microM phorbol 12-myristate 13-acetate (PMA) and 100 microM 1,2-dioctanoyl-sn-glycerol, two PKC activators, and 10 nM [Cys23] human (h)PTHrP (24-35) amide in rabbit proximal tubule cells (PTC). Both Sp-cDBIMPS and PMA, at 1 microM, also increased DNA synthesis in SV40-immortalized mouse proximal tubule cells MCT. Human PTHrP (7-34) amide [PTHrP (7-34)] dose dependently stimulated DNA synthesis in a similar manner as [34Tyr]PTHrP (1-34) amide [PTHrP (1-34)], in PTC. PMA pre-treatment for 20 h, which downregulates PKC, completely blocked the effect induced by PTHrP (7-34), but not that of PTHrP (1-34), in the latter cells. In contrast, the same PMA pre-treatment abolished the DNA synthesis stimulation by PTHrP (1-34) and PTHrP (7-34) in MCT cells, which appear to have PTH receptors mainly coupled to phospholipase C and not adenylate cyclase. Our results indicate that the stimulatory effect of PTHrP on DNA synthesis in proximal tubule cells is mediated by a cAMP- and PKC-dependent mechanism.