The paired-pulse index: a measure of hippocampal dentate granule cell modulation

This study was undertaken to assess whether the paired-pulse index (PPI) is an effective measure of the modulation of dentate granule cell excitability during normal development. Paired-pulse stimulations of the perforant path were, therefore, used to construct a PPI for 15-, 30-, and 90-day old, freely moving male rats. Significant age-dependent differences in the PPI were obtained. Fifteen-day old rats showed significantly less inhibition at short interpulse intervals [interpulse interval (IPI): 20 to 30 msec), a lack of facilitation at intermediate IPIs (50 to 150 msec), and significantly less inhibition at longer IPIs (300 to 1,000 msec) than adults.     

The effect of hippocampal dentate granule cell lesions upon the limbic seizure model of rats

The purpose of this study was to determine the role of dentate granule cells upon limbic seizure of Wistar rat caused by unilateral intra-amygdaloid administration of kainic acid (KA). Stereotactic surgery was performed in Wistar rats and stainless steel injection chemitorode was inserted in the left amygdala. Left dentate granule cells lesion were induced by microinjection of colchicine. The rats obtained recovery period for 7 days, postoperatively. The rats were divided into two groups. One group were used for observation of symptoms and electroencephalographic findings during the limbic seizure for 6 hours after the KA injection. Another group was processed for measuring local cerebral glucose utilization (LCGU) during limbic seizure status. The histological study demonstrated a selective loss of dentate granule cells in the left hippocampus 7 days after the colchicine injection. After the KA injection, initiation of the spike discharge was significantly retarded not only in the hippocampus (from 6.01 min. to 37.25 min.) but also in the amygdala (from 2.96 min. to 10.8 min.). Progression, frequency and intensity of the KA induced seizures were also inhibited by the colchicine-induced dentate granule cells lesion. During limbic seizure status, LCGU obtained by 14C-deoxyglucose autoradiography were significantly decreased not only in the hippocampus but also in the amygdala on the site of KA injection. These data suggest that hippocampal dentate granule cells play an important role on initiation and progression of the KA induced limbic seizure. The result suggested that there was an acceleration mechanism of the limbic seizure between amygdala and hippocampus.     

Temporal patterns and depolarizing actions of spontaneous GABAA receptor activation in granule cells of the early postnatal dentate gyrus

Whole cell patch-clamp recordings were used to investigate the properties of the gamma-aminobutyric acid type A (GABAA) receptor-mediated spontaneous synaptic events in immature granule cells of the developing, early postnatal day (P0-P6) rat dentate gyrus. With Cs-gluconate-filled whole cell patch pipettes at 0 mV in control medium, spontaneous inhibitory postsynaptic currents (sIPSCs) occurred in prominent bursts (peak amplitude of the bursts 406.9 +/- 58.4 pA; intraburst IPSC frequency 71.0 +/- 12.4 Hz) at 0.05 +/- 0.02 Hz in every immature granule cell younger than P7. Between the bursts of IPSCs, lower frequency (1.7 +/- 0.7 Hz), interburst IPSCs could be observed. Bicuculline and picrotoxin as well as the intracellularly applied chloride-channel blockers CsF- and 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) abolished the intraburst as well as the interburst IPSCs, indicating that the IPSCs were mediated by GABAA receptor channels. The bursts of IPSCs, but not the interburst IPSCs, were blocked by the simultaneous application of the glutamate receptor antagonists 2-amino-5-phosphovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, indicating the importance of the glutamatergic excitatory drive onto the interneurons in the early postnatal dentate gyrus. The spontaneously occurring excitatory postsynaptic currents in immature granule cells, observable after the intracellular blockade of GABAA receptor channels with CsF- and DIDS, appeared exclusively as single events at low frequencies, i.e., they did not occur in prominent bursts. Gramicidin-based perforated patch-clamp recordings determined that the reversal potential for the burst of IPSCs (-46.6 +/- 3.1 mV) was more depolarized than the resting membrane potential (-54.2 +/- 4.2 mV) but more hyperpolarized than the action potential threshold (-41. 8 +/- 1.7 mV). The depolarizing action of the bursts of synaptic events most often evoked only a single action potential per burst. Simultaneous whole cell patch recordings, with KCl-filled patch pipettes at -60 mV in current clamp from pairs of immature granule cells of the developing dentate gyrus, determined that the bursts of IPSPs took place in a similar temporal pattern but with imperfect synchrony in neighboring granule cells (average lag between the onsets of the bursts between granule cell pairs 77.7 +/- 8.6 ms). These results show that the spontaneous activation of GABAA receptors in immature dentate granule cells displays unique properties that are distinct from the temporal patterns and biophysical features of spontaneous GABAA receptor activation taking place in the developing Ammon's horn and in the adult dentate gyrus.     

Properties of spontaneous miniature GABAA receptor mediated synaptic currents in area CA3 of rat hippocampal slice cultures

Miniature, gamma-aminobutyric acid A receptor mediated inhibitory postsynaptic currents (mIPSCs) were recorded from CA3 pyramidal cells in hippocampal slice cultures using whole-cell techniques in the presence of tetrodotoxin. The kinetics and amplitudes of the mIPSCs were analyzed with the aim of determining whether subclasses of events arising from distinct populations of presynaptic interneurons could be distinguished. Histograms of mIPSC amplitude, rise time constant, and decay time constant were all positively skewed, but discrete subsets of events could not be distinguished. The positive skew did not appear to result from electrotonic filtering of distal synaptic currents because there was no correlation among mIPSC amplitudes and the kinetic parameters. Analysis of the intervals between mIPSCs indicated that each event occurred independently. The analysis of spontaneous mIPSCs does not provide evidence of the innervation of pyramidal cells by heterogeneous interneurons.     

Glutamate currents in morphologically identified human dentate granule cells in temporal lobe epilepsy

Glutamate-receptor-mediated synaptic transmission was studied in morphologically identified hippocampal dentate granule cells (DGCs; n = 31) with the use of whole cell patch-clamp recording and intracellular injection of biocytin or Lucifer yellow in slices prepared from surgically removed medial temporal lobe specimens of epileptic patients (14 specimens from 14 patients). In the current-clamp recording, low-frequency stimulation of the perforant path generated depolarizing postsynaptic potentials that consisted of excitatory postsynaptic potentials and phase-inverted inhibitory postsynaptic potentials mediated by the gamma-aminobutyric acid-A (GABA(A)) receptor at a resting membrane potential of -62.7 +/- 2.0 (SE) mV. In the voltage-clamp recording, two glutamate conductances, a fast alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor-mediated excitatory postsynaptic current (EPSC; AMPA EPSC) and a slowly developing N-methyl-D-aspartate (NMDA)-receptor-mediated EPSC (NMDA EPSC), were isolated in the presence of a GABA(A) receptor antagonist. NMDA EPSCs showed a voltage-dependent increase in conductance with depolarization by exhibiting an N-shaped current-voltage relationship. The slope conductance of the NMDA EPSC ranged from 1.1 to 9.4 nS in 31 DGCs, reaching up to twice the size of the AMPA conductance. This widely varying size of the NMDA conductance resulted in the generation of double-peaked EPSCs and a nonlinear increase of the slope conductance of up to 37.5 nS with positive membrane potentials, which resembled "paroxysmal currents," in a subpopulation of the neurons. In contrast, AMPA EPSCs, which were isolated in the presence of an NMDA receptor antagonist (2-amino-5-phosphonovaleric acid), showed voltage-independent linear changes in the current-voltage relationship and were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione. The AMPA conductance showed little variance, regardless of the size of the NMDA conductance of a given neuron. The average AMPA slope conductance was 5.28 +/- 0.65 (SE) nS in 31 human DGCs. This value was similar to AMPA EPSC conductances in normal rat DGCs (5.35 +/- 0.52 nS, mean +/- SE; n = 55). Dendritic morphology and spine density were quantified in the individual DGCs to assess epileptic pathology. Dendritic spine density showed an inverse correlation (r2 = 0.705) with a slower rise time and a longer half-width of the excitatory postsynaptic potentials mediated by the NMDA receptor. It is concluded that both AMPA and NMDA EPSCs contribute to human DGC synaptic transmission in epileptic hippocampus. However, a wide range of changes in the slope conductance of the NMDA EPSCs suggests that the NMDA-receptor-mediated conductance could be altered in human epileptic DGCs. These changes may influence the generation of chronic subthreshold epileptogenic synaptic activity and give rise to pathological excitation leading to epileptic seizures and dendritic pathology.     

Mouse cerebellar granule cell differentiation: electrical activity regulates the GABAA receptor alpha 6 subunit gene

GABAA receptor alpha6 subunit gene expression marks cerebellar granule cell maturation. To study this process, we used the Deltaalpha6lacZ mouse line, which has a lacZ reporter inserted into the alpha6 gene. At early stages of postnatal cerebellar development, alpha6-lacZ expression is mosaic; expression starts at postnatal day 5 in lobules 9 and 10, and alpha6-lacZ is switched on inside-out, appearing first in the deepest postmigratory granule cells. We looked for factors regulating this expression in cell culture. Membrane depolarization correlates inversely with alpha6-lacZ expression: granule cells grown in 25 mM [K+]o for 11-15 d do not express the alpha6 gene, whereas cultures grown for the same period in 5 mM [K+]o do. This is influenced by a critical early period: culturing for >/=3 d in 25 mM [K+]o curtails the ability to induce the alpha6 gene on transfer to 5 mM [K+]o. If the cells start in 5 mM [K+]o, however, they still express the alpha6-lacZ gene in 25 mM [K+]o. In contrast to granule cells grown in 5 mM [K+]o, cells cultured in 25 mM [K+]o exhibit no action potentials, mEPSCs, or mIPSCs. In chronic 5 mM [K+]o, factors may therefore be released that induce alpha6. Blockade of ionotropic and metabotropic GABA and glutamate receptors or L-, N-, and P/Q-type Ca2+ channels did not prevent alpha6-lacZ expression, but inhibition of action potentials with tetrodotoxin blocked expression in a subpopulation of cells.     

Arachidonic acid potentiates ACh receptor currents by protein kinase C activation but not by receptor phosphorylation

The effects of arachidonic acid on ACh-gated channel currents were examined using Torpedo nicotinic ACh receptors expressed in Xenopus oocytes. Arachidonic acid decreased ACh-evoked currents during treatment, to a greater extent in Ca(2+)-free extracellular solution. The currents were enhanced for more than 30 min after washing, reaching 150 and 170% in Ca(2+)-containing and -free extracellular solutions, respectively. The current enhancement was inhibited by the selective protein kinase C (PKC) inhibitor, GF109203X, whereas the current depression was not affected. Furthermore, arachidonic acid-evoked current depression was blocked in mutant ACh receptors with PKC phosphorylation site deletions on the alpha and delta subunits, but the long-lasting potentiation effect remained. These results indicate that arachidonic acid may decrease ACh receptor currents by a direct binding to PKC phosphorylation sites of the ACh receptors and may potentiate the currents via a novel pathway related to arachidonic acid-regulated PKC activation, but not via PKC phosphorylation of the ACh receptor itself.     

Atherogenic lipoproteins enhance mesangial cell expression of platelet-derived growth factor: role of protein tyrosine kinase and cyclic AMP-dependent protein kinase A

Mesangial cell proliferation and extracellular matrix accumulation are fundamental in the pathogenesis of glomerulosclerosis. Platelet-derived growth factor (PDGF) is a major cytokine involved in mesangial cell proliferation, and its increased expression is seen in glomerular injury. Atherogenic lipoproteins stimulate mesangial cell proliferation and induce glomerular injury in experimental animals. We examined the effect of low-density lipoprotein (LDL) and its more atherogenic oxidized forms, minimally modified LDL (mm-LDL) and oxidized LDL (ox-LDL) on mesangial cell PDGF mRNA expression. Incubation with 2.5 to 25 microg/ml LDL or mm-LDL for 1 to 4 hours stimulated mesangial cell PDGF mRNA expression (mm-LDL 2 to 3 times greater than LDL); ox-LDL had no effect. Similarly, both LDL and mm-LDL induced mesangial cell DNA synthesis (mm-LDL 1.5 to 2 times greater). In further studies evaluating key associated intracellular signal transduction mechanisms, the protein tyrosine kinase (PTK) inhibitors herbimycin and genistein markedly decreased basal and lipoprotein-induced PDGF mRNA expression. Both pertussis toxin and isoproterenol, cyclic AMP-generating substances, stimulated PDGF mRNA expression. Preincubation with H-8 or H-89, cyclic AMP-dependent protein kinase A (PKA) inhibitors, blocked the lipoprotein-induced PDGF message, whereas preincubation with calphostin C, a protein kinase C inhibitor, did not alter LDL- or mm-LDL-mediated PDGF mRNA expression. These data suggest that the accumulation of atherogenic lipoproteins and their endogenous oxidized forms within the glomerulus may regulate mesangial cell PDGF expression and related cellular responses. These events appear to be modulated by signal transduction pathways involving PTK and PKA.     

Selective inhibition of cyclic AMP-dependent protein kinase by isoquinoline derivatives

A large series of isoquinoline derivatives was synthesised including derivatives of isoquinoline, isoquinolino[3,4-c]furazan, 1,2-dihydro-1-oxoisoquinoline, 6-oxopyrimido[1,2-d]isoquinoline, benzo[c][1,8]-naphthyridine, pyrazino[2,3-c]isoquinoline and benzimidazo[2,1-a]isoquinoline as well as further structurally related isoquinoline derivatives and pyrido-2,3-furazans. Representatives of all of these classes of isoquinolines are potent and selective inhibitors of the cyclic AMP-dependent protein kinase (PKA) catalytic subunit (cAK) from rat liver. The most effective cAK inhibitors are a series of 1,3-di-substituted and 1,3,4-tri-substituted isoquinolines (IC50 values 30-50 nM) (compounds A1, A2, A3, A4 and A5) and 2-ethylcarboxy-3-amino-5,6-dihydro-6-oxobenzo[c] [1,8]naphthyridine (E1) (IC50 0.08 microM). Compounds A1-A5 inhibit cAK in a fashion that is competitive with respect to ATP as substrate. The isoquinoline inhibitors A1-A5 are ineffective or very poor inhibitors of wheat embryo Ca(2+)-dependent protein kinase (CDPK) and rat brain Ca(2+)-dependent protein kinase C (PKC), chicken gizzard myosin light chain kinase (MLCK) and potato tuber cyclic nucleotide-binding phosphatase (Pase). E1 is a moderately effective inhibitor of CDPK and PKC (IC50 values 30 and 61 microM, respectively). The bisisoquinoline-1(2H)-one compound B7 inhibits cAK, CDPK, PKC and MLCK (IC50 values 8, 95, 24 and 7 microM, respectively) as does J1 [2-(p-bromophenyl)pyrrolo-[2,3-c]isoquinoline-5(4H)-one] (IC50 values 2, 50, 44 and 7 microM, respectively). The very potent isoquinoline-derived cAK inhibitors found here involve substitution of the N-containing isoquinoline ring system and these inhibitors show high specificity for cAK.     

Cyclic AMP-dependent protein kinase (cAK) in human B cells: co-localization of type I cAK (RI alpha 2 C2) with the antigen receptor during anti-immunoglobulin-induced B cell activation

Cyclic AMP (cAMP) inhibits antigen-stimulated B cell proliferation through activation of cAMP-dependent protein kinases (cAK). We have examined the molecular composition and cellular localization of cAK in human B cells. We find that human B cells contain substantial amounts of mRNA for RI alpha, RII alpha, C alpha and C beta, barely detectable levels of RI beta mRNA, and no detectable RII beta or C gamma mRNA. At the protein level, using Western blotting and subunit-specific antibodies against the different R subunits, we find RI alpha and RII alpha, but no RI beta or RII beta. The presence of catalytic subunits was demonstrated using a nonselective anti-C antiserum. By photoaffinity labeling of R subunits with 8-azido-[32P]cAMP, followed by immunoprecipitation with subunit-specific antibodies, we were also able to demonstrate low levels of RI beta. Immunofluorescence staining of RI alpha and RII alpha demonstrates a rather homogeneous intracellular (but extranuclear) distribution of RI alpha, whereas the RII alpha subunits of cAK are localized to distinct perinuclear structures, previously identified as centrosomes in other cell types. Upon anti-Ig-mediated capping of B cells, RI alpha subunits redistribute to the cap, co-localizing with the antigen-receptors, whereas the intracellular localization of RII alpha subunits remains unchanged.     

Developmental cues modulate GABAA receptor subunit mRNA expression in cultured cerebellar granule neurons

The developmental expression of mRNAs encoding the GABAA receptor was analyzed in the rat cerebellar cortex and in cultured cerebellar granule neurons. Our studies in vivo reveal that the alpha 1-, beta 2-, and gamma 2-subunit mRNA levels in the cerebellar cortex rise dramatically during the second post-natal week, a period temporally correlated with extensive cerebellar maturation. To determine if these increases were preprogrammed or dependent on extrinsic factors, we examined subunit mRNA expression in granule cell cultures prepared at embryonic day 19 (E19) and postnatal day 10 (P10), immature and mature stages of cerebellar development, respectively. In E19 cultures, the alpha 1, beta 2, and gamma 2 GABAA receptor subunit mRNAs were present and their levels remained constant over the 21 d culture period. These results suggest that GABAA receptor gene expression is not intrinsic to the immature granule cells. A different pattern was found in P10 cultures where the three subunit mRNAs were initially present at levels approximately sixfold higher than those found at E19. The beta 2- and gamma 2-subunit mRNAs remained constant for 4 d and then increased sixfold between 4 and 7 d in culture. The magnitude and time course of these increases were similar to the developmental changes that occurred in vivo. Thus, our findings raise the possibility that signals encountered during development are necessary to induce GABAA receptor subunit mRNA expression. Moreover, these cues have been received by granule neurons prior to P10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligation of the T cell co-stimulatory receptor CD28 activates the serine-threonine protein kinase protein kinase B

The intracellular signaling pathways activated upon ligation of the co-stimulatory receptor CD28 remain relatively ill-defined, although CD28 ligation does result in the strong association with, and activation of, phosphatidylinositol (PI) 3-kinase. The downstream effector targets of the CD28-activated PI 3-kinase-dependent signaling pathway remain poorly defined, but recent evidence from other systems has shown that Akt/protein kinase B (PKB) is a major target of PI 3-kinase and have indicated that a major function of PKB is the regulation of cell survival events. Given the strong coupling of CD28 to PI 3-kinase and the known protective effects of both CD28 and PI 3-kinase against apoptosis in different cell models, we investigated the effects of CD28 on PKB activation. We demonstrate that ligation of CD28 by either anti-CD28 monoclonal antibodies or the natural ligand B7.1, results in the marked activation of PKB in both the leukemic T cell line Jurkat and freshly isolated human peripheral blood-derived normal T lymphocytes. Our data suggest therefore, that PKB may be an important intracellular signal involved in CD28 signal transduction and demonstrate CD28 coupling to downstream elements of a signaling cascade known to promote cell survival.     

Adult treatment with haloperidol increases dentate granule cell proliferation in the gerbil hippocampus

The effects of cibenzoline on transmembrane action potentials were examined in right ventricular papillary muscles and in single ventricular myocytes isolated from guinea-pig hearts. In papillary muscles, cibenzoline > or = 3 microM caused a significant decrease in the maximum upstroke velocity (Vmax) of the action potential without affecting the action potential duration. The inhibition of Vmax was enhanced at higher stimulation frequencies. In the presence of cibenzoline, trains of stimuli at rates > or = 0.2 Hz led to a use-dependent inhibition of Vmax. The time constant for Vmax recovery (tauR) from the use-dependent block was 26.2 s. The use-dependent block of Vmax with cibenzoline was enhanced and tauR was shortened when the resting potential was depolarized by high (8, 10 mM) [K+]o. The curve relating membrane potential and Vmax in single myocytes was shifted by cibenzoline (10 microM) in a hyperpolarizing direction by 7.1 mV. In myocytes treated with cibenzoline (10 microM), a 10-ms conditioning clamp to 0 mV caused a significant decrease in Vmax of the subsequent test action potential; the Vmax inhibition was enhanced modestly in association with a prolongation of the 0 mV clamp pulse duration. In the presence of cibenzoline (3 microM), application of a train of depolarizing pulses (10 ms, 200 ms) to myocytes from the resting level (-80 mV) to 0 mV resulted in a progressive Vmax reduction in a pulse number-dependent manner. Unlike glibenclamide (30 microM), cibenzoline (10 microM) did not prevent the hypoxia-induced shortening of action potential duration in papillary muscles. These findings indicate that the onset and offset kinetics of use-dependent Na+ channel block by cibenzoline are slow. Given its state dependence, cibenzoline may be a blocker of activated Na+ channels. The inhibitory action of this compound on the ATP-sensitive K+ current (I(K), ATP) would be minimal or negligible at concentrations causing sufficient Na+ channel block.     

A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells

It has been estimated that over three million workers in the USA are potentially exposed to silica or other mineral dusts. Results of epidemiological studies evaluating whether silica or glass fibers increase lung cancer risk to the exposed workers are inconclusive. Detection of DNA damage in cells exposed to genotoxic agents is being used to assess the carcinogenic potential of environmental agents. The alkaline (pH > 13) single cell gel/comet (SCG) assay was used to determine and compare DNA damage in cultured Chinese hamster lung fibroblasts (V79 cells) and human embryonic lung fibroblasts (Hel 299 cells) exposed to crystalline silica (Min-U-Sil 5), amorphous silica (Spherisorb), carbon black, and glass fibers (AAA-10). V79 or Hel 299 cells were exposed to these mineral dusts for 3 h at various concentrations. Min-U-Sil 5 and AAA-10, at almost all concentrations tested, caused a significant increase in DNA migration measured as tail length in both V79 and Hel 299 exposed cells. However, the increase was much higher in V79 then in Hel 299 cells for Min-U-Sil 5. Tail length was also increased relative to controls after amorphous silica treatment, but not to the same extent as that induced by crystalline silica. Exposure to carbon black did not induce DNA migration at any of the concentrations tested. These results indicate that silica and glass fibers, but not carbon black, can induce DNA damage in mammalian cells, and that crystalline silica has a higher DNA-damaging activity than amorphous silica. For glass fibers, induction of DNA damage in both V79 and Hel 299 cells was observed even at a concentration 10 times lower than silica and the response was similar in both cell lines. These results suggest that the SCG/comet assay is useful for the detection of DNA damage caused by occupationally related dusts/particles.     

Enhancement of recombinant gamma-aminobutyric acid type A receptor currents by chronic activation of cAMP-dependent protein kinase

alpha 1, beta 1, and gamma 2S gamma-aminobutyric acid (GABA) type A receptor (GABAR) subunit cDNAs were transiently expressed in derivative cell lines of mouse L929 fibroblasts, which possessed different levels of the catalytic subunit of cAMP-dependent protein kinase (PKA). These cell lines included L929 (intermediate levels of kinase), C alpha 12 (elevated levels of kinase), and RAB10 (low levels of kinase) cells. Pharmacological analysis of GABA-evoked whole-cell currents revealed that, compared with expression in L929 and RAB10 cells, expression of alpha 1 beta 1 gamma 2S GABARs in C alpha 12 cells produced a selective enhancement of single whole-cell current amplitudes. No other pharmacological properties (Hill slope, EC50, or diazepam sensitivity) of the expressed alpha 1 beta 1 gamma 2S GABARs were modified. The GABAR current enhancement in C alpha 12 cells was blocked by substitution of a beta 1 subunit mutated at the PKA consensus phosphorylation site, Ser409 [beta 1(S409A)], for the wild-type beta subunit. Interestingly, enhancement was specific for GABARs containing all three subunits, because it was not seen after expression of alpha 1 beta 1 or alpha 1 beta 1 (S409A) GABAR subunit combinations. Single-channel conductance and gating properties were not different for alpha 1 beta 1 gamma 2S or alpha 1 beta 1 (S409A) gamma 2S GABARs expressed in each cell line, suggesting that PKA did not enhance whole-cell currents by altering these properties of GABARs. These results suggested that unlike acute application of PKA, which has been shown to produce a decrease in GABAR current, chronic elevation of PKA activity can result in enhancement of GABAR currents. More importantly, this effect occurred only with GABARs composed of alpha 1 beta 1 gamma 2S subunits and not alpha 1 beta 1 subunits and was mediated by a single amino acid residue (Ser409) of the beta 1 subunit.     

Modulation of synaptic GABAA receptor function by PKA and PKC in adult hippocampal neurons

Several protein kinases are known to phosphorylate Ser/Thr residues of certain GABAA receptor subunits. Yet, the effect of phosphorylation on GABAA receptor function in neurons remains controversial, and the functional consequences of phosphorylating synaptic GABAA receptors of adult CNS neurons are poorly understood. We used whole-cell patch-clamp recordings of GABAA receptor-mediated miniature IPSCs (mIPSCs) in CA1 pyramidal neurons and dentate gyrus granule cells (GCs) of adult rat hippocampal slices to determine the effects of cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) activation on the function of synaptic GABAA receptors. The mIPSCs recorded in CA1 pyramidal cells and in GCs were differentially affected by PKA and PKC. In pyramidal cells, PKA reduced mIPSC amplitudes and enhanced the fraction of events decaying with a double exponential, whereas PKC was without effect. In contrast, in GCs PKA was ineffective, but PKC increased the peak amplitude of mIPSCs and also favored double exponential decays. Intracellular perfusion of the phosphatase inhibitor microcystin revealed that synaptic GABAA receptors of pyramidal cells, but not those of GCs, are continually phosphorylated by PKA and conversely, dephosphorylated, most likely by phosphatase 1 or 2A. This differential, brain region-specific phosphorylation of GABAA receptors may produce a wide dynamic range of inhibitory synaptic strength in these two regions of the hippocampal formation.     

Pseudosubstrate inhibition of cyclic AMP-dependent protein kinase in intact pancreatic islets: effects on cyclic AMP-dependent and glucose-dependent insulin secretion

Synthetic peptides derived from the endogenous protein kinase A inhibitor (PKI) offer a specific means of inhibiting cyclic AMP-dependent protein kinase A (PKA), but their use in whole cells is restricted by the plasma membrane. We have now modified PKI sequences by N-terminal myristoylation to enhance their membrane permeability, and have used the myristoylated (myr) peptides to investigate the role of PKA activation in glucose-induced insulin secretion from intact pancreatic beta-cells. The myristoylated PKI peptides, myr PKI14-22 and myrPKI6-22, were effective inhibitors in vitro of PKA activity extracted from rat islets of Langerhans. In experiments using intact islets, myr PKI14-22 caused a concentration-dependent inhibition of insulin secretion in response to the PKA activators dibutyryl cyclic AMP and forskolin, suggesting that it gained access to the cytosolic compartment of intact beta-cells and inhibited PKA in situ. However, these concentrations of myr PKI14-22 did not inhibit insulin secretion in response to glucose suggesting that the activation of PKA is not required for the initiation of glucose-induced insulin secretion.     

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.     

Inhibition of GABAA receptor function by tyrosine kinase inhibitors and their inactive analogues

The effects of tyrosine kinase inhibitors which target the ATP binding site or the substrate binding site of tyrosine kinases were assessed on murine recombinant type A gamma-aminobutyric acid (GABAA) receptors expressed in Xenopus oocytes or HEK cells using two-electrode voltage clamp or patch clamp recording. Genistein inhibited in a noncompetitive manner GABA-activated currents recorded from alpha1beta1gamma2S receptor constructs by reducing the maximum normalized response from 1.83 +/- 0.04 to 0.71 +/- 0.04 and reducing the EC50 from 35.7 +/- 2.1 microM to 15.1 +/- 3.9 microM. After mutating the two "functionally active" substrate tyrosine (Y) residues in gamma2S and expressing the mutant receptor alpha1beta1gamma2S(Y365F, Y367F), genistein still noncompetitively inhibited the responses to GABA reducing the maximum current from 1. 81 +/- 0.03 to 0.26 +/- 0.01 and the EC50 from 33.1 +/- 2.3 microM to 5.8 +/- 2.2 microM. The inactive compound, daidzein, also similarly inhibited responses to GABA on these two receptor constructs. Inhibitors targeting the substrate binding site of tyrosine kinases, the tyrphostins, also inhibited both the wild-type and the tyrosine mutant GABAA receptors. Tyrphostin A25 and the inactive tyrphostin A1 reduced the maximum normalized responses for alpha1beta1gamma2S and alpha1beta1gamma2S(Y365F, Y367F) receptors by 73 and 64%, respectively. The tyrosine kinase inhibitors and their inactive controls did not display any significant voltage sensitivity to the antagonism of GABA-activated responses. Moreover, genistein or tyrphostin A25 did not affect the potentiation of responses to GABA by pentobarbitone or diazepam. Mutating the two "functionally silent" tyrosine residues, Y370 and Y372, known to be substrates for tyrosine kinases in the beta1 subunit and coexpression in the alpha1beta1(Y370F, Y372F)gamma2S(Y365F, Y367F) construct failed to affect the inhibitory action of genistein. The study concludes that tyrosine kinase inhibitors and their inactive controls can directly interact with GABAA receptors completely independent of any effects on tyrosine kinases.