Non-selective Protein Kinase Inhibitor Research Articles

Commiphora myrrha stimulates insulin secretion from β-cells through activation of atypical protein kinase C and mitogen-activated protein kinase

Ethnopharmacological relevanceAyurvedic medicine has been used in the treatment of diabetes mellitus for centuries. In Arabia and some areas of Africa, Commiphora myrrha (CM) has been extensively used as a plant-based remedy. We have previously shown that an aqueous CM resin solution directly stimulates insulin secretion from MIN6 cells, a mouse β-cell line, and isolated mouse and human islets. However, the signaling pathways involved in CM-induced insulin secretion are completely unknown. Insulin secretion is normally triggered by elevations in intracellular Ca2+ ([Ca2+]i) through voltage gated Ca2+ channels (VGCC) and activation of protein kinases. Protein and lipid kinases such as protein kinase A (PKA), Ca2+-calmodulin dependent protein kinase II (CaMKII), phosphoinositide 3-kinases (PI3Ks), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK), specifically extracellular signal-regulated kinases (ERK1/2), may be involved in receptor-operated insulin secretion. Therefore, we hypothesized that CM may induce insulin secretion by modulating the activity of VGCC and/or one or more of the above kinases. Aim of the studyTo investigate the possible molecular mechanism of action of CM-induced insulin secretion. The effects of aqueous CM resin extract on [Ca2+]i and protein kinase activation from β-cells were examined. MethodsThe effect of aqueous CM resin solution on [Ca2+]i was assessed using Ca2+ microfluorimetry. The involvement of VGCC in CM-induced insulin secretion was investigated using static and perifusion insulin secretion experiments in the presence of either EGTA, a Ca2+ chelator, or nifedipine, a blocker of VGCC. The involvement of kinase activation in the stimulatory effect of CM on insulin secretion was examined by using static and perifusion insulin secretion experiments in the presence of known pharmacological inhibitors and/or downregulation of specific kinases. The effects of CM on phosphorylation of PKCζ and ERK1/2 were also assessed using the Wes™ capillary-based protein electrophoresis. ResultsCa2+ microfluorimetry measurements showed that exposing MIN6 cells to CM (0.5–2 mg/mL) was not associated with changes in [Ca2+]i. Similarly, incubating MIN6 cells and mouse islets with EGTA and nifedipine, respectively, did not attenuate the insulin secretion induced by CM. However, incubating mouse and human islets with CM in the presence of staurosporine, a non-selective protein kinase inhibitor, completely blocked the effect of CM on insulin secretion. Exposing mouse islets to CM in the presence of H89, KN62 and LY294002, inhibitors of PKA, CaMKII and PI3K, respectively, did not reduce CM-induced insulin secretion. However, incubating mouse and human islets with CM in the presence of Ro 31–8220, a pan-PKC inhibitor, diminished insulin secretion stimulated by CM, whereas inhibiting the action of typical PKC (with Go6976) and PLCβ (with U73122) did not affect CM-stimulated insulin secretion. Similarly, downregulating typical and novel PKC by chronic exposure of mouse islets to phorbol 12-myristate 13-acetate (PMA) was also not associated with a decrease in the stimulatory effect of CM on insulin secretion. Interestingly, CM-induced insulin secretion from mouse islets was inhibited in the presence of the PKCζ inhibitor ZIP and a MAPK inhibitor PD 98059. In addition, Wes™ capillary-based protein electrophoresis indicated that expression of the phosphorylated forms of PKCζ and ERK1/2, a MAPK, was significantly increased following exposure of INS-1832/13 cells, a rat insulinoma cell line, to CM. ConclusionsOur data indicate that CM directly stimulates insulin secretion through activating known downstream effectors of insulin-stimulus secretion coupling. Indeed, the increase in insulin secretion seen with CM is independent of changes in [Ca2+]i and does not involve activation of VGCC. Instead, the CM stimulatory effect on insulin secretion is completely dependent on protein kinase activation. Our findings indicate that CM could induce insulin exocytosis by stimulating the phosphorylation and activation of PKCζ, which in turn phosphorylates and activates ERK1/2.

Activation of the Ae4 (Slc4a9) cation-driven Cl-/HCO3- exchanger by the cAMP-dependent protein kinase in salivary gland acinar cells.

Ae4 transporters are critical for Cl- uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl-/HCO3- exchanger activity in SMG acinar cells from Ae2-/- mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to β-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH2-terminal domain according to a homology model of Ae4. NH2-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl-/HCO3- exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173.NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon β-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.

Mechanisms of the adenosine A2A receptor-induced sensitization of esophageal C fibers.

Clinical studies indicate that adenosine contributes to esophageal mechanical hypersensitivity in some patients with pain originating in the esophagus. We have previously reported that the esophageal vagal nodose C fibers express the adenosine A2A receptor. Here we addressed the hypothesis that stimulation of the adenosine A2A receptor induces mechanical sensitization of esophageal C fibers by a mechanism involving transient receptor potential A1 (TRPA1). Extracellular single fiber recordings of activity originating in C-fiber terminals were made in the ex vivo vagally innervated guinea pig esophagus. The adenosine A2A receptor-selective agonist CGS21680 induced robust, reversible sensitization of the response to esophageal distention (10-60 mmHg) in a concentration-dependent fashion (1-100 nM). At the half-maximally effective concentration (EC50: ≈3 nM), CGS21680 induced an approximately twofold increase in the mechanical response without causing an overt activation. This sensitization was abolished by the selective A2A antagonist SCH58261. The adenylyl cyclase activator forskolin mimicked while the nonselective protein kinase inhibitor H89 inhibited mechanical sensitization by CGS21680. CGS21680 did not enhance the response to the purinergic P2X receptor agonist α,β-methylene-ATP, indicating that CGS21680 does not nonspecifically sensitize to all stimuli. Mechanical sensitization by CGS21680 was abolished by pretreatment with two structurally different TRPA1 antagonists AP18 and HC030031. Single cell RT-PCR and whole cell patch-clamp studies in isolated esophagus-specific nodose neurons revealed the expression of TRPA1 in A2A-positive C-fiber neurons and demonstrated that CGS21682 potentiated TRPA1 currents evoked by allylisothiocyanate. We conclude that stimulation of the adenosine A2A receptor induces mechanical sensitization of nodose C fibers by a mechanism sensitive to TRPA1 antagonists indicating the involvement of TRPA1.

The production of vasoconstriction-induced residual NO modulates perfusion pressure in rat mesenteric vascular bed

In the presence of nitric oxide synthase (NOS) inhibitors, the contribution of residual NO to endothelium-dependent relaxation induced by chemical agonists acetylcholine and bradykinin has been documented in resistance vessels. However, the contribution of residual NO to the vasodilatation in response to pressure and fluid shear stress is not well understood. In this study, to demonstrate the activity of residual NO, we applied a NO scavenger, hydroxocobalamin (HCX), on the phenylephrine-induced increase in perfusion pressure in the presence of NOS inhibitors, Nω-nitro-L-arginine (L-NA) or Nω-nitro-L-arginine methyl ester (L-NAME) in the rat perfused mesenteric bed. The perfusion pressure was increased by phenylephrine (1-2 µM), an α1-adrenoceptor agonist. This increase was augmented by the addition of L-NA or L-NAME. In the presence of any NOS inhibitors, the application of hydroxocobalamin (100 µM) further increased the perfusion pressure. The removal of endothelium by saponin (50 mg/L) and the use of a non-selective protein kinase inhibitor, staurosporine (5 nM), and a tyrosine kinase inhibitor, erbstatin A (30 µM), but not a calmodulin inhibitor, calmidazolium (0.5 µM), inhibited the additional pressor responses induced by L-NA or L-NAME and a combination of either of them with hydroxocobalamine. These findings show that there could be a NOS inhibitor-resistant residual NO production in response to pressure in the rat mesenteric vascular bed. This residual NO production may be associated with the activation of tyrosine kinase and protein kinases, but not calmodulin. Finally, this pressure-induced residual NO exerts a modulatory role against vasoconstriction induced by phenylephrine.

Ginseng–Aconite Decoction elicits a positive inotropic effect via the reverse mode Na+/Ca2+ exchanger in beating rabbit atria

Ethnopharmacological relevanceGinseng–Aconite Decoction (GAD), a traditional oriental medicine composed of Panax ginseng C.A. Mey. (Araliaceae) and Aconitum carmichaeli Debx. (Ranunculaceae) has been used as treatment for cardiovascular diseases from Song Dynasty of China. The purpose of the present study was to elucidate the possible mechanisms of GAD-induced positive inotropic effect. Material and methodsGAD-induced changes in atrial dynamics and cAMP efflux were determined in isolated perfused beating rabbit atria. ResultsGAD significantly increased atrial dynamics such as stroke volume, pulse pressure and augmented cAMP efflux in beating rabbit atria. The inotropic effect was significantly attenuated by pre-treatment with KB-R7943, a reverse mode Na+/Ca2+ exchanger blocker. The GAD-induced increase in atrial dynamics was also markedly inhibited by staurosporine, a non-selective protein kinase inhibitor, and partly blocked by KT5720, a selective PKA inhibitor. The effect of GAD on atrial dynamics was not altered by pre-treatment with propranolol, a β-adrenergic receptor inhibitor, or diltiazem, an L-type Ca2+channel blocker. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) failed to modulate the GAD-induced increase in atrial dynamics, but markedly attenuated cAMP efflux in the beating atria. ConclusionThese results suggest that the GAD-induced positive inotropic effect in beating rabbit atria may be attributable to stimulation of the reverse mode Na+/Ca2+ exchanger, while PKA activity would, at least in part, be participated in the course.

Modulation of Purinergic Neuromuscular Transmission by Phorbol Dibutyrate is Independent of Protein Kinase C in Murine Urinary Bladder

Parasympathetic control of murine urinary bladder consists of contractile components mediated by both muscarinic and purinergic receptors. Using intracellular recording techniques, the purinergic component of transmission was measured as both evoked excitatory junctional potentials (EJPs) in response to electrical field stimulation and spontaneous events [spontaneous EJPs (sEJPs)]. EJPs, but not sEJPs, were abolished by the application of the Na(+) channel blocker tetrodotoxin and the Ca(2+) channel blocker Cd(2+). Both EJPs and sEJPs were abolished by the application of the P2X(1) antagonist 8,8'-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF279). Application of phorbol dibutyrate (PDBu) increased electrically evoked EJP amplitudes with no effect on mean sEJP amplitudes. Similar increases in EJP amplitudes were produced by PDBu in the presence of either the nonselective protein kinase inhibitor staurosporine or the specific protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X). These results suggest that PDBu increases the purinergic component of detrusor transmission through increasing neurogenic ATP release via a PKC-independent mechanism.

Activation of protein kinase A and C prevents recovery from persistent depolarization produced by oxygen and glucose deprivation in rat hippocampal neurons

Intracellular recordings were made from rat hippocampal CA1 neurons in rat brain slice preparations to investigate whether cAMP-dependent protein kinase (PKA) and calcium/phospholipid-dependent protein kinase C (PKC) contribute to the membrane dysfunction induced by oxygen and glucose deprivation (OGD). Superfusion of oxygen- and glucose-deprived medium produced a rapid depolarization ∼5 min after the onset of the superfusion. When oxygen and glucose were reintroduced immediately after the rapid depolarization, the membrane depolarized further (persistent depolarization) and reached 0 mV after 5 min from the reintroduction. The pretreatment of the slice preparation with PKA inhibitors, H-89 and Rp-cAMPS, and an adenylate cyclase inhibitor, SQ 22, 536, significantly restored the membrane toward the preexposure potential level after the reintroduction of oxygen and glucose in a concentration-dependent manner. On the other hand, a phospholipase C inhibitor, U73122, a PKC inhibitor, GF109203X, and a nonselective protein kinase inhibitor, staurosporine, also significantly restored the membrane after the reintroduction. Moreover, an inositol-1,4,5-triphosphate receptor antagonist, 2-aminoethyl diphenylborinate, and calmodulin inhibitors, trifluoperazine and W-7, significantly restored the membrane after the reintroduction, while neither an α-subunit-selective antagonist for stimulatory G protein, NF449, a Ca(2+)/calmodulin-dependent kinase II inhibitor, KN-62, nor a myosin light chain kinase inhibitor, ML-7, significantly restored the membrane after the reintroduction. These results suggest that the activation of PKA and/or PKC prevents the recovery from the persistent depolarization produced by OGD. The Ca(2+)/calmodulin-stimulated adenylate cyclase may contribute to the activation of PKA.

ER-resident Gi2protein controls sar1 translocation onto the ER during budding of transport vesicles

In our previous study, fluoride ([AlF(4) ](-) ) disturbed ER-to-Golgi transport through the activation of ER-resident heterotrimeric G protein (ER-G protein). Therefore, ER-G protein may be implicated in ER-to-Golgi transport at the early stage prior to coat protein assembly. Sar1 translocation onto the endoplasmic reticulum (ER) membrane is suppressed by non-selective protein kinase inhibitor H89, suggesting the participation of H89-sensitive kinase in this process. To investigate the involvement of ER-G protein in ER-to-Golgi transport, the effect of G(i) protein activator (mastoparan 7) was examined on Sar1 translocation onto the ER in a cell-free system consisting of microsome membrane and cytosol. Sar1 translocation onto the microsome membrane was induced by addition of GTPγS in the cell-free system. Translocation of Sar1 by GTPγS was suppressed significantly by both H89 and mastoparan 7. Mastoparan 7 suppressed the translocation of Sar1 onto the microsome membrane with dosage dependency, but mastoparan 17, the inactive analog of mastoparan 7, had no effect on Sar1 translocation. The suppressive effect of mastoparan 7 was recovered by treatment with pertussis toxin (IAP). Moreover, G(i2) protein was detected on the microsome membrane by western blotting for heterotrimeric G(i) proteins. These results indicate that ER-G(i2) protein modulated Sar1 translocation onto the ER, suggesting that ER-resident G(i2) protein is an important negative regulator of vesicular transport at the early stage of vesicle formation before coat protein assembly on the ER.

CFTR is activated through stimulation of purinergic P2Y2 receptors

It has been reported that the cystic fibrosis transmembrane conductance regulator (CFTR) can be activated through cAMP- and protein kinase A-independent pathways involving GTP-binding proteins and an unknown kinase. In this study, we further examined how G protein-coupled pathways regulate CFTR. We demonstrate that stimulation of purinergic P2Y(2) receptors in CFTR-expressing oocytes and in airway epithelial cells activates CFTR Cl(-) currents. Activation of CFTR Cl(-) currents via P2Y(2) was inhibited by CFTR(inh)-172 and was independent of intracellular Ca(2+), protein kinase C, or calmodulin-dependent kinase (CAMK). However, activation of CFTR was suppressed by inhibition of phospholipase C and by the nonselective protein kinase inhibitor staurosporine. Activation of CFTR through P2Y(2) receptors was enhanced when G(i) proteins were inhibited by pertussis toxin. Inhibition of protein kinase A and of protein kinases downstream of P2Y(2) receptors such as mitogen-activated protein kinases, tyrosine kinase, or c-src kinase did not interfere with activation of CFTR. The present results demonstrate an antagonistic regulation of CFTR by P2Y(2) receptors: CFTR is inhibited by stimulation of G(i) proteins and is activated by stimulation of G(q/11)/PLC and an unknown downstream protein kinase.

Protein kinase C inhibition attenuates hypochlorite-induced acute lung injury

Neutrophil-derived oxidative stress plays a crucial role in acute lung injury. Hypochlorite/hypochlorous acid (HOCl) is a major oxidant of neutrophils. Protein kinase C (PKC) may be an appropriate target for HOCl due to its functionally important thiols. This study investigates the role of PKC in HOCl-induced acute lung injury. Isolated lung preparations were from 30 rabbits. HOCl (1000 nmol min(-1)) or buffer (control) were infused into isolated rabbit lungs. Pulmonary artery pressure (PAP [Torr]) and lung weight were continuously measured. Capillary filtration coefficient (K(f,c)), was measured at baseline and at 30, 60, 90 min. Experiments were terminated at 105 min or when fluid retention exceeded 50 g. The non-selective protein kinase inhibitor staurosporin (100 nM) or the selective PKC inhibitor bisindolylmaleimide I (GF109203X, 10nM) were added to the perfusate 5 min prior to the start of the experiments. Staurosporin completely prevented the HOCl-induced increase in PAP (no change versus DeltaPAP(max) 5.2+/-0.78) but did not influence the increase in vascular permeability. GF109203X delayed the HOCl-induced increase in PAP and vascular permeability. PAP(max) was observed significantly later in the HOCl-GF109203X group (84.4+/-4.0 min) in comparison with the HOCl group (52.1+/-3.5 min). Termination of the experiments due to edema formation occurred significantly later in experiments with GF109203X (91.8+/-1.9 versus 79.2+/-4.1 min). Protein kinases are involved in HOCl-induced acute lung injury. Specifically PKC inhibition delayed HOCl-induced increases in PAP and vascular permeability.

Rac1 GTPase Regulates Cell Genomic Stability and Senescence

The Rho family small GTPase Rac1 has been shown to play multiple roles in cell regulation, including actin cytoskeleton organization, transcriptional activation, microtubule dynamics, and endocytosis. Here, we report a novel role of Rac1 in regulating genomic stability and cell senescence. We observed in primary mouse embryonic fibroblasts that deletion of rac1 by gene targeting, as well as expression of the constitutively active Rac1 mutant L61Rac1, led to decreased cell growth that was associated with altered cell cycle progression at both G(1)/S and G(2)/M phases, increased apoptosis, and premature senescence. The senescence induction by either loss or gain of Rac1 activity was due at least in part to an increase in cellular reactive oxygen species (ROS). rac1 gene deletion caused a compensatory up-regulation of a closely related family member, Rac3, in mouse embryonic fibroblasts, the activity of which induced ROS production independently of Rac1. Furthermore, the Rac1-regulated ROS production and senescence correlated with the extent of DNA damage in the Rac1(-/-) and L61Rac1 cells. Treatment of these cells with a ROS inhibitor inhibited phospho-H2AX-positive nuclear focus formation. Finally, phospho-Ser(15) p53 was significantly increased in L61Rac1 and Rac1(-/-) cells, and genetic deletion of p53 from these cells readily reversed the senescence phenotype, indicating that Rac1 is functionally dependent on p53 in regulating cell senescence. Taken together, our results show that Rac1 activity serves as a regulator of cell senescence through modulation of cellular ROS, genomic stability, and p53 activity.

The positive inotropic effect of the aqueous extract of Convallaria keiskei in beating rabbit atria

The positive inotropic effect of the aqueous extract of Convallaria keiskei (ACK) and the possible mechanisms responsible for this effect were investigated in beating rabbit atria. ACK significantly increased atrial stroke volume, pulse pressure, and cAMP efflux in beating rabbit atria. The effects were not altered by pre-treatment with staurosporine and diltiazem, a non-selective protein kinase inhibitor and an L-type Ca 2+ channel blocker, respectively. In addition, ACK markedly increased the K + concentration in the beating atria-derived perfusate. Convallatoxin, a well-known digitalis-like cardiac glycosidic constituent of ACK, also increased atrial stroke volume and pulse pressure but did not alter the cAMP efflux level. The increases in atrial stroke volume and pulse pressure induced by convallatoxin were not also altered by pre-treatment with diltiazem. These results suggest that the ACK-induced positive inotropic effect in beating rabbit atria may, at least in part, be due to the digitalis-like activity of convallatoxin.

Relative roles of protein kinase A and protein kinase C in modulation of transient receptor potential vanilloid type 1 receptor responsiveness in rat sensory neurons in vitro and peripheral nociceptors in vivo

The function of the transient receptor potential vanilloid type 1 capsaicin receptor is subject to modulation by phosphorylation catalyzed by various enzymes including protein kinase C and cAMP-dependent protein kinase. The aim of this study was to compare the significance of the basal and stimulated activity of protein kinase C and cAMP-dependent protein kinase in transient receptor potential vanilloid type 1 receptor responsiveness in the rat in vitro by measurement of the intracellular calcium concentration in cultured trigeminal ganglion neurons and in vivo by determination of the behavioral noxious heat threshold. KT5720, a selective inhibitor of cAMP-dependent protein kinase, reduced the calcium transients induced by capsaicin or the other, much more potent transient receptor potential vanilloid type 1 receptor agonist resiniferatoxin in trigeminal sensory neurons and diminished the drop of the noxious heat threshold (heat allodynia) evoked by intraplantar resiniferatoxin injection. Chelerythrine chloride, a selective inhibitor of protein kinase C, failed to alter either of these responses, although it inhibited the effect of phorbol 12-myristate 13-acetate in the in vitro assay. Staurosporine, a rather nonselective protein kinase inhibitor, failed to reduce the capsaicin- and resiniferatoxin-induced calcium transients but inhibited the resiniferatoxin-evoked heat allodynia. Dibutyryl-cAMP and phorbol 12-myristate 13-acetate, activator(s) of cAMP-dependent protein kinase and protein kinase C, respectively, enhanced the effect of capsaicin in the calcium uptake assay while forskolin, an activator of adenylyl cyclase, augmented that of resiniferatoxin in the heat allodynia model. None of the protein kinase inhibitors or activators altered the calcium transients evoked by high potassium, a nonspecific depolarizing stimulus. It is concluded that basal activity of cAMP-dependent protein kinase, unlike protein kinase C, is involved in the maintenance of transient receptor potential vanilloid type 1 receptor function in somata of trigeminal sensory neurons but stimulation of either cAMP-dependent protein kinase or protein kinase C above the resting level can lead to an enhanced transient receptor potential vanilloid type 1 receptor responsiveness. Similar mechanisms are likely to operate in vivo in peripheral terminals of nociceptive dorsal root ganglion neurons.

A cGMP-dependent cascade enhances an l-type-like Ca 2+ current in identified snail neurons

We studied the impact of an NO-cGMP dependent signalling pathway on the high-voltage-activated (HVA) Ca 2+ current in identified neurons of the pulmonate snail, Helix pomatia, using Ba 2+ as charge carrier. The 3′,5′-cyclic guanosine monophosphate (cGMP) analogues, dibutyryl-cGMP and 8-bromo-cGMP, consistently induced a biphasic response, consisting of an increase superseded by a decline of the Ba 2+ current. The NO donor, sodium nitroprusside (SNP), modulated only in a minority of neurons the Ba 2+ current. Blockade of protein kinase activity with 1-[5-isoquinolinesulfonyl]-2 methyl piperazine (H 7), a nonselective protein kinase inhibitor, or Rp-8-pCPT-cGMP, a selective protein kinase G (PKG) inhibitor, decreased, whereas Rp-cAMP, a selective protein kinase A (PKA) inhibitor, increased the Ba 2+ current upon application of cGMP analogues or SNP. Okadaic acid or calyculin, inhibitors of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), augmented the Ba 2+ current. Under these conditions, cGMP analogues or SNP had an additive-enhancing effect on the Ba 2+ current. When neurons were exposed to the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), cGMP analogues induced a persistent increase of the Ba 2+ current, whereas SNP induced a biphasic response. These data suggest coexistence of cGMP-PKG and cGMP-PDE pathways as well as crosstalk between cGMP and 3′,5′-cyclic adenosine monophosphate (cAMP) pathways, which converge on HVA Ca channels in Helix neurons. In this model, augmentation of the Ba 2+ current through HVA Ca channels is accomplished by PKA and PKG, whereas attenuation is mediated by PDEs, which prevent activation of protein kinases via hydrolysis of cyclic nucleotides.

Signalling pathways regulating human neutrophil migration induced by secretory phospholipases A 2

This study was designed to elucidate the signalling pathways by which secretory phospholipases A 2 (sPLA 2s) induce in vitro neutrophil migration. The cell migration assays were performed with Naja mocambique venom PLA 2 (sPLA 2 with high catalytic activity), bothropstoxin-I (sPLA 2 devoid of catalytic activity) and platelet-activating factor (PAF), using a 48-well microchemotaxis chamber. Both the non-selective protein kinase inhibitor staurosporine (30–300 nM) and the selective protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpyperazine (H7; 50–200 μM) as well as the Gi inactivator pertussis toxin (30–300 nM) caused a concentration-dependent inhibition of the neutrophil migration induced by either N. mocambique venom PLA 2 (100 μg/ml) or bothropstoxin-I (100 μg/ml). Pertussis toxin nearly abolished PAF-induced migration, while staurosporine and H7 partly (but significantly) inhibited the chemotactic responses to PAF. The dual inhibitor of cytosolic PLA 2 and Ca 2+-independent PLA 2 (iPLA 2), arachidonil-trifluoromethyl-ketone (ATK; 0.2–20 μM), or the specific iPLA 2 inhibitor bromoenol lactone (1–30 μM) caused a concentration-dependent inhibition of the migration induced by either sPLA 2s. At the maximal concentration used for each compound, the migration was almost suppressed. In contrast, both of these compounds caused only slight inhibitions of PAF-induced migration. No rise in intracellular Ca 2+ was observed in neutrophil-stimulated sPLA 2, as determined in cells preloaded with fura 2-AM. In the experimental condition used, pertussis toxin, staurosporine, H7, ATK or bromoenol lactone did not induce cytotoxic effects, according to MTT assay. Our results suggest that activation of an endogenous PLA 2 through activation of GTP-binding protein and PKC is the main mechanism by which exogenous sPLA 2s cause neutrophil migration.

Group II metabotropic glutamate receptor modulation of excitatory transmission in rat subthalamic nucleus.

Patch pipettes were used to record currents in whole-cell configuration to study the effects of group II metabotropic glutamate receptor (mGluR) stimulation on synaptic transmission in slices of rat subthalamic nucleus. Evoked glutamatergic excitatory postsynaptic currents (EPSCs) were reversibly reduced by the selective group II mGluR agonist (2'S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) in a concentration-dependent manner, with an IC50 of 0.19 +/- 0.05 microM. DCG IV (1 microM) had no effect on inhibitory postsynaptic currents mediated by GABA. DCG IV-induced inhibition of EPSCs was reversed by the selective group II mGluR antagonist LY 341495 (100 nM) and mimicked by another selective group II agonist (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Inhibition of EPSC amplitude by DCG IV and L-CCG-I was associated with an increase in the paired-pulse ratio of EPSCs. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (2 microM) reduced the inhibitory effect of DCG IV on EPSCs. However, the response to DCG IV was not affected by the protein kinase A (PKA) activator forskolin (20 microM), by the adenylyl cyclase inhibitor MDL 12230A (20 microM), or by the phosphodiesterase inhibitor Ro 20-1724 (50 microM). DCG IV-induced inhibition of EPSCs was reduced by the non-selective protein kinase inhibitors H-7 (100 microM), H-8 (50 microM) and HA-1004 (100 microM). These results suggest that group II mGluR stimulation acts presynaptically to inhibit glutamate release by a PKC-dependent mechanism in the subthalamic nucleus.

Signalling mechanism coupled to 5-hydroxytryptamine4 receptor-mediated facilitation of fast synaptic transmission in the guinea-pig ileum myenteric plexus.

5-hydroxytryptamine (HT)4 receptor agonists stimulate gastrointestinal motility partly by facilitating acetylcholine release from myenteric neurones. However, the signalling mechanisms that couple 5-HT4 receptor activation to increased transmitter release in the myenteric plexus are unknown. We used conventional intracellular electrophysiological methods to record fast excitatory postsynaptic potentials (fEPSPs) from neurones in the guinea-pig ileum myenteric plexus preparation. The substituted benzamide, renzapride, acted at 5-HT4 receptors to facilitate fEPSPs. This response was mimicked by forskolin, an activator of adenylate cyclase. Facilitation of fEPSPs by renzapride and forskolin was not blocked by treating tissues with pertussis toxin (PTX) (2 h, 2 microg mL-1). Facilitation of fEPSPs caused by renzapride was blocked by the non-selective protein kinase inhibitors, staurosporine (1 micromol L-1) and H-8 (30 micromol L-1) and by the selective protein kinase A (PKA) inhibitor, H-89 (10 micromol L-1). These data indicate that 5-HT4 receptors act via a PTX-resistant mechanism to activate PKA. Protein kinase A activation leads to an increase in transmitter release from myenteric nerve terminals and a facilitation of fast excitatory synaptic transmission.

Excitatory Effects of Orexin-A on Nucleus Tractus Solitarius Neurons Are Mediated by Phospholipase C and Protein Kinase C

Orexin (ORX)-A is a 33-amino acid peptide with demonstrated roles in the regulation of energy metabolism, autonomic control, and sleep. Orexin receptors (OXRs), OX1R and OX2R, and immunoreactive axons are present in the nucleus tractus solitarius (NTS). We demonstrated previously that bath application of ORX-A depolarizes NTS neurons through activation of a nonselective cationic conductance (NSCC) and inhibition of a sustained potassium current (IK). The present study examined the signaling pathways underlying the excitatory effects of ORX-A on NTS neurons using whole-cell patch-clamp recording techniques. Inclusion of guanosine 5'-O-(2-thiodiphosphate) in the internal pipette solution abolished the effects of ORX-A, confirming that the actions of ORX-A are mediated by G-protein-coupled receptors. The responses of ORX-A were also blocked by a phospholipase C (PLC) inhibitor, D609, and by a nonselective protein kinase (PK) inhibitor, H7, demonstrating the involvement of PLC and protein kinases. However, PKA appears not to play a role, because the depolarizing effects of ORX-A were still observed when the PKA inhibitor peptide (6-22) was included in the pipette solution, and bath application of 8-bromo-cAMP (a PKA agonist) was without effect on NTS neurons. In contrast, 12-O-tetradecanoylphorbol-13-acetate (a PKC agonist) depolarized NTS neurons, and bisindolylmaleimide (BIS), a PKC inhibitor, abolished the depolarizing effects of ORX-A. Finally, voltage-clamp experiments demonstrated that BIS also blocked the activation of NSCC and inhibition of IK by ORX-A in NTS neurons. These results therefore show that the excitatory effects of ORX-A on NTS neurons are mediated through activation of the PLC-PKC-NSCC and -IK signaling pathways, which probably result from OXR-coupled activation of Gq.

Effects of simulated microgravity on nitric oxide level in cardiac myocytes and its mechanism

The depression of cardiac contractility induced by space microgravity is an important issue of aerospace medicine research, while its precise mechanism is still unknown. In the present study, we explored effects of simulated microgravity on nitric oxide (NO) level, inducible nitric oxide synthase (iNOS) expression and related regulative mechanism using electron spin resonance (ESR) spectroscopy, immunocytochemistry and in situ hybridization. We found a remarkable increase of NO level and up-regulation of iNOS and iNOS mRNA expression in rat cardiac myocytes under simulated microgravity. Staurosporine (a nonselective protein kinase inhibitor), calphostin C (a selective protein kinase C inhibitor), partially inhibited the effect of simulated microgravity. Thus regulative effect of simulated microgravity on iNOS expression is mediated at least partially via activation of protein kinase C. These results indicate that NO system in cardiac myocytes is sensitive to simulated microgravity and may play an important role in the depression of cardiac contractility induced by simulated microgravity.

Possible involvement of energy metabolism in the change of cytoplasm organization induced by a protein phosphatase inhibitor, calyculin A, in root hair cells of Limnobium stoloniferum.

In root hair cells of Limnobium stoloniferum, transvacuolar strands disperse and cytoplasmic spherical bodies (CSBs) emerge upon treatment with a protein phosphatase inhibitor, calyculin A (CA), whose effects were previously shown to be canceled by simultaneous treatment of the cells with a nonselective protein kinase inhibitor, K-252a. CSB formation is also suppressed by latrunculin B (LB) or cytochalasin D, actin filament depolymerization drugs, or 2,3-butanedione monoxime, an inhibitor of myosin activity. To confirm the involvement of myosin activity in CSB formation induced by CA, we examined the effect of an inhibitor of energy metabolism, NaN3, on CSB formation in root hair cells pretreated simultaneously with CA and LB. In the presence of CA-LB, CSB formation was suppressed due to the depolymerization of actin filaments. When these drugs were removed, the actin filaments recovered and CSBs emerged even in the presence of K-252a. These results indicated that the phosphorylation level in the cells is elevated during the CA-LB treatment and that a phosphorylation level sufficient for the CSB formation was sustained even after CA removal. On the other hand, CSB formation after simultaneous treatment with CA and LB was significantly suppressed in the presence of NaN3. In such cells, actin filament bundles recovered, although their organization was random. The present and previous results suggested that myosin activity is necessary for CSB formation induced by CA, and that myosin regulated by phosphorylation-dephosphorylation is implicated in the organization of the actin cytoskeleton in root hair cells.