Chelerythrine Chloride Research Articles

Potentiation of Genomic Actions of Estrogen by Membrane Actions in MCF-7 Cells and the Involvement of Protein Kinase C Activation

It is now well established that estrogens (E) have at least two kinds of actions: genomic and nongenomic. But the relationship between these actions has hardly been explored. In this study we investigated this relationship in MCF-7 cells, a human breast cancer cell line, and explored the possible involvement of protein kinase C (PKC) signaling pathways. For this purpose a two-pulse paradigm was used: cells were treated with 17beta-estradiol (E), E conjugated with bovine serum albumin (E-BSA or fE'), or other test agents in the first pulse and with E in the second pulse following a 4-h interval. An E-BSA+E paradigm was used to show that replacement of E with the membrane-impermeable E-BSA in the first pulse could potentiate genomic actions of E, in the second pulse. To investigate involvement of signaling pathways, two PKC activators, phorbol 12,13-diacetate (PDAc) or phorbol 12-myristate 13-acetate (PMA), and inhibitors (chelerythrine chloride and H7-dihydrochloride) were used to replace E or E-BSA in the first pulse. PDAc was as effective as E or E-BSA in potentiating the genomic action of E in the second pulse, while PMA was almost without an effect. Conversely, the potentiating effects of E-BSA and PDAc were blocked by chelerythrine chloride but, interestingly, not by H7. The exact reason underlying these differences is not known. In summary, in MCF-7 cells a membrane action of E can potentiate a later genomic action and involves PKC signaling.

Aldosterone Rapidly Activates Na+/H+ Exchange in M-1 Cortical Collecting Duct Cells via a PKC-MAPK Pathway

Background: In this study, the mechanism of the rapid non-genomic effect of aldosterone on Na⁺/H⁺ exchanger (NHE)-mediated intracellular pH (pH_i) recovery from an acid load in murine M-1 cortical collecting duct cells was assessed. Methods: Spectrofluorescence microscopy and Western blot analysis was carried out and NH₄Cl was used to induce the acid load. Results: Aldosterone (10 nM) induced a rapid (<5 min) concentration-dependent increase in pH_i recovery in M-1 cells, an effect mimicked by its precursor deoxycorticosterone (1 nM). This response was unaffected by the mineralocorticoid receptor (MR) antagonist spironolactone (10 µM) but was significantly reduced by the NHE antagonists 5′-(N-ethyl- N-isopropyl)amiloride (EIPA) (20 µM) and cariporide (1 µM). The PKC inhibitor chelerythrine chloride (1 µM) significantly attenuated the aldosterone-induced increase in NHE1 activity. HBDDE (80 µM), a PKC_α inhibitor, inhibited the rapid aldosterone effect whereas rottlerin (15 µM), a PKC_δ antagonist, did not. The glucocorticoid receptor agonists hydrocortisone (1 µM) and dexamethasone (100 nM) decreased NHE activity, whereas the synthetic mineralocorticoid fludrocortisone (1 nM) had no significant effect. MAPK inhibition using PD98059 (25 µM) significantly attenuated the rapid aldosterone effect; Western blot analysis showed that aldosterone activation of ERK 1/2 was unaffected by pretreatment with spironolactone but was inhibited following chelerythrine chloride. Conclusion: Aldosterone causes a rapid non-genomic increase in NHE1 activity in M-1 cells via a PKC_α/MAPK pathway independent of the classical MR.

Relaxin Stimulates Protein Kinase C ζ Translocation: Requirement for Cyclic Adenosine 3′,5′-Monophosphate Production

Relaxin is a polypeptide hormone that activates the leucine-rich repeat containing G protein-coupled receptors, LGR7 and LGR8. In an earlier study, we reported that relaxin produces a biphasic time course and the second wave of cAMP is highly sensitive to phosphoinositide-3 kinase inhibitors (LY294002 and wortmannin). LY294002 inhibits relaxin-mediated increases in cAMP production by 40-50% across a large range of relaxin concentrations. Here we show that protein kinase C zeta (PKCzeta) is a component of relaxin signaling in THP-1 cells. Sphingomyelinase increases cAMP production due to the release of ceramide, a direct activator of PKCzeta. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin induced cAMP production to the same degree (approximately 40%) as LY294002. Relaxin stimulates PKCzeta translocation to the plasma membrane in THP-1, MCF-7, pregnant human myometrial 1-31, and mouse mesangial cells, as shown by immunocytochemistry. PKCzeta translocation is phosphoinositide-3 kinase dependent and independent of cAMP production. Antisense PKCzeta oligodeoxynucleotides (PKCzeta-ODNs) deplete both PKCzeta transcript and protein levels in THP-1 cells. PKCzeta-ODNs abolish relaxin-mediated PKCzeta translocation and inhibit relaxin stimulation of cAMP by 40%, as compared with mock and random ODN controls. Treatment with LY294002 in the presence of PKCzeta-ODNs results in little further inhibition. In summary, we present a novel role for PKCzeta in relaxin-mediated stimulation of cAMP.

Calcitonin Gene-Related Peptide Receptor Activation Produces PKA- and PKC-Dependent Mechanical Hyperalgesia and Central Sensitization

Calcitonin gene-related peptide (CGRP), acting through CGRP receptors, produces behavioral signs of mechanical hyperalgesia in rats and sensitization of wide dynamic range (WDR) neurons in the spinal cord dorsal horn. Although involvement of CGRP receptors in central sensitization has been confirmed, the second-messenger systems activated by CGRP receptor stimulation and involved in pain transmission are not clear. This study tested whether the hyperalgesia and sensitizing effects of CGRP receptor activation on WDR neurons are mediated by protein kinase A or C (PKA or PKC) signaling. Intrathecal injection of CGRP in rats produced mechanical hyperalgesia, as shown by paw withdrawal threshold tests. CGRP-induced hyperalgesia was attenuated significantly by the CGRP1 receptor antagonist, CGRP8-37. The effect was also attenuated significantly by a PKA inhibitor (H89) or a PKC inhibitor (chelerythrine chloride). Electrophysiological experiments demonstrated that superfusion of the spinal cord with CGRP-induced sensitization of spinal dorsal horn neurons. The CGRP effect could be blocked by CGRP8-37. Either a PKA or PKC inhibitor (H89 or chelerythrine) also attenuated this effect of CGRP. These results are consistent with the hypothesis that CGRP produces hyperalgesia by a direct action on CGRP1 receptors in the spinal cord dorsal horn and suggest that the effects of CGRP are mediated by both PKA and PKC second-messenger pathways.

Regulation of myocardial heat shock protein 70 gene expression following exercise

Post-exercise induction of myocardial heat shock protein (Hsp70) gene expression involves the activation of the heat shock transcription factor (HSF1). While the exact mechanisms governing the regulation of HSF1 are unclear, activation is believed to occur subsequent to hyperphosphorylation of specific serine residues. As two important serine kinases, protein kinase A (PKA) and protein kinase C (PKC), have been implicated in many phosphorylative events in myocardial cells, we examined the role of these kinases in the activation of Hsp70 gene expression following exercise. In this report, we show that prior administration of a PKA inhibitor, N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide 2HCl (H-89; 0.36 mg/kg), significantly suppressed the elevation in Hsp70 mRNA ( P < 0.05) and protein synthesis ( P < 0.05) in male Sprague–Dawley rats following a single bout of exercise. In contrast, this post-exercise elevation in Hsp70 mRNA and protein synthesis was not suppressed following the administration of a PKC inhibitor chelerythrine chloride (CHEL; 5 mg/kg) ( P < 0.05). Of note, inhibition of PKA did not alter the nuclear localization and binding affinity of HSF1 to the promotor region of the Hsp70 gene. These data indicate that PKA, and not PKC, plays a necessary role in the early exercise-induced regulation of Hsp70 gene expression, downstream of DNA-binding acquisition. However, the current study does not support previous observations regarding major changes in HSF1 phosphorylation and suggests that other PKA-related mechanisms mediate the activation of Hsp70 gene expression following exercise.

Effect of protein kinase C blockade on phosphorylation of NR1 in dorsal horn and spinothalamic tract cells caused by intradermal capsaicin injection in rats

We have previously reported that protein kinase A (PKA) is involved in the phosphorylation of NR1 subunits of N-methyl- d-aspartate (NMDA) receptors in dorsal horn neurons after intradermal injection of capsaicin (CAP). To see if protein kinase C (PKC) also participates in the phosphorylation of NR1, we used electron microscopic techniques to determine further where the phosphorylated NR1 subunits (pNR1) are expressed in the spinothalamic tract (STT) cells and immunohistochemistry to examine whether a PKC inhibitor, chelerythrine chloride, blocks the enhanced phosphorylation of NR1 on serine 896. The pNR1 subunits were in the soma and dendrites of STT cells and in presynaptic endings. Western blots showed that pretreatment with the PKC inhibitor caused a decrease in CAP-induced phosphorylation of NR1 protein. In immunofluorescence staining, the number of pNR1-like immunoreactive neurons was significantly decreased on the side ipsilateral to the injection when chelerythrine chloride was administered intrathecally before CAP injection. In addition, when STT cells were labeled by microinjection of the retrograde tracer, fluorogold (FG), into the thalamus, we found that the proportion of p-NR1-LI STT cells was markedly reduced after PKC inhibition. Combined with our previous findings, these results strongly suggest that NR1 subunits in spinal dorsal horn neurons are phosphorylated following CAP injection, and this phosphorylation is catalyzed by PKC, as well as by PKA.

Regulation of tumor necrosis factor-α in glioma cells by lead and lipopolysaccharide: involvement of common signaling pathway

Both lead (Pb) and lipopolysaccharide (LPS) damage nervous system, partly, by the induction of tumor necrosis factor-α (TNF-α) in glia origin. In this study, we examined the Pb- and LPS-triggered signal leading to TNF-α expression in a glioma cell line, U-373MG. Both Pb and LPS increased the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK), which depended on the activation of MAPK kinase (MEK) and protein kinase C (PKC). Selective p42/44 MAPK inhibitor could reduce the Pb- and LPS-triggered TNF-α expression in U-373MG cells. Suppressing PKC by chelerythrine chloride completely diminished the Pb- and LPS-induced TNF-α expression in glial cells in the mouse brain. Thus, our results indicated that PKC-MEK-p42/44 MAPK is a common signaling pathway for Pb- and LPS-induced TNF-α expression in glial cells.

Cytoskeletal restraints regulate homotypic ALCAM-mediated adhesion through PKCalpha independently of Rho-like GTPases.

The activated leukocyte cell adhesion molecule (ALCAM) is dynamically regulated by the actin cytoskeleton. In this study we explored the molecular mechanisms and signaling pathways underlying the cytoskeletal restraints of this homotypic adhesion molecule. We observed that ALCAM-mediated adhesion induced by cytoskeleton-disrupting agents is accompanied by activation of the small GTPases RhoA, Rac1 and Cdc42. Interestingly, unlike adhesion mediated by integrins or cadherins, ALCAM-mediated adhesion appears to be independent of Rho-like GTPase activity. By contrast, we demonstrated that protein kinase C (PKC) plays a major role in ALCAM-mediated adhesion. PKC inhibition by chelerythrine chloride and myristoylated PKC pseudosubstrate, as well as PKC downregulation by PMA strongly reduce cytoskeleton-dependent ALCAM-mediated adhesion. Since serine and threonine residues are dispensable for ALCAM-mediated adhesion and ALCAM is not phosphorylated, we can rule out that ALCAM itself is a direct PKC substrate. We conclude that PKCalpha plays a dominant role in cytoskeleton-dependent avidity modulation of ALCAM.

Protein kinase C inhibition differentially affects 3,4-methylenedioxymethamphetamine-induced dopamine release in the striatum and prefrontal cortex of the rat

The acute administration of 3,4-methylenedioxymethamphetamine (MDMA) elevates extracellular concentrations of dopamine (DA) and serotonin (5-HT) in the rat striatum and medial prefrontal cortex (mPFC). The release of DA induced by MDMA is thought to involve both transporter and impulse-mediated processes. Furthermore, the impulse-dependent release of DA in the striatum elicited by MDMA appears to involve 5-HT 2 receptor activation. Since 5-HT 2 receptors are known to utilize protein kinase C (PKC) for intracellular signaling, we examined the effects of modulators of PKC activity on DA release stimulated by MDMA. Reverse dialysis of the PKC inhibitors bisindolylmaleimide I (BIM; 30 μM) or chelerythrine chloride (100 μM) through a microdialysis probe significantly attenuated the MDMA (10 mg/kg, i.p.)-induced increase in the extracellular concentration of DA in the striatum. In contrast, BIM did not significantly alter the increase in the extracellular concentration of DA in the striatum elicited by amphetamine (5 mg/kg, i.p.). Reverse dialysis of a PKC activator, phorbol 12,13-dibutyrate (PDBu) (0.5 μM), through the microdialysis probe into the striatum, significantly increased MDMA-induced DA release. In contrast to the inhibitory effects of the PKC inhibitors on MDMA-induced DA release in the striatum, intracortical infusion of BIM enhanced MDMA-induced release of DA in the mPFC. These data suggest that PKC-mediated signaling pathways differentially modulate MDMA-induced DA release from mesocorticolimbic and nigrostriatal neurons.

Lactosylceramide recruits PKCalpha/epsilon and phospholipase A2 to stimulate PECAM-1 expression in human monocytes and adhesion to endothelial cells.

Despite the importance of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) in the adhesion and diapedesis of monocytes/lymphocytes, little is known about the mechanisms by which it is regulated. We explored the role of a glycosphingolipid, lactosylceramide (LacCer), in modulating PECAM-1 expression and cell adhesion in human monocytes. We observed that LacCer specifically exerted a time-dependent increase in PECAM-1 expression in U-937 cells. Maximal increase in PECAM-1 protein occurred after incubation with LacCer for 60 min. LacCer activated PKCalpha and -epsilon by translocating them from cytosol to membrane. This was accompanied by the activation of phospholipase A(2) (PLA(2)) and the increase of cell adhesion, which were abrogated by chelerythrine chloride, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide and 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (GO 6976) (PKC inhibitors). Similarly, bromoenol lactone (a Ca(2+)-independent PLA(2) inhibitor) and methyl arachidonyl fluorophosphonate (an inhibitor of cytosolic PLA(2) and Ca(2+)-independent PLA(2)) inhibited LacCer-induced PLA(2) activity. Bromophenacyl bromide (a PLA(2) inhibitor) abrogated LacCer-induced PECAM-1 expression, and this was bypassed by arachidonic acid. Furthermore, the arachidonate-induced up-regulation of PECAM-1 was abrogated by indomethacin [a cyclooxygenase (COX)-1 and -2 inhibitor] or N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (a COX-2 inhibitor) but not nordihydroguaiaretic acid (a lipoxygenase inhibitor). In sum, PKCalpha/epsilon are the primary targets for the activation of LacCer. Downstream activation of intracellular Ca(2+)-independent PLA(2) and/or cytosolic PLA(2) results in the production of arachidonic acid, which in turn serves as a precursor for prostaglandins that subsequently stimulate PECAM-1 expression and cell adhesion. These findings may be relevant in explaining the role of LacCer in the regulation of PECAM-1 and related pathophysiology.

Sublethal simulated ischemia promotes delayed resistance against ischemia via ATP-sensitive (K+) channels in murine myocytes: role of PKC and iNOS.

In this study, we examined whether sublethal simulated ischemia (SSI) induces delayed cellular protection in mouse cardiac myocytes, and whether the delayed cellular protection depends on the activation of protein kinase C-epsilon (PKC-epsilon), inducible nitric oxide synthase (iNOS), and ATP-sensitive K(+) (K(ATP)) channels against subsequent sustained simulated ischemia (SI). The following groups of mouse cardiac myocytes were studied: (a) SI: incubation with SI buffer for 1 h; (b) SSI: incubation with SSI buffer for 30 min; (c) SSI + PKC inhibitor, chelerythrine chloride (CCl): SSI and 1 micro M CCl; (d) SSI + iNOS inhibitor, S-methylthiourea (SMT): SSI and 100 nM SMT; (e) SSI + K(ATP) channel blocker, glibenclamide (Glb): SSI and 50 micro M Glb; (f) SSI + mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate (5-HD): SSI and 50 micro M 5-HD. The release of lactate dehydrogenase into the medium and the amount remaining in the cells was measured, and A(1) adenosine receptor, PKC-epsilon, and iNOS were detected through western blot analysis. The delayed cellular protection acquired due to SSI showed a decreased release of lactate dehydrogenase (%) from 46.51 +/- 1.60 to 37.00 +/- 1.34 (p < 0.001) and was blocked by CCl (47.08 +/- 0.95), SMT (48.08 +/- 1.18), Glb (45.88 +/- 1.31), and 5-HD (47.20 +/- 1.56). Simultaneously, SSI-induced up-regulation of A(1) adenosine receptor, PKC-epsilon, iNOS, and opening of both membrane and mitochondrial K(ATP) channels also was observed compared with controls.

Central sensitization: CGRP receptor activation produces PKA and PKC dependent mechanical hyperalgesia and central sensitization

Calcitonin gene-related peptide (CGRP), acting through CGRP1 receptors, produced behavioral signs of hyperalgesia in rats and sensitization of wide dynamic range (WDR) neuron in the spinal cord dorsal horn (Sun, et al., 2003). Although, involvement of CGRP receptors in central sensitization has been confirmed, the second messenger systems activated by CGRP receptor stimulation and that are involved in pain transmission are not clear. This study tested whether the hyperalgesic and wide dynamic range (WDR) neuron sensitizing effects of CGRP receptor activation is medicated by PKA or PKC signaling. Intrathecal injection of CGRP in rats produced mechanical hyperalgesia, as shown by paw withdrawal threshold tests. CGRP-induced hyperalgesia was attenuated significantly by the CGRP1 receptor antagonist, CGRP8-37. The effect was attenuated significantly by a protein kinase A (PKA) inhibitor (H89) or a protein kinase C (PKC) inhibitor (chelerythrine chloride). Electrophysiological experiments demonstrated that superfusion of the spinal cord with CGRP induced the sensitization of spinal dorsal horn neurons. The effect could be blocked by CGRP8-37. Either a PKA or a PKC inhibitor (H89 or chelerythrine) also attenuated this effect of CGRP. These results are consistent with the hypothesis that CGRP produces hyperalgesia by a direct action on CGRP receptors in the spinal cord dorsal horn and that this effect of CGRP is dependent on PKA and PKC signaling. In conclusion, CGRP produces mechanical hyperalgesia and sensitizes WDR neurons in dorsal horn in the absence of nerve injury via an action at CGRP1 receptors. These effects of CGRP are mediated by both PKA and PKC second-messenger pathways. Supported by NIH grants NS09743 and NS11255.

Epidermal Growth Factor Induces Fibroblast Contractility and Motility via a Protein Kinase C δ-dependent Pathway

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCdelta to promote the acute phase of EGF-induced MLC activation.

Restoration of resting sarcomere length after uniaxial static strain is regulated by protein kinase Cepsilon and focal adhesion kinase.

Physiological or pathological stresses and strains produce longer or wider muscle cells, but resting sarcomere length remains constant. Our goal was to investigate the cellular mechanisms for controlling this optimal, resting sarcomere length. To do so, we cultured neonatal rat cardiomyocytes on microfabricated peg-and-groove, laminin-coated silicone surfaces and applied a uniaxial static strain of 10%. Sarcomere length was accurately measured by fast Fourier transform analysis of images before, within 5 minutes of, and 4 to 6 hours after imposition of the strain. Sarcomere length of aligned cardiomyocytes (1.94+/-0.07 microm) was lengthened acutely (2.06+/-0.06 microm), and recovered (1.95+/-0.07 microm) by 4 hours. Puromycin, an mRNA translational inhibitor, prevented recovery of resting sarcomere length by 4 hours, thus indicating a requirement for new protein synthesis in the recovery process. Furthermore, activation of protein kinase Cepsilon (PKCepsilon) was necessary for length recovery, as nonselective PKC inhibitors [staurosporine (5 micromol/L) and chelerythrine chloride (10 micromol/L)], and a replication-defective adenovirus (Adv) encoding a dominant-negative mutant of PKCepsilon prevented the restoration of sarcomere length. To assess the importance of focal adhesion complexes, cardiomyocytes were infected with an Adv encoding a dominant-negative inhibitor of focal adhesion kinase (FAK) (Adv-GFP-FRNK). Adv-GFP-FRNK also prevented resting sarcomere length recovery, whereas a control Adv encoding only GFP did not. In conclusion, using our novel culture system, we provide evidence indicating that the length remodeling process requires new protein synthesis, PKCepsilon and FAK.

PKC inhibitor reduces airway inflammation and hyperresponsiveness by reducing MMP-9 expression

Rationale Matrix metalloproteinase-9 (MMP-9) is one of the major proteolytic enzyme that induces airway remodeling in asthma. MMP-9 also induce inflammatory cell accumulation, extracellular matrix deposition, airway smooth muscle hyperplasia/hypertrophy and airway fibrosis. We studied first the production of MMP-9 in obalbumin (OVA)-induced asthma model mice and in A549 cells treated with phorbol 12-myristate 13-acetate, and examined the effect of protein kinase C (PKC) inhibitors, calphostin C and chelerythrine chloride on MMP-9 expression in murine model of asthma and in A549 cell line. Methods Expression and activity of MMP-9 was identified by gelatin zymography, western blotting, reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analysis. Results We observed significant decreases in the accumulation of inflammatory cells and the expression of MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in administration of PKC inhibitor, calphostin C and chelerythrine chloride. Conclusions In this study, we suggest that PKC inhibitors inhibit accumulation of inflammatory cell through down -regulation of MMP-9.

Activation of VPAC1 receptors by VIP and PACAP-27 in human bronchial epithelial cells induces CFTR-dependent chloride secretion.

1. In the human airway epithelium, VIP/PACAP receptors are distributed in nerve fibers and in epithelial cells but their role in transepithelial ion transport have not been reported. Here, we show that human bronchial epithelial Calu-3 cells expressed the VPAC(1) receptor subtype which shares similar high affinity for VIP and PACAP-27. 2. The stoichiometric binding parameters characterizing the (125)I-VIP and (125)I-PACAP-27 binding to these receptors were determined. 3. We found that VIP (EC(50) approximately 7.6 nM) and PACAP-27 (EC(50) approximately 10 nM) stimulated glibenclamide-sensitive and DIDS-insensitive iodide efflux in Calu-3 cells. 4. The protein kinase A (PKA) inhibitor, H-89 and the protein kinase C (PKC) inhibitor, chelerythrine chloride prevented activation by both peptides demonstrating that PKA and PKC are part of the signaling pathway. This profile corresponds to the pharmacological signature of CFTR. 5. In the cystic fibrosis airway epithelial IB3-1 cell lacking functional CFTR but expressing VPAC(1) receptors, neither VIP, PACAP-27 nor forskolin stimulated chloride transport. 6. Ussing chamber experiments demonstrated stimulation of CFTR-dependent short-circuit currents by VIP or PACAP-27 applied to the basolateral but not to the apical side of Calu-3 cells monolayers. 7. This study shows the stimulation in human bronchial epithelial cells of CFTR-dependent chloride secretion following activation by VIP and PACAP-27 of basolateral VPAC(1) receptors.

Evidence for multiple P2Y receptors in trabecular meshwork cells.

The purpose of this study was to determine whether functional purinergic P2 receptors are present in trabecular meshwork cells. The human trabecular cell line HTM-3 and cultured bovine trabecular cells were used to assess the effects of P2 agonists on intracellular Ca(2+) levels, extracellular signal-regulated kinase (ERK1/2) activation, and P2Y receptor expression. ATP, UTP, ADP, and 2-methyl-thio-adenosine triphosphate (2-MeS-ATP) each produced a concentration-dependent increase in intracellular Ca(2+) in bovine trabecular cells and the HTM-3 cell line. The addition of UDP did not produce any detectable rise in intracellular Ca(2+). Pretreatment with the P2Y(1) receptor antagonist 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS-2179) blocked the ADP- and 2-MeS-ATP-induced rise in intracellular Ca(2+). However, the ATP- or UTP-induced rise in intracellular Ca(2+) was not inhibited by MRS-2179 pretreatment. The addition of ADP, 2-MeS-ATP, ATP, or UTP were also found to activate the ERK1/2 signaling pathway. This activation of ERK1/2 was blocked by pretreatment with the mitogen-activated protein kinase kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U-0126) or the protein kinase C inhibitor chelerythrine chloride, but not by MRS-2179. Analysis of mRNA from HTM-3 cells by reverse transcription-polymerase chain reaction revealed the expression of P2Y(1), P2Y(4), and P2Y(11) receptor subtypes. These data demonstrate that multiple P2Y receptors are present in trabecular cells. Our results are consistent with the idea that the mobilization of intracellular Ca(2+)results from the activation of P2Y(1) and P2Y(4) receptors, whereas the activation of the ERK1/2 pathway results from the activation of P2Y(4) receptors alone. However, a role for the P2Y(11) receptors in mobilization of Ca(2+), or activation of the ERK1/2 pathway, cannot be discounted.

Implications of protein kinase C in the nucleus accumbens in the development of sensitization to methamphetamine in rats

Repeated treatment with methamphetamine leads to an enhancement in the methamphetamine-induced dopamine release and its related behaviors. This phenomenon is called sensitization or reverse tolerance. Protein kinase C (PKC) controls numerous signaling cascades by virtue of its ability to phosphorylate target proteins that include other kinases. The purpose of study was then to investigate the implication of PKC in the development of sensitization to the rewarding effect and to the extracellular dopamine release induced by methamphetamine in rats. The conditioned place preference paradigm and in vivo microdialysis assay were performed in the present study. An intra-nucleus accumbens injection of a selective PKC inhibitor chelerythrine chloride abolished the enhancement of the methamphetamine-induced place preference following repeated treatment with methamphetamine. Furthermore, intra-nucleus accumbens injection of chelerythrine chloride blocked the development of sensitization to dopamine release and to the decrease in the major dopamine metabolites, 3′4-dihydroxyphenylacetic acid and homovanillic acid, in the nucleus accumbens induced by repeated methamphetamine treatment. Under these conditions, the immunoreactivity of the cytosolic phosphorylated conventional- or classic-type PKC in the limbic forebrain region including the nucleus accumbens was slightly, but significantly increased in methamphetamine-sensitized rats. The present data provide evidence for the implication of PKC in the nucleus accumbens in the development of sensitization to the methamphetamine-induced rewarding effect, dopamine release and inhibition of dopamine metabolism/re-uptake in rats.

Insulin-like growth factor-2 activation of intestinal glutamine transport is mediated by mitogen-activated protein kinases.

Insulin-like growth factor-2 (IGF-2) plays a pivotal role in regulating intestinal epithelial metabolism, growth, and proliferation, but its regulatory effects on mucosal cell amino acid transport have not been well studied. The purpose of this in vitro study was to investigate the regulatory mechanisms and intracellular signaling pathways involved in the regulation of IGF-2 on glutamine transport in cultured intestinal cells. Continuous incubation with IGF-2 stimulated glutamine transport activity in cultured IEC-6 cells in a dose- and time-dependent fashion. Prolonged incubation (up to 48 hours) resulted in a 50% increase in transport activity (0.81+/-0.21 nmole/mg protein/min in IGF-2 cells vs. 0.57+/-0.15 nmole/mg protein/min in control cells) and a threefold increase in glutamine transporter ATB(0) mRNA levels. IGF-2 stimulated transport activity by increasing transport maximal capacity (V(max) 4.31+/-0.36 nmole/mg protein/min in IGF-2 cells vs. 2.51+/-0.23 nmole/mg protein/min in control cells) without affecting the transport affinity (K(m) 0.31+/-0.03 mmol/L glutamine in IGF-2 cells vs. 0.28+/-0.03 mmol/L glutamine in control cells). This IGF-2-induced glutamine transport activity was attenuated by actinomycin-D or cycloheximide. The levels of mitogen-activated protein kinases p42/44, MEK1/2, and p38 as well as protein kinase C levels were elevated in IGF-2-treated cells and inhibitors of mitogen-activated protein kinase MEK1 (PD 98059), mitogen-activated protein kinase p38, and protein kinase C (chelerythrine chloride) individually attenuated the IGF-2-induced glutamine transport. These data suggest that IGF-2 stimulates intestinal glutamine uptake in cultured rat intestinal epithelial cells via a mechanism that involves transcription and translation of the transporter. Activation of mitogen-activated protein kinases and protein kinase C cascades are involved in the regulation. This increase in glutamine uptake may occur to support intestinal cell growth and proliferation.

N-Methyl- d-aspartate receptor and L-type voltage-gated Ca 2+ channel activation mediate proline-rich tyrosine kinase 2 phosphorylation during cerebral ischemia in rats

Cerebral ischemia induces rapid efflux of glutamate into the extracellular space contributing to excessive activation of glutamate receptors in postsynaptic cells, particularly N-methyl- d-aspartate (NMDA) receptors, which triggers the neuron lesion through calcium overload. Our studies indicated that cerebral ischemia stimulated the rapid activation of nonreceptor tyrosine kinases proline-rich tyrosine kinase 2 (Pyk2) and Src and the binding to Pyk2 activated the latter. Pyk2 activation significantly depends on the increase of the intracellular calcium level; blockage of both calcium ion channel NMDA receptors and L-type voltage-gated Ca 2+ channel (L-VGCC), respectively, could effectively inhibit phosphorylation of Pyk2 in early ischemia episodes. Moreover, pretreatment with the protein kinase C inhibitor (chelerythrine chloride) reduced the ischemia-induced activation of Pyk2. Noticeably, CaMKII, a family of calcium/calmodulin-dependent kinases, also may be involved in the regulation of Pyk2 activity because its inhibitor KN62 attenuated Pyk2 phosphorylation during ischemia. Together with previous studies, these results indicate that calcium influx elicited by active NMDA receptors and L-VGCC triggers the Pyk2-Src signaling pathway mediated by PKC, which aggravates cerebral ischemia lesions through up-regulating the function of NMDA receptors after the onset of ischemia, and also could be regulated partly by CaM-dependent kinases like CaMKII.