GF 109203X Research Articles

Protein kinase Cα mediates feedback inhibition of EGF receptor transactivation induced by Gq-coupled receptor agonists

While a great deal of attention has been focused on G-protein-coupled receptor (GPCR)-induced epidermal growth factor receptor (EGFR) transactivation, it has been known for many years that the tyrosine kinase activity of the EGFR is inhibited in cells treated with tumor-promoting phorbol esters, a process termed EGFR transmodulation. Because many GPCR agonists that elicit EGFR transactivation also stimulate the Gq/phospholipase C (PLC)/protein kinase C (PKC) pathway, we hypothesized that PKC-mediated inhibition of EGFR transactivation operates physiologically as a feedback loop that regulates the intensity and/or duration of GPCR-elicited EGFR transactivation. In support of this hypothesis, we found that treatment of intestinal epithelial IEC-18 cells with the PKC inhibitors GF 109203X or Ro 31-8220 or chronic exposure of these cells to phorbol-12,13-dibutyrate (PDB) to downregulate PKCs, markedly enhanced the increase in EGFR tyrosine phosphorylation induced by angiotensin II or vasopressin in these cells. Similarly, PKC inhibition enhanced EGFR transactivation in human colonic epithelial T84 cells stimulated with carbachol, as well as in bombesin-stimulated Rat-1 fibroblasts stably transfected with the bombesin receptor. Furthermore, cell treatment with inhibitors with greater specificity towards PKCα, including Gö6976, Ro 31-7549 or Ro 32-0432, also increased GPCR-induced EGFR transactivation in IEC-18, T84 and Rat-1 cells. Transfection of siRNAs targeting PKCα also enhanced bombesin-induced EGFR tyrosine phosphorylation in Rat-1 cells. Thus, multiple lines of evidence support the hypothesis that conventional PKC isoforms, especially PKCα, mediate feedback inhibition of GPCR-induced EGFR transactivation.

Calcium sensing receptor (CaR)‐mediated TNF production in the medullary thick ascending limb (mTAL) is G i ‐coupled

We tested the contribution of CaR-mediated Gi-coupled signaling to TNF production in mTAL cells. CaR activation induced a 3-fold increase in TNF production at 6 hr that was abolished by a selective Gi inhibitor, pertussis toxin (PTX; 100 ng/ml; n=3; p<0.001). CaR increased calcineurin (CN) activity by approximately 3-fold (n=4; p<0.01). PTX prevented CaR-mediated increases in CN activity (n=4; p<0.01), an NFAT cis-reporter construct (n=5; p<0.001), and a TNF promoter construct (n=3; p<0.01). The interaction between Gi and PKC was determined, as we previously showed that CaR-mediated TNF production was CN- and NFAT-mediated and Gq-dependent. CaR activation increased PKC activity by 2-fold, an effect abolished by transient transfection with a dominant negative CaR construct, R796W (n=4; p<0.001) or pretreatment with PTX (n=4; p<0.001). Inhibition with the pan specific PKC inhibitor, GF 109203X (20 nM) abolished CaR-mediated increases in activity of CN, an NFAT reporter (n=3; p<0.001), and a TNF promoter construct (n=3; p<0.05). PTX reversed CaR-mediated decreases in ouabain-sensitive O2 consumption, suggesting that a Gi-coupled mechanism is important in regulating mTAL transport function. Collectively, the data suggest that Gi-coupled signaling contributes to NFAT-mediated TNF production, in a CN- and PKC-dependent manner, and is part of a CaR mechanism to regulate mTAL function.

Role of PKC‐mediated calcium‐sensitization in attenuated carbachol‐induced contraction of the rat colon in TNBS‐induced colitis

Contractile agonists increase cytosolic concentration of Ca2+ by promoting influx of extracellular Ca2+ and by releasing Ca2+from intracellular stores. However, the degree of contraction and the level of MLC phosphorylation are not always proportional to Ca2+ concentration suggesting that an additional mechanism, Ca2+-sensitization, contributes to smooth muscle contraction. The objective of this study was to investigate the role of this pathway in the reduced reactivity of inflamed colonic segments to carbachol. Myeloperoxidase activity was greater in inflamed colon segments confirming inflammation in these animals. Carbachol induced concentration-dependent contractions of the colon. The maximum contraction but not sensitivity was attenuated in inflamed colon segments. CaCl2-induced contraction of permeabilized colon strips was significantly greater (P < 0.05) in inflamed tissues compared to controls, indicating greater sensitization of the contractile myofilaments to Ca2+ in colon segments from colitic rats. Carbachol-induced contractions were however greater in colon segments from control rats. Western blot analyses showed that PKC was expressed in the rat colon but the expression was significantly reduced in inflamed colon segments. GF 109203X, a selective PKC inhibitor, concentration-dependently inhibited carbachol-induced contractions of the colon in both groups. However, the effect of GF was significantly less in colon segments from TNBS-treated rats. These results would confirm a role for Ca2+ sensitization involving PKC in carbachol-induced contraction of the rat colon and that the reduced carbachol-induced contraction observed in colitis could be linked to defective PKC-mediated Ca2+ sensitization.

Rottlerin inhibits P2X7 receptor‐stimulated phospholipase D activity in chronic lymphocytic leukaemia B‐lymphocytes

Phospholipase D (PLD) is a ubiquitous enzyme that can be activated by extracellular adenosine 5'-triphosphate (ATP) or phorbol 12-myristate 13-acetate (PMA) in B-lymphocytes from subjects with chronic lymphocytic leukaemia (CLL). In this study, ATP- but not PMA-induced PLD stimulation in CLL B-lymphocytes was abolished in the presence of an anti-P2X(7) receptor monoclonal antibody, as well as in B-lymphocytes from CLL subjects homozygous for the Glu(496) to Ala loss-of-function P2X(7) polymorphism. Rottlerin, an inhibitor of novel protein kinase C (PKC) isoforms, but not GF 109203X, an inhibitor of conventional PKC isoforms, impaired the ATP-stimulated PLD activity in CLL B-lymphocytes. In contrast, both inhibitors impaired PLD activity stimulated by PMA, a known mediator of PKC activation. The inhibition of P2X(7)-stimulated PLD activity by rottlerin was attributed to a target downstream of P2X(7) activation, as the ATP-mediated (86)Rb(+) efflux from CLL B-lymphocytes was not altered in the presence of rottlerin. Our results indicate a possible role for novel PKC isoforms in the regulation of P2X(7)-mediated PLD activity.

Intracellular messengers involved in spontaneous pain, heat hyperalgesia, and mechanical allodynia induced by intrathecal dihydroxyphenylglycine

We investigated the role of two intracellular second messengers, extracellular signal-regulated protein kinase (ERK) and protein kinase C (PKC), in a model of persistent pain using intrathecal (i.t.) (R,S)-3,5-dihydroxyphenylglycine (DHPG). Spontaneous nociceptive behaviours (SNBs), mechanical allodynia (von Frey thresholds) and heat hyperalgesia (plantar test latencies) induced by DHPG were measured in animals pretreated i.t. with membrane permeable inhibitors of ERK (PD 98059) and PKC (GF 109203X). Spinal administration of PD 98059 dose-dependently reduced SNBs, and attenuated both mechanical allodynia and heat hyperalgesia induced by DHPG. GF 109203X treatment also reduced SNBs and heat hyperalgesia, but did not affect mechanical allodynia induced by DHPG. Neither PD 98059, nor GF 109203X, altered mechanical or thermal thresholds in saline-injected control rats. These results suggest that both ERK and PKC are involved in persistent pain associated with the i.t. administration of DHPG.

Phospholipase A 2-independent Ca 2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells

Melittin, a peptide from bee venom, is thought to be a phospholipase A 2 activator and Ca 2+ influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca 2+ concentration ([Ca 2+] i) and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca 2+] i and killed cells in MG63 human osteosarcoma cells. [Ca 2+] i changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 μM increased [Ca 2+] i in a concentration-dependent manner. The Ca 2+ signal was abolished by removing extracellular Ca 2+. Melittin-induced Ca 2+ entry was confirmed by Mn 2+ quenching of fura-2 fluorescence at 360 nm excitation wavelength which was Ca 2+-insensitive. The melittin-induced Ca 2+ influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PMA) and GF 109203X, or inhibition of phospholipase A 2 with AACOCF 3 and aristolochic acid; but was substantially inhibited by blocking L-type Ca 2+ channels. At concentrations of 0.5 μM and 1 μM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 μM melittin was completely reversed by pre-chelating cytosolic Ca 2+ with BAPTA. Taken together, these data showed that in MG63 cells, melittin induced a [Ca 2+] i increase by causing Ca 2+ entry through L-type Ca 2+ channels in a manner independent of protein kinase-C and phospholipase A 2 activity; and this [Ca 2+] i increase subsequently caused apoptosis.

Calcium channel and glutamate receptor activities regulate actin organization in salamander retinal neurons

Intracellular Ca2+ regulates a variety of neuronal functions, including neurotransmitter release, protein phosphorylation, gene expression and synaptic plasticity. In a variety of cell types, including neurons, Ca2+ is involved in actin reorganization, resulting in either actin polymerization or depolymerization. Very little, however, is known about the relationship between Ca2+ and the actin cytoskeleton organization in retinal neurons. We studied the effect of high-K+-induced depolarization on F-actin organization in salamander retina and found that Ca2+ influx through voltage-gated L-type channels causes F-actin disruption, as assessed by 53 +/- 5% (n = 23, P < 0.001) reduction in the intensity of staining with Alexa-Fluor488-phalloidin, a compound that permits visualization and quantification of polymerized actin. Calcium-induced F-actin depolymerization was attenuated in the presence of protein kinase C antagonists, chelerythrine or bis-indolylmaleimide hydrochloride (GF 109203X). In addition, phorbol 12-myristate 13-acetate (PMA), but not 4alpha-PMA, mimicked the effect of Ca2+ influx on F-actin. Activation of ionotropic AMPA and NMDA glutamate receptors also caused a reduction in F-actin. No effect on F-actin was exerted by caffeine or thapsigargin, agents that stimulate Ca2+ release from internal stores. In whole-cell recording from a slice preparation, light-evoked 'off' but not 'on' EPSCs in 'on-off' ganglion cells were reduced by 60 +/- 8% (n = 8, P < 0.01) by cytochalasin D. These data suggest that elevation of intracellular Ca2+ during excitatory synaptic activity initiates a cascade for activity-dependent actin remodelling, which in turn may serve as a feedback mechanism to attenuate excitotoxic Ca2+ accumulation induced by synaptic depolarization.

PKC and cAMP positively modulate alkaline‐induced exocytosis in the human mast cell line HMC‐1

We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3',5'-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used Gö6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.

Protein Kinase D Intracellular Localization and Activity Control Kinase D-interacting Substrate of 220-kDa Traffic through a Postsynaptic Density-95/Discs Large/Zonula Occludens-1-binding Motif

Protein kinase D (PKD) controls protein traffic from the trans-Golgi network (TGN) to the plasma membrane of epithelial cells in an isoform-specific manner. However, whether the different PKD isoforms could be selectively regulating the traffic of their specific substrates remains unexplored. We identified the C terminus of the different PKDs that constitutes a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif in PKD1 and PKD2, but not in PKD3, to be responsible for the differential control of kinase D-interacting substrate of 220-kDa (Kidins220) surface localization, a neural membrane protein identified as the first substrate of PKD1. A kinase-inactive mutant of PKD3 is only able to alter the localization of Kidins220 at the plasma membrane when its C terminus has been substituted by the PDZ-binding motif of PKD1 or PKD2. This isoform-specific regulation of Kidins220 transport might not be due to differences among kinase activity or substrate selectivity of the PKD isoenzymes but more to the adaptors bound to their unique C terminus. Furthermore, by mutating the autophosphorylation site Ser(916), located at the critical position -2 of the PDZ-binding domain within PKD1, or by phorbol ester stimulation, we demonstrate that the phosphorylation of this residue is crucial for Kidins220-regulated transport. We also discovered that Ser(916) trans-phosphorylation takes place among PKD1 molecules. Finally, we demonstrate that PKD1 association to intracellular membranes is critical to control Kidins220 traffic. Our findings reveal the molecular mechanism by which PKD localization and activity control the traffic of Kidins220, most likely by modulating the recruitment of PDZ proteins in an isoform-specific and phosphorylation-dependent manner.

Up-regulation of the Angiotensin II Type 1 Receptor by the MAS Proto-oncogene Is Due to Constitutive Activation of Gq/G11 by MAS

Coexpression of the MAS proto-oncogene with the angiotensin II type 1 (AT(1)) receptor in CHO-K1 cells has been reported to increase the number of [(3)H]angiotensin II-binding sites, although MAS does not bind [(3)H]angiotensin II. In HEK293 cells stably expressing AT(1) receptor-cyan fluorescent protein (CFP), MAS-yellow fluorescent protein (YFP) expression from an inducible locus caused strong up-regulation of AT(1) receptor-CFP amounts and [(3)H]angiotensin II binding levels. The time course of AT(1) receptor-CFP up-regulation was also markedly slower than that of induction of MAS expression. These effects were not mimicked by induced expression of I138D MAS-YFP, a mutant unable to cause constitutive loading of [(35)S]guanosine 5'-O-(thiotriphosphate) onto the phospholipase Cbeta-linked G protein Galpha(11). Protein kinase C (PKC) inhibitors and the selective Galpha(q)/Galpha(11) inhibitor YM-254890 fully blocked MAS-induced up-regulation of AT(1) receptor-CFP amounts, whereas the PKC activator phorbol 12-myristate 13-acetate produced strong up-regulation of AT(1) receptor-CFP without induction of MAS-YFP expression and in the presence of I138D MAS-YFP. The C-terminal tail of the AT(1) receptor is a known target for PKC-mediated phosphorylation. In cells stably expressing a C-terminally truncated version of the AT receptor, induction of MAS expression did not up-regulate the truncated construct levels. These data demonstrate that the ability of MAS to up-regulate AT(1) receptor levels reflects the constitutive capacity of MAS to activate Galpha(q)/Galpha(11) and hence stimulate PKC-dependent phosphorylation of the AT(1) receptor.

Involvement of Rho kinase and protein kinase C in carbachol‐induced calcium sensitization in β‐escin skinned rat and guinea‐pig bladders

1. The signal transduction pathways involved in carbachol (CCh)-induced calcium sensitization in beta-escin permeabilized rat and guinea-pig bladder smooth muscles were investigated and the results were compared with guinea-pig taenia caecum. 2. Calcium contractions elicited cumulatively (pCa 7.5-5) in the presence of calmodulin were significantly increased in all three tissues when CCh (50 microM) was added to the medium. 3. Under constant [Ca2+]i conditions (pCa 6), calmodulin (1 microM) and then GTP (100 microM) initiated significant contractions. CCh (50 microM) added to the bath caused a further contraction in all three tissues - calcium sensitization. This sensitization was significantly inhibited by atropine (50 microM). 4. The incubation of the tissues with the IP3-receptor blocker 2-APB (30 microM) reduced the subsequent development of calcium sensitization by CCh in rat bladder but did not affect it in guinea-pig bladder and taenia ceacum. 5. The Rho kinase (ROK) inhibitor Y-27632 (5 microM) added in the presence of CCh reversed the calcium sensitization in rat bladder, whereas a transient contraction followed by a relaxation to a level not significantly different from the CCh contraction was seen in both guinea-pig bladder and taenia caecum. Y-27632 (1 microM) continuously present significantly inhibited the CCh-induced Ca2+ sensitization in rat bladder but not in guinea-pig bladder or taenia caecum. 6. In the presence of cyclopiazonic acid (CPA) (1 microM) and calmodulin (1 microM), Y-27632 (5 microM) did not change the calcium response curve (3 x 10(-7)-10(-5) M) in rat bladder but increased the contractile responses significantly in both guinea-pig bladder and taenia caecum. 7. The protein kinase C (PKC) inhibitor GF 109203X (5 microM) added in the presence of CCh inhibited the calcium sensitization induced by this muscarinic agonist in all three tissues in different ratios. 8. In conclusion, muscarinic receptor activation induces calcium sensitization in rat and guinea-pig detrusor smooth muscles but there are differences in their pathways.

The metabotropic glutamate receptor 4 is internalized and desensitized upon protein kinase C activation

1. The metabotropic glutamate receptor 4 (mGluR4) is a Galphai-coupled receptor that modulates glutamatergic neurotransmission. As mGluR4 expression and activation have been implicated in a number of pathological conditions and because the internalization and desensitization properties of this receptor are poorly understood, studies were designed to investigate these aspects of mGluR4 biology. 2. Neither agonist activation by L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) nor L-glutamate caused mGluR4 internalization when cmyc-tagged mGluR4 was expressed in a human embryonic kidney 293 cell line as assessed by cell surface enzyme-linked immunosorbent and immunostaining assays. Instead, a modest increase in mGluR4 surface expression was observed and found to be receptor specific as the competitive antagonist alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) blocked this effect. 3. In contrast, mGluR4 internalized when the protein kinase C (PKC) pathway was activated either by phorbol-12-myristate-13-acetate (PMA) or by the activation of the Galphaq-coupled, neurokinin 3 receptor (NK3R) when co-expressed. This process was PKC-dependent as the specific PKC inhibitor GF 109203X inhibited PMA and NK3R-mediated internalization. 4. PKC activation by PMA caused desensitization of mGluR4 as measured by forskolin-stimulated cAMP inhibition, whereas agonist activation had no effect on desensitization. 5. When mGluR4's coupling was redirected from adenylyl cyclase to phospholipase C by coexpression of a chimeric Galphaqo5 protein, mGluR4 both internalized and desensitized in response to its agonists. 6. These findings demonstrate that mGluR4 internalization and desensitization are agonist-independent unless pathways leading to the activation of PKC are induced.

Adenosine A1 receptor-mediated inhibition of myocardial norepinephrine release involves neither phospholipase C nor protein kinase C but does involve adenylyl cyclase

Stimulation of adenosine A1 receptors in the heart exerts cardioprotective effects by inhibiting norepinephrine (NE) release from sympathetic nerve endings. The intraneuronal signal transduction triggered by presynaptic adenosine A1 receptors is still not completely understood. The objective of the present study was to determine whether phospholipase C (PLC), protein kinase C (PKC), and adenylyl cyclase (AC) are involved in the adenosine A1 receptor-mediated inhibition of endogenous (stimulation-induced) NE release in isolated Langendorff-perfused rat hearts as an approach to elucidate their role in the cardiovascular system. Activation of adenosine A1-receptors with 2-chloro-N6-cyclopentyladenosine (CCPA) decreased cardiac NE release by approximately 40%. Inhibition of PLC with 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U 73122) as well as inhibition of PKC with 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide (GF 109203X) slightly but significantly decreased NE release; however, the suppressive effect of CCPA on NE release was not modulated by U 73122 or GF 109203X. Blockade of AC with 9-(tetrahydro-2'-furyl)adenine (SQ 22536) reversed the inhibitory effect of CCPA on sympathetic neurotransmitter release irrespective of whether PKC was pharmacologically activated by phorbol 12-myristate 13-acetate or was not activated, indicating a PKC-independent but AC-dependent mechanism. Direct stimulation of AC with forskolin increased NE release by approximately 20%; an effect that was antagonized by either CCPA or SQ 22536. These data suggest that the adenosine A1 receptor-mediated inhibition of NE release does not involve PLC or PKC but does involve AC.

Enhancement of arachidonic acid signaling pathway by nicotinic acid receptor HM74A

HM74A is a G protein-coupled receptor for nicotinic acid (niacin), which has been used clinically to treat dyslipidemia for decades. The molecular mechanisms whereby niacin exerts its pleiotropic effects on lipid metabolism remain largely unknown. In addition, the most common side effect in niacin therapy is skin flushing that is caused by prostaglandin release, suggesting that the phospholipase A 2 (PLA 2)/arachidonic acid (AA) pathway is involved. Various eicosanoids have been shown to activate peroxisome-proliferator activated receptors (PPAR) that play a diverse array of roles in lipid metabolism. To further elucidate the potential roles of HM74A in mediating the therapeutic effects and/or side effects of niacin, we sought to explore the signaling events upon HM74A activation. Here we demonstrated that HM74A synergistically enhanced UTP- and bradykinin-mediated AA release in a pertussis toxin-sensitive manner in A431 cells. Activation of HM74A also led to Ca 2+-mobilization and enhanced bradykinin-promoted Ca 2+-mobilization through Gi protein. While HM74A increased ERK1/2 activation by the bradykinin receptor, it had no effects on UTP-promoted ERK1/2 activation.Furthermore, UTP- and bradykinin-mediated AA release was significantly decreased in the presence of both MAPK kinase inhibitor PD 098059 and PKC inhibitor GF 109203X. However, the synergistic effects of HM74A were not dramatically affected by co-treatment with both inhibitors, indicating the cross-talk occurred at the receptor level. Finally, stimulation of A431 cells transiently transfected with PPRE-luciferase with AA significantly induced luciferase activity, mimicking the effects of PPARγ agonist rosiglitazone, suggesting that alteration of AA signaling pathway can regulate gene expression via endogenous PPARs.

Distinct β-Arrestin- and G Protein-dependent Pathways for Parathyroid Hormone Receptor-stimulated ERK1/2 Activation

Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.

Effect of Anandamide on Cytosolic Ca2+ Levels and Proliferation in Canine Renal Tubular Cells

The effect of the endogenous cannabinoid anandamide on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and proliferation is largely unknown. This study examined whether anandamide altered Ca(2+) levels and caused Ca(2+)-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca(2+)](i) and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Anandamide at concentrations above 5 muM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 78% by removing extracellular Ca(2+). The anandamide-induced Ca(2+) influx was insensitive to L-type Ca(2+) channel blockers and the cannabinoid receptor antagonist AM 251, but was inhibited differently by aristolochic acid, WIN 55,212-2 (a cannabinoid receptor agonist), phorbol ester, GF 109203X and forskolin. After pretreatment with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), anandamide-induced Ca(2+) release was inhibited. Inhibition of phospholipase C with U73122 did not change anandamide-induced Ca(2+) release. At concentrations of 100 muM and 200 muM, anandamide killed 50% and 95% cells, respectively. The cytotoxic effect of 100 muM anandamide was completely reversed by pre-chelating cytosolic Ca(2+) with BAPTA. Collectively, in MDCK cells, anandamide induced [Ca(2+)](i) rises by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, anandamide can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.

An essential role of ERK signalling in TPA-induced reactivation of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated causally in the development of several human malignancies, including primary effusion lymphoma (PEL). PEL cells serve as tools for KSHV research, as most of them are latently infected and allow lytic virus replication in response to various stimuli. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) is the most potent inducer of lytic KSHV reactivation; nevertheless, the exact mechanism by which it induces reactivation remains unknown. It has previously been reported by our group that the protein kinase C (PKC) delta isoform plays a crucial role in TPA-mediated KSHV reactivation. Here, the activation pathway was dissected and it was demonstrated that TPA induces KSHV reactivation via stimulation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway. Western blot analysis revealed a rapid phosphorylation of ERK1/2. Cells treated with MAPK/ERK inhibitors before TPA addition demonstrated repression of ERK1/2 phosphorylation, which was associated with a block of KSHV lytic-gene expression. This inhibition prevented c-Fos accumulation, yet increased c-Jun phosphorylation. Similar results were obtained in response to rottlerin, a selective PKCdelta inhibitor. Notably, the PKC inhibitor GF 109203X reduced ERK1/2 phosphorylation, c-Fos accumulation, c-Jun phosphorylation and KSHV reactivation. It is proposed that TPA induces KSHV reactivation through at least two arms. The first involves PKCdelta, ERK phosphorylation and c-Fos accumulation, whilst the second requires another PKC isoform that induces the phosphorylation of c-Jun. c-Fos and c-Jun jointly form an active AP-1 complex, which functions to activate the lytic cascade of KSHV.

Regulation of a1‐adrenoceptor‐mediated contractions of the uterine artery by PKC in pregnant sheep

Little is known about the adaptation of contractile mechanisms of uterine arterial smooth muscle to pregnancy. We hypothesize that PKC plays an important role in the regulation of α1-adrenoceptor-mediated contractions of the uterine artery and their adaptation to pregnancy. Uterine arteries were isolated from pregnant ewes (~140 days gestation). α1-adrenoceptor-mediated contractions were measured with increasing concentrations of phenylephrine (PE) in half-log increments in the absence or presence of the PKC activator phorbol 12, 13-dibutyrate (PDBu) and/or PKC inhibitor GF-109203X (GF). PE produced dose-dependent contractions of uterine arteries. PDBu (0.3 and 1 μM) significantly decreased pD2 value and maximum contraction (Emax) induced by PE. GF itself (0.1, 0.3, 1 μM) markedly decreased pD2 value and Emax induced by PE in a dose dependent pattern. However, treatment with PDBu and 1 μM GF significantly increased pD2 and Emax as compared with PDBu treatment alone without GF. These results suggest that activation of PKC plays an inhibitory role in the regulation of α1-adrenoceptor-mediated contraction of the uterine arteries during late pregnancy, which is likely to be important in the adaptation of α1-adrenoceptor-mediated contractions to pregnancy. (Supported in part by NIH grants HL57787 and HD 31226)

Bradykinin augments EGF-induced airway smooth muscle proliferation by activation of conventional protein kinase C isoenzymes

This study aims to investigate the effects of bradykinin, alone and in combination with growth factors on proliferation of cultured bovine tracheal smooth muscle cells. Bradykinin did not induce mitogenic responses by itself, but concentration-dependently augmented growth factor-induced [ 3H]thymidine incorporation and cell proliferation. The bradykinin effect was mediated by bradykinin B 2 receptors, and not dependent on cyclo-oxygenase. Bradykinin-induced synergism with epidermal growth factor (EGF) could be suppressed by the protein kinase C (PKC) inhibitors GF 109203X (Bisindolylmaleimide I; specific for conventional and novel PKCs) and Gö 6976 (12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; specific for conventional PKCs). In addition, sole activation of PKC using Phorbol 12-myristate 13-acetate (PMA) was sufficient for a synergistic interaction with EGF. In contrast to bradykinin however, PMA was mitogenic by itself which was not at all affected by Gö 6976, but abolished by GF 109203X. Bradykinin transiently activated the p42/p44 MAP kinase pathway, whereas PMA-induced activation of p42/p44 mitogen activated protein (MAP) kinase was sustained. Neither the combination of bradykinin and EGF nor that of PMA and EGF induced synergistic activation of p42/p44 MAP kinase, however. These results show that bradykinin B 2 receptor-stimulation augments growth factor-induced mitogenic responses of airway smooth muscle cells through activation of conventional PKC isozymes. In addition, the results show that PKC isozyme-specificity underlies stimulus-specific differences in mitogenic capacity for bradykinin and PMA.

Sphingosylphosphorylcholine enhances calcium entry in thyroid FRO cells by a mechanism dependent on protein kinase C

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca 2+] i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca 2+] i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca 2+] i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca 2+] i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl- sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.