Epidermal growth factor and angiotensin II regulation of extracellular signal-regulated protein kinase in rat liver epithelial WB cells

Activation of extracellular signal-regulated protein kinase (ERK) is considered essential for mitogenesis. In the present study, rat liver epithelial WB cells were used to investigate the relative roles of Ca2+, protein kinase C (PKC), and protein tyrosine phosphorylation in mitogenesis and activation of the ERK pathway stimulated by epidermal growth factor (EGF) and angiotensin II (Ang II). The sensitivity of the ERK pathway to Ca2+ was studied by using 1,2-bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) to chelate intracellular Ca2+ and a low extracellular Ca2+ concentration to prevent Ca2+ influx. Agonist-induced PKC activation was diminished by inhibition of PKC by GF-109203X (bisindolylmaleimide) or by down-regulation of PKC by long-term treatment of the cells with phorbol myristate acetate (PMA). Our results show that although activation of PKC was critical for mitogenesis induced by Ang II or EGF, the initial activation of ERK by both agonists in these cells was essentially independent of PKC activation and was insensitive to Ca2+ mobilization. This is in contrast to the findings in some cell types that exhibit a marked dependency on mobilization of Ca2+ and/or PKC activation. On the other hand, an obligatory tyrosine phosphorylation step for activation of ERK was indicated by the use of protein tyrosine kinase inhibitors, which profoundly inhibited the activation of ERK by EGF, Ang II, and PMA. Additional experiments indicated that tyrosine phosphorylation by a cytosolic tyrosine kinase may represent a general mechanism for G-protein coupled receptor mediated ERK activation.     

Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK

Growth factors activate mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs) and Jun kinases (JNKs). Although the signaling cascade from growth factor receptors to ERKs is relatively well understood, the pathway leading to JNK activation is more obscure. Activation of JNK by epidermal growth factor (EGF) or nerve growth factor (NGF) was dependent on H-Ras activation, whereas JNK activation by tumor necrosis factor alpha (TNF-alpha) was Ras-independent. Ras activates two protein kinases, Raf-1 and MEK (MAPK, or ERK, kinase) kinase (MEKK). Raf-1 contributes directly to ERK activation but not to JNK activation, whereas MEKK participated in JNK activation but caused ERK activation only after overexpression. These results demonstrate the existence of two distinct Ras-dependent MAPK cascades--one initiated by Raf-1 leading to ERK activation, and the other initiated by MEKK leading to JNK activation.     

Stimulation of Jun N-terminal kinase (JNK) by gonadotropin-releasing hormone in pituitary alpha T3-1 cell line is mediated by protein kinase C, c-Src, and CDC42

The signaling of ligands operating via heterotrimeric G proteins is mediated by a complex network that involves sequential phosphorylation events. Signaling by the G protein-coupled receptor GnRH was shown to include elevation of Ca2+ and activation of phospholipases, protein kinase C (PKC) and extra-cellular signal-regulated kinase (ERK). In this study, GnRH was shown to activate Jun N-Terminal Kinase (JNK)/SAPK in alpha T3-1 cells in a PKC- and tyrosine kinase-dependent manner. GnRH as well as tumor-promoting agent (TPA) also increased c-Src activity, which peaked at 2 min after GnRH stimulation and was sensitive both to PKC and to tyrosine kinase inhibitors. Coexpression of Csk, which serves as a Src-dominant interfering kinase, and constitutively active forms of Src, together with JNK, confirmed the involvement of c-Src downstream of PKC in the GnRH-JNK pathway. Coexpression of dominant negative and constitutively active forms of CDC42, Rac1, Ras, MEKK1, and MEK1 with JNK indicated that JNK activation by GnRH and TPA is mediated by CDC42 and MEKK1. Ras and MEK1, which are involved in a related mitogen-activated protein kinase (MAPK) pathway, did not affect JNK activation in alpha T3-1 cells. Taken together, our results suggest that GnRH stimulation of JNK activity is mediated by a unique pathway that includes sequential activation of PKC, c-Src, CDC42, and probably also MEKK1.     

Alpha1A-adrenergic receptors mediate vasoconstriction of the isolated spiral modiolar artery in vitro

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [32P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [32P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.     

Thrombopoietin potentiates the protein-kinase-C-mediated activation of mitogen-activated protein kinase/ERK kinases and extracellular signal-regulated kinases in human platelets

The thrombopoietin (TPO) receptor is expressed in the megakaryocytic lineage from late progenitors to platelets. We investigated the effect of TPO on the extracellular signal-regulated kinase (ERK) activation pathway in human platelets. TPO by itself did not activate ERK1, ERK2 and protein kinase C (PKC), whereas TPO directly enhanced the PKC-dependent activation of ERKs induced by other agonists including thrombin and phorbol esters, without affecting the PKC activation by those agonists. TPO did not activate the mitogen-activated protein kinase/ERK kinases, MEK1 and MEK2, but activated Raf-1 and directly augmented the PKC-mediated MEK activation, suggesting that TPO primarily potentiates the ERK pathway through regulating MEKs or upstream steps of MEKs including Raf-1. The MEK inhibitor PD098059 failed to affect not only thrombin-induced or phorbol ester-induced aggregation, but also potentiation of aggregation by TPO, denying the primary involvement of ERKs and MEKs in those events. ERKs and MEKs were located mainly in the detergent-soluble/non-cytoskeletal fractions. ERKs but not MEKs were relocated to the cytoskeleton following platelet aggregation and actin polymerization. These data indicate that TPO synergizes with other agonists in the ERK activation pathway of platelets and that this synergy might affect functions of the cytoskeleton possibly regulated by ERKs.     

Coupling of store-operated Ca++ entry to contraction in rat aorta

The purpose of this study was to test whether the elevated intracellular Ca++ level ([Ca++]i) resulting from store-operated Ca++ entry was associated with vascular smooth muscle contraction. Cyclopiazonic acid (CPA), a selective inhibitor of sarcoplasmic reticulum Ca(++)-ATPase, concentration-dependently (1-10 microM) elevated [Ca++]i in rat aorta, as indicated by an increase in the fura-2 340/380 ratio. Simultaneous measurement of contraction demonstrated that 1 and 10 microM CPA induced insignificant and variable amounts of contraction, respectively. Verapamil (10 microM) had relatively little effect on the 1 and 10 microM CPA-elevated [Ca++]i. In contrast, Ni++ (0.1 mM), in the presence of verapamil, abolished the 1 microM CPA-elevated [Ca++]i. Ni++ (0.1 mM) also partially decreased the 10 microM CPA-elevated [Ca++]i and, furthermore, abolished the associated contraction. A higher Ni++ concentration (1 mM) abolished the 10 microM CPA-elevated [Ca++]i that remained after verapamil and 0.1 mM Ni++. Phorbol dibutyrate (10 nM), a protein kinase C activator, potentiated contractions to 1 and 10 microM CPA in the presence of verapamil. Ni++ (0.1 mM) abolished the enhanced contractions, and decreased the elevated [Ca++]i. These results suggest that 1) elevated [Ca++]i due to store-operated Ca++ entry is dissociated from contraction; 2) the elevated [Ca++]i is restricted to at least two noncontractile compartments that can be differentiated by their relative sensitivities to blockade by low (0.1 mM) and higher (1 mM) Ni++ concentrations, and 3) [Ca++]i elevation within the compartment sensitive to blockade by 0.1 mM Ni++ can be coupled to contraction via protein kinase C activation.     

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.     

Evidence for the involvement of protein kinase C isoforms in alpha-1 adrenergic activation of phospholipase A2 in FRTL-5 thyroid cells

BACKGROUND: FRTL-5 thyroid cells are a cell line extensively used for the investigation of thyroid functions. Activation of alpha-1 adrenergic receptors stimulates both arachidonic acid (AA) release and cytosolic Ca2+ increase in this cell line. Cytosolic Ca2+ and arachidonic acid are known to be important second messengers regulating a variety of thyroid functions. The generation of these messengers is regulated primarily by two different types of phospholipases, phospholipase C (PLC) and phospholipase A2 (PLA2). METHODS: Norepinephrine (NE, 10 mumol/L) was used as an alpha-1 adrenergic activator, and cytosolic-free Ca2+ concentration ([Ca2+]i) was determined using the fluorescent dye indo-1. Arachidonic acid release was measured as an indicator of PLA2 activation, and protein kinase C (PKC) activity determination and isoforms identification were performed using commercial kits. RESULTS: Norepinephrine increased [Ca2+]i and AA release. Prevention of NE-induced cytosolic Ca2+ influx, either by removal of extracellular Ca2+ or by use of Ca2+ channel blockers, NiCl2 or CoCl2, inhibited AA generation entirely. Inhibition of NE-induced increase in [Ca2+]i by the Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), also significantly suppressed NE-induced AA release. Inhibition of PKC activity by PKC inhibitors (H-7 or staurosporine) or downregulation induced by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) or thyleametoxin (TX) significantly blocked the NE-induced AA release, which indicates PKC is involved in mediating NE-induced AA release. Protein kinase C activity measurement indicated that NE induced an activation of PKC in 5 minutes. To further characterize the role of PKC or Ca2+ in regulation of AA release, we identified PKC isoforms by immunoblotting with specific antibodies against 8 different Protein kinase C isoforms. PKC-alpha, -beta I, -beta II, -gamma, delta, -epsilon, -zeta, and -eta isoforms were identified. Norepinephrine induced translocation of PKC-alpha, -beta I, -beta II, -gamma, -delta, and -epsilon isoforms but not -zeta and -eta from cytosol to membrane. Chelation of intracellular Ca2+, prevention of Ca2+ influx, or prolonged treatment with thymeleatoxin (TX) completely blocked the NE-induced translocation of PKC-alpha. CONCLUSIONS: These results, taken together with data obtained from AA experiments, suggest that PKC plays a critical role in alpha-1 adrenergic receptor mediated PLA2 activation and subsequent AA release. Extracellular Ca2+ influx is a prerequisite for both PKC-alpha translocation and AA release. Whether Ca2+ acts directly upon the PLA2, or via PKC-alpha, to regulate AA generation is an intriguing question that remains to be clarified.     

Atypical protein kinase C isozyme zeta mediates carbachol-stimulated insulin secretion in RINm5F cells

Carbachol-stimulated insulin release in the RINm5F cell is associated with elevation of the cytosolic Ca2+ concentration ([Ca2+]i) through mobilization of Ca2+ from thapsigargin-sensitive intracellular stores and with the generation of diacylglycerol (DAG). Thus carbachol activates phospholipase C, and this was thought to be the means by which it stimulates insulin secretion. However, when the elevation of [Ca2+]i was blocked by thapsigargin, the effect of carbachol to stimulate insulin release was unchanged. Thus the effect of carbachol to increase [Ca2+]i was dissociated from the stimulation of release. When the role of protein kinase C (PKC) was examined, carbachol-stimulated insulin release was found to be unaffected by phorbol ester-induced downregulation of PKC, using 12-O-tetradecanoylphorbol-13-acetate (TPA), and by the PKC inhibitors staurosporine, bisindolylmaleimide, and 1-O-hexadecyl-2-O-methylglycerol (AMG-C16). These treatments abolished the stimulation of release by TPA. Thus the carbachol activation of PKC appeared also to be dissociated from the stimulation of insulin release. However, when the activation of several different PKC isozymes was studied, an atypical PKC isozyme, zeta, was found to be translocated by carbachol. By Western blotting analysis, carbachol selectively translocated the conventional PKC isozymes alpha and beta (the activation of which is dependent on Ca2+ and DAG) from the cytosol to the membrane. Carbachol also translocated the atypical PKC isozyme zeta, which is insensitive to Ca2+, DAG, and phorbol esters. The PKC inhibitors staurosporine, bisindolylmaleimide, and AMG-C16 blocked the stimulated translocation of PKC-alpha and -beta, but not that of PKC-zeta. Prolonged treatment of the cells with TPA downregulated PKC-alpha and -beta, but not PKC-zeta. Under all these conditions, carbachol-stimulated insulin release was unaffected. However, a pseudosubstrate peptide inhibitor specific for PKC-zeta inhibited the translocation of PKC-zeta and 70% of the carbachol-stimulated insulin secretion. The data indicate that carbachol-stimulated insulin release in RINm5F cells is mediated to a large degree by the activation of the atypical PKC isozyme zeta.     

Protein kinase C, but not tyrosine kinases or Ras, plays a critical role in angiotensin II-induced activation of Raf-1 kinase and extracellular signal-regulated protein kinases in cardiac myocytes

Angiotensin II (AngII) induces cardiac hypertrophy through activating a variety of protein kinases. In this study, to understand how cardiac hypertrophy develops, we examined AngII-evoked signal transduction pathways leading to the activation of extracellular signal-regulated protein kinases (ERKs), which are reportedly critical for the development of cardiac hypertrophy, in cultured cardiac myocytes isolated from neonatal rats. Inhibition of protein kinase C (PKC) with calphostin C or down-regulation of PKC by pretreatment with a phorbol ester for 24 h abolished AngII-induced activation of Raf-1 and ERKs, and addition of a phorbol ester conversely induced a marked increase in the activities of Raf-1 and ERKs. Pretreatment with two chemically and mechanistically dissimilar tyrosine kinase inhibitors, genistein and tyrphostin, did not attenuate AngII-induced activation of ERKs. In contrast, genistein strongly blocked insulin-induced ERK activation in cardiac myocytes. Although pretreatment with manumycin, a Ras farnesyltransferase inhibitor, or overexpression of a dominant-negative mutant of Ras inhibited insulin-induced ERK activation, neither affected AngII-induced activation of ERKs. Overexpression of a dominant-negative mutant of Raf-1 completely suppressed ERK2 activation by AngII, endothelin-1, and insulin. These results suggest that PKC and Raf-1, but not tyrosine kinases or Ras, are critical for AngII-induced activation of ERKs in cardiac myocytes.     

Identification of a retinoic acid responsive aldoketoreductase expressed in HL60 leukaemic cells

We evaluated the role of protein kinase C (PKC) in the regulation of apoptosis triggered by singlet oxygen. Activation of PKC by short-term 12-O-tetradecanoyl phorbol 13-acetate (TPA) treatment inhibited apoptosis, whereas inhibition of PKC with several inhibitors potentiated this process. The antiapoptotic effect of TPA was accompanied by phosphorylation of extracelluar signal-regulated kinase 1/2 (ERK1/2). Pretreatment of cells with MEK inhibitor, PD98059, inhibited TPA-induced phosphorylation of ERK1/2 and the cytoprotective ability of TPA. These results suggest that activation of PKC in HL-60 cells confers protection against apoptosis induced by singlet oxygen and that ERK1/2 mediates antiapoptotic signaling of PKC.     

Alpha adrenergic receptor subtypes involved in prostaglandin synthesis are coupled to Ca++ channels through a pertussis toxin-sensitive guanine nucleotide-binding protein

Previously, we have shown that alpha-2C and alpha-1A adrenergic receptors (AR) stimulate prostacyclin (PGI2) synthesis through a pertussis toxin-sensitive guanine nucleotide-binding protein (G protein) in vascular smooth muscle cells (VSMC). The purpose of this study was to assess the role of Ca++ in PGI2 production elicited by alpha-AR activation and to investigate the modulation of the Ca++ channel by G proteins coupled to these alpha-AR in VSMC. PGI2 was measured as immunoreactive 6-keto-PGF1 alpha by radioimmunoassay and cytosolic calcium ([Ca++]i) by spectrofluorometry using fura-2. Norepinephrine, methoxamine and UK-14304 enhanced 6-keto-PGF1 alpha production and [Ca++]i, which was inhibited by depletion of extracellular Ca++ and by Ca++ channel antagonists (verapamil, nifedipine and PN 200-110). Moreover, the Ca++ channel activator Bay K 8644 increased 6-keto-PGF1 alpha production in a nifedipine-sensitive manner, indicating the involvement of dihydropyridine-sensitive Ca++ channels in VSMC. Pertussis toxin inhibited AR agonist-induced 6-keto-PGF1 alpha production and the increase in [Ca++]i. Alpha AR agonists increase Ca++ influx in the presence of guanosine 5'-0-(2- thiodiphosphate) (GTP-gamma-S), and this effect was blocked in the presence of guanine 5'-O-(2-thiodiphosphate) (GDP-beta-S) and antiserum against Gi alpha 1-2 protein in reversibly permeabilized cells with beta-escin. VSMC of rabbit aortae contain a G protein(s) that was recognized by Gi alpha 1-2 but not Gi alpha 3 or G0 antibodies at 1:200 dilution. The calmodulin inhibitor W-7 blocked AR agonist and Bay K 8644-stimulated 6-keto-PGF1 alpha production. The phospholipase A2 inhibitors 7,7-dimethyleicosadienoic acid and oleoyloxyethyl phosphocholine but not phospholipase C inhibitor U-73122 reduced 6-keto-PGF1 alpha production in VSMC. These data suggest that a pertussis toxin-sensitive G protein, probably Gi alpha 1-2, coupled to alpha AR regulates Ca++ influx, which, in turn, by interacting with calmodulin, increases phospholipase A2 activity to release arachidonic acid for PGI2 synthesis in VSMC of rabbit aortae.     

The role of self-treatment guidelines in self-management education for adult asthmatics

In cardiac fibroblasts, angiotensin II (Ang II) induced a rapid increase in extracellular signal regulated kinase (ERK) activity in a pertussis toxin insensitive manner. This ERK activation was abolished by the Gq-associated phospholipase C inhibitor U73122 but was insensitive to protein kinase C (PKC) inhibitors or PKC downregulation by phorbol ester. Intracellular Ca2+ chelation by BAPTA-AM or TMB-8 abolished Ang II induced ERK activation, whereas treatment with EGTA or nifedipine did not affect it. Ca2+ ionophore A23187 also induced a rapid increase in ERK activity to an extent similar to that of Ang II stimulation. Calmodulin inhibitors (W7 and calmidazolium) and tyrosine kinase inhibitors (genistein and ST638) completely blocked ERK activation by Ang II and A23187. Both Ang II and A23187 caused a rapid increase in the binding of GTP to p21(Ras), which was nearly abolished by genistein and calmidazolium. Transfection with the dominant negative mutant of Ras and the Ras inhibitor manumycin completely inhibited Ang II induced ERK activation. It was also found for the first time that cardiac fibroblasts abundantly expressed Ca2+-sensitive tyrosine kinase Pyk2/CAKbeta/RAFTK and that Ang II markedly induced its activation in a Ca2+/calmodulin-sensitive manner. Overexpression of the dominant negative mutant of Pyk2 significantly attenuated Ang II or A23187-induced ERK activities (36% and 38% inhibition compared with that in mock-transfected cells, respectively) and ERK tyrosine phosphorylation levels, as well as an increase in the binding of GTP to p21(Ras). These findings demonstrate that in cardiac fibroblasts, Ang II induced Ras/ERK activation is dominantly regulated by Gq-coupled Ca2+/calmodulin signaling and that Pyk2 plays an important role in the signal transmission for efficient activation of the Ang II induced Ras/ERK pathway.     

Adrenocorticotropin induction of stress-activated protein kinase in the adrenal cortex in vivo

A broad array of stressors induce ACTH release from the anterior pituitary, with consequent stimulation of the adrenal cortex and release of glucocorticoids critical for survival of the animal. ACTH stimulates adrenocortical gene expression in vivo and inhibits adrenocortical cell proliferation. Binding of ACTH to its G-protein-coupled receptor stimulates the production of cAMP and activation of the protein kinase A pathway. The stress-activated protein kinases (SAPKs) (or c-Jun N-terminal kinases) and the extracellular signal-regulated kinases (ERKs) are members of the mitogen-activated protein kinase family of serine/threonine kinases, which have recently been implicated in G-protein-coupled receptor intracellular signaling. The SAPKs are preferentially induced by osmotic stress and UV light, whereas the ERKs are preferentially induced by growth factors and proliferative signals in cultured cells. In these studies, ACTH stimulated SAPK activity 3-4-fold both in the adrenal cortex in vivo and in the Y1 adrenocortical cell line. 12-O-Tetradecanoylphorbol-13-acetate but not cAMP induced SAPK activity in Y1 cells. The isoquinolinesulfonamide inhibitors H-8 and H-89 blocked ACTH induction of SAPK activity at protein kinase C inhibitory doses but not at protein kinase A inhibitory doses. The calcium chelating agent EGTA inhibited ACTH-induced SAPK activity and the calcium ionophore A23187 induced SAPK activity 3-fold. In contrast with the induction of SAPK by ACTH, ERK activity was inhibited in the adrenal cortex in vivo and in Y1 adrenal cells. Together these findings suggest that ACTH induces SAPK activity through a PKC and Ca+2-dependent pathway. The induction of SAPK and inhibition of ERK by ACTH in vivo may preferentially regulate target genes involved in the adrenocortical stress responses in the whole animal.     

Formyl peptide receptors are coupled to multiple mitogen-activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase

Formyl peptide receptor activation of three mitogen-activated protein kinase (MAPK) cascades, extracellular signal-regulated kinases (ERKs), N-terminal kinases (JNKs), and p38 MAPK was examined in differentiated HL-60 granulocytes. FMLP stimulated a concentration- and time-dependent increase in ERK, JNK, and p38 MAPK activities, all of which were dependent on a pertussis toxin-sensitive G protein. Pharmacologic inhibitors were used to examine the roles of tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C. FMLP-stimulated ERK activity was dependent on tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C; p38 MAPK activation was dependent on phosphatidylinositol 3-kinase and phospholipase C; while JNK activation was independent of all of these signaling components. The mitogen-activated protein kinase/ERK kinase inhibitor PD098059 reduced ERK activation by 90%, while an inhibitor of p38 MAPK, SB203580, inhibited p38 MAPK activation by 80%. Both PD098059 and SB203580 inhibited FMLP-stimulated superoxide release, as did inhibitors directed against protein kinase C, tyrosine kinases, and phosphatidylinositol 3-kinase. We conclude that formyl peptide receptors are coupled to three MAPK cascades by Gi proteins. ERKs, p38 MAPK, and JNKs are each activated by distinct proximal signal transduction pathways. Activation of p38 MAPK is necessary for FMLP stimulation of respiratory burst activity; however, a second signal that may involve ERK is also required for this activity.     

Carbachol stimulates c-fos expression and proliferation in oligodendrocyte progenitors

To determine if muscarinic receptor-activation plays a role in oligodendrocyte development, the effect of carbachol a stable acetylcholine analog, on gene expression and proliferation was investigated. Using Northern blot analysis we showed that carbachol caused a time and concentration-dependent increase in c-fos mRNA. This effect was blocked by atropine, a non-selective muscarinic antagonist. In addition, the muscarinic-stimulated c-fos increase was inhibited by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C (PKC), but not by N-2-(p-bromocinnamylamino)-ethyl-5-isoquinoline-sulfonamide (H-89), a potent inhibitor of protein kinase A, suggesting the involvement of PKC in mediating the response. Down-regulation of PKC by overnight pre-treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) blocked only the phorbol ester-stimulated c-fos accumulation while no effect was observed in the carbachol-induced response. These results suggested that carbachol stimulated an H-7 sensitive PKC pathway which may be different than that activated by TPA. Further evidence for two separate mechanisms of proto-oncogene induction was provided by the additive effect of carbachol and TPA. Induction of c-fos mRNA by carbachol was dependent on both influx of extracellular Ca2+ and release from intracellular stores, as both EDTA and BAPTA blocked the response. Since activation of muscarinic receptors can affect cell division in other cellular systems, the effect of carbachol on [3H]thymidine and bromodeoxyuridine incorporation into oligodendrocyte DNA was measured. Carbachol stimulated DNA synthesis in oligodendrocyte progenitors. This effect was mediated by muscarinic receptors as [3H]thymidine incorporation was prevented or significantly reduced by the addition of atropine. In conclusion, the present findings suggest that, the neurotransmitter, acetylcholine may act as a trophic factor in developing oligodendrocytes, regulating their growth and development in the central nervous system.     

Effects of low density lipoprotein on cytosolic [Ca++] in cultured rat mesangial cells

BACKGROUND: Recently, a low density lipoprotein (LDL) receptor has been identified in mesangial cells. However, the nature of intracellular signals in mesangial cells after exposure to LDL is unclear. METHODS: We studied the effect of LDL on cultured rat mesangial cell [Ca++]i using spectrofluorometry. RESULTS: Addition of LDL (15 micrograms/mL) produced a rapid, transient, and dose-dependent rise in [Ca++]i within seconds, returning to baseline in 6 minutes. No further rise was observed at higher LDL concentrations. No significant rise in [Ca++]i was observed with LDL in cells placed in a Ca(++)-free medium. The [Ca++]i rise was greatly attenuated in magnitude and duration when lanthanum was used. In contrast, verapamil failed to block the LDL-induced rise in [Ca++]i. Addition of LDL did not alter production of inositol 1,4,5-triphosphate (IP3) by mesangial cells. CONCLUSIONS: Low density lipoprotein caused a transient rise in [Ca++]i in cultured rat mesangial cells. The observed rise in [Ca++]i was largely caused by influx of extracellular Ca++ through receptor-gated channels. Mobilization from intracellular stores and activation of IP3 were not involved. The rise in [Ca++]i may mediate the effects of LDL on mesangial cell function.