Protein Kinase C-independent Pathway Research Articles

Nicotinic Stimulation of Catecholamine Synthesis and Tyrosine Hydroxylase Phosphorylation in Cervine Adrenal Medullary Chromaffin Cells

The synthesis and secretion of catecholamines by the adrenal medulla is of major importance in the stress response. Tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, has been extensively studied in adrenal medullary chromaffin cells from a number of species. Cervine chromaffin cells are of interest because the deer is known to be a relatively stress-prone reactive species. We report the first characterisation of tyrosine hydroxylase regulation in cervine chromaffin cells. Nicotinic receptor activation resulted in a time- and concentration-dependent increase in catecholamine synthesis, which was significantly reduced by the extracellular signal-regulated kinase (ERK)1/2 signalling pathway inhibitor PD98059 and the calcium/calmodulin protein kinase II inhibitor KN-93, but not by H89 or bisindolylmaleimide I, inhibitors of protein kinase A and C, respectively. Nicotinic stimulation also increased the phosphorylation of ERK1/2 and tyrosine hydroxylase. This latter response occurred on serine residues 19, 31 and 40 of the enzyme. The nicotinic-induced phosphorylation of ERK1/2 and serine 31 of tyrosine hydroxylase was suppressed by PD98059 but not bisindolylmaleimide I. These data indicate that nicotinic stimulation of tyrosine hydroxylase involves the phosphorylation of serine 31 via an ERK1/2-dependent, protein kinase C-independent pathway. Protein kinase C activation by phorbol 12-myristate 13-acetate also caused an ERK1/2-dependent increase in the serine 31 phosphorylation of tyrosine hydroxylase but, in contrast to the nicotinic response, was not accompanied by an increase in enzyme activity. Thus, ERK1/2-mediated serine 31 phosphorylation of tyrosine hydroxylase appears necessary but not sufficient for nicotinic activation of catecholamine synthesis in cervine chromaffin cells. These data present potentially important similarities and differences between the regulation of catecholamine synthesis in cervine and the more widely studied bovine adrenal medulla.

Diabetes insipidus and cognitive function

It has been well known that several neuropeptides may affect human behavior, and that some endocrinopathies are associated with impaired higher function of the brain. There have been increasing evidences that vasopressin has both peripheral and central effects, the latter of which is involved in memory. In experimental animals, male mice with a null mutation in the V1a receptor (V1aR) exhibit a profound impairment in social recognition and changes in anxiety-like behavior. An AVP fragment analog has been reported to facilitate memory retention and recall in mice through protein kinase C-independent pathways. In human, a few recent reports have suggested that a familial central diabetes insipidus, caused by a heterozygous mutation in the gene for vasopressin prohormone, have minor disturbances in central nervous system. Taken together, it is hypothesized that the subject with central diabetes insipidus may frequently present with an impaired cognitive ability. It is justified to examine the cognitive function, when we make a diagnosis of central diabetes insipidus and to perform a clinical study to investigate whether central diabetes insipidus may be associated with impairment of higher brain functions.

Mitogen-activated protein kinase activation by hydrogen peroxide is mediated through tyrosine kinase-dependent, protein kinase C-independent pathways in vascular smooth muscle cells: upregulation in spontaneously hypertensive rats

To investigate the putative molecular mechanisms underlying mitogen-activated protein (MAP) kinase activation by hydrogen peroxide (H(2)O(2)) in vascular smooth muscle cells (VSMC) and to evaluate whether H(2)O(2)-induced actions are altered in VSMC from spontaneously hypertensive rats (SHR). VSMC from mesenteric arteries of Wistar-Kyoto rats (WKY) and SHR were stimulated with H(2)O(2) (2-30 min). The phosphorylation of extracellular signal-regulated kinases (ERK)1/2 and p38MAP kinase was determined by immunoblotting. The involvement of tyrosine kinase and protein kinase C (PKC) was evaluated using pharmacological inhibitors, tyrphostin (A23 and A9) and GF109203X, respectively. The role of receptor tyrosine kinases (RTK) was assessed with AG1478, AG1296 and AG1024, selective inhibitors of epidermal growth factor receptor, platelet-derived growth factor receptor and insulin-like growth factor receptor, respectively. Non-receptor tyrosine kinases (NRTK) were studied using AG490 (JAK2 inhibitor) and PP2 (Src inhibitor). H(2)O(2) stimulated phosphorylation of ERK1/2 and p38MAP kinase in a time-dependent manner. This increase was significantly greater in SHR versus WKY (P < 0.01). The activation of MAP kinases was unaffected by GF109203X but was decreased by tyrphostins (P < 0.01). The inhibition of NRTK attenuated H(2)O(2)-mediated phosphorylation of ERK1/2 (P < 0.001) but not of p38MAP kinase, whereas Src and JAK2 inhibition significantly decreased phosphorylation of both MAP kinases (P < 0.01). These data indicate that H(2)O(2) increases ERK1/2 and p38MAP kinase activation through tyrosine kinase-dependent, PKC-independent mechanisms. Whereas ERK1/2 is regulated by both RTK and NRTK, p38MAP kinase is regulated by NRTK. Our findings identify an important role for tyrosine kinases, but not PKC, in H(2)O(2)-induced phosphorylation of ERK1/2 and p38MAP kinase in VSMC. The upregulation of these processes may contribute to enhanced redox-dependent MAP kinase signaling in SHR VSMC.

Phosphatidylinositol-4-phosphate 5-Kinase Regulates Fission Yeast Cell Integrity through a Phospholipase C-mediated Protein Kinase C-independent Pathway

Fission yeast its3-1 mutant is an allele of the essential gene its3+ that encodes a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) that produces phosphatidylinositol 4,5-bisphosphate. We found that the its3-1 mutant is sensitive to micafungin, a (1,3)-beta-D-glucan synthase inhibitor, suggesting a cell wall integrity defect. Consistently, its3-1 mutation caused synthetic lethality with a (1,3)-beta-D-glucan synthase mutant, bgs1-i2, and its3-1 mutant cells showed aberrant localization of green fluorescent protein-Bgs1. Similar aberrant localization of green fluorescent protein-tagged Rgf1, a putative phosphatidylinositol 4,5-bisphosphate-binding guanine nucleotide exchange factor for Rho protein, in its3-1 mutants was observed, suggesting a defective Rgf1/Rho pathway. To unravel the molecular mechanism(s), putative downstream components of PIP5K signaling were analyzed. Unexpectedly, overexpression of phospholipase C (Plc1), but not that of protein kinase C (PKC; Pck1 and Pck2), suppressed the phenotypes of the its3-1 mutant. These findings indicate that PKCs are not involved in the suppression, and further analysis revealed that PKCs are not downstream of Plc1 in fission yeast. Also, the enzymatic activity of Plc1 is essential for the suppression of the phenotypes and for the viability of the its3-1 mutant. These findings suggest that Its3 PIP5K regulates cell integrity through a Plc1-mediated PKC-independent pathway, in addition to the Rho/PKC pathway.

Protein kinase C-independent pathway for NADPH oxidase activation in guinea pig peritoneal polymorphonuclear leukocytes by cytochalasin D

Cytochalasin D (CD) induced production of the superoxide radical ( O 2 − ) in guinea pig polymorphonuclear leukocytes (PMNs). The protein kinase C (PKC) inhibitor GF109203X (GFX) was rarely without effect on CD-induced O 2 − production. CD as well as PMA induced the translocation of p47 phox to the membrane fraction, and this translocation was slightly decreased by GFX. Moreover, the inhibitory effect of a PKCζ antagonist with sequences based on the endogenous PKCζ pseudosubstrate region was weaker than the inhibitory effect on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced O 2 − production. On the other hand, the production of O 2 − induced by CD was more strongly suppressed by the PLD inhibitor ethanol and phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin than that induced by fMLP, and the activation of phospholipase D (PLD) by CD was restrained by wortmannin. These findings suggest that NADPH oxidase is activated by CD through a PKC-independent signaling pathway in PMNs, and this pathway involves the activation of PLD through PI3-K.

Gαq-coupled Receptor Internalization Specifically Induced by Gαq Signaling: REGULATION BY EBP50

In the present report, we investigated the effect of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) expression on the agonist-induced internalization of the thromboxane A(2) beta receptor (TPbeta receptor). Interestingly, we found that EBP50 almost completely blocked TPbeta receptor internalization, which could not be reversed by overexpression of G protein-coupled receptor (GPCR) kinases and arrestins. Because we recently demonstrated that EBP50 can bind to and inhibit Galpha(q), we next studied whether Galpha(q) signaling could induce TPbeta receptor internalization, addressing the long standing question about the relationship between GPCR signaling and their internalization. Expression of a constitutively active Galpha(q) mutant (Galpha(q)-R183C) resulted in a robust internalization of the TPbeta receptor, which was unaffected by expression of dominant negative mutants of arrestin-2 and -3, but inhibited by expression of EBP50 or dynamin-K44A, a dominant negative mutant of dynamin. Phospholipase Cbeta and protein kinase C did not appear to significantly contribute to internalization of the TPbeta receptor, suggesting that Galpha(q) induces receptor internalization through a phospholipase Cbeta- and protein kinase C-independent pathway. Surprisingly, there appears to be specificity in Galpha protein-mediated GPCR internalization. Galpha(q)-R183C also induced the internalization of CXCR4 (Galpha(q)-coupled), whereas it failed to do so for the beta(2)-adrenergic receptor (Galpha(s)-coupled). Moreover, Galpha(s)-R201C, a constitutively active form of Galpha(s), had no effect on internalization of the TPbeta, CXCR4, and beta(2)-adrenergic receptors. Thus, we showed that Galpha protein signaling can lead to internalization of GPCRs, with specificity in both the Galpha proteins and GPCRs that are involved. Furthermore, a new function has been described for EBP50 in its capacity to inhibit receptor endocytosis.

The effect of chelerythrine on depolarization-induced force responses in skinned fast skeletal muscle fibres of the rat.

1 We examined the effect of the protein kinase C (PKC) inhibitor chelerythrine on depolarization-induced force responses (DIFRs) and sarcoplasmic reticulum (SR) function in single, mechanically skinned skeletal muscle fibres of the rat. 2 In this study, the DIFRs in the skinned fibres normally underwent an irreversible loss of excitation-contraction coupling (ECC) after 10-15 responses. Chelerythrine (12 micro M) was shown to restore ECC in these fibres. Restored force responses were similar in peak (control 50.8+/-6.4%, chelerythrine 56.9+/-12.4% of maximum force, P=0.42, n=21), but significantly broadened compared to initial control responses (full-width at half maximum, control; 3.7+/-0.3 s, chelerythrine; 13.3+/-1.1 s, P<0.001). Early exposure to chelerythrine prevented run-down of DIFRs. Chelerythrine also induced spontaneous force responses in some fibres. 3 The PKC inhibitors calphostin C and staurosporine did not restore ECC, and the PKC activator phorbol 12-myristate 13-acetate did not promote loss of ECC in the skinned fibres. 4 Chelerythrine significantly increased SR Ca(2+) loading by 8.4+/-1.7% (P=0.02, n=9) and SR Ca(2+) release by at least 14.1+/-2.7% (P=0.004, n=11) in the skinned fibres. 5 Chelerythrine had no significant effect on maximum force production or the [Ca(2+)] producing half maximal activation of the myofilaments. However, chelerythrine did have a small effect on the slope of the force-Ca(2+) relationship (P=0.02, n=10). 6 Chelerythrine reverses the use-dependent loss of excitation-contraction coupling in skinend skeletal muscle fibres by a PKC independent pathway. Chelerythrine may be an important pharmacological probe for examining the mechanisms of contraction-induced muscle injury.

Activation of extracellular-regulated kinase by 5-hydroxytryptamine(2A) receptors in PC12 cells is protein kinase C-independent and requires calmodulin and tyrosine kinases.

5-Hydroxytryptamine (5-HT)(2A) receptors have been implicated to play a role in both the treatment and pathophysiology of a number of psychiatric disorders. Therefore, the coupling of this receptor to signals, such as extracellular signal-regulated kinase (ERK), that elicit long-term neuronal changes may be relevant. In the present study we examined the coupling of the G(q)-coupled receptor to ERK in PC12 cells, a cell line commonly used as a neuronal model system. Activation of ERK occurred through a pathway different than the protein kinase C-dependent pathways described previously in studies of non-neuronal cells. Activation of ERK, in PC12 cells, was inhibited by both chelation of extracellular Ca(2+) and by depletion of intracellular Ca(2+) stores. Surprisingly, activation was not inhibited, but actually potentiated, by a variety of protein kinase C inhibitors covering all known protein kinase C isoforms. In contrast, the coupling of receptor to activation of ERK was found to be sensitive to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7) and N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (W13), inhibitors of calmodulin, but not to 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN62) and 2-[N-(2-hydroxyethyl)]-N-4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN93), inhibitors of calmodulin-dependent protein kinase. Additionally, the general tyrosine kinase inhibitor genistein, as well as the Src inhibitor PP1 and the epidermal growth factor receptor kinase inhibitor 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG 1478), inhibited receptor-mediated activation of ERK, suggesting a role for tyrosine kinases. In fact, 5-HT was found to stimulate tyrosine phosphorylation of a number of proteins, and this phosphorylation was inhibited by W7. 5-HT(2A) receptor-activation of ERK through a protein kinase C-independent pathway requiring Ca(2+)/calmodulin/tyrosine kinases represents a pathway distinct from those described in studies of non-neuronal cells.

Tamoxifen-induced growth arrest and apoptosis in pituitary tumor cells in vitro via a protein kinase C-independent pathway

Protein kinase C (PKC), a kinase family involved in cell signal transduction, is overexpressed in most pituitary adenoma cells. We studied the effect of tamoxifen, an estrogen receptor antagonist and also a protein kinase inhibitor, on pituitary tumor cell proliferation and the induction of apoptosis; and we compared its effects with those of another PKC inhibitor, staurosporine. Tamoxifen induced growth arrest and apoptosis in a mouse pituitary adenoma cell line, AtT20, and in low-passage human primary pituitary tumor cell cultures. Staurosporine also inhibited pituitary tumor cell growth. PKC activity in AtT20 cells was inhibited by staurosporine and by prolonged treatment with phorbol myristate acetate, which down-regulates PKC activity, but not by tamoxifen, at the dosages used to induce apoptosis. Our findings suggest that tamoxifen induces apoptosis in AtT20 cells independent of a classical PKC isozyme pathway.

The C Terminus of the Metabotropic Glutamate Receptor Subtypes 2 and 7 Specifies the Receptor Signaling Pathways

There is accumulating evidence that the specificity of the transduction cascades activated by G protein-coupled receptors cannot solely depend on the nature of the coupled G protein. To identify additional structural determinants, we studied two metabotropic glutamate (mGlu) receptors, the mGlu2 and mGlu7 receptors, that are both coupled to G(o) proteins but are known to affect different effectors in neurons. Thus, the mGlu2 receptor selectively blocks N- and L-type Ca(2+) channels via a protein kinase C-independent pathway, whereas the mGlu7 receptor selectively blocks P/Q-type Ca(2+) channels via a protein kinase C-dependent pathway, and both effects are pertussis toxin-sensitive. We examined the role of the C-terminal domain of these receptors in this coupling. Chimeras were constructed by exchanging the C terminus of these receptors and transfected into neurons. Different chimeric receptors bearing the C terminus of mGlu7 receptor blocked selectively P/Q-type Ca(2+) channels, whereas chimeras bearing the C terminus of mGlu2 receptor selectively blocked N- and L-type Ca(2+) channels. These results show that the C terminus of mGlu2 and mGlu7 receptors is a key structural determinant that allows these receptors to select a specific signaling pathway in neurons.

Endothelium-derived endothelin-1 reduces cerebral artery sensitivity to nitric oxide by a protein kinase C-independent pathway.

Nitric oxide (NO) reduces endothelin-1 (ET-1) production and blunts ET-1 dependent vasoconstriction. The direct effects of smooth muscle ET(A) receptor stimulation on NO-mediated relaxation are unknown. We hypothesized that endothelium-derived ET-1 regulates vascular tone by reducing smooth muscle sensitivity to NO, possibly through activation of protein kinase C (PKC). Rings of rabbit middle cerebral artery were mounted on microvessel myographs to measure isometric tension. Dose-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP; an NO donor) were obtained with or without ET-1 receptor blockade. Experiments were performed in the presence of indomethacin (10 micromol/L). Results are expressed as mean+/-SEM. In depolarized conditions (40 mmol/L KCl physiological solution), ACh-induced relaxation was entirely NO-dependent, as indicated by its suppression by N(omega)-nitro-L-arginine (P<0.05). Arterial sensitivity (pD(2)) to ACh (6.32+/-0.11, n=6) was increased (P<0.05) to 6.77+/-0.10 (n=6) by BQ123 (ET(A) receptor antagonist, 5 micromol/L) but not by BQ788 (ET(B) receptor antagonist, 5 micromol/L; 6.08+/-0.22, n=5). Consistent with this finding, blockade of ET(A) receptors increased (P<0.05) vascular sensitivity to SNP (6.95+/-0.10, n=8), whereas BQ788 had no influence on arterial sensitivity to SNP (6.17+/-0.07, n=7) compared with control (6.43+/-0.13, n=11). In denuded arteries, the sensitivity to SNP (7.10+/-0.08, n=8) was reduced by exogenous ET-1 (6.51+/-0.35, n=7, P<0.05). Chelerythrine, a PKC inhibitor, did not alter smooth muscle sensitivity to NO, whereas phorbol 12-myristate 13-acetate, a PKC activator, strongly increased it. Blockade of ET(A) but not ET(B) receptors sensitizes vascular smooth muscle to exogenous and endothelium-derived NO. This suggests that ET-1 regulates smooth muscle sensitivity to NO by a PKC-independent pathway. This represents an alternative pathway by which NO and ET-1 interact to regulate vascular tone.

Fumarylacetoacetate, the metabolite accumulating in hereditary tyrosinemia, activates the ERK pathway and induces mitotic abnormalities and genomic instability.

Patients suffering from the metabolic disease hereditary tyrosinemia type I (HT1), caused by fumarylacetoacetate hydrolase deficiency, have a high risk of developing liver cancer. We report that a sub-apoptogenic dose of fumarylacetoacetate (FAA), the mutagenic metabolite accumulating in HT1, induces spindle disturbances and segregational defects in both rodent and human cells. Mitotic abnormalities, such as distorted spindles, lagging chromosomes, anaphase/telophase chromatin bridges, aberrant karyokinesis/cytokinesis and multinucleation were observed. Some mitotic asters displayed a large pericentriolar material cloud and/or altered distribution of the spindle pole-associated protein NuMA. FAA-treated cells developed micronuclei which were predominantly CREST-positive, suggesting chromosomal instability. The Golgi complex was rapidly disrupted by FAA, without evident microtubules/tubulin alterations, and a sustained activation of the extracellular signal-regulated protein kinase (ERK) was also observed. Primary skin fibroblasts derived from HT1 patients, not exogenously treated with FAA, showed similar mitotic-derived alterations and ERK activation. Biochemical data suggest that FAA causes ERK activation through a thiol-regulated and tyrosine kinase-dependent, but growth factor receptor- and protein kinase C-independent pathway. Pre-treatment with the MEK inhibitor PD98059 and the Ras farnesylation inhibitor B581 decreased the formation of CREST-positive micronuclei by approximately 75%, confirming the partial contribution of the Ras/ERK effector pathway to the induction of chromosomal instability by FAA. Replenishment of intracellular glutathione (GSH) with GSH monoethylester abolished ERK activation and reduced the chromosomal instability induced by FAA by 80%. Together these results confirm and extend the previously reported genetic instability occurring in cells from HT1 patients and allow us to speculate that this tumorigenic-related phenomenon may rely on the biochemical/cellular effects of FAA as a thiol-reacting and organelle/mitotic spindle-disturbing agent.

Prolactin-releasing Peptide Activation of the Prolactin Promoter Is Differentially Mediated by Extracellular Signal-regulated Protein Kinase and c-Jun N-terminal Protein Kinase

Regulation of the mitogen-activated protein kinase (MAPK) family by prolactin-releasing peptide (PrRP) in both GH3 rat pituitary tumor cells and primary cultures of rat anterior pituitary cells was investigated. PrRP rapidly and transiently activated extracellular signal-regulated protein kinase (ERK) in both types of cells. Both pertussis toxin, which inactivates G(i)/G(o) proteins, and exogenous expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase I, which specifically blocks signaling mediated by the betagamma subunits of G proteins, completely blocked the PrRP-induced ERK activation, suggesting the involvement of G(i)/G(o) proteins in the PrRP-induced ERK activation. Down-regulation of cellular protein kinase C did not significantly inhibit the PrRP-induced ERK activation, suggesting that a protein kinase C-independent pathway is mainly involved. PrRP-induced ERK activation was not dependent on either extracellular Ca(2+) or intracellular Ca(2+). However, the ERK cascade was not the only route by which PrRP communicated with the nucleus. JNK was also shown to be significantly activated in response to PrRP. JNK activation in response to PrRP was slower than ERK activation. Moreover, to determine whether a MAPK family cascade regulates rat prolactin (rPRL) promoter activity, we transfected the intact rPRL promoter ligated to the firefly luciferase reporter gene into GH3 cells. PrRP activated the rPRL promoter activity in a time-dependent manner. Co-transfection with a catalytically inactive form of a MAPK construct or a dominant negative JNK, partially but significantly inhibited the induction of the rPRL promoter by PrRP. Furthermore, co-transfection with a dominant negative Ets completely abolished the response of the rPRL promoter to PrRP. These results suggest that PrRP differentially activates ERK and JNK, and both cascades are necessary to elicit rPRL promoter activity in an Ets-dependent mechanism.

Phorbor myristate acetate induces NADPH oxidase activity of cytochalasin B-primed neutrophils through the protein kinase C-independent pathway.

We examined the effect of cytochalasin B (CB) or granulocyte colony stimulating factor (GCSF) on superoxide radical (O2-) production of neutrophils by phorbor myristate acetate (PMA)-stimulation. It was observed that O2- generation of intact and GCSF-treated neutrophils by PMA-stimulation showed a lag during the early stage, and was largely inhibited by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (200 microm) or GF109203X (GFX) (0.2 microM), but not by ethanol (1%) and wortmannin (100 nM). In contrast, O2- generation of CB-pretreated neutrophils by PMA-stimulation did not show a lag, but was less than that of intact cells, and was only minimally depressed by the above inhibitors, but was markedly depressed by the simultaneous addition of GFX and ethanol or GFX and wortmannin. Although translocation of p47phox and p67phox to the membrane fraction by PMA-stimulation of intact and GCSF-treated neutrophils occurred in parallel with O2- production, that of CB-treated neutrophils by PMA-stimulation was not always proportional to O2- production. These findings suggest that pretreatment of neutrophils with CB dramatically alters the PMA response of the cells; that is, the protein kinase C-dependent pathway is largely depressed, and a phospholipase D-dependent one for NADPH oxidase activation appears in CB-treated cells.

Characterization of cellular response to thiol‐modified gold surfaces implanted in mouse peritoneal cavity

The early inflammatory reaction in vivo to three well defined surfaces—gold, gold coated with glutathione (GSH), and 3-mercapto-1,2-propanediol (MG)—was assessed as manifested by the adherence and activation of inflammatory cells during implantation intraperitoneally in mice. Evaluation of cell adhesion and activation was done by immunohistochemistry using specific monoclonal antibodies directed against cell differentiation antigens CD11b/CD18, CD74, and CD25 or by measurement by chemoluminescence of reactive oxygen radical species produced by adhering cells. Cell recruitment and activation was slow on the GSH-coated gold surfaces. These surfaces also had the highest percentage of adhering cells with an intact cell membrane. The MG-coated surfaces, on the other hand, rapidly recruited and activated cells and also caused cell membrane leakage to propidium iodide, suggesting cell membrane damage or cell death. The respiratory burst of adhering cells was stimulated by phorbol-myristate acetate on the GSH-coated surface but not on the MG-coated surface and by opsonized zymosan on the Mg-coated surface but only to a small degree on the GSH-coated surface. The respiratory burst following zymosan activation of cells adhering to the MG-coated surface was inhibited by treatment with 2.3-diphosphoglycerate, a phospholipase D inhibitor. The presented data suggest that peritoneal leukocytes adhering to foreign materials may raise a respiratory burst response via a phospholipase D-dependent and protein kinase C-independent pathway. © 1999 John Wiley & Sons, Inc. J Biomed Mater Res, 45, 117–124, 1999.

Characterization of cellular response to thiol-modified gold surfaces implanted in mouse peritoneal cavity.

The early inflammatory reaction in vivo to three well defined surfaces-gold, gold coated with glutathione (GSH), and 3-mercapto-1, 2-propanediol (MG)-was assessed as manifested by the adherence and activation of inflammatory cells during implantation intraperitoneally in mice. Evaluation of cell adhesion and activation was done by immunohistochemistry using specific monoclonal antibodies directed against cell differentiation antigens CD11b/CD18, CD74, and CD25 or by measurement by chemoluminescence of reactive oxygen radical species produced by adhering cells. Cell recruitment and activation was slow on the GSH-coated gold surfaces. These surfaces also had the highest percentage of adhering cells with an intact cell membrane. The MG-coated surfaces, on the other hand, rapidly recruited and activated cells and also caused cell membrane leakage to propidium iodide, suggesting cell membrane damage or cell death. The respiratory burst of adhering cells was stimulated by phorbol-myristate acetate on the GSH-coated surface but not on the MG-coated surface and by opsonized zymosan on the Mg-coated surface but only to a small degree on the GSH-coated surface. The respiratory burst following zymosan activation of cells adhering to the MG-coated surface was inhibited by treatment with 2. 3-diphosphoglycerate, a phospholipase D inhibitor. The presented data suggest that peritoneal leukocytes adhering to foreign materials may raise a respiratory burst response via a phospholipase D-dependent and protein kinase C-independent pathway.

Transforming G Protein-Coupled Receptors Block Insulin andras-Induced Adipocytic Differentiation in 3T3-L1 Cells: Evidence for a PKC and MAP Kinase Independent Pathway

We have used the expression of muscarinic m1 receptors in the preadipocytic 3T3-L1 cell line for dissecting the nature of the G protein-linked pathways governing adipocytic differentiation, a complex process controlled by many stimuli and their downstream targets. 3T3-L1 cells can be differentiated by insulin or byrasoncogenes, and MAP kinase has been implicated in this process. However, m1 stimulation failed to induce differentiation of 3T3-L1 cells. Furthermore, it prevented insulin orv-ras-induced adipocytic differentiation, utilizing a protein kinase C-independent pathway. m1 stimulation did not alter the phosphorylation state of the insulin receptor substrates IRS-1 and SHC, nor the recruitment of Grb-2. Interestingly, whereas m1 receptors potently activated MAP kinase, another differentiation-inhibitor, TNF α, did not affect it. These results suggest that the control of adipocytic differentiation can occur utilizing a biochemical route independent of protein kinase C, and acting downstream, or independently from the Ras-MAP kinase pathway.

Indication of a Protein Kinase C-Independent Pathway for NADPH Oxidase Activation in Human Neutrophils

A potent tyrosine phosphatase inhibitor, pervanadate, induced (i) translocation of the cytosolic NADPH oxidase factors, p47-phoxand p67-phox,to the plasma membrane; and (ii) O−2production in human neutrophils. However, the translocation of p47-phoxand p67-phoxwas inhibited by H-7, a protein kinase C (PKC) inhibitor without markedly affecting O−2production in whole neutrophils. Results from the plasma membrane fraction showed that NADPH oxidase activity in neutrophils treated with pervanadate did not vary in the presence or absence of H-7, despite a lower content of p47-phoxand p67-phoxin H-7-treated neutrophils. These findings suggest that in addition to the well-known PKC-dependent pathway, there may exist another PKC-independent pathway to activate NADPH oxidase in human neutrophils. This pathway involves protein tyrosine phosphorylation but does not seem to necessitate translocation of p47-phoxand p67-phoxto the plasma membrane.

Bombesin and neuromedin B stimulate the activation of p42(mapk) and p74(raf-1) via a protein kinase C-independent pathway in Rat-1 cells.

The mechanisms by which seven transmembrane receptors activate p42-(mapk)/p44(mapk) are not well defined although p21ras- and protein kinase C (PKC)-dependent pathways have been implicated, typically for Gi- and Gq-coupled receptors, respectively. Here, we demonstrate that in Rat-1 cells transfected with the Gq-coupled bombesin/gastrin releasing peptide receptor, bombesin stimulated activation of p42(mapk) that was not inhibited by the specific PKC inhibitor GF 109203X or by down regulation of phorbol ester-sensitive PKC isoforms. In addition, bombesin rapidly stimulated p74(raf-1) activity that was also independent of PKC activity and insensitive to inhibition by pertussis toxin. Furthermore, addition of neuromedin B to Rat-1 cells transfected with the neuromedin B preferring receptor also activated p42(mapk) and p74(raf-1) in a PKC-independent and pertussis toxin-insensitive manner. Finally we show that addition of bombesin to Rat-1 cells stimulated the GTP loading of p21ras. Our results reveal a novel PKC-independent pathway in the action of Gq-coupled receptors and stress the importance of cell context in defining the signal transduction pathway(s) that link specific receptors to the activation of the mitogen-activated protein kinase cascade.

Tyrosine dephosphorylation of glycogen synthase kinase-3 is involved in its extracellular signal-dependent inactivation

We examined whether extracellular signals regulate glycogen synthase kinase-3 (GSK-3) activity through tyrosine dephosphorylation of GSK-3. In resting Chinese hamster ovary cells overexpressing the human insulin receptor (CHO-IR cells), GSK-3 was tyrosine-phosphorylated and active. Insulin and 12- O-tetradecanoylphorbol 13-acetate (TPA) induced inactivation and tyrosine dephosphorylation of GSK-3. It is known that Ser-9 of GSK-3β is phosphorylated in response to insulin and that the phosphorylation of this amino acid residue causes inactivation of GSK-3β. However, the ectopically expressed GSK-3β Δ9, in which the N-terminal nine amino acids of GSK-3β were deleted, was still inactivated and tyrosine-dephosphorylated in response to insulin. Protein phosphatase 2A treatment partially reversed insulin-induced GSK-3β inactivation, but did not change GSK-β Δ9 inactivation. In CHO-IR cells where protein kinase C was down-regulated, TPA neither inactivated nor tyrosine-dephosphorylated GSK-3. However, insulin inactivated and tyrosine-dephosphorylated GSK-3, although to a lesser degree than in the control cells. These results suggest that in addition to serine phosphorylation, tyrosine dephosphorylation of GSK-3 is also important for the regulation of GSK-3 activity in response to extracellular signals and that insulin regulates GSK-3 activity through both protein kinase C-dependent as well as protein kinase C-independent pathways.