ADP-ribosylation factor proteins mediate agonist-induced activation of phospholipase D

The role of small G proteins of the ADP-ribosylation factor (ARF) and Rho families on the activation of phospholipase D (PLD) by platelet-derived growth factor (PDGF) and phorbol esters (PMA) has been investigated. The activation of PLD by PDGF and PMA was blocked by brefeldin A (BFA), an inhibitor of ARF activation, but not by Clostridium botulinum C3 exotoxin, an inhibitor of the activity of Rho. PDGF and PMA, in the presence of GTPgammaS, promoted the association of ARF and RhoA with cell membranes. Cells depleted of ARF and Rho by digitonin permeabilization showed a significant reduction of the activity of phospholipase D. Recombinant ARF was sufficient to restore agonist-induced PLD activity to digitonin-permeabilized, cytoplasm-depleted cells. In contrast, isoprenylated recombinant RhoA had no effects in this reconstitution assay. HIRcB cells were transiently transfected with wild-type and dominant-negative mutants of ARF1 and ARF6. Neither wt-ARF1 nor wt-ARF6 had any effects on agonist-dependent PLD activity. However, dominant-negative ARF1 and ARF6 mutants blocked the stimulation of PLD by PDGF but only partially inhibited the effects of PMA. These results demonstrate that ARF rather than Rho proteins mediate the activation of PLD by PDGF and phorbol esters in HIRcB fibroblasts.     

Increased prevalence of luteinizing hormone beta-subunit variant in Japanese infertility patients

To determine the frequency of a common luteinizing hormone variant in a Japanese population and to evaluate its significance in infertility, serum samples were collected from 169 healthy non-pregnant Japanese women, 105 healthy adult Japanese men and 97 female Japanese infertility patients. The luteinizing hormone variant includes two point mutations in the beta-subunit gene (Trp8 to Arg8 and Ile15 to Thr15). DNA from blood cells was studied in 10 healthy women, 10 men and five patients using polymerase chain reaction and direct sequencing. In immunoassays, results with a monoclonal antibody recognizing only the wild-type hormone and a polyclonal antibody recognizing the variant as well were compared as a ratio; ratios in heterozygotes and in individuals with only wild-type alleles ranged from 0.19 to 0.50 and from 0.56 to 1.21, respectively, and 0.50 was considered a 'cut-off' value for identifying individuals with the variant. For the larger subject groups, the frequency of the variant was 9.5% in normals. The mean ratio (0.80 +/- 0.35) in infertility patients was significantly lower (P < 0.01) than in healthy women (1.09 +/- 0.56), and the variant occurred more frequently in infertility patients (16.5%) than in healthy women (8.3%; P < 0.05). The variant was more frequent in patients with ovulatory disorders (43.8%) than other patients (16.0%; P < 0.05).     

A comparison of phorbol myristate acetate with phytohemgglutinin as a stimulator of in vitro colony growth of peripheral human leucocytes

Peripheral blood mononuclear cells from normal individuals were cultured in soft agar (0.3%) with either phytohaemagglutinin (PHA) or the tumor promoter phorbol myristate acetate (PMA). PMA was found to induce colony growth under these conditions as well as that achieved by PHA. The colonies from PHA stimulation were entirely lymphoid but with PMA the type of cell was more varied with some cells that appeared to be of the granulocytic-macrophage line. PMA thus appears to have the ability to promote growth in cells other than lymphocytes in the peripheral blood under these conditions.     

Activation of protein kinase C increases neuronal excitability by regulating persistent Na+ current in mouse neocortical slices

Effects of the protein kinase C activating phorbol ester, phorbol 12-myristate 13-acetate (PMA), were studied in whole cell recordings from layer V neurons in slices of mouse somatosensory neocortex. PMA was applied intracellularly (100 nM to 1 microM) to restrict its action to the cell under study. In current-clamp recordings, it enhanced neuronal excitability by inducing a 10- to 20-mV decrease in voltage threshold for action-potential generation. Because spike threshold in neocortical neurons critically depends on the properties of persistent Na+ current (INaP), effects of PMA on this current were studied in voltage clamp. After blocking K+ and Ca2+ currents, INaP was revealed by applying slow depolarizing voltage ramps from -70 to 0 mV. Intracellular PMA induced a decrease in INaP at very depolarized membrane potentials. It also shifted activation of INaP in the hyperpolarizing direction, however, such that there was a significant increase in persistent inward current at potentials more negative than -45 mV. When tetrodotoxin (TTX) was added to the bath, blocking INaP and leaving only an outward nonspecific cationic current (Icat), PMA had no apparent effect on responses to voltage ramps. Thus PMA did not affect Icat, and it did not induce any additional current. Intracellular application of the inactive PMA analogue, 4 alpha-PMA, did not affect INaP. The specific protein kinase C inhibitors, chelerythrine (20 microM) and calphostin C (10 microM), blocked the effect of PMA on INaP. The data suggest that PMA enhances neuronal excitability via a protein kinase C-mediated increase in INaP at functionally critical subthreshold voltages. This novel effect would modulate all neuronal functions that are influenced by INaP, including synaptic integration and active backpropagation of action potential from the soma into the dendrites.     

Evaluating the options for developing databases to support research-based medicine at the NHS Centre for Reviews and Dissemination

The internalisation of metabotropic glutamate receptor (mGluR1alpha) and its splice variant (mGluR1beta), in response to agonist and phorbol esters (PMA), has been studied. Both mGluR1alpha and mGluR1beta exhibit a similar rate of internalisation following PMA treatment, with a shift in their distribution from plasma membrane to endosome-enriched membrane fractions. Agonist challenge however caused a rapid loss, within 5-10 min, of mGluR1beta but not mGluR1alpha from the cell surface. These results show that the two forms of mGluR1 show different internalisation responses to agonist and suggest that the C-terminal region of the molecule plays an important role in this phenomenon.     

Involvement of protein kinase C in agonist-stimulated goldfish ovulation

The effects of two protein kinase C (PKC) inhibitors, calphostin C and staurosporine, on the in vitro ovulation of goldfish (Carassius auratus) oocytes were investigated. Ovulation was stimulated by prostaglandin (PG) F2 alpha (PGF2 alpha, 2.0 micrograms/ml), by sodium orthovanadate (0.1 mM), by a combination of the phorbol ester phorbol 12-myristate-13-acetate (PMA, 0.1 micrograms/ml) and calcium ionophore A23187 (0.05 micrograms/ml), by thapsigargin (0.2 micrograms/ml), and by elevated pH (8.1). In addition, the effects of these inhibitors on the PKC activity of the goldfish follicle wall was determined by use of a specific peptide substrate phosphorylation assay. At 0.1 microM, staurosporine significantly blocked ovulation induced by all agents. However, at lower (0.01 microM) levels it blocked only PMA/A23187-induced ovulation. In contrast, calphostin significantly blocked only PMA/A23187-induced ovulation, although there was a decrease in pH-induced ovulation at lower calphostin concentrations. Both calphostin and staurosporine blocked follicular PKC activity at levels that were inhibitory to ovulation. In addition, staurosporine significantly blocked PKC activity at levels even lower than those needed to block ovulation. The combined results suggest that orthovanadate, PGF2 alpha, and thapsigargin do not require PKC activation for the induction of ovulation, whereas PMA/A23187 does.     

P-glycoprotein-independent decrease in drug accumulation by phorbol ester treatment of tumor cells

The effect of a change in the phosphorylation state of the drug transporter P-glycoprotein (P-gp) on its drug transport activity was studied for the substrates daunorubicin (DNR), etoposide (VP-16), and calcein acetoxymethyl ester (Cal-AM). Phorbol ester (PMA), added to stimulate phosphorylation of P-gp by protein kinase C (PKC), caused a decrease in the cellular accumulation of DNR and VP-16, both in multidrug-resistant (MDR) P-gp-overexpressing cells and in wild-type cells. Since treatment of cells with kinase inhibitor staurosporine (ST) reversed this effect of PMA and the non-PKC-stimulating phorbol ester 4alpha-phorbol, 12,13-didecanoate (4alphaPDD) did not result in a decreased DNR accumulation, we conclude that this effect is the result of kinase activity. The concentration dependence of the inhibition of P-gp by verapamil (Vp) was not influenced by PMA. Accumulation of the P-gp substrate Cal-AM was not influenced by PMA in wild-type cells. Therefore, Cal-AM was used to study the effect of PMA-induced phosphorylation of P-gp on its transport activity. Activation of PKC with PMA or inhibition of protein phosphatase 1/2A (PP1/PP2A) with okadaic acid (OA) did not affect the accumulation of Cal-AM in the MDR cells or wild-type cells. The kinase inhibitor ST increased the Cal-AM accumulation only in the MDR cells. Neither stimulating PKC with PMA nor inhibiting PP1/PP2A with OA led to a decreased inhibition of P-gp by ST, indicating that ST inhibits P-gp directly. From these experiments, we conclude that PKC and PP1/PP2A activity do not regulate the drug transport activity of P-gp. However, these studies provide evidence that PMA-induced PKC activity decreases cellular drug accumulation in a P-gp-independent manner.     

PMA inhibits both spontaneous and glucocorticoid-mediated leptin secretion by human omental adipose tissue explants in vitro

The activation of PKC by the acute administration of the phorbol ester PMA (1 microM, 2h) to omental adipose tissue explants in vitro resulted in a marked (about 75%) and persistent (up to at least 96 h) inhibition of leptin secretion. This PKC-mediated inhibition was not observed after the administration of an inactive phorbol ester (phorbol 12,13-dicecanoate). The inhibition by PMA of leptin secretion was not restricted to the spontaneous secretion, but blocked also effectively the leptin response to a powerful stimulus, such as the glucocorticoid dexamethasone. As the PKC activity has been shown to be elevated during fasting, the negative relation here described between PKC activity and leptin secretion could be of physiological relevance.     

Cyclosporin A inhibits the decrease of CD4/CD8 expression induced by protein kinase C activation

Cyclosporin A (CsA) is a powerful immunosuppressive drug widely used in transplantation medicine. A major effect of CsA is inhibition of the differentiation of immature double-positive (DP) CD4+ CD8+ thymocytes into mature single-positive (SP) CD4+ CD8- or CD4- CD8+ thymocytes. The mechanisms underlying the changes in CD4/CD8 expression during normal differentiation of thymocytes and the way CsA interferes with this differentiation process are still unknown. Here we show that protein kinase C (PKC) activation by phorbol 12-myristate 13-acetate (PMA) causes a decrease of both CD4 and CD8 expression at the cell surface level and at the mRNA level in a CD4+ CD8+ T cell line and in freshly isolated thymocytes. A PKC inhibitor, staurosporin, interferes with the differentiation from DP to SP in fetal thymus organ culture system. These data suggest that the alternation of CD4/CD8 expression from DP to SP is dependent on PKC activation. CsA blocks this decrease of CD4/CD8 expression by PMA in vitro. Moreover, this PMA effect is also blocked by treatment with cycloheximide. These results suggest that the reduction of CD4/CD8 expression requires de novo synthesis of a protein(s) induced in response to a signal conveyed by activated PKC. CsA may block the transition from DP to SP by inhibition of CD4/CD8 down-regulation induced by PKC activation.     

Activation of protein kinase C pathway by phorbol ester results in a proteoglycan synthesis increase in peritubular cells from immature rat testis

In cultured peritubular cells (PT) from rat testis, protein kinase C (PKC) was activated by phorbol 12-myristate 13-acetate (PMA). PMA enhanced the synthesis of proteoglycans (PG) and to a lesser extent their catabolism; the stimulation of the synthesis appeared to be due to an increase in PG protein moiety production and, at the same time, to an increase in the glycanation process as revealed by the use of an exogenous acceptor, p-nitrophenyl-beta-d-xyloside. In the presence of PMA, the molecular weight of neosynthesized PG and the length of their constitutive glycosaminoglycan chains were not modified. Moreover, the distribution of proteochondroitin sulfate and proteoheparan sulfate in medium and in cell layer remained unchanged. However, PMA reduced the sulfation level of chondroitin sulfate and heparan sulfate chains, suggesting that PKC activation resulted in an independent modulation of the sugar chain formation and of the sulfate residue transfer. PMA effect on the synthesis of hyaluronan was also determined: PMA dramatically enhanced its production by PT cells.     

Differential expression of human histone deacetylase mRNAs in response to immune cell apoptosis induction by trichostatin A and butyrate

Hepatocellular mitogen (HGF and EGF) inhibited lipopolysaccharide and cytokine mixture (referred as LPS/CM)-induced NO synthesis and cellular injury in hepatocytes. Mitogenic inhibitors such as hydroxyurea and Wortmannin could not reverse EGF or HGF-inhibited NO production, whereas both of them showed some inhibitory effect on hepatocyte NO synthesis. Although activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) had no effect on hepatocyte NO synthesis, deletion of PKC activity by long-term treatment of hepatocytes with PMA abolished LPS/CM-induced NO production. In addition, pretreatment of hepatocytes with HGF and EGF also blocked LPS/CM-induced NO synthesis in the hepatocyte. These results suggest that proliferating signal is not directly involved in mitogen-inhibited NO synthesis in the hepatocyte, and LPS/CM-mediated NO synthesis is associated with the metabolic/redox state of hepatocytes.     

Stimulatory and inhibitory effects of a phorbol ester on chloride secretion by rat epididymal epithelium

The effects of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), on Cl- secretion by rat cauda epididymal epithelium were studied through use of the short-circuit current (Isc) technique. PMA alone could stimulate the Isc in a dose-dependent manner. The PMA-induced Isc was blocked by the Cl channel blocker, diphenylamine-2-carboxylate, but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. PMA also exerted an inhibitory effect on the subsequent Ca(2+)-activated Isc. ATP or ionomycin-induced Isc was significantly reduced by treatment with PMA (5-10 min). Both stimulatory and inhibitory effects of PMA could be mimicked by a diacylglycerol analog, 1,2-dioctanoyl-sn-glycerol, but not an inactive analog of PMA, 4 alpha-phorbol 12,13-didecanote (4 alpha D). Down-regulation of protein PKC by prolonged treatment of epididymal cells with PMA (12 h) diminished both stimulatory and inhibitory effects of PMA on Isc. These results suggest that the dual effect of PMA on Isc was mediated by PKC. However, the PKC inhibitor, calphostin C, could block the inhibitory effect of PMA on ATP-induced Isc but not the stimulatory effect of PMA alone on Isc. The stimulatory effect of PMA was apparent only when PMA was applied to the apical aspect; in contrast, the inhibitory effect of PMA on ATP-induced Isc was readily seen with application of PMA to either side of the epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells

Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.     

Binding profile of the novel 5-HT1B/1D receptor antagonist, [3H]GR 125,743, in guinea-pig brain: a comparison with [3H]5-carboxamidotryptamine

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M3 subtype. Acute exposure to phorbol esters (1 microM phorbol 12,13-dibutyrate, PDB, or 0.1 microM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 microM) and H7 [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 microM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [3H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Parathyroid hormone-related protein increases DNA synthesis in proximal tubule cells by cyclic AMP- and protein kinase C-dependent pathways

The present study was performed to characterize the possible involvement of cAMP synthesis and protein kinase C (PKC) activation in the DNA synthesis-stimulating effect of parathyroid hormone-related protein (PTHrP) in proximal tubule cells. We found that DNA synthesis was stimulated by 10 microM 8BrcAMP, and 1 microM Sp-cDBIMPS, two cAMP analogs, and also by 1 microM phorbol 12-myristate 13-acetate (PMA) and 100 microM 1,2-dioctanoyl-sn-glycerol, two PKC activators, and 10 nM [Cys23] human (h)PTHrP (24-35) amide in rabbit proximal tubule cells (PTC). Both Sp-cDBIMPS and PMA, at 1 microM, also increased DNA synthesis in SV40-immortalized mouse proximal tubule cells MCT. Human PTHrP (7-34) amide [PTHrP (7-34)] dose dependently stimulated DNA synthesis in a similar manner as [34Tyr]PTHrP (1-34) amide [PTHrP (1-34)], in PTC. PMA pre-treatment for 20 h, which downregulates PKC, completely blocked the effect induced by PTHrP (7-34), but not that of PTHrP (1-34), in the latter cells. In contrast, the same PMA pre-treatment abolished the DNA synthesis stimulation by PTHrP (1-34) and PTHrP (7-34) in MCT cells, which appear to have PTH receptors mainly coupled to phospholipase C and not adenylate cyclase. Our results indicate that the stimulatory effect of PTHrP on DNA synthesis in proximal tubule cells is mediated by a cAMP- and PKC-dependent mechanism.     

Adhesion-dependent degranulation of neutrophils requires the Src family kinases Fgr and Hck

Polymorphonuclear neutrophils (PMN) adherent to integrin ligands respond to inflammatory mediators by reorganizing their cytoskeleton and releasing reactive oxygen intermediates. As Src family tyrosine kinases are implicated in these responses, we investigated their possible role in regulating degranulation. Human PMN incubated on fibrinogen released lactoferrin in response to TNF-alpha and this response was inhibited by PP1, a Src family tyrosine kinase inhibitor. This drug had no effect on lactoferrin secretion induced by PMA, an adhesion-independent agonist of PMN degranulation. However, PP1 blocked secretion in PMN plated on plain tissue culture plastic, a surface inducing PMN spreading in the absence of any stimulus. Double knockout hck-/- fgr-/- PMN adherent to collagen or fibrinogen failed to release lactoferrin in response to TNF-alpha but responded to PMA as wild-type PMN. Degranulation induced by spreading over tissue culture plastic was also defective in hck-/- fgr-/- PMN. Defective adhesion-dependent degranulation required the absence of both kinases, because single knockout fgr-/- or hck-/- PMN responded as wild-type cells. Analysis of lactoferrin secretion in hck-/- fgr-/- or PP1-treated, suspended PMN showed that Src kinases are not implicated in degranulation dependent on activation of protein kinase C or increase in intracellular free Ca2+ but may play a role in the response to FMLP of cytochalasin B-treated PMN. These findings identify a role for Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and suggest that Fgr and Hck would be targets for pharmacological control of adhesion-dependent degranulation in the inflammatory site.     

Visual hallucinations associated with Parkinson disease

Lysophosphatidic acid (LPA, 1-acyl-sn-glycerol 3-phosphate), at a concentration of 1-40 microM, was found to induce the formation of [3H]inositol-labelled phosphatidylinositol-4-phosphate (PIP) without significantly altering the levels of either phosphatidylinositol (PI) or phosphatidylinositol bisphosphate (PIP2) in washed human platelets. Preincubation of platelets with the cyclooxygenase/lipoxygenase inhibitor, BW755C at 100 microM, did not alter the LPA-induced formation of PIP. Activation of platelets with the phorbol ester, phorbol 12-myristate 13-acetate (PMA), elicited a similar response (induction of PIP formation). The specific protein kinase C (PKC) inhibitor, GF109203X (10 microM), completely blocked the effect of PMA but not the LPA-induced generation of PIP. The present results indicate that LPA can induce PIP formation via PI-4-kinase activation, through processes which are independent of the eicosanoid/TxA2 pathway and are not PKC-dependent.     

Gene-polymorphisms of angiotensin converting enzyme and endothelial nitric oxide synthase in patients with primary glomerulonephritis

The human pre-B acute lymphoblastic leukemia cell line REH6 was utilized for characterization of CD45 glycoprotein by monoclonal antibodies (mAb) recognizing four distinct CD45 antigen specificities, i.e. nonrestricted CD45, restricted CD45RA, CD45RB and CD45R0. Immunoprecipitation revealed two antigen specificities on REH6 cells of m.w. 220 kDa and 190 kDa, both presenting wide range of isoelectric point pI approximately 6.0-7.5. Nonrestricted CD45 epitopes were not affected by the sialyl acid cleavage with sodium metaperiodate or neuraminidase, but were sensitive to both, tunicamycin, the N-glycosylation inhibitor and monensin, an inhibitor of protein transport through the Golgi compartment. O-sialoglycoprotein endopeptidase from Pasteurella haemolytica A1 partially cleaved CD45RA and CD45RB epitopes, while nonrestricted CD45 determinants were not affected by this enzyme. Limited proteolysis of this antigen resulted in the appearance of 160-180 kDa peptide domains which retained CD45 epitopes. Further, the treatment of cells with phorbol myristate acetate (PMA) induced marked down-regulation of 220 and 190 kDa isoforms and the appearance of new 210, 180 and 170 kDa variant glycoprotein forms which were not found on parental cells. This PMA effect was not accompanied by the programmed cell death and was markedly blocked by a nonselective protein kinase (PK) inhibitor isoquinoline sulfonamide H7. Modulation of CD45 by phorbol esters might serve as an in vitro model for an additional insight into the function of CD45 in hematopoietic cells.     

Morphine inhibits signal transduction subsequent to activation of protein kinase C in mouse splenocytes

Morphine administered as a subcutaneous implant was previously reported to inhibit the mitogen-induced initial increases in cytoplasmic free calcium concentrations ([Ca2+]i) in mouse splenocytes. The present studies were initiated to determine whether morphine affects signal transduction subsequent to activation of protein kinase C (PKC) in immune cells. Administration of morphine significantly inhibited the phorbol myristate acetate (PMA)-stimulated increase in interleukin-2 receptor (IL-2R) expression in both CD4+ and CD8+ mouse T cells. In contrast, morphine treatment had no effect on PMA/calcium ionophore (A23187)-induced increase in IL-2 secretion, suggesting a selective inhibition of IL-2R expression. Simultaneous administration of morphine and the opiate antagonist naltrexone blocked the effect of morphine on CD4+ cells. The inhibition of PMA-stimulated IL-2R expression was not reproduced by incubating splenocytes with morphine (10(-8)-10(-5) M). These results suggest that this effect of morphine was mediated through opiate-receptors, but not directly via opiate receptors located on T cells. Moreover, adrenalectomy abolished this effect of morphine in CD4+ but not CD8+ T cells, suggesting that the inhibitory effect of morphine on IL-2R expression in CD4+ T cells may be mediated through a morphine-induced increase in corticosteroid levels. Thus, opiate-induced immunosuppression may involve an inhibition of post-PKC events, especially IL-2R expression, as well as impairment of earlier events in the activation of immune cells such as the increase in [Ca2+]i.     

Calcium- and calmodulin-dependent PMA-activation of the CD44 adhesion molecule

The ability of the CD44 adhesion molecule to interact with its ligand hyaluronic acid (HA) is tightly regulated. CD44-positive mouse LB lymphoma cells are unable to bind HA unless activated by the tumor promoter phorbol 12-myristate 13-acetate (PMA). PMA causes a dose-dependent increase in both CD44 expression level and HA-binding capacity, with the binding of HA observed only above a threshold amount of CD44 molecules. This induction of HA-binding as well as the increase in CD44 expression are prevented by cycloheximide, suggesting a requirement for new additional CD44 molecules on the cell surface and/or cooperating proteins. In the present study, we have investigated which of the signal transduction pathways activated by PMA leads to the increased CD44 expression with subsequent acquisition of HA-binding capacity. By comparing the influence of each inhibitory agent on PMA-activated LB lymphoma cells versus that on a constitutive HA-binder cell line derived from LB cells (designated HA9 cells), we could distinguish between an effect on the PMA-activation phase and a one on the HA-binding phase. Our data show that the PMA-induced HA-binding could not be blocked by agents inhibiting protein kinase C (PKC) (staurosporine, sphingosine, polymyxin B, quercetin) or genestein, an inhibitor of tyrosine protein kinases. However, this PMA response was strongly inhibited by calmodulin antagonists (chlorpromazine, trifluoperazine, W-7) and the calcium blocker verapamil. The calmodulin antagonists inhibited the PMA-induced increase in CD44 expression on LB cells, but had no influence on the ability of the constitutive HA-binder HA9 cell line to interact with HA, indicating an effect on the PMA induction phase rather than on the binding itself. Verapamil also blocked the PMA-induced increase in CD44 expression on LB cells, but in addition it slightly reduced the ability of the HA9 cells to bind HA without affecting their CD44 expression level. In conclusion, our data suggest that CD44 activation by PMA is calcium and calmodulin dependent, rather than mediated by protein kinase C.