Pulmonary alveolar macrophage function during acute inflammatory lung injury

Pulmonary alveolar macrophages (PAM) are present during acute lung inflammation, yet the functional role of these cells in both the initiation and resolution of lung injury is not well defined. To better understand the relationship between PAM functional responses and the evolution of acute reversible lung injury, we examined the ability of both unstimulated and stimulated (PMA, zymosan) PAM to secrete reactive oxygen metabolites (superoxide anion O2-) and lysosomal enzymes (lysozyme, N-acetyl-B-D-glucosaminidase) at specific time points (0, 6, 12, 24, 48, and 72 h) after initiation of acute lung injury via reverse passive Arthus reaction in pathogen-free Sprague-Dawley rats. After acute lung injury, stimulated PAM produced increasing amounts of O2- compared with PAM from noninjured lungs. Maximal O2- production by PAM occurred at 24 h after lung injury, at which time a 3.5-fold and 50% increase in O2- production by PAM was observed when PAM were stimulated with PMA and zymosan, respectively. The amount of O2- generated by these cells slowly decreased during the next 48 h. Enhanced generation of O2- by PAM from injured lungs was not due to altered enzymatic activity of the O2--producing NADPH oxidase, nor was it due to an absolute increase in the NADPH oxidase in "activated" PAM. These observations suggest that increased O2- generation by PAM from injured lungs is due to enhancement of mechanisms responsible for induction of oxidase activity. In addition, a differential accumulation and secretion of lysozyme and N-acetyl-B-D-glucosaminidase activity by PAM was observed after acute lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of protein kinase-C in regulation of insulin-like growth factor-binding protein-1 production by HepG2 cells.

Insulin-like growth factor binding protein-1 (IGFBP-1) is a liver-derived protein that modulates the mitogenic actions of the insulin-like growth factors (IGFs). IGFBP-1 production is potently inhibited by insulin both in vivo and in HepG2 human hepatoma cells. To further define the pathways of IGFBP-1 regulation, we studied the effects of modulators of protein kinase-C (PKC) on HepG2 cell IGFBP-1 production. Phorbol 12-myristate 13-acetate (PMA) stimulated IGFBP-1 production in a time- and dose-dependent manner, with maximal stimulation occurring at 10-100 nmol/L. The degree of stimulation was dependent on cell density, ranging from about 2-fold in confluent to more than 10-fold in sparse cultures. Preincubation with PMA abolished the inhibitory effect of insulin, while preincubation with insulin did not inhibit PMA stimulation. The transient PKC activator diC8 had no effect, while studies with the PKC inhibitors sphinganine and H-7 were limited by solvent vehicle cytotoxicity. Staurosporine (STS), a potent PKC inhibitor, stimulated IGFBP-1 production 2- to 4-fold and augmented the stimulatory effect of PMA. Concanavalin-A, an inhibitor of PMA-stimulated PKC translocation and down-regulation, inhibited the effects of PMA and STS. Our findings indicate that PKC is involved in the regulation of hepatic IGFBP-1 production. The effects of PMA, which causes rapid activation, followed by membrane translocation and down-regulation of PKC, are similar to those of STS and are countered by Concanavalin-A. These data suggest that PKC activity may mediate tonic inhibition of IGFBP-1 production, while PKC downregulation stimulates the production of this regulatory protein.     

Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose- dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose- dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the "translocation" of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.     

Evidence for participation of vicinal dithiols in the activation sequence of the respiratory burst of human neutrophils

Phenylarsine oxide (PAO) specifically forms a stable ring complex with vicinal dithiols that can be reversed with 2,3-dimercaptopropanol (DMP). Pretreatment of human neutrophils with micromolar concentrations of PAO inhibited release of superoxide anion (O2-) stimulated by N- formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol 12-myristate 13- acetate (PMA); the inhibition was reversed with DMP, but not with 2- mercaptoethanol. PAO did not affect O2- release in previously stimulated cells. PAO did not affect the FMLP-induced Ca2+ response, suggesting that PAO affects a postreceptor event that does not modulate the Ca2+ transient. Treatment of isolated membrane or cytosolic fractions with PAO did not change the rates of arachidonate-stimulated O2- production in a cell-free system. Pretreatment of unstimulated neutrophils with PAO inactivated cytosolic protein kinase C (PKC); the inactivation was reversed with DMP. However, PAO did not affect PMA- induced translocation of beta-PKC protein or reduce the PKC activity translocated to the membrane. PAO had no effect on tyrosine kinase activity but inactivated phosphotyrosine phosphatase; stimulus-induced tyrosine phosphorylation of several proteins was markedly enhanced. These results suggest that vicinal dithiols play an essential role in activation of the respiratory burst oxidase. Possible sites for the activity of these essential vicinal dithiols include PKC and the regulatory balance of tyrosine phosphatase activity and tyrosine phosphorylation.     

Increased polymorphonuclear leukocyte respiratory burst function in type 2 diabetes

The predisposition to infection and chronic inflammation in diabetes may in part be related to the effects of hyperglycemia or other metabolic abnormality on polymorphonuclear leukocytes (PMN). We evaluated oxidative respiratory burst activity (superoxide production) in non-stimulated and stimulated PMN from 70 stable type 2 Hispanic diabetic patients, as compared to 70 healthy Hispanic individuals without diabetes. The influences of protein kinase C (PKC) inhibitors and certain antibiotics on superoxide production were examined. Both resting and stimulated (PMA, zymosan) PMN from diabetic individuals produced more superoxide than PMN from controls. Inhibitors of PKC, a possible mediator of the augmented respiratory burst activity, decreased superoxide production in all (resting and stimulated) diabetic and control PMN. Azithromycin, which is markedly concentrated by PMN, profoundly inhibited superoxide generation in all groups of diabetic and control cells. PMN from Hispanic diabetic patients produced greater quantities of superoxide than non-diabetic controls. This increased oxidative respiratory burst activity may predispose to infection and chronic inflammation in diabetes. PKC inhibitors and azithromycin inhibited this respiratory burst response. The possible role of PKC (especially PKC beta) as the mediator of this augmented respiratory burst response requires further evaluation, and may lead to therapeutic studies with appropriate inhibitors.     

Human peripheral blood and pleural fluid eosinophils can be induced by immune complexes to release IgG immune complexes and aggregated IgE

The ability of IgG and IgE immune complexes and of phorbol myristate acetate (PMA), a soluble membrane activator, to stimulate hydrogen peroxide (H2O2) release and to induce oxygen radical-mediated cytotoxic activity by human peripheral blood (PBL) eosinophils and by PBL neutrophils was evaluated in normal volunteers and patients with hypereosinophilic malignant pleural effusions due to lung cancer. PMA stimulated a significant respiratory burst. Similar results were obtained with IgG IC stimulation, although the levels of H2O2 were lower. Agg IgE induced H2O2 release only by PBL and PE eosinophils and not by neutrophils. PMA stimulation resulted in detectable cytotoxic activity. IgG IC generated both PBL and PE eosinophil and PBL neutrophil cytotoxicity. Agg IgE induced significant cellular cytotoxicity in both PBL and PE eosinophils. This study suggests that eosinophil oxidative metabolic burst and cytotoxic activity stimulated by IgG and IgE immune complexes could represent a possible mechanism of parenchymal injury in eosinophilic disorders.     

Characteristics of the phagocytically induced respiratory burst in leukocytes from young adult and aged beagle dogs

Phagocytically stimulated canine leukocyte suspensions obtained from 12 young adult and 43 aged individuals were examined for several physiological manifestations of the phagocytically induced respiratory burst. There was considerable variation in levels of oxygen consumption, glucose oxidation via the hexose monophosphate shunt (HMPS), and chemiluminescence response by both resting and phagocytically stimulated leukocytes from different individual animals in each age-group. Leukocyte suspensions from specific individuals in each age-group exhibited a weakly responsive respiratory burst. Chronological age could not be used as a predictor of either the specific (oxygen consumption and HMPS activity) or nonspecific (chemiluminescence response) manifestations of the respiratory burst. The kinetics of the chemiluminescence response were similar for all age-groups. Collectively, the results suggest that there is not an age-related alteration in the phagocytically induced respiratory burst of canine neutrophils and that cells from young adult and aged dogs have a comparable capacity to generate levels of highly reactive antimicrobial oxidizing agents. The increased relative susceptibility of aging dogs to microbial agents is apparently not related to an absent, abbreviated, or reduced leukocyte respiratory burst.     

Monocyte-associated tissue factor is suppressed by phorbol myristate acetate

The monocyte is the only normal circulating cell type capable of initiating blood coagulation through the expression of tissue factor. Recently isolated peripheral blood monocytes that contain no demonstrable tissue factor activity can be induced to express tissue factor activity by a number of stimulatory agents. Monocyte-associated tissue factor activity transiently increases in response to adherence to tissue culture plates and, consistent with other reports, markedly increases after the isolated monocytes are treated with endotoxin. Phorbol myristate acetate (PMA) induced an increase in tissue factor activity at low doses (10(-11) to 10(-12) mol/L). Conversely, concentrations of PMA that stimulate release of oxygen metabolites or that cause the cytosol-to-membrane translocation of protein kinase C (PKC) (10(-9) to 10(-7) mol/L) resulted in a rapid decrease in both adherence-induced and endotoxin-induced monocyte tissue factor activity. The effects of PMA on monocytes were time- and dose-dependent with respect to PKC translocation, release of oxygen metabolites, and changes in tissue factor activity. Immunofluorescent staining of monocytes with monoclonal antibody (MoAb) HTF1-7B8, directed against human tissue factor, revealed that tissue factor antigen was induced concurrently with tissue factor activity by adherence and endotoxin and that tissue factor antigen decreased after PMA stimulation.     

Antimicrobial and respiratory burst characteristics of pulmonary alveolar macrophages recovered from smokers of marijuana alone, smokers of tobacco alone, smokers of marijuana and tobacco, and nonsmokers.

Rodent studies indicate that marijuana smoke can adversely affect the antimicrobial function of pulmonary alveolar macrophages (PAM). To evaluate whether marijuana smoke similarly affects human PAM, we compared phagocytosis, fungistatic/fungicidal activity, and superoxide anion (O2-) production of PAM recovered from marijuana smokers (MS), tobacco smokers (TS), marijuana and tobacco smokers (MTS), and nonsmokers (NS). Although PAM from the four groups were equally capable of ingesting Candida albicans, the macrophages from smokers differed from normal PAM in their ability to restrict intracellular yeast germination (ungerminated candida in smokers' PAM = 68 +/- 3% [46] versus NS = 54 +/- 6% [17], mean +/- SEM [n], p = 0.022). Despite heightened fungistatic activity, PAM from MS and TS destroyed significantly fewer intracellular yeast (28 +/- 2 and 29 +/- 2%, respectively) after 4 h than did macrophages recovered from NS (40 +/- 4%, p less than 0.05). Both basal and stimulated (dihydrocytochalasin B + opsonized zymosan or phorbol myristate acetate) O2- production were similar in PAM from MS and NS, but significantly increased in PAM from TS (p less than 0.05). Our findings indicate that marijuana smoking does not alter the phagocytic behavior or the respiratory burst of human PAM, but marijuana smoking does decrease the ability of human PAM to destroy ingested Candida albicans. These findings contrast with the effects of tobacco smoking, which not only decreases the fungicidal activity of human PAM but also increases their respiratory burst.     

Insulin, oxytocin, and vasopressin stimulate protein kinase C activity in adipocyte plasma membranes.

Incubation of isolated rat adipocytes with insulin, vasopressin, or oxytocin increased plasma membrane-bound protein kinase C (PKC) activity by 100-400%. PKC activity was assayed by a procedure that is virtually background-free, thus permitting assay of protein kinase activity in highly diluted samples of solubilized membranes. Hormone-dependent increases in PKC activity were limited to plasma membranes. Stimulation of the kinase was half-maximal with 70 pM insulin, and the hormone effect was rapid. Oxytocin and vasopressin produced effects on PKC similar to insulin, but the magnitude of the vasopressin stimulation exhibited seasonal variations. Treatment of cells with phorbol 12-myristate 13-acetate (PMA) resulted in a loss of PKC activity from the cytosol and a gain in plasma membrane activity, indicative of translocation of the enzyme. With activity measurements it was not possible to determine if insulin stimulated a translocation of the kinase. However, Western blot analysis of plasma membranes with polyclonal antibodies directed against PKC suggest that at least some of the insulin-stimulated PKC activity resulted from enzyme translocation.     

Ceramide inhibition of phospholipase D and its relationship to RhoA and ARF1 translocation in GTP gamma S-stimulated polymorphonuclear leukocytes

Phospholipase D (PLD) regulates the polymorphonuclear leukocyte (PMN) functions of phagocytosis, degranulation, and oxidant production. Ceramide inhibition of PLD suppresses PMN function. In streptolysin O-permeabilized PMNs, PLD was directly activated by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulation of adenosine diphosphate (ADP)-ribosylation factor (ARF) and Rho, stimulating release of lactoferrin from specific granules of permeabilized PMNs; PLD activation and degranulation were inhibited by C2-ceramide but not dihydro-C2-ceramide. To investigate the mechanism of ceramide's inhibitory effect on PLD, we used a cell-free system to examine PLD activity and translocation from cytosol to plasma membrane of ARF, protein kinase C (PKC)alpha and beta, and RhoA, all of which can activate PLD. GTP gamma S-activated cytosol stimulated PLD activity and translocation of ARF, PKC alpha and beta, and RhoA when recombined with cell membranes. Prior incubation of PMNs with 10 microM C2-ceramide inhibited PLD activity and RhoA translocation, but not ARF1, ARF6, PKC alpha, or PKC beta translocation. However, in intact PMNs stimulated with N-formyl-1-methionyl-1-leucyl-1-phenylalamine (FMLP) or permeabilized PMNs stimulated with GTP gamma S, C2-ceramide did not inhibit RhoA translocation. Exogenous RhoA did not restore ceramide-inhibited PLD activity but bound to membranes despite ceramide treatment. These observations suggest that, although ceramide may affect RhoA in some systems, ceramide inhibits PLD through another mechanism, perhaps related to the ability of ceramide to inhibit phosphatidylinositol-bisphosphate (PIP2) interaction with PLD.     

Regulation of Phosphatidylinositol 4-phosphate 5-kinase by Protein Kinase C in Human Platelet Membranes

Phorbol myristate acetate (PM A) increased the formation of |(32)P | PI 4,5-P, in (32)P-prelabeled human platelet. In saponin-permeabilized platelets, in which (32)P from exogenous |γ-(32)P| ATP was incorporated into PI 4-P and PI 4,5-P(2), addition of 10 nM PMA resulted in increased formation of |(32)P|PI 4,5-P(2) and |(32)P|PI 4-P. In order to distinguish whether increased [(32)P]PI 4,5-P(2) formation by PMA reflected merely an increase of [(32)P]PI 4-P, the substrate for PI 4-P 5-kinase, or activation of PI 4-P Skinase, we examined the membrane fraction in which most of the kinase activity was located. Although PMA itself did not affect the PI 4-P 5-kinase activity in the control membranes, the kinase activity was increased nearly 2-fold in membranes pretreated with 10 nM PMA but not 4α-phorbol didecanoate which does not activate protein kinase C (PKC). These results suggested that membrane PI 4-P 5-kinase activity was stimulated by the activation of PKC. However, 100 nM PMA did not stimulate [(32)P]PI 4,s-P(2) formation in saponin-permeabilized platelets, and the PI 4-P 5-kinase activity in membranes from platelets pretreated with 100 nM PMA was almost the same as that in control membranes. This can be explained by product inhibition, since PI 4,5-P(2) inhibited concentration-dependently the membrane PI 4-P 5-kinase activity. The Ca(2+) -dependent PKC fraction partially purified from the platelet cytosol stimulated the membrane PI 4-P 5-kinase activity, whereas the Ca(2)'-independent PKC fraction inhibited the kinase activity. Taken together, the present results suggest that the platelet membrane PI 4-P 5-kinase activity is stimulated by Ca(2+) -dependent PKC (cPKC) and is negatively regulated by PI 4,s-p(2) and Ca(2+) -independent PKC(nPKC).     

N-acetylcysteine effect on the luminol-dependent chemiluminescence pattern of reactive oxygen species generation by human polymorphonuclear leukocytes

The evidence of the effect of N-acetylcysteine on reactive oxygen species produced by human polymorphonuclear leukocytes (PMNs) is often contradictory, as thiol compounds may react with not only reactive oxygen and nitrogen species but also they may influence intracellular glutathione levels. The effect of 20, 100 and 200 microM N-aceylcysteine (NAC) on luminol dependent chemiluminescence (LDCL) of human PMNs (10(6) cells/ml phospate buffered saline (PBS)) and whole blood to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol-12-myristate-13-acetate (PMA) was studied. Further, the ability of NAC to increase PMNs intracellular thiols and affect subsequent PMNs, stimulation was assessed. NAC 100 and 200 microM, but not 20 microM, was found to attenuate the kinetic parameters of initial phase of fMLP-stimulated PMNs oxidative burst. NAC at the concentration of 100 and 200 microM decreased the rate, maximum and integrated value of PMNs response to fMLP. The integrated value of PMA-induced PMNs and fMLP-induced whole blood LDCL response was also decreased by 100 and 200 microM NAC. Furthermore, all tested NAC concentrations decreased LDCL of resting PMNs suspension.The chemiluminescence pattern of reactive oxygen species (ROS) generation by PMNs stimulated with fMLP or PMA did not differ significantly from those preincubated with either 20, 100, or 200 microM NAC. Similarly, NAC did not increase the concentration of intracellular thiols in resting PMNs. However, addition of 200 microM NAC to PMA-stimulated PMNs prevented the decline in intracellular thiols pool. Both PMA- and fMLP-activated PMNs oxidized extracellular NAC. These results indicate that NAC decreases the intensity of PMNs oxidative burst by direct scavenger activity.     

Mycophenolic acid inhibits PMA-induced activation of the neutrophil respiratory burst

Abstract: Mycophenolate mofetil (MMF) is commonly used in immunosuppressive regimens for solid organ transplantation. There is evidence that the hydrolyzed active agent mycophenolic acid (MPA) causes the endothelial depletion of intracellular guanosine 5'-triphosphate (GTP) levels. This depletion may cause inactivation of nicotinamide adenine dinucleotide phosphate oxidase. The purpose of the present study was to examine the impact of MPA on the neutrophil respiratory burst and phagocytic activity using flow cytometry. In whole blood of healthy volunteers, 2 different doses of MPA (1 and 10 μmol/L) did not alter hydrogen peroxide production of neutrophils induced by receptor-dependent activators. In contrast, MPA inhibits the protein kinase C (PKC)-mediated hydrogen peroxide production by phorbol 12-myristate 13-acetate (PMA) in a time-dependent manner (negative: 21.17 ± 1.64 vs. 120 min: 14.46 ± 1.28 mean fluorescence intensity, incubation with 1 μmol/L MPA). In conclusion, our results corroborated that the neutrophil respiratory burst activity of healthy volunteers, induced by either formyl-methionyl-leucylphenylalanine (fMLP), priming with tumor necrosis factor alpha followed by fMLP or Escherichia coli and neutrophil phagocytic capacity, were not significantly affected after MPA treatment. We also could demonstrate that the hydrogen peroxide production of neutrophils decreased in response to the PKC activator PMA in a time-dependent manner.     

Involvement of protein kinase C in regulating tumor necrosis factor alpha-stimulated progesterone production in rat preovulatory follicles in vitro

Tumor necrosis factor alpha (TNF) increased progesterone production in preovulatory rat follicles in vitro. More than 1 h in the presence of TNF was needed to enhance progesterone secretion, which was only seen after 24 h of culture. Neither cAMP nor cGMP levels in media and follicles increased either at short (5-20 min) or long periods (6-24 h) after TNF stimulation. The protein kinase C (PKC) inhibitor, H-7, blocked TNF-stimulated progesterone in a dose-dependent manner (1-300 mM), with 50% inhibition corresponding to 5.2 microM H-7, it also blocked LH-stimulated progesterone production, but higher doses were needed (50% inhibition corresponding to 54.5 microM H-7). However, the cAMP- and cGMP-dependent protein kinase inhibitor, HA1004, did not block TNF stimulated progesterone. The PKC activator, phorbol 12-myristate 13-acetate (PMA), increased progesterone maximally at 32 nM and above. Low doses of PMA in combination with TNF increased progesterone levels above that stimulated by PMA alone; however with the highest does of PMA (320 nM), TNF was unable to increase follicular progesterone secretion. The time course of progesterone stimulation by PMA was similar to that of TNF. H-7 also blocked PMA and PMA + TNF stimulated progesterone accumulation, with a 50% inhibition corresponding to 4.2 and 4.1 microM H-7, respectively. These results indicate that PKC may be a mediator of TNF-stimulated progesterone secretion in preovulatory rat follicles.     

Effect of phorbol ester and platelet-derived growth factor on protein kinase C in rat hepatic stellate cells.

BACKGROUND/AIMS: Hepatic stellate cells (HSC) play a key role in hepatic fibrogenesis and thus, it is important to understand the intracellular signalling pathways that influence their behaviour. This study investigated the expression and regulation of protein kinase C (PKC) in HSC. RESULTS: Western blot analysis indicates that rat HSC express at least four PKC isoforms, PKC-alpha, PKC-delta, PKC-epsilon and PKC-zeta. PKC-alpha and PKC-zeta were located predominantly in the cytosol and were redistributed to the membrane by the PKC agonist, phorbol 12-myristate 13-acetate (PMA), while PKC-delta and PKC-epsilon were highly membrane-bound and did not undergo translocation by PMA. PKC-alpha, PKC-delta and PKC-zeta were rapidly downregulated by PMA. However, PKC-epsilon was resistant to downregulation. We also examined phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS), a specific substrate of PKC, as another approach to assess activation of PKC. Platelet-derived growth factor (PDGF) and PMA increased the phosphorylation of MARCKS, suggesting that PDGF can induce PKC activation. PDGF-induced stimulation of extracellular signal-regulated kinase, phosphatidylinositol 3-kinase and p70-S6 kinase was not abrogated by downregulation of PKC-alpha, PKC-delta and PKC-zeta. Prolonged PKC inhibition did not inhibit the fibrogenic phenotype. CONCLUSION: Multiple PKC isoforms are expressed in rat HSC and are differentially regulated by PMA. PDGF activates certain mitogenic signalling pathways independent of PKC-alpha, PKC-delta and PKC-zeta. Specific PKC isoforms may modulate different cell functions in HSC.     

Effect of protein kinase C and insulin on Na+/H+ exchange in red blood cells of essential hypertensives.

The kinetic properties of sodium-proton exchange are abnormal in human red blood cells of hypertensive patients and it has been demonstrated that the transport protein undergoes post-translational modifications able to affect its kinetic properties. Protein kinase C (PKC) activation decreases the affinity constant for intracellular protons while insulin increases the maximal rate of proton translocation. The present study therefore aimed to examine the relationships among PKC activity, fasting insulin levels and the kinetic behaviour of sodium-proton exchange in red blood cells from 20 normotensives and 36 hypertensives. In comparison with normotensive subjects, hypertensive patients had higher body mass index (26.2 +/- 0.7 vs 23.6 +/- 0.6 kg/m2, P < 0.05), higher fasting insulin levels (93.2 +/- 10.8 vs 38.6 +/- 2.9 pmol/L), increased maximal velocity of proton translocation (37.9 +/- 2.7 vs 27.6 +/- 1.9 mmol/L per cell x h, P < 0.05), and reduced Hill's coefficient (1.6 +/- 0.1 vs 2.0 +/- 0.1, P < 0.01) of sodium-proton exchange. Basal PKC activity of the cytosol and membrane was similar in the study groups. However, after treatment with 1 micromol/L phorbol 12-myristate 13-acetate (PMA) for 10 min, membrane PKC activity was stimulated to a larger extent in hypertensives (to 181 +/- 8 pmol/min/mg protein) than in normotensives (to 136 +/- 6 pmol/min/mg protein, P < 0.01). The PMA stimulated PKC activity was positively correlated to fasting insulin levels (r = 0.59, P < 0.01). Stimulation of membrane PKC by PMA corrected the low Hill's coefficient for H(i)+ activation of sodium-proton exchange in the hypertensives, while the constant for half maximal activation for intracellular protons (ie, the affinity for intracellular protons) decreased to a similar extent in both groups. The maximal transport rate was unaffected by PMA. These results indicate that the abnormal proton activation of red blood cell sodium-proton exchange in hypertensives reflects an abnormal regulation of PKC translocation to the cell membrane, associated to hyperinsulinaemia and probably insulin resistance. Therefore, post-translational modifications of the transport protein(s) account for the altered kinetic behaviour of sodium-proton exchange in hypertensives.     

Defective stimulus-response coupling in human monocytes infected with Leishmania donovani is associated with altered activation and translocation of protein kinase C.

Stimulus-response coupling through protein kinase C (PKC) was shown to be defective in mononuclear phagocytes (M phi) infected with Leishmania donovani. Phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst activity and protein phosphorylation were markedly attenuated in infected M phi. These results were not explained either by quantitative alterations in amounts of PKC or by altered phorbol ester binding but were related to defects in kinase activation. Analysis in vitro of the kinetic properties of PKC from infected M phi revealed an approximately 2-fold increase in the concentration of 1,2-dioleoyl-rac-glycerol required to achieve half-maximal kinase activation. Evidence for abnormal PKC activation in vivo was reflected by attenuation of PMA-induced translocation of enzyme to the particulate fraction of infected cells. These results provide direct evidence that infection with Leishmania inhibits activation of, and therefore intracellular signaling dependent on, PKC. Inhibition of stimulus-response coupling through PKC provides a basis for understanding impairment of cellular activation by Leishmania and may contribute to chronic infection.     

Protein kinase C, an endogenous regulator of hormone-induced cyclic AMP induction in porcine luteal cells.

LH, in addition to increasing cyclic AMP (cAMP) in ovarian cells, stimulates phosphoinositide hydrolysis producing inositol trisphosphate and diacylglycerol (DG). DG activates phospholipid- and calcium-dependent protein kinase (PKC). In the present study, we have used both PKC activators and inhibitors to examine the interactions of the PKC pathway on hormone-induced cAMP production in porcine luteal cells. Phorbol 12-myristate 13-acetate (PMA) enhanced LH- and forskolin-induced cAMP production. A time-course study indicated that the facilitatory effect of PMA was greater when added to incubation tubes following addition LH or forskolin. The non-tumour-promoting phorbol ester 4 alpha-phorbol 12,13-didecanoate, which does not stimulate PKC activation, did not facilitate hormone-induced cAMP induction. PKC inhibitors polymyxin B, sphingosine and 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) antagonized the facilitatory effect of PMA on LH-induced cAMP production. The cAMP induction by both LH and forskolin was inhibited in the presence of PKC inhibitors. Polymyxin E, which differs from polymyxin B by a single amino acid and does not inhibit PKC activation, did not inhibit LH- or forskolin-induced cAMP induction. The results of this study provide evidence for a facilitative action of the PKC effector system on hormonally stimulated cAMP production. Furthermore, PKC may be an important endogenous regulator of adenylate cyclase activity in porcine luteal cells.     

Studies on the intracellular mechanism of action of alpha-melanocyte-stimulating hormone on rat adrenal zona glomerulosa.

The intracellular mechanisms of action of alpha-MSH in rat adrenocortical cells were examined. When rat adrenal capsule (largely glomerulosa) cells were stimulated with a range of concentrations of alpha-MSH there was significant stimulation of aldosterone secretion at 10(-10) mol/l, although cyclic AMP was not increased until high concentrations of alpha-MSH were used (10(-6) mol/l and above). However, cells incubated with ACTH showed an increase in aldosterone secretion at 10(-11) mol/l and levels of cyclic AMP were elevated at 10(-9) mol ACTH/l. When rat adrenal whole capsules were incubated with alpha-MSH, membrane-bound protein kinase C (PKC) activity was increased and cytosolic enzyme activity decreased, showing PKC activation. Stimulation with angiotensin II also induced translocation of PKC activity, but ACTH did not. When [3H]inositol-loaded glomerulosa cells were stimulated with alpha-MSH there was significant generation of [3H]inositol trisphosphate (IP3) at concentrations of alpha-MSH which stimulated secretion of aldosterone. Significantly increased levels of [3H]IP3 were also measured when loaded cells were exposed to angiotensin II. ACTH did not cause any significant stimulation of [3H]IP3 production at any concentration used. These results indicate that activation of PKC and phospholipase C is important in modulating the steroidogenic effect of alpha-MSH.