In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase C mu

To investigate whether thromboxane and/or platelet activating factor (PAF) mediate the pulmonary vasoconstrictive response to antigen in vivo, we intra-arterially injected human erythrocytes as antigen into sensitized rabbits after administration of putative inhibitors: a cyclooxygenase synthetase inhibitor (indomethacin, 5 mg.kg-1), a thromboxane synthetase inhibitor (OKY 046, 10 mg.kg-1 + 100 micrograms.kg-1.min-1), and a PAF blocker (CV6209, .1 mg.kg-1). Pulmonary artery and airway pressures significantly increased after the antigen challenge in sensitized rabbits, but did not in nonsensitized rabbits. Both indomethacin and OKY046 significantly inhibited the increase in pulmonary artery pressure after the antigen challenge, while CV6209 did not. CV6209 significantly attenuated the decrease in femoral artery pressure after the antigen challenge, while neither indomethacin nor OKY046 did. There were no significant differences in the increase in airway pressure among the groups. We conclude that thromboxane rather than PAF mediates the pulmonary vasoconstriction after the antigen challenge and that mediators other than thromboxane and PAF mediate bronchoconstriction after the antigen challenge in sensitized rabbits.     

Effects of a protein kinase C inhibitor, Ro 31-7549, on the activation of human leukocytes by particulate stimuli

1. A new specific protein kinase C (PKC) inhibitor, Ro 31-7549, was used to explore the mechanisms by which particulate stimuli, quartz and chrysotile, stimulate human polymorphonuclear leukocytes (PMNL) to produce reactive oxygen metabolites (ROM). Also soluble stimuli, formyl-Methionyl-Leucyl-Phenylalanine (fMLP) and phorbol myristate acetate (PMA) were used. 2. Ro 31-7549 inhibited chrysotile-induced free intracellular calcium ([Ca2+]i) elevations but did not have an effect on quartz-induced elevations of [Ca2+]i. Both quartz and chrysotile induced production of ROM were partially inhibited by Ro 31-7549. fMLP-induced elevation of [Ca2+]i was inhibited by Ro 31-7549 whereas PMA did not affect [Ca2+]i. Ro 31-7549 strongly inhibited fMLP-induced ROM production, and completely abolished that induced by PMA. 3. These result suggest that PKC may have an important role in the activation of PMNL to produce ROM by particulate and soluble stimuli. However, the inhibition of chrysotile-, but not of quartz-induced [Ca2+]i elevations by Ro 31-7549 provides evidence that both PKC-dependent and -independent mechanisms may play a role in the activation of human leukocytes to produce ROM.     

Vasopressin-induced activation of protein kinase C in renal epithelial cells

A method of collecting hypophyseal portal blood (HPB) in conscious pigs was used to show the relationship between GRF and somatostatin (SRIF) concentration and peripheral GH response. Six male castrate pigs (approximately 63 kg body weight) had HPB and jugular blood collected individually for an average of 175 min each. Twenty-seven spontaneous GH pulses were detected in the 1050 min of total HPB collection. Of the associations examined, the only significant finding was that GH pulse maxima occurred nonrandomly within periods of SRIF descent (63%; P = 0.005). Although 48% (13/27) of GH pulse maxima were associated with an ascent in portal GRF concentration, these associations were not determined to be nonrandom (P = 0.14). Only 7 of 27 (26%) GH pulse maxima were associated with an ascent in portal GRF concentration and a descent in SRIF concentration occurring simultaneously. A saline infusion given approximately 120 min after beginning blood collection resulted in an increase in SRIF pulse frequency and a decrease in GH-AUC and GRF-AUC. The cause of this saline effect is unknown, but it may have been related to acclimation of the pigs to the blood collection procedure. These data show the complexity of the relationship between SRIF and GRF concentrations and GH secretion and may indicate a close relationship with SRIF in GH pulse generation in the pig. In addition, these data support the hypothesis that, in the pig, mediation of GH release cannot be explained simply by antagonism between GRF and SRIF.     

Multiple signaling pathways for the activation of JNK in mast cells: involvement of Bruton's tyrosine kinase, protein kinase C, and JNK kinases, SEK1 and MKK7

Stimulation of the high affinity IgE receptor (FC epsilonRI) as well as a variety of stresses induce activation of c-Jun N-terminal protein kinases (JNKs) stress-activated protein kinases in mast cells. At least three distinct signaling pathways leading to JNK activation have been delineated based on the involvements of Bruton's tyrosine kinase (Btk), protein kinase C (PKC), and the JNK-activating cascades composed of multiple protein kinases. The PKC-dependent pathway, which is inhibited by a PKC inhibitor Ro31-8425 and can be activated by PMA, functions as a major route in FC epsilon RI-stimulated mast cells derived from btk gene knockout mice. On the other hand, wild-type mouse-derived mast cells use both PKC-dependent and PKC-independent pathways for JNK activation. A PKC-independent pathway is regulated by Btk and SEK1 via the PAK-->MEKK1-->SEK1-->JNK cascade, and is sensitive to phosphatidylinositol 3-kinase inhibitors, wortmannin and LY-294002, while the PKC-dependent pathway is affected to a lesser extent by both wortmannin treatment and overexpression of wild-type and dominant negative mutant SEK1 proteins. Another PKC-independent pathway involves Btk and MKK7, a recently cloned direct activator of JNK. Among the stresses tested, UV irradiation seems to activate Btk and JNK via the PKC-independent pathways.     

Expression of protein kinase C isozymes in human basophils: regulation by physiological and nonphysiological stimuli

The expression of protein kinase C (PKC) isozymes in human basophils and the regulation of PKC isozymes during basophil activation by phorbol 12-myristate 13-acetate (PMA) +/- ionomycin, f-met-leu-phe (FMLP), and anti-IgE antibody were examined. In human basophils (> 98% purity), PKCbetaI, betaII, delta, and were expressed, PKCalpha was difficult to detect, and PKCgamma and eta were undetectable. In unstimulated basophils, PKCbetaI and betaII were found primarily in the cytosol fraction (95% +/- 3% of total and 98% +/- 1%, respectively). Within 5 minutes of stimulation with PMA (100 ng/mL), both PKCbetaI and betaII were translocated to the membrane fraction (85% +/- 4% and 83% +/- 6%, respectively). In resting cells, 48% +/- 3% and 61% +/- 10% of PKCdelta and , respectively, existed in the membrane fraction. Within 1 minute of stimulation with PMA, 90% +/- 6% of PKC was found in the membrane fraction, however, no translocation of PKCdelta was apparent. Stimulation with FMLP caused modest translocation ( approximately 20%) of all PKC isozymes by 1 minute, whereas stimulation with anti-IgE antibody led to no detectable changes in PKC location throughout a 15-minute period of measurement. However, concentrations of PMA and ionomycin that alone caused no PKC translocation and little histamine release, together caused significant histamine release but no apparent PKC translocation. Studies with bis-indolylmaleimide analogs showed inhibition of PMA-induced, but not anti-IgE-induced, histamine release. These pharmacological studies suggest that PKC does not play a prodegranulatory role in human basophil IgE-mediated secretion.     

Expression and activation of protein kinase C isoforms in a human megakaryocytic cell line

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. We have shown previously that phorbol 12-myristate 13-acetate (PMA) induces the Dami human megakaryocytic cell line to become polyploid and to express platelet-specific proteins, including von Willebrand factor (vWF) and glycoprotein Ib (GpIb). Phorbol esters are thought to regulate gene expression principally through the activation of protein kinase C (PKC), a family of structurally related kinases with potentially unique activation requirements and substrate specificities. A survey of PKC isoforms in Dami cells revealed that, by both Western and Northern analyses, PKC isoforms alpha, beta, delta, epsilon, eta, theta, and zeta were reproducibly detected. PKC-gamma was not detected. In order to define the role of individual PKC isoforms in megakaryocytic maturation, PMA and 2-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a putative selective activator of the PKC-beta 1 isotype, were compared for their effects on Dami cell maturation. Treatment with either dPPA or PMA caused Dami cells to cease proliferating, to become polyploid, and to express vWF. We also examined dPPA and PMA for their ability to activate and to downregulate expression of different PKC isoforms. Fifteen-minute treatment with PMA resulted in the translocation of PKC isoforms alpha, epsilon, and theta from the cytosolic to the membrane fraction; twenty-four hour treatment resulted in the downregulation of these isoforms. In contrast, dPPA was found to be a potent activator of PKC-epsilon alone and exhibited weaker effects on alpha and theta. These data suggest that PKC isoforms beta, delta, eta, and zeta, which appear not to be activated by either phorbol ester, are unlikely to be primarily involved in megakaryocytic maturation in response to these agents. The isoforms that are translocated by both phorbol esters-PKC isoforms alpha and theta, and particularly epsilon-are more likely to transduce the signals that stimulate Dami cell differentiation.     

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.     

Activation and interaction with protein kinase C of a cytoplasmic tyrosine kinase, Itk/Tsk/Emt, on Fc epsilon RI cross-linking on mast cells

Cross-linking of the high-affinity IgE receptor (Fc epsilon RI) on mast cells induces rapid phosphorylation on serine, threonine, and tyrosine residues and increases the enzymatic activity, of a Tec subfamily tyrosine kinase, Itk/Tsk/Emt (Emt). The pleckstrin homology domain of Emt at its amino-terminal interacts directly with multiple isoforms of protein kinase C (PKC) in vitro. In addition, a portion of Emt is physically associated with multiple isoforms of PKC in intact mast cells. PKC phosphorylates a bacterial fusion protein containing the pleckstrin homology domain of Emt in vitro. Coexpression of Emt in COS-7 cells with Ca(2+)-dependent PKC isoforms (alpha, beta I, or beta II) induces an enhancement in tyrosine phosphorylation of Emt. In vivo inhibition of PKC expression or activity attenuates tyrosine phosphorylation and enzymatic activity of Emt induced upon Fc epsilon RI cross-linking. These data collectively suggest that PKC phosphorylates Emt and activates its autophosphorylating activity. Alternatively, PKC could activate another tyrosine kinase that phosphorylates Emt, or PKC-mediated phosphorylation of Emt may render it a target for another tyrosine kinase. In any case, PKC appears to play a major role in the activation of Emt induced upon Fc epsilon RI cross-linking.     

Glucose- and phorbol ester-induced insulin secretion in human insulinoma cells--association with protein kinase C activation

To investigate the incidence and demographics of gastric hypoacidity among persons infected with human immunodeficiency virus (HIV), 146 asymptomatic subjects were evaluated with use of a radiotelemetry device (Heidelberg capsule). Gastric hypoacidity (minimum gastric pH of > or = 3) occurred in 24 subjects (17%). Demographic characteristics, CD4 cell counts, and Helicobacter pylori serological status were evaluated for an association with gastric pH. Subjects with hypoacidity were more likely to have positive H. pylori serology than were subjects without hypoacidity (15 of 24 vs. 23 of 74, respectively; P = .004). Multivariate analysis indicated that a positive H. pylori serology was the most significant predictor of hypoacidity, accounting for an increase in gastric pH of 39%. A history of injection drug use, heterosexual transmission of HIV, and male gender were also associated with an elevated gastric pH. CD4 cell counts did not contribute to predictions of gastric pH. A history of H. pylori infection is relatively common in HIV-positive black and Hispanic populations and is a predictor of gastric pH.     

PD153035, a tyrosine kinase inhibitor, prevents epidermal growth factor receptor activation and inhibits growth of cancer cells in a receptor number-dependent manner

PD153035 is reported to be a specific and potent inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase and, to a lesser degree, of the closely related HER2/neu receptor. We show that PD153035 inhibits EGF-dependent EGF receptor phosphorylation and suppresses the proliferation and clonogenicity of a wide panel of EGF receptor-overexpressing human cancer cell lines. EGF receptor autophosphorylation in response to exogenous EGF was completely inhibited at PD153035 concentrations of >75 nM in cells overexpressing the EGF receptor. In contrast, PD153035 only reduced heregulin-dependent tyrosine phosphorylation in HER2/neu-overexpressing cell lines at significantly higher concentrations (1400-2800 nM). PD153035 exposure did not affect the expression of either EGF receptors or HER2/neu. PD153035 caused a dose-dependent growth inhibition of EGF receptor-overexpressing cell lines at low micromolar concentrations, and the IC50 in monolayer cultures was less than 1 microM in most cell lines tested. At doses of up to 2.5 microM, the IC50 for HER2/neu-overexpressing cells was not reached. In colony-forming assays, the PD153035 growth-inhibitory activity in cultures driven by endogenous (autocrine) ligand was correlated with EGF receptor number, with higher activity in cells expressing higher numbers of EGF receptors and only minimal activity in cells expressing normal numbers of EGF receptors but high HER2/neu levels. PD153053 also abolished all growth effects mediated by the addition of exogenous EGF; this condition could be reversed upon removal of the compound. Cotreatment with C225, an anti-EGF receptor-blocking monoclonal antibody, further enhanced the antitumor activity of PD153035, suggesting mechanisms of action for C225 other than competition with ligand binding. This latter finding also suggests that combined anti-EGF receptor strategies may be of enhanced benefit against tumors with high levels of EGF receptor expression.     

Effect of PD 098059, a specific inhibitor of mitogen-activated protein kinase kinase, on urokinase expression and in vitro invasion

The elevated expression of the urokinase-type plasminogen activator gene, which is necessary for the invasive phenotype of several types of cancers, is controlled by growth factors such as epidermal growth factor, transforming growth factor a, and fibroblast growth factor which bind to and activate protein tyrosine kinase transmembrane receptors. Since these activated receptors communicate with the nucleus via a signaling pathway in which c-Raf-1, mitogen-activated protein kinase kinase 1 (MEK1), and the extracellular signal-regulated kinases are sequentially activated, we determined the effect of a specific MEK1 inhibitor (PD 098059) on urokinase expression in two squamous cell carcinoma cell lines (UM-SCC-1 and MDA-TU-138) characterized as avid secretors of the plasminogen activator. PD 098059 treatment of either cell line reduced the amount of secreted urokinase in a dose-dependent manner. In contrast, a compound (daidzein) chemically unrelated to PD 098059 had little effect on urokinase secretion. The effect of PD 098059 on urokinase secretion in UM-SCC-1 cells was reversible and correlated with decreased extracellular signal-regulated kinase 1 activity. PD 098059 caused a dose-dependent reduction in the in vitro invasiveness of UM-SCC-1 cells whereas it had little effect on proliferation rates. Transient transfection assays with a chloramphenicol acetyl transferase reporter driven by the urokinase promoter indicated that diminished secretion of the protease was largely a consequence of reduced promoter activity. These findings suggest that interfering with MEK1 may provide a novel means of controlling the invasiveness of tumors in which this signaling cascade is activated by autocrine and/or paracrine growth factors.     

Ethanol inhibits mitogen activated protein kinase activity and growth of vascular smooth muscle cells in vitro

The intrathecal (i.t.) injection of endothelins to conscious rats was found to cause respiratory arrest. To gain some insights into this central phenomenon, peripheral vascular permeability and lung oedema were measured after i.t. and i.v. injections of these peptides. When injected at T-8 spinal cord level, endothelin-1 (65 and 650 pmol) and endothelin-3 (650 pmol) enhanced vascular permeability in the lungs by 22-fold and 7-fold, respectively, and caused sudden death at the highest dose. Less prominent increases (between 1.4- and 2.2-fold) of vascular permeability were observed in other tissues (trachea, kidney, ears, skin of hind paws and back skin) with endothelin-1. Endothelin-1 (650 pmol) caused a similar increase (27-fold) in lung vascular permeability when injected at T-2, although the response was significantly less (P < 0.05) if injected at the L-4 (15-fold) spinal cord level. Only endothelin-1 produced lung oedema when injected at the T-2 or T-8 level. In contrast, intravenous injection of endothelins-1 and -3 (650 pmol) did not produce lung oedema and the lung vascular permeability was increased by only 1.4-1.6-fold and all rats survived. The prior i.t. injection of 6.5 nmol BQ-123 (cyclo[D-Trp, D-Asp, L-Pro, D-Val, L-Leu]), a selective endothelin ET(A) receptor antagonist, prevented the increases of lung vascular permeability and oedema and the mortality induced by i.t. endothelin-1 (650 pmol). Whereas i.v. treatment with phentolamine (2 mg/kg) or pentolinium (25 mg/kg + 50 mg/kg per h x 15 min) abolished the lung vascular permeability changes evoked by endothelin-1 (650) pmol), atropine (1 mg/kg), NG-nitro-L-arginine (50 mg/kg) or indomethacin (5 mg/kg) had no effect. Moreover, the effects of endothelin-1 were attenuated in capsaicin pretreated rats (125 mg/kg, 10 days earlier) and almost abolished in rats subjected to sympathectomy with 6-hydroxydopamine (100 mg/kg, 24-48 h earlier). All these treatments except atropine and NG-nitro-L-arginine prevented the endothelin-1-induced lung oedema and reduced the lethality by around 50%. These results suggest that the increases of pulmonary vascular permeability and oedema induced by i.t. endothelin-1 are due to an intense pulmonary vasoconstriction mediated by alpha-adrenoceptors following the release of catecholamines in response to the activation of endothelin ET(A) receptor in the spinal cord. This central phenomenon seems to be reflexogenic, including the involvement of primary afferent C-fibers and spinal cord ascending fibers to the brain. Thus, endothelin-1 could play a role in neurogenic pulmonary oedema through a central mechanism.     

HA1077, a protein kinase inhibitor, inhibits calponin phosphorylation on Ser175 in porcine coronary artery

Calponin is a thin filament-associated protein which has been implicated in the modulation of the contractile state of smooth muscle via its interaction with actin and inhibition of the actin-activated myosin Mg-ATPase. This inhibitory effect is alleviated by phosphorylation of calponin at Ser175 in vitro by protein kinase C. The issue of calponin phosphorylation in intact smooth muscle in response to agonists that activate protein kinase C is controversial. We have produced a monoclonal antibody that specifically recognizes calponin phosphorylated at Ser175 and used it to analyze calponin phosphorylation in porcine coronary arterial smooth muscle stimulated with prostaglandin F2alpha or phorbol 12,13-dibutylate (PDB). Calponin phosphorylation increased rapidly in response to prostaglandin F2alpha concomitant with the increase in tension. Calponin was then dephosphorylated while force was maintained. Tension development in response to PDB was significantly slower, but again calponin phosphorylation paralleled force development. In this case, calponin dephosphorylation was very slow, consistent with prolonged activation of protein kinase C. The protein kinase inhibitors, HA1077 (1-5-(isoquinoline sulfonyl)-homopiperazine HCl) and HA1100 (1-hydroxy HA1077; 1-(hydroxy-5-isoquinoline sulfonyl-homopiperazine), inhibited tension development and calponin phosphorylation in a concentration-dependent manner with similar ED50 values in response to prostaglandin F2alpha and PDB. These results support physiological roles for calponin in force development in smooth muscle in response to agonists which trigger protein kinase C activation and in the latch state, i.e., force maintenance at low energy cost. Furthermore, the vasodilator effect of HA1077 and HA1100 is more likely due to inhibition of protein kinase C than of myosin light chain kinase.     

Decay-accelerating factor is a component of subendothelial extracellular matrix in vitro, and is augmented by activation of endothelial protein kinase C

The vasculature is protected from complement activation by regulatory molecules expressed on endothelial cells. However, complement fixation also occurs on subendothelial extracellular matrix (ECM) in vitro, and is initiated simply by retraction or removal of overlying cells. To investigate mechanisms controlling vascular complement activation, we examined subendothelial ECM for the presence of complement regulatory proteins. Decay-accelerating factor (DAF) was found on both human umbilical vein endothelial cells (HUVEC) and in their ECM; in contrast, membrane cofactor protein was found only on cells. ECM and HUVEC DAF were distinguishable based on several properties. While HUVEC DAF is anchored to cell membranes by a phospholipase C-sensitive glycosylphosphatidylinositol linkage. DAF was removed from ECM only by proteolytic digestion. Cytokines (TNF-alpha, IL-1 beta, IL-4) increased HUVEC DAF expression, but had minimal effect on ECM DAF; in contrast, phorbol 12-myristate 13-acetate (PMA) and wheat germ agglutinin markedly increased DAF on both HUVEC and ECM. The effect of PMA was mediated by activation of protein kinase C. The complement regulatory potential of ECM DAF was assessed by evaluating the effect of DAF-neutralizing antibodies on C3 deposition on HUVEC ECM, as well as on HeLa cell ECM, which had a considerably higher DAF content. DAF blockade enhanced C3 deposition on HeLa ECM, but had no effect on HUVEC ECM. As ECM DAF is likely to be immobile, i.e. able to interact only with C3 convertases forming in the immediate vicinity, its ability to regulate complement activation may be particularly density dependent, and contingent on endothelial-dependent up-regulation.     

Identification of a novel cDNA clone encoding protein tyrosine kinase in murine skin

Tyrosine phosphorylation is widely recognized as playing important roles in cell differentiation, proliferation, and carcinogenesis. We have used the polymerase chain reaction (PCR) method to identify protein tyrosine kinases that are expressed in the skin. Mixed oligonucleotide probes were used to amplify and screen a neonatal murine skin cDNA pool for clones encoding amino acid contiguities whose conservation is characteristic of the protein tyrosine kinase family. When the PCR products were sequenced, 13 distinct clones were found, of which one is novel to date and has provisionally been named tks (for tyrosine kinase identified from skin). Sequence homology comparison showed that the tks gene is homologous to the src and fes/fps families. Northern blotting using PCR products of tks as a probe revealed that the mRNA of tks is detected ubiquitously and weakly in other tissues such as brain, lung, liver, thymus and kidney. This fact suggests that the tks gene is expressed in widely distributed cell types.     

Transactivation of human hepatitis B virus X protein, HBx, operates through a mechanism distinct from protein kinase C and okadaic acid activation pathways

Human hepatitis B virus (HBV) X protein, HBx, transactivates virus and host genes through a wide variety of cis-elements. Expression of HBx is controlled by HBV enhancer 1 (Enh1). Both Enh1 and the core sequence of Enh1, which consists of an AP-1 related site (cFAP1) and a C stretch, respond to HBx and a phorbol ester (TPA). Biochemical pathways of the responses to HBx and TPA are still controversial. We therefore asked whether HBx and TPA stimulate Enh1 core activity through a common process. Protein kinase C (PKC) inhibitors, H-7 and staurosporin, did not inhibit HBx transactivation at concentrations sufficient to abolish the TPA effects in HepG2 cells. Although HBx transactivation synergized independently with TPA or a phosphoprotein phosphatase inhibitor, okadaic acid (OA), the PKC inhibitors eliminated only the TPA contribution. HBx transactivation required both the cFAP1 and the C stretch of the Enh1 core region; however, mutations in either or both of the two cis-elements demonstrated that TPA augmentation required only cFAP1. These results imply that HBx transactivation operates through a mechanism distinct from the PKC and OA activation pathways.     

Cholinergic inhibition of Na-K-ATPase via activation of protein kinase C in Madin-Darby canine kidney cells

Recently, it was reported that muscarinic-type cholinergic receptors coupled to the phosphoinositide messenger system are present in the rabbit inner medullary collecting duct and Madin-Darby canine kidney (MDCK) cells. The receptor density in MDCK cells is 50 times more than that in inner medullary collecting duct cells. To examine if muscarinic receptor activation influences Na-K-ATPase, the effects of a cholinergic agonist, carbachol, on Na-K-ATPase activity in MDCK cells were measured. Carbachol inhibited Na-K-ATPase activity in a time- and concentration-dependent manner. A maximum of approximately 80% of the enzyme activity was inhibited in 160 min with an EC50 of 5 microM carbachol. The inhibition of Na-K-ATPase activity was reversible; up to 80% of the enzyme activity was recovered within 4 h after carbachol was removed. The inhibitory effect of carbachol was blocked by a muscarinic antagonist atropine and by inhibitors of protein kinase C (PKC), 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine HCl, and N-(2-(methylamino)ethyl)-5-isoquinoline sulfonamide HCl. Direct activators of PKC, phorbol 12-myristate 13-acetate, N(n-heptyl)-5-chloro-1-naphthalene sulfonamide, and phosphatidyl serine, also inhibited Na-K-ATPase activity in MDCK cells, and their effect was also blocked by PKC inhibitors. These results indicate that cholinergic agonists inhibit Na-K-ATPase activity in MDCK cells by the activation of PKC. It is concluded that the inhibition of Na-K-ATPase by PKC may, in part, be responsible for the natriuretic action of cholinergic agonists, which have been shown to stimulate phosphoinositide hydrolysis in renal collecting duct cells.