Ethanol sensitizes NF-kappaB activation in pancreatic acinar cells through effects on protein kinase C-epsilon

Although ethanol abuse is the most common cause of pancreatitis, the mechanism of alcohol's effect on the pancreas is not well understood. Previously, we demonstrated that in vitro ethanol treatment of pancreatic acinar cells augmented the CCK-8-induced activation of NF-kappaB, a key signaling system involved in the inflammatory response of pancreatitis. In the present study, we determine the role for individual PKC isoforms in the sensitizing effect of ethanol on NF-kappaB activation. Dispersed rat pancreatic acini were treated with and without ethanol and then stimulated with CCK-8; 100 nM CCK-8 caused both NF-kappaB and PKC-delta, -epsilon, and -zeta activation, whereas 0.1 nM CCK-8 did not increase PKC-epsilon, PKC-zeta, or NF-kappaB activity. CCK-8 (0.1 nM) did activate PKC-delta. PKC-epsilon activator alone did not cause NF-kappaB activation; however, together with 0.1 nM CCK-8, it caused NF-kappaB activation. Ethanol activated PKC-epsilon without affecting other PKC isoforms or NF-kappaB activity. Of note, stimulation of acini with ethanol and 0.1 nM CCK-8 resulted in the activation of PKC-delta, PKC-epsilon, and NF-kappaB. The NF-kappaB activation to 0.1 nM CCK-8 in ethanol-pretreated acini was inhibited by both PKC-delta inhibitor and PKC-epsilon inhibitor. Taken together, these results demonstrate the different modes of activation of PKC isoforms and NF-kappaB in acini stimulated with ethanol, high-dose CCK-8, and low-dose CCK-8, and furthermore suggest that activation of both PKC-epsilon and -delta is required for NF-kappaB activation. These results suggest that ethanol enhances the CCK-8-induced NF-kappaB activation at least in part through its effects on PKC-epsilon.     

The novel protein kinase C isoforms -delta and -epsilon modulate caerulein-induced zymogen activation in pancreatic acinar cells

Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein, concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC-delta and PKC-epsilon reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC-delta and -epsilon, reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC-delta and -epsilon isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC-epsilon redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC-epsilon overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC-delta and -epsilon isoforms translocate to specific acinar cell compartments and modulate zymogen activation.     

Agonist-stimulated pathways of calcium signaling in pancreatic acinar cells.

In pancreatic acinar cells stimulation of different intracellular pathways leads to different patterns of Ca2+ signaling. Bombesin induces activation of both phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and phospholipase D (PLD). The latter leads to generation of diacylglycerol (DAG) in addition to that produced by activation of PIP2-PLC. Strong activation of protein kinase C (PKC) results in inhibition of Ca(2+)-induced Ca2+ release from Ca2+ pools arranged in sequence to the luminally located IP3-sensitive Ca2+ pools. Consequently the Ca2+ wave which starts in the luminal cell pole is slower in the presence of bombesin (5 microm/s) as compared to that in the presence of acetylcholine (17 microm/s) which activates PIP2-PLC but not PLD. Activation of high-affinity CCK-receptors triggers a Ca2+ wave with slow propagation (5 microm/s) due to stimulation of phospholipase A2 (PLA2) and generation of arachidonic acid, which in turn leads to inhibition of Ca(2+)-induced Ca2+ release. Low-affinity CCK-receptors are coupled to both PIP2-PLC and PLD.     

Redistribution of protein kinase C in pancreatic acinar cells stimulated with caerulein or carbachol

We examined phospholipid/calcium-dependent protein kinase (protein kinase C) activity and amylase secretion in isolated pancreatic acinar cells, when exposed to caerulein or carbachol. Upon stimulation with 10(-10) M caerulein or 10(-6) M carbachol cytosolic protein kinase C activity was increased in accordance with amylase secretion. Effect of carbachol on increase in membrane-associated protein kinase C activity was maximal at 10(-6) M where the rate of amylase secretion was highest. On the other hand, caerulein showed the maximal secretion of amylase at 10(-9) M, but the activity of the protein kinase C associated with membranes increased progressively with increasing concentration of caerulein. These results indicate different profiles of redistribution of protein kinase C upon stimulation of pancreatic acinar cells with carbachol or caerulein, and they were discussed in terms of amylase secretion.     

PKC-delta and -epsilon regulate NF-kappaB activation induced by cholecystokinin and TNF-alpha in pancreatic acinar cells

Although NF-kappaB plays an important role in pancreatitis, mechanisms underlying its activation remain unclear. We investigated the signaling pathways mediating NF-kappaB activation in pancreatic acinar cells induced by high-dose cholecystokinin-8 (CCK-8), which causes pancreatitis in rodent models, and TNF-alpha, which contributes to inflammatory responses of pancreatitis, especially the role of PKC isoforms. We determined subcellular distribution and kinase activities of PKC isoforms and NF-kappaB activation in dispersed rat pancreatic acini. We applied isoform-specific, cell-permeable peptide inhibitors to assess the role of individual PKC isoforms in NF-kappaB activation. Both CCK-8 and TNF-alpha activated the novel isoforms PKC-delta and -epsilon and the atypical isoform PKC-zeta but not the conventional isoform PKC-alpha. Inhibition of the novel PKC isoforms but not the conventional or the atypical isoform resulted in the prevention of NF-kappaB activation induced by CCK-8 and TNF-alpha. NF-kappaB activation by CCK-8 and TNF-alpha required translocation but not tyrosine phosphorylation of PKC-delta. Activation of PKC-delta, PKC-epsilon, and NF-kappaB with CCK-8 involved both phosphatidylinositol-specific PLC and phosphatidylcholine (PC)-specific PLC, whereas with TNF-alpha they only required PC-specific PLC for activation. Results indicate that CCK-8 and TNF-alpha initiate NF-kappaB activation by different PLC pathways that converge at the novel PKCs (delta and epsilon) to mediate NF-kappaB activation in pancreatic acinar cells. These findings suggest a key role for the novel PKCs in pancreatitis.     

Mitochondrial Dok-4 recruits Src kinase and regulates NF-kappaB activation in endothelial cells

The downstream of kinase (Dok) family of adapter proteins consists of at least five members structurally characterized by an NH2-terminal tandem of conserved pleckstrin homology and phosphotyrosine binding domains linked to a unique COOH-terminal region. To determine the role of the novel adapter protein Dok-4 in endothelial cells, we first investigated the cell localization of Dok-4. Most surprisingly, immunofluorescence microscopy, cell fractionation studies, and studies with enhanced green fluorescent protein chimeras showed that wild type Dok-4 (Dok-4-WT) specifically localized in mitochondria. An NH2-terminal deletion mutant of Dok-4 (Dok-4-(deltaN11-29)), which lacks the mitochondrial targeting sequence, could not accumulate in mitochondria. Co-immunoprecipitation revealed an interaction of c-Src with Dok-4-WT in endothelial cells. Most interestingly, overexpression of Dok-4-WT, but not Dok-4-(deltaN1-99), increased mitochondrial c-Src expression, whereas knock-down of endogenous Dok-4 with a small interfering RNA vector greatly inhibited mitochondrial localization of c-Src, suggesting a unique function for Dok-4 as an anchoring protein for c-Src in mitochondria. Dok-4-WT significantly decreased 39-kDa subunit complex I expression. PP2, a specific Src kinase inhibitor, prevented the Dok-4-mediated complex I decrease, suggesting the involvement of Src kinase in regulation of complex I expression. Dok-4-WT enhanced tumor necrosis factor-alpha (TNF-alpha)-mediated reactive oxygen species (ROS) production, supporting the functional relevance of a Dok-4-Src-complex I/ROS signaling pathway in mitochondria. Finally, Dok-4 enhanced TNF-alpha-mediated NF-kappaB activation, whereas this was inhibited by transfection with Dok-4 small interfering RNA. In addition, Dok-4-induced NF-kappaB activation was also inhibited by transfection of a dominant negative form of c-Src. These data suggest a role for mitochondrial Dok-4 as an anchoring molecule for the tyrosine kinase c-Src, and in turn as a regulator of TNF-alpha-mediated ROS production and NF-kappaB activation.     

Substance P treatment stimulates chemokine synthesis in pancreatic acinar cells via the activation of NF-kappaB

Acinar cell injury early in acute pancreatitis leads to a local inflammatory reaction and to the subsequent systemic inflammatory response, which may result in multiple organ dysfunction and death. Inflammatory mediators, including chemokines and substance P (SP), are known to play a crucial role in the pathogenesis of acute pancreatitis. It has been shown that pancreatic acinar cells produce the chemokine monocyte chemoattractant protein-1 (MCP-1) in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Similarly, SP levels in the pancreas and pancreatic acinar cell expression of neurokinin-1 receptor, the primary receptor for SP, are both increased during secretagogue-induced experimental pancreatitis. This study aims to examine the functional consequences of exposing mouse pancreatic acinar cells to SP and to determine whether it leads to proinflammatory signaling, such as production of chemokines. Exposure of mouse pancreatic acini to SP significantly increased synthesis of MCP-1, macrophage inflammatory protein-1alpha (MIP-1alpha), as well as MIP-2. Furthermore, SP also increased NF-kappaB activation. The stimulatory effect of SP was specific to chemokine synthesis through the NF-kappaB pathway, since the increase in chemokine production was completely attenuated when pancreatic acini were pretreated with the selective NF-kappaB inhibitor NF-kappaB essential modulator-binding domain peptide. This study shows that SP-induced chemokine synthesis in mouse pancreatic acinar cells is NF-kappaB dependent.     

Ethanol differentially regulates G proteins in neural cells

Long-term incubation of clonal neural cell lines with ethanol differentially reduces the stimulation of cAMP accumulation by hormones and cholera toxin. In the NG108-15 neuroblastoma chi glioma hybrid cell line, this heterologous desensitization was associated with a 42% reduction in the expression of Gs alpha and no significant change in Gi alpha. By contrast, ethanol treatment of the parental neuroblastoma cell line N18TG2 caused little loss of response to hormones or cholera toxin and no significant change in Gs alpha or Gi alpha. Ethanol induced heterologous desensitization in N1E-115 neuroblastoma cells; however, this cell line showed a dose-dependent increase in Gi alpha and a later decrease in Gs alpha. Thus, ethanol causes heterologous desensitization of hormone-stimulated cAMP accumulation by different mechanisms in related neural cell lines.     

Ethanol exerts dual effects on calcium homeostasis in CCK-8-stimulated mouse pancreatic acinar cells

Background  

A significant percentage of patients with pancreatitis often presents a history of excessive alcohol consumption. Nevertheless, the patho-physiological effect of ethanol on pancreatitis remains poorly understood. In the present study, we have investigated the early effects of acute ethanol exposure on CCK-8-evoked Ca²⁺ signals in mouse pancreatic acinar cells. Changes in [Ca²⁺]_i and ROS production were analyzed employing fluorescence techniques after loading cells with fura-2 or CM-H₂DCFDA, respectively.     

Hormonal regulation of lactate dehydrogenase-A through activation of protein kinase C pathways in MCF-7 breast cancer cells

Lactate dehydrogenase A (LDH-A) is hormonally regulated in rodents, and increased expression of LDH-A is observed during mammary gland tumorigenesis. The mechanisms of hormonal regulation of LDH-A were investigated using a series of deletion and mutant constructs derived from the rat LDH-A gene promoter. Results of these studies show that constructs containing the -92 to -37 region of the LDH-A promoter are important for basal and E2-induced transactivation, and mutation of the consensus CRE motif within this region results in significant loss of basal activity and hormone-responsiveness. Gel mobility shift assays using nuclear extracts from MCF-7 cells show that both CREB and ATF-1 interact with the CRE. Studies with kinase inhibitors show that E2-induced activation of this CRE is dependent on protein kinase C, and these data indicate that LDH-A is induced through a non-genomic pathway of estrogen action.     

Ethanol augments elevated-[Ca2+]C induced trypsin activation in pancreatic acinar zymogen granules

It has been long recognized that significant percentage of patients with acute pancreatitis often presents with a history of excessive alcohol consumption; however, the patho-physiological effect of ethanol on acute pancreatitis remains poorly understood. Abnormally elevated cytosolic Ca²⁺ ([Ca²⁺]_C) has been found to be a shared phenomenon in acute pancreatitis that could induce trypsin premature activation. Here, we present the effects of ethanol to sensitize zymogen granules (ZGs) of pancreas acinar cells to elevated [Ca²⁺]_C leading to zymogen premature activation that could result in acute pancreatitis. The pH fluctuations ([pH]_G), Ca²⁺ concentration ([Ca²⁺]_G), and premature trypsin activation inside the ZGs were monitored directly with specific fluorescence indicators. Our results showed that ethanol could act directly on ZGs and cause ZGs more receptive to elevated [Ca²⁺]_C that could induce premature activation of zymogen (trypsin). This alcohol-induced effect is concentration dependent and strongly influenced by the surrounding [Ca²⁺]_C. The K⁺ channels on ZGs membranes are required in the sensitization process. Our observations provide a mechanistic understanding of the role of ethanol in the initiation phase of alcoholic pancreatitis.     

Inhibition of electrical coupling in pairs of murine pancreatic acinar cells by OAG and isolated protein kinase C

Summary Gap junctional coupling was studied in pairs of murine pancreatic acinar cells using the double whole-cell patch-clamp technique. During stable electrical coupling, addition of OAG (1-oleoyl-2-acetyl-sn-glycerol) induced a progressive reduction of the junctional conductance to the detectable limit (3 pS). Prior to complete electrical uncoupling, varius discrete single channel conductances between 20 and 100 pS could be observed. Polymyxin B, a potent inhibitor of the protein kinase C (PKC) system, completely suppressed OAG-stimulated electrical uncoupling. Dialysis of cell pairs with solutions containing PKC. isolated from rat brain, also caused electrical uncoupling. The presence of 0.1mm dibutyryl cyclic AMP and 5mm ATP in the pipette solution, which serves to stabilize the junctional conductance, did not suppress the effects of OAG or isolated PKC. We conclude that an increase of protein kinase C activity leads to the closure of gap junction channels, presumably via a PKC-dependent phosphorylation of the junctional peptide, and that this mechanism is dominant over cAMP-dependent upregulatory effects in the experimental time range (1 hr). A correlation of the observed single channel conductances with the appearance of channel subconductance states or various channel populations is discussed.     

Cholecystokinin-stimulated protein kinase C-delta kinase activation,tyrosine phosphorylation,and translocation are mediated by Src tyrosine kinases in pancreatic acinar cells

Protein kinase C-delta (PKC-delta) is involved in growth, differentiation, tumor suppression, and regulation of other cellular processes. PKC-delta activation causes translocation, tyrosine phosphorylation, and serine-threonine kinase activity. However, little is known about the ability of G protein-coupled receptors to activate these processes or the mediators involved. In the present study, we explored the ability of the neurotransmitter/hormone, CCK, to stimulate these changes in PKC-delta and explored the mechanisms. In rat pancreatic acini under basal conditions, PKC-delta is almost exclusively located in cytosol. CCK and TPA stimulated a rapid PKC-delta translocation to membrane and nuclear fractions, which was transient with CCK. CCK stimulated rapid tyrosine phosphorylation of PKC-delta and increased kinase activity. Using tyrosine kinase (B44) and a tyrosine phosphatase inhibitor (orthovanadate), changes in both CCK- and TPA-stimulated PKC-delta tyrosine phosphorylation were shown to correlate with changes in its kinase activity but not translocation. Both PKC-delta tyrosine phosphorylation and activation occur exclusively in particulate fractions. The Src kinase inhibitors, SU6656 and PP2, but not the inactive related compound, PP3, inhibited CCK- and TPA-stimulated PKC-delta tyrosine phosphorylation and activation. In contrast, PP2 also had a lesser effect on CCK- but not TPA-stimulated PKC-delta translocation. CCK stimulated the association of Src kinases with PKC-delta, demonstrated by co-immunoprecipitation. These results demonstrate that CCKA receptor activation results in rapid translocation, tyrosine phosphorylation, and activation of PKC-delta. Stimulation of PKC-delta translocation precedes tyrosine phosphorylation, which is essential for activation to occur. Activation of Src kinases is essential for the tyrosine phosphorylation and kinase activation to occur and plays a partial role in translocation.