Lysosomal enzymes and pancreatitis.
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Acute Disease↗
Biomedical subjects
F S Gorelick
Publications and source records attributed to F S Gorelick.
Ca2+/calmodulin-dependent protein kinase II activation and regulation of adrenal glomerulosa Ca2+ signaling.
We recently reported that elevations in the intracellular Ca2+ concentration ([Ca2+]i) enhance low-voltage-activated, T-type, Ca2+ channel activity via Ca2+/calmodulin-dependent protein kinase II (CaMKII). Here, we document CaMKII activity in bovine adrenal glomerulosa (AG) cells and assess the importance of CaMKII in depolarization-induced Ca2+ signaling. AG cell extracts exhibited kinase activity toward a CaMKII-selective peptide substrate that was dependent on both Ca2+ [half-maximal concentration for Ca2+ activation (K0.5) = 1.5 microM] and calmodulin (K0.5 = 46 nM) and was sensitive to a calmodulin antagonist and a CaMKII peptide inhibitor. On cell treatment with elevated extracellular potassium (10-60 mM) or angiotensin II, Ca(2+)-independent CaMKII activity increased to 133-205% of basal activity. Ca(2+)-independent kinase activity in agonist-stimulated extracts was inhibited by the CaMKII inhibitor peptide, 1(-)[N,O-bis(1,5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a cell-permeable inhibitor of CaMKII, reduced the agonist-induced stimulation of Ca(2+)-independent CaMKII activity. KN-62 also diminished depolarization-induced increases in [Ca2+]i without affecting the membrane potential. These observations suggest that CaMKII is activated in situ by aldosterone secretagogues and augments Ca2+ signaling through voltage-gated Ca2+ channels.
Angiotensin II↗
Calmodulin-dependent protein kinases in rat glioblastoma.
The mitogenic activity of several growth factors is mediated by calcium-dependent signal transduction. Calmodulin (CaM) binding proteins such as CaM-dependent protein kinases are important components of this pathway and may be altered in diseases characterized by abnormal cell growth. CaM kinase II is believed to regulate the phosphorylation of microtubular-associated proteins and control the initiation of DNA synthesis. Furthermore, drugs that inhibit CaM-mediated signal transduction also inhibit cellular proliferation and are cytotoxic to numerous malignant cell lines, including those established from malignant gliomas. Yet, little is known about CaM-dependent protein kinases in these tumors. Therefore, we have investigated the activity and distribution of CaM-dependent protein kinase II in normal and malignant glial tissues, a kinase believed to play a critical role in cell cycle regulation. C6 and 9L cells contained kinase activities that were activated by Ca2+/CaM and inhibited by trifluoperazine. Tissue extracts from these cell lines and from rat brain white matter phosphorylated exogenous synapsin I in a pattern consistent with the presence of CaM kinase II activity as determined by phosphopeptide mapping. CaM kinase II activity was confirmed using a specific peptide substrate and inhibitor. An unexpected finding was that glioma lines, but not rat brain white matter, also contained a CaM-dependent protein kinase detected by the phosphorylation of a M(r) 100,000 protein, subsequently identified as elongation factor 2, the only known substrate for CaM kinase III.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Cell cycle-dependent and kinase-specific regulation of the apical Na/H exchanger and the Na,K-ATPase in the kidney cell line LLC-PK1 by calcitonin.
Calcitonin (CT), which regulates serum calcium through its actions in bone and the kidney tubule, also has a potent natriuretic effect in vivo. Na reabsorption in the proximal kidney tubule is mostly dependent on the activity of the Na,K-ATPase and the apical Na/H exchanger. We have previously shown that CT regulates the activity of the Na,K-ATPase in the proximal kidney tubule cell line LLC-PK1 in a cell cycle-dependent manner. We report here that, in the same cells, CT also regulates the Na/H exchanger through a cell cycle-specific activation of the Ca/calmodulin-dependent protein kinase II. In G2 phase, no changes in ethylisopropyl amiloride-sensitive 22Na uptake is observed, despite an increase in cAMP. In contrast, the hormone inhibits the apical exchanger when the cells are in S phase, resulting in an 80% inhibition of 22Na uptake. These results demonstrate that CT affects the activity of the two major proximal tubule Na transport systems and may help clarify the mechanisms by which CT regulates Na+ reabsorption.
Animals↗
Effect of buprenorphine on pancreatic enzyme synthesis and secretion in normal rats and rats with acute edematous pancreatitis.
Pancreatic enzyme secretion is inhibited during acute pancreatitis, resulting in an increase in acinar zymogen content. Since the premature activation of zymogens has been assigned a central role in the pathogenesis of acute pancreatitis, minimizing the amount of stored zymogens might lead to less severe acute pancreatitis. Inhibition of enzyme synthesis or stimulation of enzyme secretion would result in reduction of zymogen stores. Opiates have a varying effect on pancreatic secretion, depending on the dosage, site of administration, and presence of pancreatic stimulants. The effect of opiates and acute pancreatitis on individual pancreatic enzyme synthesis is unknown. The following study was undertaken in order to examine the effects of an opiate on pancreatic enzyme secretion and synthesis during experimental acute pancreatitis. Four groups of rats were studied. Group I received cerulein (25 micrograms/kg); group II received an opiate, buprenorphine (BPN, 0.5 mg/kg); and group III received cerulein and BPN. Drugs were dissolved in gelatin/saline and injected subcutaneously. A control group (group IV) received only gelatin/saline. Rats were sacrificed 4 hr after injection, and pancreatic mass was measured. Pancreatic acini were prepared and assayed for amylase and DNA content. Amylase, trypsinogen, chymotrypsinogen and lipase synthesis, and amylase secretion were measured for 2 hr. Results showed that, compared to controls, acini of rats with AP had increased amylase content, a finding consistent with decreased in vivo amylase secretion. Total protein and individual enzyme synthesis rates were significantly lower in the acini of the rats with AP than in those of the controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Acute Disease↗
Calcium/calmodulin-dependent protein kinase-II activation in rat pituitary cells in the presence of thyrotropin-releasing hormone and dopamine.
PRL release from rat lactotrophs in response to TRH is Ca2+ dependent. TRH-induced PRL release is inhibited either after repeated pulses of TRH or in the presence of dopamine. TRH, however, generates increases in intracellular Ca2+ concentrations ([Ca2+]i) in both conditions. Calcium/calmodulin-dependent protein kinase-II (CaM kinase-II) is a ubiquitous enzyme implicated in secretion. To determine whether down-regulation of CaM kinase-II activity caused the lack of responsiveness to increases in [Ca2+]i, we measured the generation of calcium/calmodulin-independent kinase activity. Anterior pituitary cells contain a 50-kilodalton form of CaM kinase-II, determined by immunoblot, and the enzyme is in lactotrophs, determined by immunocytochemistry. TRH rapidly and transiently increased calcium/calmodulin-independent kinase activity; the increase was maximal by 15 sec and returned to basal by 2 min. When TRH pulses (1 microM, 15 sec) were applied every 10 min, each pulse caused an increase in calcium/calmodulin-independent kinase activity of similar magnitude, and the activity returned to basal values between pulses. Pretreatment of cells with dopamine (1 microM; 30 min) inhibited PRL release, but did not prevent the increase in calcium/calmodulin-independent kinase activity. These results indicate that TRH still activates CaM kinase-II when PRL release is inhibited. Dopamine and repeated pulses of TRH must inhibit PRL release at a site after the TRH-induced increase in [Ca2+]i and at a site other than CaM kinase-II.
Animals↗
Antibodies to an epitope on synapsin I detect a protein associated with the endocytic compartment in non-neuronal cells.
To detect potential substrate proteins for Ca2+/calmodulin-dependent protein kinase II outside the central nervous system, antibodies were made to a synthetic peptide corresponding to a sequence within synapsin I which is phosphorylated by this enzyme. In neural tissues, this antibody (212) identified an 86/80 kDa doublet corresponding to synapsin I. In rat liver, intestinal enterocytes and the clone 9 cell line this antibody identified two proteins of 170 and 85 kDa. These proteins were present in the particulate fraction of liver postnuclear supernatant, and were released into the soluble fraction when extracted with 100 mM NaCl. In liver, enterocytes, and clone 9 cells, these antigens were localized by immunocytochemical techniques to small intracellular vesicles. The endocytic compartment of clone 9 cells was labeled by continuous uptake of horseradish peroxidase; antibody 212-labeled vesicles exhibited overlap with the compartment. To confirm the identity of this compartment as endosomal, rat liver endosomes were labeled in vivo by intravenous injection of horseradish peroxidase. Horseradish peroxidase-containing endosomes of approximately 80 nm were recognized by antibody 212. Occasionally, larger endosomes (approximately 300-500 nm) were also labeled. In clone 9 cells, partial overlap was observed between the 212 antigen and a transferrin receptor-positive, brefeldin A-sensitive compartment. In clone 9 cells double-labeled with anti-tubulin and antibody 212, then imaged using confocal microscopy, these vesicles appeared to be associated with microtubules. This antigen has properties similar to that of CLIP-170, a membrane-associated endosomal phosphoprotein. These findings demonstrate that a 170/85 kDa antigen containing an epitope for the Ca2+/calmodulin-dependent protein kinase II phosphorylation sequence is associated with an endocytic compartment.
Animals↗
Phosphorylation of elongation factor 2 in normal and malignant rat glial cells.
Certain calmodulin (CaM)-dependent protein kinases phosphorylate substrates have been implicated in regulating cellular proliferation. In this study, CaM-dependent phosphorylation has been examined in normal and tumor tissue from rat brain to determine whether differences exist. Using in vitro phosphorylation reactions, we compared endogenous substrates for Ca2+/CaM-dependent protein kinases in rat brain white matter (RBWM), a tissue rich in normal glia, to those of C6 rat glioma cells. A major phosphoprotein having a M(r) of 100,000 was observed in proliferating C6 cells that was not present in RBWM or in nonproliferating cells. Phosphorylation was stimulated by Ca2+ and CaM and inhibited by trifluoperazine. An antibody to elongation factor 2 (EF-2) immunoprecipitated the M(r) 100,000 protein from C6 cells. EF-2 was present in RBWM but was not phosphorylated. Homogenates of RBWM did not phosphorylate exogenous EF-2, which suggested the absence of CaM kinase III activity in normal glial tissue. Furthermore, the addition of purified, exogenous CaM kinase III to homogenates of RBWM resulted in EF-2 phosphorylation. These data demonstrate that a basal level of EF-2 phosphorylation exists in proliferating glioma cells that is markedly diminished or absent in normal glial tissue and is due to the activity of CaM kinase III.
Animals↗
Characterization of cAMP-dependent protein kinase activation by CCK in rat pancreas.
This study reports on the use of a new sensitive assay of cAMP-dependent protein kinase activity to examine the effect of cholecystokinin (CCK) on the cAMP second messenger cascade in rat pancreatic acini. Treatment of acini with both low (pM) and high (nM) concentrations of CCK was associated with an increase in cAMP-dependent protein kinase activity. The increases in kinase activity were detected in the absence of phosphodiesterase inhibition, a condition required to detect a measurable increase in cellular cAMP in these cells. Furthermore, the cAMP cascade was dissociated from the secretory effects of CCK, since the CCK analogue, OPE, mediates enzyme secretion but does not increase cellular cAMP levels or kinase activity.
1-Methyl-3-isobutylxanthine↗
Influence of chloroquine on diet-induced pancreatitis.
Recent investigations have suggested that digestive zymogens may become activated within the acinar cell during acute pancreatitis. While the molecular events responsible for intracellular zymogen activation remain unknown, several potential enzymatic pathways require an acidic pH to optimally proceed. We therefore proposed that manipulation of subcellular pH might alter the course of experimental pancreatitis. Chloroquine, a weak base that raises the pH of acidic subcellular compartments, was administered to young female mice in which pancreatitis was induced by a choline-deficient, ethionine-supplemented (CDE) diet. Control animals were maintained on regular laboratory chow. Examination of isolated pancreatic acini using acridine orange cytofluorescence demonstrated expansion of acidic subcellular compartments in animals fed the CDE diet. These compartments were effectively neutralized in animals receiving chloroquine. Animals receiving continuous infusions of high-dose chloroquine demonstrated a significant (p < 0.05) decrease in free pancreatic tryptic activity as well as improved survival. These changes were also associated with decreased trypsinogen content in animals treated with high-dose chloroquine, suggesting an additional potential effect of chloroquine on zymogen synthesis and accumulation. One explanation of these findings is that a low-pH compartment may be important in the pathogenesis of diet-induced pancreatitis.
Acute Disease↗
Distribution of calcium/calmodulin-dependent protein kinase II in rat ileal enterocytes.
Ca2+/calmodulin (CaM)-dependent protein kinase II is a major effector of the Ca2+ signaling pathway. It has a wide tissue distribution and phosphorylates multiple substrates. Villus enterocytes from rat ileum contain a Ca2+/CaM-dependent kinase activity that phosphorylates the exogenous neural substrate synapsin I. This phosphorylation is blocked by a specific peptide inhibitor. Antibodies made to rat brain Ca2+/CaM-dependent protein kinase II label a single band with a relative molecular mass of approximately 50 kDa in isolated rat enterocytes by immunoblot. Almost one-half of this immunoreactive protein is preferentially found in a particulate compared with a soluble subcellular fraction of the enterocytes. Virtually all of the 50-kDa band in the particulate fraction is insoluble in nonionic detergent, suggesting that the kinase is associated with the enterocyte cytoskeleton. Antibodies to Ca2+/CaM-dependent protein kinase II immunocytochemically detect fibrillar structures concentrated in the terminal web region of intestinal epithelial cells that colocalized with myosin II. This enzyme may have a role in regulating the intestinal epithelial cytoskeleton.
Animals↗
Synaptic vesicle-associated Ca2+/calmodulin-dependent protein kinase II is a binding protein for synapsin I.
Synapsin I is a synaptic vesicle-associated phosphoprotein that is involved in the modulation of neurotransmitter release. Ca2+/calmodulin-dependent protein kinase II, which phosphorylates two sites in the carboxy-terminal region of synapsin I, causes synapsin I to dissociate from synaptic vesicles and increases neurotransmitter release. Conversely, the dephosphorylated form of synapsin I, but not the form phosphorylated by Ca2+/calmodulin-dependent protein kinase II, inhibits neurotransmitter release. The amino-terminal region of synapsin I interacts with membrane phospholipids, whereas the C-terminal region binds to a protein component of synaptic vesicles. Here we demonstrate that the binding of the C-terminal region of synapsin I involves the regulatory domain of a synaptic vesicle-associated form of Ca2+/calmodulin-dependent protein kinase II. Our results indicate that this form of the kinase functions both as a binding protein for synapsin I, and as an enzyme that phosphorylates synapsin I and promotes its dissociation from the vesicles.
Amino Acid Sequence↗
Scientific advances in cystic fibrosis.
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Chloride Channels↗
New perspectives on acute pancreatitis.
The past decade has witnessed considerable changes in the clinical management of acute pancreatitis. Simultaneously, significant advances have been made in understanding the cellular and biochemical events involved in the initiation of this disease. This review summarizes recent clinical and scientific progress regarding acute pancreatitis and suggests areas for future investigation.
Acute Disease↗
Agonist-regulated phosphorylation of the pancreatic cholecystokinin receptor.
The present study was undertaken to determine if the cholecystokinin (CCK) receptor may be phosphorylated, and to gain insight into its regulation. For this, the ATP pool of rat pancreatic acini was prelabeled with 32P, and the cells were stimulated with various secretagogues. CCK receptors from treated cells were enriched by sequential fractionation to produce plasmalemma, and subsequent solubilization and lectin-affinity chromatography. This protocol detected a phosphorylated Mr = 85,000-95,000 plasma membrane glycoprotein with features similar to the CCK receptor. Phosphorylation of this protein occurred rapidly (less than 2 min) and in a concentration-dependent manner in response to CCK, and was inhibited by the CCK receptor antagonist L-364,718. Further evidence that this represented the CCK receptor included comigration of phosphorylated and CCK radioligand affinity-labeled proteins on sodium dodecyl sulfate-polyacrylamide gels, both in native forms and after endoglycosidase F deglycosylation, and the specific adsorption of the phosphoprotein to a CCK analogue affinity resin. Phosphorylation occurred predominantly on serine residues of the receptor protein. Phosphorylation of this protein was also enhanced in response to other secretagogues which, like CCK, stimulate a cascade leading to protein kinase C activation, and in response to direct activation of this enzyme by 12-O-tetradecanoylphorbol 13-acetate. Thus, the pancreatic CCK receptor is phosphorylated in a regulated manner, in response to both homologous and heterologous secretagogues, and to protein kinase C activation.
Adenosine Triphosphate↗
Intracellular activation of digestive zymogens in rat pancreatic acini. Stimulation by high doses of cholecystokinin.
The mechanism by which digestive zymogens become activated during acute pancreatitis remains poorly understood. Given the ability for cholecystokinin (CCK) to induce pancreatitis in vivo, the effects of high dose CCK on preparations of isolated pancreatic acini were examined. Using an immunologic technique for the detection of zymogen activation, CCK was found to stimulate the conversion of procarboxypeptidase A1 to a 35-kD form having the same net charge and electrophoretic mobility as purified recombinant carboxypeptidase A1. This enhanced conversion was proportional to the dose of CCK (maximal at 100 nM), and time dependent. CCK also produced changes in the electrophoretic mobility of procarboxypeptidase B and chymotrypsinogen 2 immunoreactivity, consistent with activation of these zymogens. These events were detectable only within acinar cell pellets and not in the incubation medium, suggesting an intracellular site of conversion. The conversion of procarboxypeptidase A1 to its active form was inhibited by pretreatment with the weak base chloroquine (40 microM) and the protonophore monensin (10 microM). This conversion was also inhibited by pretreatment with the serine protease inhibitor benzamidine (10 mM) but not the cysteine protease inhibitor E64 (100 microM). The results suggest that high dose CCK stimulates the intracellular activation of digestive zymogens within isolated pancreatic acini. This event appears to require an acidic subcellular compartment and serine protease activity.
Animals↗
Localization of the cystic fibrosis transmembrane conductance regulator in pancreas.
Cystic fibrosis (CF) is characterized by an abnormality in cAMP-regulated chloride transport that results from a primary defect in the protein product of the CF gene, the CF transmembrane conductance regulator (CFTR). In this report, antibodies against CFTR peptides were used to localize the CFTR protein in human pancreas. An affinity purified antibody (alpha-1468) raised against a synthetic CFTR peptide identified a 155-170-kD protein on immunoblot. Cytochemical studies with alpha-1468 localized CFTR to small branching, tubular structures. The same structures were recognized by two other antibodies raised against different regions of the CFTR molecule. To identify the cells being stained, double-label immunofluorescence studies were performed using alpha-1468 and a monoclonal antibody which stains pancreatic centroacinar and intralobular duct cells. Both antibodies localized to the same population of cells, with alpha-1468 being confined to the apical domain of these cells. No conclusive staining of acinar cells was evident. These findings suggest that proximal duct epithelial cells play a key role in the early events leading to pancreatic insufficiency in CF, and imply that apical chloride transport by these cells is essential for normal pancreatic secretory function.
Amino Acid Sequence↗
Acute pancreatitis at its centenary. The contribution of Reginald Fitz.
1989 represents the 100th anniversary of Reginald Fitz's initial characterization of acute pancreatitis. Our current understanding of this disease has advanced little beyond Fitz's early description. While survival from pancreatitis has improved largely through advancements in critical care techniques, no medical or surgical therapy exists that can limit pancreatic autodigestion and inflammation. Recent investigations have suggested that pancreatitis may result from a disruption of normal stimulus-secretion coupling within the acinar cell. Future research based on these observations may ultimately result in successful therapy for this disease.
Acute Disease↗