Alloxan inhibition of a Ca2+- and calmodulin-dependent protein kinase activity in pancreatic islets

Alloxan was found to inhibit a Ca2+- and calmodulin-dependent protein kinase recently identified in pancreatic islets. This effect of alloxan may be specifically related to the inhibitory action of alloxan on insulin secretion from islets since: 1) in islet-cell subcellular fractions, alloxan at micromolar concentrations irreversibly inhibits the Ca2+- and calmodulin-dependent protein kinase activity; 2) pretreatment of intact islets with alloxan at concentrations that inhibit insulin secretion similarly inhibits the protein kinase activity; and 3) alloxan inhibition of both insulin secretion and protein kinase activity in intact islets can be prevented by D-glucose. This inhibition by alloxan appears to be a direct effect on the enzyme since alloxan treatment of either the islet homogenate or the microsomal fraction enriched in protein kinase activity inhibited the kinase activity with similar concentration dependence. These results suggest that alloxan-induced inhibition of a Ca2+- and calmodulin-dependent protein kinase may represent a critical inhibitory site which mediates alloxan-induced inhibition of insulin secretion.     

Biochemical basis for the specificity of alloxan inactivation of calmodulin-dependent protein kinase II.

The specificity and biochemical basis of inactivation of calmodulin-dependent protein kinase II by alloxan was studied in dispersed rat brain cells and a partially purified kinase preparation from an insulin-secreting tumor-cell line, RINm5f. When mechanically dispersed rat brain cells were incubated with [32P]-phosphate to label endogenous ATP, depolarization with 44 mM KCl produced a significant (P = 0.03) increase in phosphorylation of endogenous synapsin (132 +/- 8% of basal). Pre-treatment of the brain cells with 1.5 mM alloxan reduced depolarization-sensitive synapsin phosphorylation (109 +/- 5%). Phosphopeptide mapping of depolarization-phosphorylated synapsin showed that alloxan pre-treatment reduced phosphorylation specifically at synapsin sites phosphorylated by calmodulin-dependent protein kinase II. The results demonstrate selective inactivation of calmodulin-dependent protein kinase II activity by alloxan in an intact cell system, which may be useful in the study of the Type II kinase in cells and tissues. Using a partially purified kinase preparation from RINm5f cells, alloxan (100 microM) inactivated 76 +/- 1% calmodulin-dependent protein kinase II activity in 5 min at 37 degrees C. Subsequent incubation with dithiothreitol restored most of the activity. 5,5'-Dithiobis (2-nitrobenzoic acid) (I50 = 2.5 microM) also inactivated the kinase. These results suggested that a sulfhydryl group was involved at the inactivation site. Iodoacetamide (1.0 mM) had no inhibitory effect; however, preincubation with iodoacetamide protected the kinase activity from subsequent inactivation by alloxan. Covalent binding of [14C]-alloxan to calmodulin-dependent protein kinase was demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase

We have previously shown that diabetogenic antibiotic streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), inhibits the enzyme O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase) which is responsible for the removal of O-GlcNAc from proteins. Alloxan, another beta-cell toxin is a uracil analog. Since the O-GlcNAc transferase (OGT) uses UDP-GlcNAc as a substrate, we investigated whether alloxan might interfere with the process of protein O-glycosylation by blocking OGT, a very abundant enzyme in beta-cells. In isolated pancreatic islets, alloxan almost completely blocked both glucosamine-induced and STZ-induced protein O-GlcNAcylation, suggesting that alloxan indeed was inhibiting (OGT). In order to show definitively that alloxan was inhibiting OGT activity, recombinant OGT was incubated with 0-10 mM alloxan, and OGT activity was measured directly by quantitating UDP-[(3)H]-GlcNAc incorporation into the recombinant protein substrate, nucleoporin p62. Under these conditions, OGT activity was completely inhibited by 1 mM alloxan with half-maximal inhibition achieved at a concentration of 0.1 mM alloxan. Together, these data demonstrate that alloxan is an inhibitor of OGT, and as such, is the first OGT inhibitor described.     

DNA strand breaks in pancreatic islets by in vivo administration of alloxan or streptozotocin

Administration of diabetogenic doses of alloxan or streptozotocin to rats caused extensive DNA strand breaks in pancreatic islets. DNA of pancreatic exocrine cells was not affected by either alloxan or streptozotocin. hepatocyte DNA was fragmented by streptozotocin but not by alloxan. Intracellular NAD level was decreased in tissues whose DNA was fragmented. The results may raise a novel aspect concerning the mechanisms of action of the diabetogenic agents as well as concerning the organotropisms of the agents.     

Depolarizing stimuli reduce Ca(2+)/calmodulin-dependent protein kinase II activity in islets of Langerhans

Elevations in intracellular Ca(2+) ([Ca(2+)](i)) initiate insulin secretion from pancreatic beta-cells, but the secretory responses become rapidly desensitised to maintained elevations in [Ca(2+)](i). We have investigated the mechanisms underlying the Ca(2+) desensitization of insulin secretion using electrically permeabilized rat islets of Langerhans. Measurements of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) enzyme activity and immunoreactivity in permeabilized islets demonstrated Ca(2+)-induced reductions in enzyme activity which could not be attributed to reductions in CaMK II immunoreactive protein. Measurements in intact islets demonstrated that the Ca(2+)-induced reduction of CaMK II activity was also operative in intact cells, suggesting that this mechanism may have pathophysiological implications for beta-cell function.     

Calmodulin-dependent protein kinase in acini from lactating rat mammary tissue: subcellular locale, characterization, and solubilization

Acini isolated from lactating rat mammary tissue were used as the starting material to determine the subcellular location and characteristics of a calcium and calmodulin-dependent protein kinase. The kinase activity phosphorylated a 53,600-Da endogenous protein, required Mg2+, and was stimulated only by the simultaneous presence of calcium and calmodulin. Fractionation by differential and sucrose gradient centrifugation demonstrated the enzyme activity in acinar homogenates to be largely particulate; yet the activity did not co-fractionate with markers for nuclei, secretory vesicles, endoplasmic reticulum, mitochondria, lysozymes, Golgi or plasma membranes. The addition of dephosphorylated K-casein to these preparations resulted in a calcium and calmodulin-dependent phosphorylation of the exogenous substrate. A combination of differential centrifugation and equilibrium sucrose density gradient centrifugation purified the kinase 15-fold and revealed a density for the kinase activity between 1.33 and 1.27 g/cm3, suggesting that the kinase was associated with a particle composed largely or entirely of protein. Gel chromatography on Sephacryl S-1000 also purified the activity significantly, and provided a molecular weight of approximately 10(6). In both procedures, the enzymatic activity and principal endogenous protein substrate were enriched indicating that the kinase was associated with the 53,600-Da substrate. Sodium dodecyl sulfate-gel electrophoresis of the fractions enriched in kinase activity by either gel-exclusion chromatography or equilibrium density gradient centrifugation revealed a discrete set of proteins common to both preparations. These included proteins with molecular weights of approximately 32, 35, 54, 70, 94, 100 and 103 K. The calmodulin-dependent protein kinase of mammary acini may be associated in a large complex with these protein species or may represent a polymer of one or several of the proteins. Despite no apparent association with the common phospholipid membranous organelles, the kinase activity was solubilized by treatment with a mixture of phospholipases C and D. After phospholipase treatment and chromatography on Sephacryl S-1000, calcium and calmodulin-dependent phosphorylation was no longer detectable, indicating separation of enzyme and endogenous substrate. Phospholipase treatment of the kinase preparation may be useful in future studies as a method to solubilize the activity.     

Protein Kinase C Participates in Up-Regulation of Dihydropyridine-Sensitive Calcium Channels by Ethanol

Exposure to ethanol for several days increases the expression of dihydropyridine-sensitive, voltage-dependent Ca2+ channels in brain and in the neural cell line PC12. Since protein phosphorylation is a major mechanism by which ion channels are regulated, we used protein kinase inhibitors to investigate whether ethanol-induced up-regulation of Ca2+ channels involves activation of a protein kinase. Sphingosine and polymixin B, which inhibit protein kinase C and calmodulin-dependent kinases, prevented the enhancement of 45Ca2+ uptake induced by exposure of PC12 cells to ethanol for 4 days. In addition, sphingosine blocked the ability of ethanol to increase the number of [3H]dihydropyridine binding sites in PC12 cell membranes. Sphingosine's effect was prevented by simultaneous exposure to phorbol 12,13-dibutyrate, a potent activator of protein kinase C. Therefore, protein kinase C appears to be involved in the up-regulation of dihydropyridine-sensitive Ca2+ channels during prolonged exposure to ethanol.     

Purification and characterization of a calmodulin-dependent myosin heavy chain kinase from intestinal brush border

A calcium- and calmodulin-dependent kinase that represents the majority of the myosin heavy chain kinase activity in chicken intestinal brush borders has been highly purified. The purification steps include gel filtration, high performance chromatography on anion and cation exchangers, and affinity chromatography on calmodulin-Sepharose. The purified kinase consists of a single major, apparently autophosphorylatable polypeptide of 50,000 daltons. The Stokes radius (68 A) and sedimentation coefficient (17.5 S) indicate that it has a molecular weight of approximately 490,000. The kinase catalyzed the incorporation of a maximum of 0.8 mol of phosphate/mol of heavy chain, and essentially no phosphate was incorporated into the light chains. This kinase is distinct from other myosin kinases, but has a number of properties in common with the type II calmodulin-dependent protein kinases.     

Effect of superoxide dismutase, catalase, chelating agents, and free radical scavengers on the toxicity of alloxan to isolated pancreatic islets in vitro.

The effect of superoxide dismutase, catalase, metal-chelating agents and hydroxyl radical scavengers on the toxicity of alloxan to isolated ob/ob mouse pancreatic islets in vitro has been compared with the reported ability of such substances to protect against alloxan diabetes in vivo. Superoxide dismutase and catalase protected beta-cells of isolated pancreatic islets against alloxan cytotoxicity, as did the hydroxyl radical scavengers dimethyl sulfoxide (DMSO) and butanol. However, 1,3-dimethylurea and thiourea, that are recognised as effective hydroxyl radical scavengers and that protect animals against the diabetogenic effects of alloxan, were without effect. Similarly, desferrioxamine, that inhibits hydroxyl radical formation from alloxan in chemically defined systems, did not protect against alloxan toxicity. Diethylenetriamine pentaacetic acid, which does not inhibit hydroxyl radical formation from alloxan, also gave no significant protection. The results indicate a role for superoxide radical and hydrogen peroxide in the mechanism of toxicity of alloxan but do not support the involvement of the hydroxyl radical in this process. Alternative explanations must be sought for the ability of hydroxyl radical scavengers and metal-chelating agents to protect against alloxan toxicity in vivo.     

Phosphorylation of rabbit liver glycogen synthase by multiple protein kinases

Purified rabbit liver glycogen synthase was found to be a substrate for six different protein kinases: (i) cyclic AMP-dependent protein kinase, (ii) two Ca2+-stimulated protein kinases, phosphorylase kinase (from muscle) and a calmodulin-dependent glycogen synthase kinase, and (iii) three members of a Ca2+ and cyclic nucleotide independent class, PC0.7, FA/GSK-3, and casein kinase-1. Greatest inactivation accompanied phosphorylation by cyclic AMP-dependent protein kinase (to 0.5-0.7 phosphate/subunit, +/- glucose-6-P activity ratio reduced from approximately 1 to 0.6) or FA/GSK-3 (to approximately 1 phosphate/subunit, activity ratio, 0.46). Phosphorylation by the combination FA/GSK-3 plus PC0.7 was synergistic, and more extensive inactivation was achieved. The phosphorylation reactions just described caused significant reductions in the Vmax of the glycogen synthase with little effect on the S0.5 (substrate concentration corresponding to Vmax/2). Phosphorylase kinase achieved a lesser inactivation, to an activity ratio of 0.75 at 0.6 phosphate/subunit. PC0.7 acting alone, casein kinase-1, and the calmodulin-dependent protein kinase did not cause inactivation of liver glycogen synthase with the conditions used. Analysis of CNBr fragments of phosphorylated glycogen synthase indicated that the phosphate was distributed primarily between two polypeptides, with apparent Mr = 12,300 (CB-I) and 16,000-17,000 (CB-II). PC0.7 and casein kinase-1 displayed a decided specificity for CB-II, and the calmodulin-dependent protein kinase was specific for CB-I. The other protein kinases were able, to some extent, to introduce phosphate into both CB-I and CB-II. Studies using limited proteolysis indicated that CB-II was located at a terminal region of the subunit. CB-I contains a minimum of one phosphorylation site and CB-II at least three sites. Liver glycogen synthase is therefore potentially subject to the same type of multisite regulation as skeletal muscle glycogen synthase although the muscle and liver enzymes display significant differences in both structural and kinetic properties.     

Effects of an inhibitor of myosin light chain kinase on amylase secretion from rat pancreatic acini

Ca(2+)/calmodulin-dependent protein (CaM) kinases play an important role in Ca(2+)-mediated secretory mechanisms. Previously, we demonstrated that a CaM kinase II inhibitor KN-62 had a small inhibitory effect on amylase secretion stimulated by CCK. In the present study, we investigated the effects of a myosin light chain kinase (MLCK) inhibitor on amylase secretion and Ca(2+) signaling in rat pancreatic acini. A specific inhibitor of MLCK, wortmannin, inhibited amylase secretion stimulated by CCK-8 (30 pM) in a concentration-dependent manner. Wortmannin (10 microM) had no effects on basal secretion but reduced amylase secretion stimulated by CCK-8 (30 pM) by 67 +/- 3%. Wortmannin inhibited amylase secretion stimulated by calcium ionophore (A23187) and phorbol ester (TPA). Wortmannin also inhibited amylase response to thapsigargin by 76 +/- 8% and to both thapsigargin and TPA by 52 +/- 10%. Ca(2+) oscillations evoked by CCK-8 (10 pM) were inhibited by wortmannin (10 microM). Wortmannin had a little inhibitory effect on an initial rise in [Ca(2+)](i), and abolished a subsequent sustained elevation of [Ca(2+)](i) evoked by 1 nM CCK-8. In conclusion, MLCK plays a crucial role in amylase secretion from pancreatic acini and regulates Ca(2+) entry from the extracellular space.     

Parallel effects of arachidonic acid on insulin secretion, calmodulin-dependent protein kinase activity and protein kinase C activity in pancreatic islets.

A potential role of arachidonic acid in the modulation of insulin secretion was investigated by measuring its effects on calmodulin-dependent protein kinase and protein kinase C in islet subcellular fractions. The results were interpreted in the light of arachidonic acid effects on insulin secretion from intact islets. Arachidonic acid could replace phosphatidylserine in activation of cytosolic protein kinase C (K0.5 of 10 microM) and maximum activation was observed at 50 microM arachidonate. Arachidonic acid did not affect the Ca2+ requirement of the phosphatidylserine-stimulated activity. Arachidonic acid (200 microM) inhibited (greater than 90%) calmodulin-dependent protein kinase activity (K0.5 = 50-100 microM) but modestly increased basal phosphorylation activity (no added calcium or calmodulin). Arachidonic acid inhibited glucose-sensitive insulin secretion from islets (K0.5 = 24 microM) measured in static secretion assays. Maximum inhibition (approximately 70%) was achieved at 50-100 microM arachidonic acid. Basal insulin secretion (3 mM glucose) was modestly stimulated by 100 microM arachidonic acid but in a non-saturable manner. In perifusion secretion studies, arachidonic acid (20 microM) had no effect on the first phase of glucose-induced secretion but nearly completely suppressed second phase secretion. At basal glucose (4 mM), arachidonic acid induced a modest but reproducible biphasic insulin secretion response which mimicked glucose-sensitive secretion. However, phosphorylation of an 80 kD protein substrate of protein kinase C was not increased when intact islets were incubated with arachidonic acid, suggesting that the small increases in insulin secretion seen with arachidonic acid were not mediated by protein kinase C. These data suggest that arachidonic acid generated by exposure of islets to glucose may influence insulin secretion by inhibiting the activity of calmodulin-dependent protein kinase but probably has little effect on protein kinase C activity.     

Effects of phorbol ester and cholecystokinin on the intracellular distribution of protein kinase C in rabbit pancreatic acini.

Treatment of rabbit pancreatic acini with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), resulted in a time- and dose-dependent decrease of soluble protein kinase C activity coinciding with an increase of protein kinase C activity in the particulate fraction. After 5 min, soluble protein kinase C activity had decreased to almost 10% of the corresponding control. Total extractable protein kinase C activity, however, remained unchanged, indicating that the decrease of soluble protein kinase C activity was not due to TPA-induced inactivation of the enzyme. The biologically inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, did not induce such a translocation of protein kinase C. The half-maximal concentration for TPA-induced translocation of protein kinase C was 40 nM, and was equal to that for TPA-induced amylase secretion from isolated acini. This suggests that translocation of protein kinase C to the particulate fraction is an important step in TPA-induced activation of protein kinase C and enzyme secretion. On the other hand, cholecystokinin, a secretagogue of the calcium-mobilizing type, whose secretory action is thought to be mediated, at least in part, by protein kinase C, did not change the subcellular distribution of protein kinase C. In the presence of R59022 6-(2-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl ) ethyl-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one, an inhibitor of diacylglycerol kinase activity, cholecystokinin produced a small but significant translocation of protein kinase C, suggesting that the inability of the hormone to induce translocation is not due to a rapid conversion of the diacylglycerol formed into phosphatidic acid.     

Diabetes in the Cohen Rat Intensifies the Fetal Pancreatic Damage Induced by the Diabetogenic High Sucrose Low Copper Diet

Intrauterine hyperglycemic environment could harm the fetus making it more susceptible to develop postnatal glucose intolerance. A possible mechanism is compromise of the fetal pancreatic development. We previously found that a high sucrose low copper diabetogenic diet induces type 2 diabetes in the Cohen diabetic sensitive rats, but not in the Sabra control rats. However, oxidative stress was observed in the placenta and term fetal liver of diabetic and nondiabetic controls. We now investigated whether the fetal pancreas is affected by this diet and whether the effects result from oxidative stress, maternal hyperglycemia, or both. Term fetal pancreases were evaluated for morphology, beta cells, oxidative stress, apoptosis, and DNA methylation. There were no microscopic changes in hematoxylin and eosin stained sections and beta cells immunostaining in the pancreas of fetuses of both strains. Fetuses of the sensitive strain fed diabetogenic diet had significantly higher activity of superoxide dismutase and catalase, elevated levels of low molecular weight antioxidants, and more intense immunostaining for nuclear factor kappa‐B and hypoxia inducing factor‐1α. Both strains fed diabetogenic diet had increased immunostaining for Bcl‐2‐like protein and caspase 3 and decreased immunostaining for 5‐methylcytosine in their islets and acini. Our data suggest that maternal diabetogenic diet alters apoptotic rate and epigenetic steady states in the term fetal pancreas, unrelated to maternal diabetes. Maternal hyperglycemia further increases pancreatic oxidative stress, aggravating the pancreatic damage. The diet‐induced insults to the fetal pancreas may be an important contributor to the high susceptibility to develop diabetes following metabolic intrauterine insults     

Structure-activity study of cytotoxicity and microtubule assembly in vitro by taxol and related taxanes

Fractionation of bovine brain cytosol by DEAE cellulose chromatography revealed the presence of a calcium-dependent protein kinase. This soluble neuronal protein kinase selectively phosphorylated several endogenous substrates. The most prominent substrate was a polypeptide with an apparent M_r of 45,000 which was stimulated 20-fold by addition of both calcium and calmodulin. Activation was dose-dependent, with half-maximal phosphorylation occurring at 0.9 μM free Ca²⁺ and 60nM calmodulin. The effect of calmodulin was competitively inhibited by a variety of calmodulin inhibitors, in a manner characteristic of most calmodulin-dependent enzymes. This calcium- and calmodulin-dependent protein kinase is distinct from any previously described protein kinase.     

Characterization of the phosphorylation of rat mammary ATP-citrate lyase and acetyl-CoA carboxylase by Ca2+ and calmodulin-dependent multiprotein kinase and Ca2+ and phospholipid-dependent protein kinase

ATP-citrate lyase and acetyl-CoA carboxylase purified from lactating rat mammary gland are phosphorylated stoichiometrically by the calmodulin-dependent multiprotein kinase from rabbit skeletal muscle. The reactions are completely dependent on the presence of both Ca2+ and calmodulin. ATP-citrate lyase and acetyl-CoA carboxylase are also phosphorylated stoichiometrically by the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) purified from bovine brain. Phosphorylation of these substrates is stimulated 6-fold and 40-fold respectively by Ca2+ and phosphatidylserine. The calmodulin-dependent and phospholipid-dependent protein kinases phosphorylate the same serine residue on ATP-citrate lyase that is phosphorylated by cyclic-AMP-dependent protein kinase. The sequence of the tryptic peptide containing this site on the mammary enzyme is identical with the sequence of the peptide containing the site on ATP-citrate lyase that is phosphorylated in isolated hepatocytes in response to insulin and/or glucagon. The calmodulin-dependent, phospholipid-dependent and cyclic-AMP-dependent protein kinases phosphorylate distinct sites on acetyl-CoA carboxylase. However, one of the three phosphorylated tryptic peptides derived from enzyme treated with the phospholipid-dependent kinase is identical with the major phosphopeptide (T1) derived from enzyme treated with cyclic-AMP-dependent protein kinase. Phosphorylation of acetyl-CoA carboxylase by the phospholipid-dependent protein kinase inactivates acetyl-CoA carboxylase in a similar manner to cyclic-AMP-dependent protein kinase. With either protein kinase slightly greater phosphorylation and inactivation is seen after pretreatment of acetyl-CoA carboxylase with protein phosphatase-2A, but the effects of the protein phosphatase treatment are not completely reversed. Inactivation by the phospholipid-dependent protein kinase is Ca2+- and phospholipid-dependent, is reversed by protein phosphatase-2A, and correlates with the degree of phosphorylation. The relevance of these findings to insulin- and growth-factor-promoted phosphorylation of ATP-citrate lyase and acetyl-CoA carboxylase in intact cells is discussed.     

Epoxyeicosatrienoic acids stimulate glucagon and insulin release from isolated rat pancreatic islets

Metapyrone and eicosatetraynoic acid but not indomethacin are effective inhibitors of the secretory response of isolated rat pancreatic islets to arginine and glucose. Epoxyeicosatrienoic acids, products of the cytochrome P-450-NADPH dependent arachidonic acid epoxygenase activity, are potent and selective mediators for the in vitro release of either insulin or glucagon from preparations of isolated rat pancreatic islets.     

Diabetogenic action of alloxan in short-term termination of the blood supply to the pancreas and spleen

It has been shown that the development of alloxan diabetes in rats and the appearance of diabetogenic factor in blood is caused by the direct alloxan action on pancreas and spleen--the organs supplying by blood through the spleen artery. The stopping of blood circulation in that artery preserves rat's organism from the development of general toxic effect of alloxan. The inactivation of alloxan as a diabetogenic agent has been shown after its 5-minute at 37 degrees C incubation with blood. It has been established that the half activity of intravenous injected alloxan disappears in rat's organism during 50 s. and does not depend on alloxan sensitivity of animals.     

Regulation of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase (lyso-PAF-acetyltransferase) in exocrine glands. Evidence for an activation via phosphorylation by calcium/calmodulin-dependent protein kinase

Stimulation of secretion in guinea pig exocrine cells is associated with an enhanced synthesis in these cells of 1-O-alkyl-2-sn-acetyl-glycero-3-phosphocholines (PAF) from 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) (S?ling, H-D., and Fest, W. (1986) J. Biol. Chem. 261, 13916-13922). This results from a stimulation of the activity of lyso-1-alkylglycerophosphocholine acetyltransferase (EC 2.3.1.67). Here we have analyzed the effects of various agonists on the activity of this enzyme in guinea pig parotid gland microsomes. Carbamoylcholine leads within less than 30 s to a 2- to 4-fold activation of lyso-PAF-acetyltransferase, which persists after solubilization of the microsomal enzyme with octyl glucoside. The calcium ionophore A23187 has a similar though smaller effect. Neither isoproterenol (2 X 10(-5) M), which stimulates exocytosis more than carbachol, nor phorbol ester significantly affected lyso-PAF-acetyltransferase activity. Incubation of microsomes from unstimulated parotid gland acini with cAMP-dependent and calcium/calmodulin-dependent protein kinase resulted in a 4-fold and 2.9-fold activation of lyso-PAF-acetyltransferase activity, respectively. Protein kinase C had no significant effect. Activation with calcium/calmodulin-dependent protein kinase was inhibited by 40 microM trifluoperazine. When microsomes from carbachol-stimulated glands were used, in vitro activation of the enzyme by calcium/calmodulin-dependent protein kinase was almost abolished. Protein phosphatase 2A in vitro strongly reduced lyso-PAF-acetyltransferase activity in microsomes from both stimulated and unstimulated glands, whereas alkaline phosphatase and protein phosphatase 1 had only small effects. Following treatment with protein phosphatase 2A, enzyme activity in microsomes from stimulated glands could be enhanced more than 8-fold by subsequent incubation with calcium/calmodulin-dependent protein kinase. Although unsuccessful attempts have made it impossible so far to demonstrate directly the incorporation of phosphate into lyso-PAF-acetyltransferase, the results reported here strongly suggest that the enzyme in exocrine cells is regulated by phosphorylation-dephosphorylation and that a calcium/calmodulin-dependent protein kinase is responsible for the activation of the enzyme and type-2 protein phosphatases for its inactivation.     

Inhibition of glucokinase in hepatocytes by alloxan

The effect of alloxan on glucokinase in isolated rat hepatocytes was studied. Exposure of hepatocytes to alloxan (3 mM) at 30 degrees C for 5 min produced a marked inhibition (77%) of glucokinase activity and altered slightly the phosphofructokinase activity (32% inhibition). Pyruvate kinase and glucose 6-phosphate dehydrogenase, however, were not inhibited at all. Alloxan induced a concentration-dependent inhibition of glucokinase activity with a detectable inhibition at an alloxan concentration of 1 mM. The inhibition of glucokinase activity by alloxan was protected by the simultaneous presence of 15 mM hexose such as D-glucose, 3-O-methylglucose, or D-mannose. D-Galactose showed no protective effect. These results suggest that alloxan may exert its cytotoxic action through the inhibition of glucokinase activity not only in the liver but also in the pancreatic islets, since liver and islet glucokinases are known to be quite similar in various properties.