Effects of insulin secretagogues on protein kinase C-catalyzed phosphorylation of an endogenous substrate in isolated pancreatic islets

The influence of the insulin secretagogues, carbachol and glucose, on protein kinase C activation in isolated pancreatic islets has been examined by determination of the phosphorylation state of an endogenous 80-kDa protein substrate of protein kinase C. The islet 80-kDa protein was identified as the myristoylated alanine-rich C kinase substrate previously described (Stumpo D. J., Graff, J. M., Albert, K. A., Greengard, P., and Blackshear, P. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4012-4016) by immunoprecipitation studies. The muscarinic agonist, carbachol (500 microM), induced insulin secretion and a time-dependent increase in the phosphorylation state of this protein in islets. This phosphorylation was maximal (220 +/- 24% of control) at 5 min and was suppressed by the protein kinase C inhibitor, staurosporine. Concentrations of glucose (28 mM) which induce maximal insulin secretion did not induce a statistically significant increase in 80-kDa phosphorylation. The combination of carbachol and a submaximally stimulatory concentration of glucose (10 mM), when added simultaneously, exerted a marked synergistic effect on insulin secretion and a synergistic effect on the phosphorylation of the 80-kDa protein kinase C substrate. These data suggest that the activation of protein kinase C may play an important role in carbachol-induced insulin secretion and in the potentiation by carbachol of insulin secretion induced by glucose. However, the activation of protein kinase C does not appear to be a primary determinant of insulin secretion induced by glucose alone.     

5'-Nucleotide phosphodiesterase: features of the substrate binding site as deduced from specificity and kinetics of some novel substrates

Phosphonate monoesters and phosphate diesters with systematically varied substituents and leaving groups were synthesized and tested as substrates for homogeneous 5'-nucleotide phosphodiesterase from bovine intestine. The enzyme was shown to hydrolyze phosphorothioate and phosphonoamidate compounds but at significantly lower rates than comparable oxy compounds. The effects of bulk and structure of the ester or phosphonate substituents were also investigated. Dibenzyl phosphate, an ester of an aliphatic alcohol, was a poor substrate. The enzyme did not hydrolyze aliphatic monoesters of phosphonates, regardless of bulk. Kinetic parameters of several nitrophenyl phosphonomonoesters and phosphodiesters are presented. The results suggest that synthetic nonnucleotide substrates can bind in two different modes, only one of which is productive. Incidence of nonproductive binding, with consequent kinetic effects, is increased by increasing the symmetry of the substrates.     

Glucose-stimulated protein phosphorylation in the pancreatic islet

The effect of inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism on steroidogenesis in rat testis Leydig cells and rat tumour Leydig cells has been investigated. In the presence of nordihydroguaiaretic acid [NDGA; 4,4'-(2,3- dimethylbutan -1,4- diyl )bis[1,2- benzendiol ]], 5,8,11,14-eicosatetraynoic acid (ETYA), BW 755C [3-amino-1-[3-(trifluoromethyl)phenyl]-2-pyrazoline hydrochloride] and benoxaprofen [ Opren ; 2-(2-p-chlorophenyl- benzoxazol -5-yl)propionic acid)] (which inhibit lipoxygenase activity), but not indomethacin and aspirin (which inhibit cyclo-oxygenase activity), a dose-related inhibition of lutropin (LH)-stimulated testosterone and pregnenolone production was obtained (ID50 values of 2.5, 30, 25 and 30 microM for NDGA, ETYA, BW 755C and benoxaprofen were obtained, respectively). BW 755C and benoxaprofen had no significant effect on LH-stimulated cyclic AMP production except at the highest concentrations examined (330 and 380 microM, respectively), whereas NDGA and ETYA inhibited LH-stimulated cyclic AMP production in a dose-dependent manner (ID50 7.0 and 22 microM respectively). However, NDGA and ETYA also caused a dose-dependent inhibition of dibutyryl cyclic AMP-stimulated testosterone and pregnenolone production. The metabolism of exogenous ( 22R )-hydroxycholesterol or pregnenolone to testosterone by Leydig cells was not inhibited by either NDGA, ETYA or indomethacin. At low concentrations of NDGA and ETYA a significant increase in the conversion of both pregnenolone and ( 22R )-hydroxycholesterol to testosterone was obtained. Studies in which the metabolism of [14C]arachidonic acid by purified rat tumour Leydig cells was investigated indicate that products are formed by tumour Leydig cells that have similar mobilities in a thin layer chromatography system to 5-L-hydroxy-6,8,11,14-eicosatetraenoic acid, 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid and leukotriene B4. The formation of these products was inhibited to varying degrees by NDGA, BW 755C and benoxaprofen but not by aspirin and indomethacin. These studies demonstrate for the first time that inhibition of lipoxygenase activity but not cyclo-oxygenase activity causes an inhibition of LH- and dibutyryl cyclic AMP-stimulated steroid production and suggest a stimulatory role for products of the lipoxygenase pathway of arachidonic acid metabolism in steroidogenesis. The site of this stimulation is apparently distal to the production of cyclic AMP and before the side chain cleavage of cholesterol.     

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.     

Comparison of Ca2+ -dependent phosphorylation in viable dispersed brain cells with calmodulin-dependent protein kinase activity in cell-free preparations of rat brain.

Using two depolarizing agents, veratrine and high concentrations of extracellular KCl, we studied depolarization-stimulated phosphorylations in 32P-labelled dispersed brain tissue in order to identify phosphoprotein substrates for Ca2+ - and calmodulin-dependent protein kinase activity at the cellular level, for comparison with findings in cell-free preparations. In intact brain cells, the only prominent depolarization-stimulated phosphorylation was a 77 kDa protein separated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This phosphorylation was dependent on external Ca2+, since chelation of Ca2+ in media with 6 mM-EGTA or the presence of verapamil (a Ca2+ -channel blocker) in the incubation media inhibited depolarization-stimulated phosphorylation of the 77 kDa protein. Phosphorylation of the 77 kDa protein also appeared to be dependent on calmodulin, because depolarization-stimulated phosphorylation was significantly decreased (P less than 0.05) when 100 microM-trifluoperazine was present in the incubation media. Polymyxin B, an inhibitor of Ca2+- and phospholipid-dependent phosphorylation, and 12-O-tetradecanoylphorbol 13-acetate, the phorbol ester enhancing Ca2+- and phospholipid-dependent phosphorylation, had no effect on the phosphorylation of the 77 kDa protein. The 77 kDa phosphoprotein was identified as a protein previously named synapsin I [Ueda, Maeno & Greengard (1973) J. Biol. Chem 248, 8295-8305] on the basis of similar migration of native and proteolytic fragments of the 77 kDa protein with those of authentic synapsin I on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Whereas several studies with cell-free preparations showed that 57 kDa and 54 kDa endogenous phosphoproteins were the most prominent species phosphorylated in a Ca2+ and calmodulin-dependent manner, these results indicate that synapsin is the most prominent Ca2+-and calmodulin-dependent phosphorylation in intact cells. The phosphorylations of 54 kDa and 57 kDa proteins may not be as important in vivo, but instead occur as a result of the disruption of cellular integrity inherent in preparation of cell-free subfractions of brain tissue.     

Regulation of pancreatic islet-cell plasma membrane Ca2+ + Mg2+-ATPase by Calmodulin

The carboxyl group reagent dicyclohexylcarbodiimide inhibits the electrogenic entry of Cl^? and NO₃^? into rat liver mitochondria at alkaline pH. The inhibition is time dependent and 50% inhibition is obtained by the addition of 3–4 nmol DCCD/mg protein. The blockage of the pH-dependent anion-conducting pore appears to be unrelated to the other known actions of DCCD on rat liver mitochondria but seems similar to its effect on the uncoupling protein of brown adipose tissue.     

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.     

Apoptotic cells overexpress vinculin and induce vinculin-specific cytotoxic T-cell cross-priming.

Here we show that apoptotic cells overexpress vinculin and are ingested by dendritic cells, which subsequently cross-prime vinculin-specific cytotoxic T lymphocytes (CTLs). Successful cross-priming requires that the apoptotic cells provide maturation signals to dendritic cells through CD40-CD40 ligand (CD40L) interactions. If apoptotic cells are CD40L-, the help of a third T cell is needed for priming, indicating a regulatory role for apoptotic cells in determining priming or tolerance. Vinculin-specific CTL priming is also related to apoptosis in vivo, given that in HIV-seropositive individuals, the frequency of specific CTLs depends on the proportion of peripheral CD40L+ apoptotic cells.