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Enzymatic sorting of bacterial colonies on filter paper replicas: detection of labile activities.

To utilize autoradiographic colony-sorting techniques (C. R. H. Raetz, Proc. Natl. Acad. Sci. U.S.A. 72:2274-2278, 1975) for the isolation of mutants with unstable enzymes, we report a new desiccation-induced lysis method, compatible with low temperatures. Furthermore, a general, two-step protocol is presented for clonal detection of hydrolytic reactions. The advantages of these critical modifications are demonstrated with the membrane enzymes glycerol 3-phosphate acyltransferase and cytidine 5'-diphosphate-diglyceride hydrolase.

Acyltransferases↗

Modification, processing, and subcellular localization in Escherichia coli of the pCloDF13-encoded bacteriocin release protein fused to the mature portion of beta-lactamase.

A fusion between the pCloDF13-derived bacteriocin release protein and beta-lactamase was constructed to investigate the subcellular localization and posttranslational modification of the bacteriocin release protein in Escherichia coli. The signal sequence and 25 of the 28 amino acid residues of the mature bacteriocin release protein were fused to the mature portion of beta-lactamase. The hybrid protein (Mr, 31,588) was expressed in minicells and whole cells and possessed full beta-lactamase activity. Immunoblotting of subcellular fractions revealed that the hybrid protein is present in both the cytoplasmic and outer membranes of E. coli. Radioactive labeling experiments in the presence or absence of globomycin showed that the hybrid protein is modified with a diglyceride and fatty acids and is processed by signal peptidase II, as is the murein lipoprotein. The results indicated that the pCloDF13-encoded bacteriocin release protein is a lipoprotein which is associated with both membranes of E. coli cells.

Bacterial Proteins↗

Studies on cardiolipin biosynthesis in Mycobacterium smegmatis.

Supplementation of a growth medium with 5% glucose has been found to stimulate the formation of cardiolipin and phosphatidylethanolamine five- and threefold, respectively, in Mycobacterium smegmatis. The presence of both cytidine diphosphate diglyceride and phosphatidylglycerol pathways of biosynthesis of cardiolipin in cell-free extracts has been demonstrated. The enzymes were localized in the fractions which contained membranes. Isonicotinic acid hydrazide and streptomycin sulfate inhibited the formation of cardiolipin.

Cardiolipins↗

Nonpolar lipids of a halotolerant species of Staphylococcus epidermidis.

The nonpolar lipids of a halotolerant Staphylococcus epidermidis, isolated in pure culture from a growth medium for extreme halophiles containing 25% sodium chloride, were found to contain squalene, menaquinone-7, free fatty acids (mainly anteiso-15:0 and anteiso-17:0), undecaprenol, nonaprenol with predominately cis-isoprene residues, heptaprenol, with predominately trans-isoprene residues, and 1,2- and 1,3-diglycerides containing anteiso- 15:0 and anteiso-17:0 branched chain fatty acid residues. The above compounds were isolated in pure form by column and thin-layer chromatography and were characterized by ultraviolet, proton magnetic resonance, and mass spectra. Fatty acid moieities were characterized by gas-liquid chromatographic retention times of their methyl esters.

Chromatography, Thin Layer↗

Adipose tissue glyceride synthesis in patients with hyperapobetalipoproteinemia.

Adipose tissue was obtained at thoracotomy in five control patients with valvular heart disease, all of whom were free of coronary artery disease and all of whom were normolipidemic with normal low density lipoprotein apolipoprotein B levels, and eight patients with coronary artery disease, all of whom had hyperapobetalipoproteinemia. In both groups, the rates at which linoleic acid and palmitic acid were incorporated into diglyceride and triglyceride were determined in vitro. The data indicate that fatty acid incorporation into adipose tissue glycerides was twice as rapid in controls as in patients with hyperapobetalipoproteinemia. By contrast there was no difference between the groups in the rate of net lipolysis of adipocyte glyceride. The data at hand do not establish the mechanism responsible for the difference in synthesis between normal subjects and patients with hyperapobetalipoproteinemia, but this may explain the delayed chylomicron triglyceride clearance previously observed in the disorder.

Adipose Tissue↗

Effect of ANP on sustained aldosterone secretion stimulated by angiotensin II.

The sustained aldosterone secretory response to angiotensin II (ANG II) depends on receptor-mediated increases in membrane diglyceride (DG) and an increase in calcium influx rate. These signals serve to activate membrane-associated protein kinase C (PKC) and result in enhanced phosphorylation of a unique set of proteins. These events can be mimicked by the addition of a phorbol ester, 12-O-tetra decanoyl phorbol 13-acetate (TPA), and a calcium ionophore, A23187, that bypass the initial receptor-associated events. We studied the inhibitory action of atrial natriuretic peptide (4-28 hANP) on the sustained secretory response to ANG II in isolated bovine adrenal glomerulosa cells. Although 10 nM ANP inhibited aldosterone secretion, it did not significantly alter the ANG II-elicited rise in 45Ca2+ influx rate [control (CON): 0.44 +/- 0.06; ANG II: 1.11 +/- 0.12 (P less than 0.001); ANG II + ANP: 1.18 +/- 0.14], the steady-state level of aequorin luminescence [intracellular [Ca2+] ([Ca2+]i)], or the rise in cellular DG content [CON: 0.132 +/- 0.01; ANG II: 0.194 +/- 0.01 (P less than 0.005); ANG II + ANP: 0.202 +/- 0.01 nmol/10(6) cells]. IN addition, ANP was able to inhibit aldosterone secretion stimulated by the combined addition of A23187 + TPA. When protein phosphorylation in the ANP-inhibited cells was evaluated, ANG II-induced protein phosphorylation events were preserved. In contrast to the effect of ANP, the calcium channel blocker nitrendipine abolished the ANG II-induced rise in 45Ca2+ influx rate, reduced the steady-state level of [Ca2+]i, and returned the phosphoproteins to their control states.(ABSTRACT TRUNCATED AT 250 WORDS)

Aequorin↗

Calcium-activated phosphatidylcholine-specific phospholipase C and D in MDCK epithelial cells.

Calcium ionophore exposure generates diglycerides (DAG) from phosphatidylcholine (PC) hydrolysis in Madin-Darby canine kidney (MDCK) epithelial cells. This study compares calcium ionophore-activated PC hydrolysis with the previously described phorbol ester-stimulated PC hydrolysis pathway using MDCK cells labeled with [14C]-linoleic acid. Lipid species were measured using thin-layer chromatography. DAG resulted in part from PC hydrolysis because DAG increased in cells labeled with [palmitoyl-2-14C]phosphatidylcholine. Neither protein kinase C (PKC) inhibitors nor PKC depletion affected the ionomycin (IONO)-induced increase in DAG. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prevented the increased DAG after IONO but not after phorbol 12,13-dibutyrate (PDBu) exposure. The EGTA effect was reversed by adding excess calcium but was not reversed by adding excess Mg2+. IONO exposure also increased phosphatidic acid (PA) production. The PA was produced by phospholipase D (PLD) because phosphatidylethanol was produced when IONO was added to the cells in the presence of ethanol. Although increasing concentrations of ethanol resulted in progressively less PA, it had no effect on increased DAG after IONO exposure at any time point tested. These data are consistent with both increased phospholipase C (PLC) and increased PLD activity following ionomycin. In contrast to IONO exposure, ethanol completely prevented the increase in DAG after PDBu exposure, consistent with DAG produced by PLD activation. These results demonstrate that calcium activates both PC-specific PLC and PLD in MDCK cells and that the calcium-activated pathway is independent of the previously described PKC activation pathways.

Animals↗

Phenylephrine and endothelin differentially stimulate cardiac PI hydrolysis and ANF expression.

In ventricular myocytes, phenylephrine (PE) and endothelin (ET) stimulate phosphoinositide (PI) hydrolysis, cell growth, and expression of several genes [e.g., atrial natriuretic factor (ANF)] often associated with cardiac hypertrophy. In this study the production of inositol monophosphate (InsP) and diglyceride (DG), both products of PI hydrolysis, and ANF were monitored during long-term exposure (up to 72 h) to PE or ET. For PE, InsP production increased and continued unabated throughout the time course; DG levels also increased and remained elevated, and similar rates of ANF production were observed from 24 to 72 h. For ET, the initial InsP and DG responses equaled those to PE, but diminished by 24 h. ET-stimulated ANF production equaled the PE response at 24 h but subsided by 72 h. Adding PE to cells previously desensitized to ET elicited maximal InsP formation, indicating the desensitization was ET specific. These data emphasize that while phenylephrine and endothelin have similar initial effects on cardiac second messenger production and ANF expression, hormonally specific patterns develop over extended periods of exposure.

Animals↗

The role of protein kinase C in alpha-adrenergic regulation of NaCl(K) cotransport in human airway epithelial cells.

alpha 1-Adrenergic (alpha 1-AR) agents stimulate NaCl(K) cotransport and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]-specific phospholipase C in human trachea and nasal polyp epithelial cells. One second messenger generated by PtdIns(4,5)P2 degradation is inositol trisphosphate. We now show that diglycerides (DG) are also generated during alpha 1-AR stimulation. In cells prelabeled with [3H]arachidonic acid, alpha 1-AR agents produced a biphasic DG generation in normal and cystic fibrosis (CF) cells that is blocked by pertussis toxin. The early DG peak closely paralleled PtdIns(4,5)P2 degradation, stimulation of cotransport by phorbol 12-myristate 13-acetate (PMA), and inhibition of cotransport by the protein kinase C (PKC) inhibitor staurosporine. This suggests that cotransporter activation requires PKC-protein phosphorylation. This possibility was tested using the protein phosphatase inhibitor okadaic acid. Okadaic acid elevated bumetanide-sensitive Cl efflux. Staurosporine also blocked > 63% of okadaic-acid-stimulated Cl transport. The late DG peak did not support hormone-stimulated cotransport. The results demonstrate that DGs are a pivotal link between alpha 1-AR stimulation and NaCl(K) cotransport activation with a role for PKC and protein phosphorylation. alpha 1-AR intracellular signaling mechanisms apparently operate normally in CF cells.

Alkaloids↗

Endothelin stimulates phosphatidylcholine hydrolysis through both PLC and PLD pathways in mesangial cells.

Endothelin (ET) is a recently characterized vasoconstrictor hormone that has potent effects on glomerular function. Many vasoconstrictors, like ET, that stimulate phospholipase C (PLC) hydrolysis of polyphosphoinositides also stimulate phosphatidylcholine (PtdCho) hydrolysis via both PLC and phospholipase D (PLD) pathways. We have previously reported that ET stimulates a protein kinase C (PKC)-regulated, intracellular calcium-insensitive PLD activity that forms phosphatidic acid (PA) in rat mesangial cells (MC). We now ask whether ET-induced diglyceride (DG) production is also, in part, a result of either PLC- or PLD-induced hydrolysis of PtdCho. ET induced both a time- and dose-dependent stimulation in DG as measured by radioflux and mass assays. ET-stimulated DG production was still elevated even at time points where inositol polyphosphates had returned to basal levels. In addition, using [3H]choline-labeled cells, ET stimulated [3H]phosphocholine accumulation, suggesting a PLC-mediated hydrolysis of PtdCho. Stimulation of DG was unaffected by the presence of ethanol or propranolol, suggesting that ET-stimulated DG were not a result of a sequential PLD/PA phosphohydrolase activity. We further dissociated PtdCho-dependent PLC and PLD activities because, in contrast to ET-induced stimulation of PLD, the effect of ET on DG formation was mimicked with ionomycin and was inhibited with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid but not ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ET stimulation of DG could not be mimicked by phorbol myristate acetate and was not blocked by PKC inhibition or depletion. Together, these data suggest that ET stimulates multiple signaling pathways in MC that hydrolyze PtdCho via separate PLC and PLD mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Incorporation in vivo of 1-14C-palmitic acid into placental and fetal liver lipids of the rabbit.

The incorporation of free fatty acid into the placental and fetal liver lipids of rabbits was studied after fetal injections of albumin-bound 1-14C-palmitic acid. The fetuses were killed either 5--10 or 10--20 min after the injection. The placentas and livers were extracted for lipids and the specific activities of triglycerides (TG), phospholipids (PL), free fatty acids (FFA), monoglycerides (MG) and diglycerides (DG) measured. The lipids of the liver and placenta took up 17.0 and 3.6% of the dose, respectively, and of that liver TG accounted for 74% and the placental TG 34% of the label in each tissue. Most of the remaining counts were in the PL fraction with the rest more or less evely distributed between the FFA, DG and MG fractions. No activity was recorded in the cholesterol esters. The placental TG, PL, DG and MG specific activities reached the same level as that of the placental FFA, while in the liver these esters had higher specific activities (than the liver FFA). The liver TG, DG and PL had higher specific activities when compared with those of the placenta. The specific activity of the placental FFA was lower at 10--20 min than at 5--10 min; the opposite was seen for the placental TG. No time-related changes were seen in the liver lipids. It is concluded that (i) both placenta and fetal liver incorporate FFA into glycerides and PL; (ii) the liver incorporates FFA more rapidly and to a greater extent than the placenta; (iii) most of the FFA is incorporated into TG and to a lesser extent (PL; (iv) in both organs hydrolysis of PL or TG occurs. These results are discussed with reference to placental transport of FFA and fetal fat metabolism.

Animals↗

Cellular mechanism of endothelin-1 release by angiotensin and vasopressin.

Release of endothelin-1, a novel potent vasoconstrictor peptide originally isolated from endothelial cells, from cultured bovine endothelial cells has been shown to be stimulated by arginine vasopressin and angiotensin II. To elucidate the cellular mechanism by which endothelin-1 is released by these vasoconstrictors, we tested the effects of several compounds on the agonist-induced endothelin-1 release and studied the changes of cytosolic free Ca2+ concentrations and phosphoinositide breakdown by these agonists in cultured bovine endothelial cells. Protein kinase C inhibitors (H-7, staurosporine), an intracellular Ca2+ chelator, and an inhibitor of phospholipase C (neomycin), all abolished the agonist-induced endothelin-1 release, whereas the Ca2+ channel blocker nicardipine was ineffective. Although synthetic 1,2-diglyceride (diolein) dose dependently stimulated endothelin-1 release, downregulation of protein kinase C after pretreatment with phorbol ester resulted in decreased effects to increase endothelin-1 release by the agonists. Both arginine vasopressin and angiotensin II induced immediate and transient increases in intracellular Ca2+ levels of fura-2-loaded endothelial cells as well as formation of inositol trisphosphate; the agonist-induced intracellular Ca2+ increases were not affected either by nicardipine or by chelating extracellular Ca2+. The arginine vasopressin- and angiotensin II-induced intracellular Ca2+ increases, inositol trisphosphate formation, and endothelin-1 release were completely abolished by V1-receptor antagonist and saralasin, respectively. It is concluded that arginine vasopressin and angiotensin II stimulate the release of endothelin-1 by a common mechanism, involving receptor-mediated mobilization of intracellular Ca2+ and activation of protein kinase C in endothelial cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Pharyngeal lipase and digestion of dietary triglyceride in man.

Lipolytic activity was studied in esophageal and gastric aspirates obtained with a nasogastric tube from 14 healthy adult subjects. Samples were collected from esophagus, first at 30-35 cm and then at 40-45 cm from the nose, as the subject, after drinking 15-30 ml of a cream-milk mixture, swallowed small amounts of water. The samples from stomach were taken last and usually contained a small amount of cream-milk mixture. Lipolytic activity was assayed using chylomicron, milk, and corn oil triglyceride as substrate. Esophageal and gastric samples both contained lipolytic activity which hydrolyzed long-chain triglyceride to diglyceride, monoglyceride, and FFTA, had a pH optimum of 5.4, and was not affected by either had a pH optimum of 5.4, and was not affected by either 0.5 M NaCl or 4 mM sodium taurodexycholate. The activity, expressed as nanomoles of chylomicron triglyceride hydrolyzed per milliter per minute, ranged from 0 to 145 in upper esophageal, 5 to 303 in lower esophageal, and 50 to 357 in gastric samples. Only a trace of lipolytic activity was found at pH 5.4 in saliva collected from the parotid, submandibular, and sublingual glands, thus excluding those tissues as a source of the activity found in esophageal and gastric aspirates. The findings suggest that in man glands in or near the pharynx secrete a lipase that acts in the stomach to hydrolyze long-chain triglyceride to partial glycerides and FFA. It is proposed this reaction is the first step in the digestion of dietary fat and that the amphiphilic lipids formed by lipolysis facilitate the emulsification of triglyceride in the stomach.

Adult↗

Sex differences in long chain fatty acid utilization and fatty acid binding protein concentration in rat liver.

Female sex and estrogen administration are associated with increased hepatic production of triglyceride-rich lipoproteins; the basis for this has not been fully elucidated. Inasmuch as hepatic lipoprotein production is also influenced by FFA availability and triglyceride biosynthesis, we investigated sex differences in FFA utilization in rat hepatocyte suspensions and in the components of the triglyceride biosynthetic pathway. Isolated adult rat hepatocyte suspensions were incubated with albumin-bound [(14)C]oleate for up to 15 min. At physiological and low oleate concentrations, cells from females incorporated significantly more (14)C into glycerolipids, especially triglycerides, and into oxidation products than did male cells, per milligram cell protein. At 0.44 mM oleate, incorporation into triglycerides in female cells was approximately twice that in male cells. Comparable sex differences were observed in cells from fasted animals and when [(14)C]-glycerol incorporation was measured. At higher oleate concentrations, i.e., fatty acid:albumin mole ratios in excess of 2:1, these sex differences were no longer demonstrable, suggesting that maximal rates of fatty acid esterification and oxidation were similar in female and male cells. In female and male hepatic microsomes, specific activities of long chain acyl coenzyme A synthetase, phosphatidate phosphohydrolase, and diglyceride acyltransferase were similar, but glycerol-3-phosphate acyltransferase activity was slightly greater in females at certain substrate concentrations. Microsomal incorporation of [(14)C]oleate into total glycerolipids was not significantly greater in females. In further contrast to intact cells, microsomal incorporation of [(14)C]oleate into triglycerides, although significantly greater in female microsomes, accounted for only a small fraction of the fatty acid esterified.The binding affinity and stoichiometry of partially purified female hepatic fatty acid binding protein (FABP) were similar to those of male FABP. In contrast, the concentration of FABP, per milligram cytosolic protein, was 44% greater in female liver than in male, as indicated by measurement of [(14)C]oleate binding and of 280 nm OD in the FABP fraction of 105,000 g supernate after gel filtration chromatography. These experiments demonstrate profound sex differences in hepatocyte utilization of long chain fatty acids at concentrations within and below the physiological range, and suggest that these are attributable at least in part to corresponding differences in cytosolic FABP concentration. At higher FFA concentrations, sex differences in hepatocyte FFA utilization are virtually eliminated, suggesting that under these conditions, differences in FABP concentration are not rate determining. Sex differences in hepatic lipoprotein production may largely reflect these important differences in the initial stages of hepatocyte FFA utilization.

Albumins↗

Dietary omega-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils.

Earlier studies demonstrated that dietary omega-3 polyunsaturated fatty acid (PUFA) supplementation attenuates the chemotactic response of neutrophils and the generation of leukotriene (LT) B4 by neutrophils stimulated with calcium ionophore; however, the mechanisms and relationship of these effects were not examined. Neutrophils and monocytes from eight healthy individuals were examined before and after 3 and 10 wk of dietary supplementation with 20 g SuperEPA daily, which provides 9.4 g eicosapentaenoic acid (EPA) and 5 g docosahexaenoic acid. The maximal neutrophil chemotactic response to LTB4, assessed in Boyden microchambers, decreased by 69% after 3 wk and by 93% after 10 wk from prediet values. The formation of [3H]inositol tris-phosphate (IP3) by [3H]inositol-labeled neutrophils stimulated by LTB4 decreased by 71% after 3 wk (0.033 +/- 0.013% [3H] release, mean +/- SEM) and by 90% after 10 wk (0.011 +/- 0.011%) from predict values (0.114 +/- 0.030%) as quantitated by beta-scintillation counting after resolution on HPLC. LTB4-stimulated neutrophil chemotaxis and IP3 formation correlated significantly (P < 0.0001); each response correlated closely and negatively with the EPA content of the neutrophil phosphatidylinositol (PI) pool (P = 0.0003 and P = 0.0005, respectively). Neither the affinities and densities of the high and low affinity LTB4 receptors on neutrophils nor LTB4-mediated diglyceride formation changed appreciably during the study. Similar results were observed in neutrophils activated with platelet-activating factor (PAF). The summed formation of LTB4 plus LTB5 was selectively inhibited in calcium ionophore-stimulated neutrophils and was also inhibited in zymosan-stimulated neutrophils. The inhibition of the summed formation of LTB4 plus LTB5 in calcium ionophore-stimulated neutrophils and in zymosan-stimulated neutrophils did not correlate significantly with the EPA content of the PI pool. The data indicate that dietary omega-3 PUFA supplementation inhibits the autoamplification of the neutrophil inflammatory response by decreasing LTB4 formation through the inactivation of the LTA epoxide hydrolase and independently by inhibiting LTB4- (and PAF) stimulated chemotaxis by attenuating the formation of IP3 by the PI-selective phospholipase C. This is the initial demonstration that dietary omega-3 PUFA supplementation can suppress signal transduction at the level of the PI-specific phospholipase C in humans.

Adult↗

Enhanced superoxide release and elevated protein kinase C activity in neutrophils from diabetic patients: association with periodontitis.

Inflammation and oxidative stress are important factors in the pathogenesis of diabetes and contribute to the pathogenesis of diabetic complications. Periodontitis is an inflammatory disease that is characterized by increased oxidative stress, and the risk for periodontitis is increased significantly in diabetic subjects. In this study, we examined the superoxide (O(2)(-))-generating reduced nicotinamide adenine dinucleotide phosphate-oxidase complex and protein kinase C (PKC) activity in neutrophils. Fifty diabetic patients were grouped according to glycemic control and the severity of periodontitis. Neutrophils from diabetic patients with moderate [amount of glycated hemoglobin (HbA(1c)) between 7.0% and 8.0%] or poor (HbA(1c) >8.0%) glycemic control released significantly more O(2)(-) than neutrophils from diabetic patients with good glycemic control (HbA(1c) <7.0%) and neutrophils from nondiabetic, healthy individuals upon stimulation with 4beta-phorbol 12-myristate 13-acetate or N-formyl-Met-Leu-Phe. Depending on glycemic status, neutrophils from these patients also exhibited increased activity of the soluble- and membrane-bound forms of PKC, elevated amounts of diglyceride, and enhanced phosphorylation of p47-phox during cell stimulation. In addition, we report a significant correlation between glycemic control (HbA(1c) levels) and the severity of periodontitis in diabetic patients, suggesting that enhanced oxidative stress and increased inflammation exacerbate both diseases. Thus, hyperglycemia can lead to a novel form of neutrophil priming, where elevated PKC activity results in increased phosphorylation of p47-phox and O(2)(-) release.

Diabetes Mellitus↗

Response surface methodology for the optimization of ubiquinone self-nanoemulsified drug delivery system.

The aim of the present study was to prepare and evaluate an optimized, self-nanoemulsified drug delivery system of ubiquinone. A 3-factor, 3-level Box-Behnken design was used for the optimization procedure with the amounts of Polyoxyl 35 castor oil (X1), medium-chain mono- and diglyceride (X2), and lemon oil (X3) as the independent variables. The response variable was the cumulative percentage of ubiquinone emulsified in 10 minutes. Different ubiquinone release rates were obtained. The amount released ranged from 11% to 102.3%. Turbidity profile revealed 3 regions that were used to describe the progress of emulsion formation: lag phase, pseudolinear phase, and plateau turbidity. An increase in the amount of surfactant decreased turbidity values and caused a delay in lag time. Addition of cosurfactant enhanced the release rates. Increasing the amount of the eutectic agent was necessary to overcome drug precipitation especially at higher loading of surfactants and cosurfactants. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The regression equation generated for the cumulative percentage emulsified in 10 minutes was Y1 = 90.9 - 22.1X1 + 5.03X2 + 13.95X3 + 12.13X1X2 + 15.13X1X3 - 14.40X1(2) - 6.25X3(2). The optimization model predicted a 93.4% release with X1, X2, and X3 levels of 35, 35, and 30 respectively. The observed responses were in close agreement with the predicted values of the optimized formulation. This demonstrated the reliability of the optimization procedure in predicting the dissolution behavior of a self-emulsified drug delivery system.

Castor Oil↗

Involvement of protein kinase C in the regulation of adrenocorticotropin release from rat anterior pituitary cells.

The involvement of protein kinase C in normal corticotroph function was studied by analysis of the effects of the phorbol ester derivative phorbol 12-myristate-13-acetate (PMA) and the synthetic diacylglycerol dioctanoylglycerol (DOG) on basal and stimulated ACTH release in cultured rat anterior pituitary cells. Incubation of rat pituitary cells with increasing concentrations of PMA or DOG caused dose-related increases in ACTH release up to 13.4 +/- 2.1- and 10.1 +/- 0.9-fold, respectively, similar to that caused by CRF (9.8 +/- 1.6-fold). Also, stimulation of endogenous diglyceride formation by phospholipase C (100 mU/ml) stimulated ACTH release by 2.5 +/- 0.1-fold. In cells incubated with maximum stimulatory concentrations of CRF (10 nM) or 8-bromo-cAMP (8-Br-cAMP; 5 mM), addition of either 100 microM DOG or 100 nM PMA caused significantly higher ACTH responses than those obtained with CRF, 8-Br-cAMP, DOG, or PMA alone. 8-Br-cAMP (5 mM) and 10 nM CRF significantly increased the effect of 100 nM PMA by 1.4 +/- 0.2- and 1.5 +/- 0.1-fold, respectively. Combinations of 10 nM CRF with either vasopressin (VP) or angiotensin II (AII) increased ACTH secretion to values higher than those produced by CRF, VP, or AII alone. However, addition of maximal stimulatory concentrations of VP or AII (10 nM) did not further increase the effects of either PMA alone or PMA/CRF combinations, indicating that their mechanisms of action may be similar to that of PMA. These results indicate that in addition to the established cAMP-dependent mechanism, stimulation of ACTH release in normal pituitary cells may be elicited by activation of protein kinase C. The evidence also suggests that protein kinase C is involved during stimulation of ACTH release by the cAMP-independent regulators VP and AII and in the synergistic effects of VP and AII with CRF on the corticotroph.

8-Bromo Cyclic Adenosine Monophosphate↗