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PubMed1981

[Enzymatic determination of serum glycerophosphatides, using phospholipase C (author's transl)].

A simple method is described for the determination of serum glycerophosphatides, with the exception of lyso-phosphatide. Two determinations are necessary. The usual "triglyceride" determination is performed on native serum, and a further determination is performed after incubation of the serum with phospholipase C. Glycerophosphatides are hydrolysed to diglycerides and phosphorylcholine by phospholipase C. The resulting diglycerides are determined in addition to the mono-, di-, triglycerides and free glycerol of the native serum. The difference between the two glyceride determinations corresponds to the concentration of glycerophosphatides. Involvement of glycerophosphatides in blood coagulation and their increased levels in certain liver illnesses are two reasons for the importance of the present method. Compared with the usual method of measuring phospholipid phosphorus, the present method involves considerably less work, and, as a fully enzymatic method, it has a higher specificity.

Glycerophosphates↗
PubMed1980

Properties of phospholipase C isolated from rat liver lysosomes.

Phospholipase C (EC 3.1.4.3) has been identified in a soluble, delipidated protein fraction isolated from rat liver lysosomes. Lysosomal phospholipase C is active against all phospholipids tested, including phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine. It has an acid pH optimum, does not require divalent cations, and is not inhibited by EDTA. With [1-14C]dioleoylphosphatidylcholine as the substrate, 14C-labeled monoglyceride and diglyceride are the reaction products. Monoglyceride is formed rapidly from diglyceride by a lysosomal acid lipase, although some monoglyceride may be formed directly by phospholipase C hydrolysis of lysophosphatidylcholine. The other product, phosphocholine, has been identified by its behavior during Dowex 1-formate anion exchange chromatography. This appears to be the first demonstration in mammalian systems ofa phospholipase C which is active against all phosphoglycerides.

Animals↗
PubMed1976

Metabolic fate of 3,4-dihydroxybutyl-1-phosphonate in Escherichia coli.

3,4-Dihydroxy[3-(3)H]butyl-1-phosphonate, and analogue of glycerol 3-phosphate, is incorporated into a very polar lipid material by cultures of Escherichia coli strain 8 and in vitro by CDP-diglyceride:sn-glycerol-3-phosphate phosphatidyltransferase. These labeled lipids have been fractionated by column chromatography on DEAE-cellulose, revealing that only one labeled compound is formed in vitro, while four are synthesized in vivo. The main component of the material formed by intact cells has been shown to be identical with that produced enzymatically. This species has been identified as the phosphonic acid analogue of phosphatidylglycerophosphate [(1,2-diacyl)-sn-glyceryl-D-4'-phosphoryloxy-3'-hydroxybutyl-1'-phosphonate]. Hydrolysis of this novel lipid with phospholipase C resulted in the production of diglyceride and a water-soluble derivative of 3,4-dihydroxybutyl-1-phosphonate and inorganic phosphate in a molar ratio of 1.03/1. Enzymatic analysis of the phosphonate liberated in this manner showed it to be the D enantiomer, thereby confirming the proposed structure of the lipid analogue. The analogue of phosphatidylglycerophosphate did not turn over and appeared to have no precursor-product relationship to the other labeled lipids derived from 3,4-dihydroxy[3-(3)H]butyl-1-phosphonate in vivo. Analysis of the other three labeled products revealed the tritium to be present on glycerol 3-phosphate and not intact phosphonate, indicating some metabolic degradation of the latter. Examination of cell components other than lipids revealed little incorporation of label, while a significant amount of tritium was found to be present in a distillable form, 3H2O. Experiments with mutants of E. coli lacking the known glycerol-3-phosphate dehydrogenases indicated that these enzymes are not responsible for the removal of tritium from from 3,4-dihydroxy[3-(3)H]butyl-1-phosphonate in vivo. Indirect evidence suggests that the inhibition of cell growth by this analogue is not due to its catabolic products.

Butylene Glycols↗
PubMed1996

Phospholipase D activity regulates the binding of leukotriene B4 to human polymorphonuclear leukocytes.

Unsaturated fatty acids, such as arachidonic acid, have been implicated as second messengers of various cellular responses. In this paper, we demonstrate that the release of arachidonate from human polymorphonuclear leukocytes (PMNL) via a pathway initiated by phospholipase D (PLD) mediates phorbol myristate acetate (PMA)-induced desensitization of leukotriene B4 (LTB4) receptors. PMA caused a delayed release of arachidonic acid from PMNL, which occurred within minutes of the generation of the PLD products, phosphatidic acid and diglyceride, from [3H]lysophosphatidylcholine prelabeled PMNL. Moreover, stimulating PMNL with PMA in the presence of ethanol resulted in the formation of phosphatidylethanol at the expenses of phosphatidic acid, diglyceride and arachidonic acid. The PMA-induced generation of these three PLD products was inhibited by mepacrine which, in parallel, significantly blocked PMA-induced desensitization of [3H]LTB4 binding, which suggested that elevation of one or more of these products played a role in desensitization. In contrast, under basal conditions mepacrine reduced levels of these three lipids in a dose-related manner and, in parallel, increased basal [3H]LTB4 binding to PMNL. The reduction of PMA-induced LTB4 receptor desensitization by mepacrine could be overcome to various degrees by adding back PLD-derived lipids such as arachidonic acid. These data demonstrate that [3H]LTB4 binding to PMNL is decreased by PLD-derived lipids and suggests that intracellular arachidonic acid may modulate PMNL responsiveness to LTB4 activation.

Binding, Competitive↗
PubMed1977

Studies on some unusual lipids of the preputial glands of rats and mice and their hormonal influence.

Major neutral lipid fractions of the preputial glands of mice have been identified by analytical and preparative thin layer chromatography (TLC) and gas liquid chromatography (GLC) as wax esters, neutral plasmalogen, alkyl diglyceride, triglyceride and cholesterol. An unusual lipid fraction between wax esters and neutral plasmalogens was identified as alkyl acetate, which has been reported earlier to function as sex attractant. The lipid pattern of the preputial glands of rats in both sexes was similarly studied. Three unknown bands of lipids were identified by TLC between wax ester and alk-1-enyl diglycerides, which on further analysis by GLC and infrared spectroscopy gave preliminary indication of aliphatic diester waxes. Administration of androgen stimulated the biosynthesis of these substances.

Animals↗
PubMed1997

Enhancing effects of monohexanoin and two other medium-chain glyceride vehicles on intestinal absorption of desmopressin (dDAVP).

The intestinal absorption enhancement of the nonapeptide [Mpa1,D-Arg8]vasopressin (dDAVP) by medium-chain glyceride vehicles was studied using an in vivo rat model. Rats were gavaged with dDAVP formulated with three different lipid vehicles: (1) monohexanoin, (2) mixed monoglycerides, diglycerides and triglycerides of hexanoic acid and (3) monoglycerides, diglycerides and triglycerides of octanoic and decanoic acids, and with saline as control. The marker absorption into blood and urine was followed for 24 hr. All lipid vehicles enhanced the oral bioavailability of dDAVP, but monohexanoin gave the highest increase, approximately 10 times that of control. In contrast to dDAVP, the stable and more lipophilic nonapeptide analog [Mpa1,D-Tyr(ethyl)2,Val4,D-Arg8]oxytocin did not show increased urine recovery when formulated with monohexanoin. A 2-fold increase in urine recovery of the inert low-molecular-weight marker [51Cr]EDTA was observed when formulated with monohexanoin. With use of the fluorescent marker Evans blue formulated with monohexanoin, an elevated accumulation of Evans blue in the mucus layer was observed after incubation in in situ loops. No mucosal damage after lipid vehicle gavage was observed by light microscopic evaluation. Medium-chain glycerides functioned well as oral absorption enhancers of the model peptide dDAVP, and monohexanoin showed the highest enhancement capacity. The mechanisms of this enhancement appear to be related to a protection against luminal dDAVP degradation, mucoadhesive properties of the vehicle and, possibly, an altered epithelial absorption pathway.

Animals↗
PubMed1997

Myocardial fatty acid pattern in rats fed on an erucic acid enriched diet.

Individual lipid classes and their fatty acid pattern in myocardium of rats fed on a diet containing 10% erucic acid ethyl ester (cis-13-docosenoic acid ethyl ester) were investigated and compared to rats fed on a normal diet. Two groups of rats were treated for 10 consecutive days with the erucic acid ethyl ester diet and the standard diet, respectively. After extracting total lipids from the myocardium of the rats, the individual lipid classes and the percentage of fatty acids in phospholipids, free fatty acids, diglycerides and triglycerides were measured. The data obtained demonstrate that the erucic acid ethyl ester diet induces a marked increase in free fatty acids and in triglycerides and marked differences in fatty acid pattern in triglycerides, free fatty acids and diglycerides, but only marginal differences in phospholipids, which seem to be carefully preserved as fundamental components of cell and mitochondria membranes.

Animals↗
PubMed1997

Phospholipid metabolism in boar spermatozoa and role of diacylglycerol species in the de novo formation of phosphatidylcholine.

We have investigated pathways of lipid metabolism in boar spermatozoa sperm cells incubated for up to 3 days with [14C]palmitic acid, [14C]glycerol, [14C]choline, or [14C]arachidonic acid or incorporated these precursors into diglycerides and/or phospholipids. When spermatozoa were incubated with [14C]palmitic acid or [14C]glycerol, there was first an incorporation into phosphatidic acid, followed by labelling of 1,2-diacylglycerol (DAG) and then phosphatidylcholine (PC). This indicates that the de novo pathway of phospholipid synthesis is active in these cells. However, not all DAG was converted to PC. A pool of di-saturated DAG, which represented a considerable proportion of the high basal levels of DAG, accumulated the majority of label. Another DAG pool, containing saturated fatty acids in position 1 and unsaturated fatty acids in position 2 and representing the remaining basal DAG, was in equilibrium with PC. When spermatozoa were incubated with [14C]arachidonic acid, there was a considerable incorporation of label into PC, which indicates the presence of an active deacylation/ reacylation cycle. The behaviour of certain lipid pools varied depending on the temperature at which spermatozoa were incubated. For example, in the presence of [14C]palmitic acid or [14C]arachidonic acid, there was more incorporation of label into PC when spermatozoa were incubated at 25 degrees C than when incubated at 17 degrees C. Taken together, these results indicate that spermatozoa have an active lipid synthetic capacity. It may therefore be possible to design methods to evaluate the metabolic activity of boar spermatozoa based on the incorporation of lipid precursors under standardized conditions.

Animals↗
PubMed2003

Lipase regio- and stereoselectivities toward three enantiomeric pairs of didecanoyl-deoxyamino-O methyl glycerol: a kinetic study by the monomolecular film technique.

A kinetic study was carried out on the regio- and stereoselectivities of 12 lipases of animal and microbial origin. For this purpose, monomolecular films consisting of three pairs of enantiomers (didecanoyl-deoxyamino-O methyl glycerol, DDG) containing a single hydrolyzable decanoyl ester bond and two lipase-resistant groups were spread at the air-water interface. Each of the lipases tested displayed a particular type of behavior, on the basis of which they were classified in two groups, depending on their ability to hydrolyze the sn-2 position. From the qualitative point of view, the sn-2 preference measured on triacylglycerides and DDG were in good agreement. The inductive chemical effect might explain why a greater level of hydrolytic activity was observed with the diglycerides than with DDG. With most of the lipases tested, it was observed that the enantiomeric pair having two distal acyl chains was more clearly differentiated stereochemically than the two homologous pairs with two adjacent acyl chains. This finding is consistent with the hypothesis that during the chiral recognition process two of the three attachment points may be the external (distal) hydrophobic chains, which is in line with the hypothesis of a tuning fork conformation of a triglyceride in the lipase active site.

Animals↗
PubMed1990

1,2-Dioctanoylglycerol accelerates or retards mitotic progression in Tradescantia stamen hair cells as a function of the time of its addition.

We have treated living, intact stamen hair cells from the spiderwort plant, Tradescantia virginiana, with 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol, a potent and permeant activator of protein kinase C, and have observed the rates of progression of mitosis from prophase through anaphase. We have found that in addition to the concentration used, the time of initial treatment with 1,2-dioctanoylglycerol defines the response by the cells. The cells rapidly undergo nuclear envelope breakdown when this diglyceride is added in very late prophase, 0 to approximately 8 min prior to the time of normal nuclear envelope breakdown. Anaphase onset occurs 28 min after nuclear envelope breakdown, rather than after the 33 min interval observed in untreated cells. Rapid progression through metaphase is also observed if cells are treated with 0.5 microgram/ml 1,2-dioctanoylglycerol during prometaphase, up to 15 min after nuclear envelope breakdown. The addition of 0.5 microgram/ml 1,2-dioctanoylglycerol in late metaphase, approximately 26 min after nuclear envelope breakdown, results in sister chromatid separation slightly ahead of its normal time, 33 min after nuclear envelope breakdown, and in precocious cell plate vesicle aggregation, 3-5 min earlier than that observed in untreated cells. Treatment of cells with 60 micrograms/ml of 1,2-dioctanoylglycerol at any point during the interval from 0 to approximately 5 min prior to nuclear envelope breakdown results in precocious entry into anaphase. If cells are treated with either 0.5 microgram/ml or 60 micrograms/ml 1,2-dioctanoylglycerol earlier than 20 min before nuclear envelope breakdown, they do not enter mitosis, but instead revert to interphase without dividing. When 1,2-dioctanoylglycerol is added at other times during mitosis, the rate of subsequent mitotic progression is dramatically slowed; the cells require greater than 55 min to progress from nuclear envelope breakdown to anaphase onset, though once in anaphase, the cells progress onward to cytokinesis at normal rates. Treatments o of cells with 1,3-dioctanoylglycerol at any point during prophase, prometaphase, or metaphase are without effect on the rate of subsequent mitotic progression. The shifts in response by cells treated at specific times with 1,2-dioctanoylglycerol during mid- and late metaphase may be indicative of the existence of one or more regulatory switch points (i.e., checkpoints) just prior to anaphase onset.

Anaphase↗
PubMed1994

Separation of agonist-stimulated arachidonate mobilization from subsequent leukotriene B4 synthesis in human neutrophils: different effects of oleoylacetylglycerol and phorbol myristate acetate as priming agents.

Preincubation of human neutrophils with phorbol esters or soluble diglycerides enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization and leukotriene B4 (LTB4) synthesis. We have recently reported that 1,3-dioctanoylglycerol (1,3-diC8) is equipotent with 1,2-sn-dioctanoylglycerol (1,2-diC8) as priming agent, thus suggesting that the priming effects of diacylglycerols are protein kinase C (PKC) independent (Rosenthal et al., 1993, Biochim. Biophys. Acta 1177:79-86). In order to further investigate this question, the present study has directly compared the effects of oleoylacetylglycerol (OAG) and the PKC activator, phorbol 12-myristate 13-acetate (PMA), on agonist-stimulated lipid metabolism. The results indicate that both OAG and PMA dose dependently enhance f-Met-Leu-Phe (fMLP)-stimulated release of [3H]arachidonate. Optimal concentrations of OAG (5 microns) and PMA (10 nM) are equipotent in increasing fMLP-stimulated arachidonate mobilization as quantitated either with total radioactivity or by mass measurements of free arachidonate. By contrast OAG is sixfold more effective than PMA in enhancing synthesis of 5-lipoxygenase (5-LO) metabolites by mass and two to threefold more effective than PMA in enhancing synthesis of [3H]eicosanoids. Furthermore, OAG, but not PMA, enhances fMLP-stimulated synthesis of platelet-activating factor. By contrast, PMA directly stimulates [3H]arachidonate mobilization, while OAG (20 microM) does not; despite these differences, the combined effects of PMA + OAG on subsequent agonist-stimulated arachidonate release are not greater than those of PMA alone. In cells challenged with subthreshold concentrations (< 0.1 microM) of the calcium ionophore A23187, both OAG and PMA stimulate [3H]arachidonate release but not [3H]LTB4 synthesis. These findings suggest that OAG does not directly activate 5-LO, but instead couples arachidonate mobilization to leukotriene synthesis in a PKC-independent manner.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1995

Phorbol ester-induced priming of superoxide generation by phosphatidic acid-stimulated neutrophils and granule-free neutrophil cytoplasts.

This study was undertaken to examine the mechanisms involved in polymorphonuclear leukocyte superoxide release stimulated by exogenous phosphatidic acid (PA). Unlike the immediate burst of superoxide release affected by membrane-permeable dioctanoylglycerol (DiC8-DAG), dioctanoyl phosphatidic acid (DiC8-PA) induced superoxide release after a lag period of 5-20 min. This period was considerably reduced or eliminated when cells were primed by substimulatory levels of phorbol myristate acetate (PMA). Granule-depleted neutrophil cytoplasts also responded to DiC8-PA with a burst of superoxide generation. Activation of the cytoplast superoxide generating system in response to DiC8-PA was also significantly faster after cells had been preexposed to substimulatory levels of PMA, indicating that at least a portion of the priming mechanism was independent of PMA-induced degranulation. To further examine the potential mechanism of PMA priming of responses to PA, we evaluated the activity of neutrophil ecto-phosphatidic acid phosphohydrolase (ecto-PA phosphohydrolase), which generates diacylglycerol from exogenous PA. PMA priming had no discernable effect on the activity of this enzyme. In addition, propranolol, an inhibitor of PA phosphohydrolase, did not selectively inhibit PMA priming of neutrophil responses to DiC8-PA, indicating that priming did not result from acceleration of DiC8-PA hydrolysis. We therefore investigated the possibility that activation of protein kinase C was the basis of the primed response. Several semiselective protein kinase C inhibitors (calphostin C, H-7, and acylmethylglycerol) inhibited DiC8-DAG- and DiC8-PA-induced superoxide release as well as PMA-primed responses to approximately the same extent. These results are consistent with the hypothesis that neutrophil responses to phosphatidate are mediated by diglyceride generated by the action of ecto-PA phosphohydrolase. PMA priming does not result from increased catalytic activity of ecto-PA phosphohydrolase but rather seems to result from potentiation of an intermediate involved in the cells' response to multiple stimuli.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Oligodendrocyte-substratum adhesion activates the synthesis of specific lipid species involved in cell signaling.

Ovine oligodendrocytes (OLGs) undergo biochemical and morphological changes following attachment to polylysine. Autoradiographs of two-dimensional thin-layer chromatograms of [14C]Gal-labeled OLG cultures revealed that attachment of OLGs to a polylysine substratum and their subsequent morphological differentiation is accompanied by an increased synthesis of multiple forms of galactosylceramide, sulfogalactosylceramide, and both sulfogalactosyl- and galactosyl-diglycerides, together with an array of complex sialoglycosphingolipids, predominantly GM2 ganglioside. As previously reported, overall lipid synthesis measured by [14C]acetate incorporation into glycerophosphatides, sphingomyelin, and neutral lipids also increased dramatically for up to 60 days (last time point examined) following OLG-substratum adhesion, reflecting membrane growth. Attachment was associated with a rapid augmentation in the synthesis of ethanolamine plasmalogen from 12 to 27% within 24 hr to reach a 35% plateau at 30 days and remain constant thereafter. In contrast, the plasmalogen content of phosphatidylcholine remained constant at 3-5%. This rapid increase in lipid synthesis (especially in the ethanolamine plasmalogen content following attachment) closely paralleled increased diacylglycerol (DAG) production and protein kinase C-dependent phosphorylation of both myelin basic protein and 2',3'-cyclic nucleotide phosphohydrolase. Labeling studies indicated that the major source of [3H]arachidonate-labeled DAG following attachment was from phosphatidylinositol turnover (and to a lesser extent phosphatidylcholine) rather than polyphosphoinositides or plasmalogens. Enhanced lipid synthesis is not only required for the production of membranes in these myelin-producing cells but is also a source of second messengers required in the posttranslational modification of key myelin and cellular proteins.

Animals↗
PubMed1979

Lipid-protein interactions in membranes: effect of lipid composition on mobility of spin-labeled cysteine residues in yeast plasma membrane.

In order to gain direct evidence for lipid-dependent protein conformation in membrane, effects of modification of lipid composition on mobility of spin-labeled cysteine residues were investigated in the plasma membrane of the yeast Saccharomyces cerevisiae. Conversion of the bulk of phospholipids to diglycerides by treatment of the membrane with phospholipase C substantially enhanced spectral anisotropy. However, alterations of the viscosity of the lipid-bilayer by enriching the membrane with palmitelaidic or oleic acid had no effect on mobility of spin-labeled cysteine residues. These observations indicate that while the spin-labeled residues are not in direct contact with the lipid core of the membrane, there are lipid-protein interactions to the extent that removal of the polar portion of the bulk of phospholipids induces conformational changes in proteins, which in turn restrict mobility of these residues. It is concluded that conformation of membrane proteins on lipid structure and that phospholipids have a role in preserving the native conformation of proteins.

Adenosine Triphosphatases↗
PubMed1993

sn-1,2-diacylglycerol cholinephosphotransferase from pig liver: mixed micellar assay and kinetic analysis of the partially pure enzyme.

sn-1,2-Diacylglycerol cholinephosphotransferase from pig liver microsomes was partially purified through a procedure involving solubilization with sodium cholate and chromatography on Sepharose 6B. The resulting preparation was 19-fold enriched with respect to microsomes and was shown to be very sensitive to different detergents. Sodium cholate gave the best yields in activity. In a mixed micellar assay with Triton X-100 a strong dependence of the enzyme activity on the concentration of mixed micelles was observed, due to Triton X-100 acting as an inactivator. Soja phosphatidylcholine added exogenously protected the enzyme against detergent inactivation and stimulated the enzyme activity. Dioleoyl-phosphatidylcholine had a similar stimulatory effect, whereas didecanoyl- or dioctanoyl-phosphatidylcholine did not; thus long-chain phosphatidylcholines seem to be essential in the activation of cholinephosphotransferase. In a mixed micellar assay with sodium cholate no inactivation of the enzyme could be detected and it was found that soja phosphatidylcholine stimulates the activity in a greater extent than in Triton X-100 mixed micelles. The phospholipid activates the enzyme in a noncompetitive way with an activation constant of 176 mol%. Km was estimated as 1.54 mol% with a Vmax = 30 nmol/min/mg protein. Those results support an activation mechanism by phosphatidylcholine interacting at sites different from the active center. The high activation constant led to the conclusion that cholinephosphotransferase requires a lipidic boundary for full activation. No activation by substrate was observed. Short-chain diacylglycerides such as dihexanoyl-, dioctanoyl-, or didecanoylglycerol can be used as substrates although the enzyme in this case has only 5 to 10% of the activity it has for dioleoylglycerol or egg diglycerides.

Animals↗
PubMed1994

Phosphatidylcholine-specific phospholipase D activity is elevated in v-Fps-transformed cells.

Activating the protein-tyrosine kinase of v-Fps results in a rapid increase in diglyceride (DG) in rat fibroblasts. The v-Fps-induced increases in DG were detected only when phospholipids were prelabeled with [3H]-myristate, which is incorporated primarily into phosphatidylcholine (PC). Inhibition of phosphatidic acid (PA) phosphatase (PAP), which converts PA to DG, blocked v-Fps-induced DG production. PA is a primary metabolite of type D phospholipases (PLD). Consistent with these observations, PLD activity was activated in response to the kinase activity of v-Fps. The increased PLD activity was detected only when the cells were prelabeled with the PC-specific [3H]-myristate. These data support the hypothesis that v-Fps-induced DG is derived from PC via the PLD/PAP pathway.

Animals↗
PubMed1996

Interactions between signaling pathways in mediating GnRH-stimulated GTH release from goldfish pituitary cells: protein kinase C, but not cyclic AMP is an important mediator of GnRH-stimulated gonadotropin secretion in goldfish.

In the goldfish, it has been proposed that gonadotropin (GTH) release induced by GTH-releasing hormone (GnRH) involves Ca2+ entry through voltage-sensitive Ca2+ channels (VSCC), protein kinase C (PKC) activation, and arachidonic acid (AA) metabolism, but not cyclic AMP (cAMP) action. However, cAMP appears to mediate GnRH action in other teleosts. In this study, the relative importance of PKC and cAMP in mediating GnRH action in goldfish was studied using primary cultures of dispersed pituitary cells. Consistent with an involvement of PKC in GnRH action, the GTH responses to the PKC activating tetradecanoyl phorbol acetate (TPA), salmon (s)GnRH, and chicken (c)GnRH-II were inhibited by two selective PKC inhibitors, calphostin C, and staurosporine. Furthermore, GTH release responses induced by sGnRH or cGnRH-II were not additive to responses stimulated by the PKC-activating diglyceride DiC8, in either long-term static incubation or acute perifusion experiments. In static incubation studies, the GTH responses to sGnRH and DiC8 were potentiated by the VSCC agonist Bay K 8644, suggesting that VSCC participates in both PKC and GnRH action. Concentrations of K+ < 100 mM did not elicit GTH secretion when tested alone, but were effective in stimulating GTH release in the presence of subthreshold doses of DiC8 or TPA. This suggests that minimal activation of PKC greatly enhances the effectiveness of Ca2+ influx to increase GTH secretion. Taken together, these results indicate that PKC is an important mediator of GnRH-induced, VSCC-dependent GTH release. In contrast to the involvement of PKG, cAMP-dependent mechanisms showed no evidence of direct participation in GnRH-induced GTH release in goldfish. In static incubation studies, the GTH responses to sGnRH and cGnRH-II were not affected by H89, a cAMP-dependent protein kinase (PKA) inhibitor. Furthermore, the GTH release stimulated by cAMP was additive to the response to sGnRH, cGnRH-II, DiC8, TPA, or AA. However, compared to the response to forskolin or TPA alone, combinations of forskolin and TPA resulted in a potentiated increase in GTH release. The acute GTH response to forskolin was also enhanced by DiC8. Thus, cAMP-dependent mechanisms may constitute an independent pathway that interacts positively with GnRH-dependent mechanisms in the regulation of GTH release.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1985

Platelet protein phosphorylation.

As can be seen from this review, protein phosphorylation appears involved in both positive and negative regulation of platelets. To date, good evidence has been presented for the involvement of protein phosphorylation in the regulation of granule centralization (i.e. myosin light chain phosphorylation). It is probable that protein phosphorylation may also be involved in granule labilization, pseudopod formation and ATP synthesis. Protein phosphorylation in association with platelet activation appears mediated through calcium flux, in the case of myosin light chain phosphorylation, and through diglyceride or other substances in the case of 47P phosphorylation. A summary scheme is shown in Figure 1.

Actin Cytoskeleton↗