Clement J. Welsh

The phosphatidylcholine pathway of diacylglycerol formation stimulated by phorbol diesters occurs via phospholipase D activation

Agonist‐induced degradation of phosphatidylcholine (PC) is of interest as this pathway of diacylglycerol (DG) generation may provide added opportunities for the regulation of protein kinase C (PKC). In REF52 cells [3H]myristic acid is preferentially incorporated into PC; this, coupled with the use of [3H]choline, allows for quantitation of both the water‐soluble and the lipid products generated when PC is degraded. In cells prelabeled with [3H]choline, TPA stimulated a time‐dependent release, into the medium, of choline and not phosphocholine or glycerophosphocholine. Treatment of [3H]myristic acid‐labeled cells with either phorbol diesters, sn‐1,2‐dioctanoylglycerol, or vasopressin elicited the formation of labeled phosphatidate (PA) and DG. The temporal pattern of PC hydrolysis in cells treated with TPA is indicative of a precursor (PA)‐product (DG) relationship for an enzymatic sequence initiated by phospholipase D. Adding propranolol, a phosphatidate phosphohydrolase inhibitor, eliminated TPA‐induced DG formation, whereas PA generation was unaffected. From these data we conclude that TPA elicits DG formation from PC by the sequential actions of phospholipase D and phosphatidate phosphohydrolase.

Evidence for a protein kinase C-directed mechanism in the phorbol diester-induced phospholipase D pathway of diacylglycerol generation from phosphatidylcholine

In this study we provide evidence for the involvement of protein kinase C (PKC) in phorbol diester‐induced phosphatidylcholine (PC) hydrolysis by the phospholipase D pathway. Rat embryo fibroblasts (REF52) were prelabeled with either tritiated choline or myristic acid; these compounds are preferentially incorporated into cellular PC. Phorbol diester‐induced PC degradation was determined by measuring the release of [3H]choline, and the formation of [3H]myristoyl‐containing phosphatidate (PA), diacylglycerol (DG), and phosphatidylethanol (PE). Staurosporine, a PKC inhibitor, blocked from 73 to 90% of the phorbol diester‐induced PC hyrolysis. The inhibition of phorbol diester‐induced choline release by staurosporine was dose dependent with an approximate ED50 of 150 nM. Pretreatment of cells with phorbol diester inhibited subsequent phorbol diester‐induced PC degradation by 78–92%. A close correlation between the ED50 for phorbol diester‐stimulated choline release and the K d for phorbol diester binding was demonstrated. Neither forskolin nor dibutyryl cAMP elicited cellular PC degradation. In vitro experiments using phospholipase D from Streptomyces chromofuscus showed that staurosporine did not inhibit and TPA did not stimulate enzyme activity.

Vasopressin, phorbol diesters and serum elicit choline glycerophospholipid hydrolysis and diacylglycerol formation in nontransformed cells: transformed derivatives do not respond

REF52, a rat embryo cell line, and several transformed derivatives were used to examine the lipid-related events associated with agonist treatment (phorbol diesters, vasopressin, fetal bovine serum). Exposure of cells, prelabeled with [3H]glycerol, to TPA (12-O-tetradecanoylphorbol 13-acetate) resulted in 3-4-fold increase in the amount of intracellular diacyl[3H]glycerols as early as 10 min after treatment. Continued incubation (up to 60 min) revealed that the diacyl[3H]glycerol formed was under dynamic metabolic regulation as shown by the production of triacyl[3H]glycerols and free [3H]glycerol. Serum and vasopressin likewise induced the generation of intracellular diacyl[3H]glycerol, thereby illustrating that physiological agents provoke a similar reaction. In the three SV-40-transformed variants examined, the diacylglycerol generative-response to TPA, serum and vasopressin, was greatly diminished or totally absent. Experiments employing REF52 cells prelabeled with [3H]choline demonstrated that both TPA and vasopressin induce the hydrolysis of cellular choline-containing glycerophospholipids; this was measured by both a decrease in cell-associated phosphatidylcholine radioactivity and an increase in the production of water-soluble [3H]choline-containing metabolites in the culture medium. 92-97% of the tritium released to the medium was identified as [3H]choline. Vasopressin treatment of REF52 cells prelabeled with [3H]arachidonic acid elicited an increase of more than 11-fold in the amount of cellular diacyl[3H]glycerol and a concomitant release of arachidonic acid to the culture medium that was 12-fold higher than controls. These data demonstrate that tumor-promoting phorbol esters (agonists of protein kinase C), serum and vasopressin, increase the levels of cellular diacylglycerol by stimulating the hydrolysis of choline-containing glycerophospholipids. This agonist-directed mechanism is inoperable in transformed cells. Further, collateral with vasopressin-induced phosphatidylcholine hydrolysis, the cellular release of arachidonic acid occurs. The participation of these lipid-related responses in the signaling of agonist-directed events and their relation to cellular homeostasis is currently being explored.

Vasopressin stimulates phospholipase D activity against phosphatidylcholine in vascular smooth muscle cells

It is now clear that various hormones and agonists can stimulate the production of lipid mediators from nonphosphoinositide phospholipids. We have investigated the production of diacylglycerol from nonphosphoinositide sources, and we demonstrated that vasopressin and other vasoactive agents stimulate hydrolysis of phosphatidylcholine in a variety of cultured vascular smooth muscle cells of rat and human origin. We used vasopressin to characterize this response and found that vasopressin stimulates phospholipase D activity against phosphatidylcholine in A-10 vascular smooth muscle cells. The vasopressin-stimulated phosphatidylcholine hydrolysis is both time- and concentration-dependent. The half-maximal dose of vasopressin required for phosphatidylcholine hydrolysis (ED50∼1 nM) correlates well with vasopressin binding to A-10 cells (Kd∼2 nM). The phosphatidylcholine in A-10 cells can be preferentially radiolabeled with [3H]myristic acid; subsequent treatment with vasopressin stimulates a rapid increase in3H-labeled phosphatidate (∼4×control values at 3 min), and after a short lag,3H-labeled diacylglycerol rises and reaches maximal levels at 10 min (∼2×control values). Similar temporal elevations of phosphatidate and diacylglycerol occur in A-10 cells labeled with [3H]glycerol. In A-10 cells radiolabeled with [3H]choline the elevation of cellular phosphatidate and diacylglycerol is concomitant with the release of [3H]choline metabolites (predominately choline) to the culture medium. The temporal production of phosphatidate and diacylglycerol as well as the release of choline to the culture medium are consistent with vasopressin activating phospholipase D. In addition, vasopressin stimulates a transphosphatidylation reaction that is characteristic of phospholipase D. The transphosphatidylation reaction is detected by the production of phosphatidylethanol that occurs when A-10 cells are incubated with ethanol and stimulated with vasopressin. The phospholipase D is active in the absence of extracellular Ca++ whereas the vasopressin-stimulated mobilization of arachidonic acid is dependent on extracellular Ca++. The data indicate that vasopressin stimulates phospholipase D which hydrolyzes phosphatidylcholine to phosphatidate. The phosphatidate is then metabolized, presumably by a phosphatidate phosphohydrolase, to produce sustained levels of cellular diacylglycerol. These sustained levels of diacylglycerol may activate protein kinase C and theraby function in the “sustained phase” of cellular responses.

Effects of proximate cholesterol precursors and steroid hormones on mouse myeloma growth in serum-free medium

SummaryThe proximate cholesterol precursors lathosterol, 7-dehydrocholesterol and desmosterol supported the growth of NS-1 and X63 mouse myeloma cells. These cells and X63.653 cells are cholesterol auxotrophs, yet each was able to convert [3H]lathosterol to [3H]cholesterol. These results are consistent with the conclusion that cholesterol auxotrophy in these myeloma cells is due to a deficiency in 3-ketosteroid reductase activity. The steroid hormones testosterone, progesserone and hydrocortisone could not replace cholesterol as a medium supplement. These results provide a greater understanding of the cholesterol auxotrophy characteristic of cell lines clonally-derived from the MOPC 21 myeloma tumor, and they provide a rational basis for the use of sterols in defined culture medium for mouse myeloma cells.

Assays for investigations of signal transduction mechanisms involving phospholipase D: Mass measurements of phosphatidate, phosphatidylethanol, and diacylglycerol in cultured cells

With the intent of achieving a better understanding of agonist-induced phospholipase D activity, we have developed simple, rapid assays for quantitating the mass of phosphatidate, phosphatidylethanol, and diacylglycerol. Crude lipid extracts of cultured cells are used; preliminary sample cleanup or derivatization procedures are not necessary. The assays resolve the particular lipids by short-bed/continuous-development thin-layer chromatography. Quantitative assessments are derived from photodensitometric analysis of charred lipid spots. The assays may be employed for as little as 45 pmol of diacylglycerol and 50 pmol of phosphatidate or phosphatidylethanol. The newly developed assays are compared to other procedures for quantitating lipid mediators. The utility of the assays is illustrated in experiments that use a variety of cultured cells to demonstrate the agonist activation of the phospholipase D pathway. In addition, experiments designed to screen various agonists for signal-response coupling to phospholipase D are described.

Platelet-Derived growth factor activates phospholipase D and chemotactic responses in vascular smooth muscle cells

SummaryPlatelet-derived growth factor (BB dimer; PDGF-BB) stimulates a mitogenic response in A-10 vascular smooth muscle cells. In addition, PDGF-BB stimulates phospholipase D activity against phosphatidylcholine in A-10 cells. This response was observed as a rapid metabolism of phosphatidylcholine to phosphatidate and choline; a subsequent metabolism generates sustained levels of diacylglycerol. The accumulation of phosphatidylethanol, a transphosphatidylation product of phospholipase D, was obvious in PDGF-treated cells. PDGF-BB also stimulates a chemotactic response in A-10 cells. The concentrations of PDGF-BB required to stimulate mitogenesis, phospholipase D activity and chemotaxis are similar. This finding shows that PDGF induces a variety of cellular responses and suggests that these responses may share common metabolic pathways. That conception was tested by investigating the activity of the different PDGF dimers. PDGF-AA had little or no activity in A-10 cells for any of the responses measured. PDGF-AB and PDGF-BB were equally potent in stimulating mitogenic responses. However, the AB heterodimer was only half as active as PDGF-BB with respect to activation of phospholipase D and chemotactic responses. These results demonstrate that PDGF stimulates phospholipase D in vascular smooth muscle cells. In addition, the data indicate that different PDGF dimers can transduce varying signals and suggest a link between the mechanisms by which PDGF-BB activates phospholipase D and the chemotactic response.

Improved method for thin-layer chromatographic resolution and photodensitometric assessment of the major classes of phospholipids

Abstract Thin-layer chromatography and photodensitometry were used to resolve and quantitate trace levels of the major phospholipid classes. A solvent system of chloroform/methanol/ethyl acetate/40% methyl amine (20/20/20/10; v/v) resolved the phospholipids. After spraying with 10% CuS04 in 10% H3P04 and charring, photodenstiometric assesments were reliably achieved with as little as lOOng of phospholipid. To illustrate the ease and reliability of the methodology, analyses of various biological materials are presented.