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C J Welsh

Publications and source records attributed to C J Welsh.

At least 37 records · Page 2Linked to original sources
PubMed1991

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.

Absorptiometry, Photon↗
PubMed1991

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

Platelet-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.

Animals↗
PubMed1990

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 non-phosphoinositide 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 approximately 1 nM) correlates well with vasopressin binding to A-10 cells (Kd approximately 2 nM). The phosphatidylcholine in A-10 cells can be preferentially radiolabeled with [3H]myristic acid; subsequent treatment with vasopressin stimulates a rapid increase in 3H-labeled phosphatidate (approximately 4 X control values at 3 min), and after a short lag, 3H-labeled diacylglycerol rises and reaches maximal levels at 10 min (approximately 2 X 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 (predominantly 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 thereby function in the "sustained phase" of cellular responses.

Animals↗
PubMed1989

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 hydrolysis. 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 Kd 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.

Alkaloids↗
PubMed1988

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.

Animals↗
PubMed1988

Vasopressin is the only component of serum-free medium that stimulates phosphatidylcholine hydrolysis and accumulation of diacylglycerol in cultured REF52 cells.

Vasopressin stimulates phosphatidylcholine hydrolysis in REF52 cells, and this phosphatidylcholine hydrolysis results in increases in choline containing metabolites in the culture medium (2.3 x control levels) and accumulation of cellular diacylglycerol (6.5 x control levels). Vasopressin is the only component of a 6-component mixture of the serum-free medium for REF52 cells that induces the phosphatidylcholine hydrolysis response. The effect of vasopressin is both time- and concentration-dependent. Maximal levels of both phosphatidyl-choline hydrolysis and accumulation of diacylglycerol are observed between 10 and 20 min after treatment with vasopressin. Effects are maximal at vasopressin concentrations of 100 ng/ml; the ED50 for vasopressin-stimulated phosphatidyl-choline hydrolysis is approximately 0.7 ng/ml. The evolution of diacylglycerol occurs in a time frame that is consistent with the diacylglycerol activating protein kinase C in a "second phase" agonist response.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

Observations on demyelinating lesions induced by Theiler's virus in CBA mice.

We examined demyelinating lesions in the spinal cord of CBA mice infected with the BeAn strain of Theiler's virus to see if it was possible to document the sequence of changes which result in demyelination. It was found that the lesions which develop in the late stages of the disease were progressive. Therefore, by examining the different zones of a single lesion, it was possible to follow a sequence of changes which lead to demyelination. There was a clear progression from normal myelin, to vacuolated myelin, to myelin phagocytosis, to demyelinated axons, to remyelinated axons. Virus was detected in degenerating oligodendrocytes in the area showing myelin vacuolation by both electron microscopy and immunocytochemistry, a finding which indicated that virus infection precedes demyelination. The area of normal myelin which surrounded the zone of vacuolated myelin was infiltrated by lymphocytes, indicating that lymphocytic infiltration preceded viral replication and oligodendrocyte degeneration. Our observations indicate that cells of the immune system may play a role in the initiation of virus replication which appears to be a prerequisite for demyelination.

Animals↗
PubMed1988

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

The 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, progesterone 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.

3-Hydroxysteroid Dehydrogenases↗
PubMed1988

Hormonal regulation of preputial gland function in male Microtus montanus, the montane vole.

1. Preputial gland function in male Microtus montanus is androgen-dependent, both in terms of preputial weights and in the production of a series of lipids which are present in M. montanus and absent from Microtus pennsylvanicus. 2. Production of these species-typical lipids is decreased but not eliminated in castrates, as well as in adrenalectomized castrates treated with corticosterone. Therefore, in the total absence of androgens, a low level of these lipids is still produced. 3. 5 alpha-dihydrotestosterone and 17 beta-estradiol enanthate have limited effects on maintenance of preputial weight, suggesting that testosterone itself is the active steroid in the preputial gland of this species.

Adrenalectomy↗
PubMed1987

The detection of heat-aggregated IgG (as a model for immune complexes) by reverse passive haemagglutination using a human monoclonal rheumatoid factor coupled to erythrocytes.

A human monoclonal IgM rheumatoid factor (RF) produced in vitro by an Epstein-Barr virus (EBV)-immortalized cell line was purified by protein A-Sepharose adsorption and coupled by the chromic chloride method to human erythrocytes. The RF-coupled cells were incorporated in reverse passive haemagglutination (RPH) assays to detect immune complexes (IC) using heat-aggregated human IgG as a model system. The sensitivity of the RPH was comparable to an enzyme-linked immunosorbent assay (ELISA) using sheep C1q for the detection of ICs.

Antibodies, Monoclonal↗
PubMed1987

sn-1,2-diacylglycerols and phorbol diesters: uptake, metabolism, and subsequent assimilation of the diacylglycerol metabolites into complex lipids of cultured cells.

The cell-permeable diacylglycerol mediators have been shown to mimic partially the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured cells. In order to evaluate the metabolic stability of the lipid mediators, several radiolabeled diacylglycerols were synthesized and their uptake and intracellular fate in cultured HL-60 (human promyelocytic leukemia) cells was compared with TPA. In addition to whole cell assessment, the stability of diacyl lipids and TPA was evaluated in a buffer/water system and in the presence of serum and subcellular fractions. The compounds studied include 1,2-dioleoyl-sn-glycerol (DiOG), 1-oleoyl-2-acetyl-sn-glycerol (OaG), 1-palmitoyl-2-acetyl-sn-glycerol (PaG), the ether-linked analog 1-palmityl-2-acetyl-sn-glycerol (ePaG), and TPA. TPA was comparatively stable to lipase hydrolysis in all systems examined. First, the data show that within 5 min at pH 7.9, nearly 50% of the PaG (originally greater than 92% 1,2-isomer) had isomerized, and rapid formation of the 1,3-isomer also occurred with OaG and ePaG. The metabolism of OaG and PaG by serum hydrolases, using a reaction medium containing 10% serum, was chiefly by acetate hydrolysis; however, fatty acid was also liberated. After a 60-min incubation 68% of the [14C]OaG was converted, by serum enzymes, to monooleoylglycerol plus oleic acid. Heat-inactivation of serum reduced the enzymatic formation of fatty acid by 60-70%. ePaG was also metabolized by serum enzymes, but the ether-linked alkylglycerol product was stable. The results of cell-free studies (postmitochondrial supernatant) showed that cellular enzymes were present that could, like serum, convert the diacylglycerols to monoacylglycerols and free fatty acids. Studies using cultured cells showed that radiolabeled OaG, PaG, and ePaG were rapidly taken up by the cells and metabolized. Labeled metabolic products from the diacylglycerols appeared, in a time-dependent manner, in cellular phospholipids and triacylglycerols. The results from experiments employing 1-acyl-2-acetyl-sn-[3H]glycerol and [3H]acyl-2-acetyl-sn-glycerol indicate that the intracellular mode of mediator metabolism is via complete hydrolysis with subsequent incorporation of 3H-acyl groups into complex lipids. Data are also presented which show that a substantial amount of cellular lipid acyl group modification occurs and large amounts of glycerol are produced when cells are cultured with OaG. Collectively, these results demonstrate that the diacylglycerol mediators, when compared with TPA, are not stable and are metabolized by both serum and cellular enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell-Free System↗
PubMed1987

The effect of L3T4 T cell depletion on the pathogenesis of Theiler's murine encephalomyelitis virus infection in CBA mice.

Theiler's murine encephalomyelitis virus (TMEV) gives rise to a biphasic disease of the central nervous system (CNS) following intracranial inoculation of susceptible strains of mice. The early phase, during the first month, resembles poliomyelitis and in the late phase the mice suffer from inflammatory demyelination reminiscent of multiple sclerosis. In order to investigate the role of helper T cells in the acute and chronic phases of the disease we depleted mice of their L3T4 T cells in vivo with rat monoclonal antibodies, prior to infection and prior to the onset of clinical signs of demyelination. Mice depleted of their helper cells failed to produce antibodies to TMEV and consequently were unable to clear virus from the CNS and died within the first month of infection. Depletion of T cells before the demyelinating phase of the disease resulted in a marked decrease in the incidence of disease from 77% of the immunocompetent animals with clinical signs of paralysis to 36%. Immunocompetent TMEV-infected mice also developed antibodies against myelin suggesting that autoimmune mechanisms may play a role in TMEV-induced demyelination.

Animals↗
PubMed1986

A flow cytometric method for intracellular labeling and purification of rare neuronal populations: isolation of fixed neurophysin neurons.

A method is described for flow cytometric analysis and fluorescence-activated cell sorting of small populations of neurons following dissociation of fixed brain tissue and immunofluorescent labeling of intracellular antigens. This method has been successfully applied to neurophysin-containing magnocellular neurons of the rat supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. These neurons constitute a rare population in the context of flow cytometry, comprising less than 2% of all cells present in dissociated tissue punches of SON and PVN. Following labeling with anti-neurophysins sera and fluorescein-conjugated second antibody, a highly enriched population containing 80-85% neurophysin-positive neurons was isolated by fluorescence-activated cell sorting. Recovery of 29% of all neurophysin-containing neurons in the SON/PVN was achieved. Perikarya were recovered largely intact, frequently with attached proximal dendritic processes. Applications of this method include purification of specific neuronal types for use as immunogens in production of monoclonal antibodies to cell-type-specific antigens, and rapid surveys of fluorescent lectin or other ligand binding to cell populations identified by the presence of particular intracellular antigens.

Animals↗