Platelet-derived growth factor-induced alterations in vinculin distribution in porcine vascular smooth muscle cells

Exposure of porcine vascular smooth muscle cells to platelet-derived growth factor (PDGF; 18-180 ng/ml) but not epidermal growth factor (EGF; 30 ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 microM), results in a rapid, reversible, time- and concentration-dependent disappearance of vinculin staining in adhesion plaques; actin-containing stress fibers also become disrupted following exposure of cells to PDGF. Disappearance of vinculin staining from adhesion plaques is also caused by 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 200-400 nM), though the time course of the disappearance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF-induced removal of vinculin from adhesion plaques was inhibited in a concentration-dependent fashion by 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8; 0.25-4 microM) and leupepetin (2-300 microM), and by n-alpha-tosyl-L-lysine chloromethylketone (TLCK; 100 microM) and trifluoperazine (TFP; 2.5 microM). Addition of PDGF to vascular smooth muscle cells caused a rapid, transient increase in cytosolic free calcium, from a basal resting level of 146 +/- 6.9 nM (SEM, n = 62) to 414 +/- 34 nM (SEM, n = 22) as determined using the calcium-sensitive indicator Fura-2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB-8 but not leupeptin. This rise in cytosolic free calcium was found to occur in approximately 80% of the sample population and displayed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF-induced disruption of vinculin from adhesion plaques, as determined using the pH-sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose-dependent fashion. Both could be inhibited by leupeptin or TMB-8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF-induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium-independent mechanism, and 4) the cellular response to PDGF-stimulated increases in cytosolic free calcium is heterogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of early platelet-derived growth factor responses in BALB/c-3T3 cells by interferon

Stimulation of total inositol phosphate production, alteration of cytosolic free calcium [( Ca++]i), vinculin disruption from adhesion plaques, and DNA synthesis caused by PDGF were examined in normal and INF pretreated density arrested BALB/c-3T3 fibroblasts. In normal cells, PDGF caused an increase in total inositol phosphates, a rapid, transient increase in [Ca++]i, disappearance of vinculin from adhesion plaques, and stimulation of DNA synthesis. Pretreatment of cells with INF inhibited PDGF-stimulated increases in [Ca++]i, vinculin disruption from adhesion plaques, and DNA synthesis, but had no effect on PDGF-induced increase in total inositol phosphate levels. These findings suggest that INF prevents entry of quiescent BALB/c-3T3 cells into G1 by inhibiting PDGF-induced release of Ca++ from intracellular stores.     

Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells

Exposure of BALB/c-3T3 cells (clone A31) to platelet-derived growth factor (PDGF) results in a rapid time- and dose-dependent alteration in the distribution of vinculin and actin. PDGF treatment (6-50 ng/ml) causes vinculin to disappear from adhesion plaques (within 2.5 min after PDGF exposure) and is followed by an accumulation of vinculin in punctate spots in the perinuclear region of the cell. This alteration in vinculin distribution is followed by a disruption of actin-containing stress fibers (within 5 to 10 min after PDGF exposure). Vinculin reappears in adhesion plaques by 60 min after PDGF addition while stress fiber staining is nondetectable at this time. PDGF treatment had no effect on talin, vimentin, or microtubule distribution in BALB/c-3T3 cells; in addition, exposure of cells to 5% platelet-poor plasma (PPP), 0.1% PPP, 30 ng/ml epidermal growth factor (EGF), 30 ng/ml somatomedin C, or 10 microM insulin also had no effect on vinculin or actin distribution. Other competence-inducing factors (fibroblast growth factor, calcium phosphate, and choleragen) and tumor growth factor produced similar alterations in vinculin and actin distribution as did PDGF, though not to the same extent. PDGF treatment of cells for 60 min followed by exposure to EGF (0.1-30 ng/ml for as long as 8 h after PDGF removal), or 5% PPP resulted in the nontransient disappearance of vinculin staining within 10 min after EGF or PPP additions; PDGF followed by 0.1% PPP or 10 microM insulin had no effect. Treatment of cells with low doses of PDGF (3.25 ng/ml), which did not affect vinculin or actin organization in cells, followed by EGF (10 ng/ml), resulted in the disappearance of vinculin staining in adhesion plaques, thus demonstrating the synergistic nature of PDGF and EGF. These data suggest that PDGF-induced competence and stimulation of cell growth in quiescent fibroblasts are associated with specific rapid alterations in the cellular organization of vinculin and actin.     

Reorganization of alpha-actinin and vinculin induced by a phorbol ester in living cells

We have used fluorescent analogue cytochemistry, image intensification, and digital image processing to examine the redistribution of alpha-actinin and vinculin in living cultured African green monkey kidney (BSC-1) cells treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Before treatment, microinjected alpha-actinin shows characteristic distribution along stress fibers and at adhesion plaques; vinculin is localized predominantly at adhesion plaques. Soon after the addition of TPA, highly dynamic membrane ruffles begin to form. These incorporate a large amount of alpha-actinin but little vinculin. Alpha-actinin is subsequently depleted, more or less uniformly, from stress fibers. Disrupted stress fibers often fragment into aggregates and move into the perinuclear region. Careful analyses of fluorescence intensity distribution indicate that alpha-actinin is depleted more rapidly from adhesion plaques than from stress fibers. Furthermore, the depletion of alpha-actinin from adhesion plaques is also faster than either the depletion of vinculin or the disappearance of focal contacts. These observations indicate that TPA may initiate disruption of stress fibers by interfering with a link between alpha-actinin and vinculin, causing alpha-actinin to be preferentially depleted from adhesion plaques.     

Stimulation by melittin of adrenocorticotropin and beta-endorphin release from rat adenohypophysis in vitro

The effect of melittin on the release of adrenocorticotropin (ACTH) and beta-endorphin from the corticotropic cells of the rat adenohypophysis was examined in vitro. Anterior pituitary quarters were perifused or incubated in vitro and ACTH- (ACTH-IR) or beta-endorphin-like immunoreactivity (beta-End-IR) in the medium was measured by radioimmunoassays. Melittin stimulated ACTH-IR and beta-End-IR release. This effect was rapid in onset, reversible, and concentration-related (50-5000 ng/ml) and depended on the presence of calcium ions in the incubation medium. Melittin also elevated the tissue content of unesterified 3H-arachidonic acid that had previously been incorporated into lipids. Purported phospholipase A2 inhibitors, mepacrine (up to 1 mM), dexamethasone (0.5 mg/kg in vivo, 50 nM in vitro), or p-bromophenacylbromide (100 microM), did not decrease the melittin (500 ng/ml) - induced beta-End-IR release, although mepacrine and dexamethasone may have inhibited phospholipase A2 activity as indicated by an inhibition of melittin-evoked prostaglandin E2 formation. After stimulation by melittin (500 ng/ml), beta-End-IR release was not affected by the cyclooxygenase inhibitor indomethacin (up to 140 microM), whereas nordihydroguaiaretic acid (100 microM), a lipoxygenase inhibitor, or BW755C (250 microM), an inhibitor of both cyclooxygenase and lipoxygenase, abolished melittin-induced hormone secretion. We conclude that melittin generates a signal in the corticotropic cells of the rat adenohypophysis which induces hormone secretion by exocytosis. This signal may be unrelated to the activation by melittin of phospholipase A2.     

Inhibition by protein kinase C activation of melittin-induced arachidonic acid release in PC12 pheochromocytoma cells.

In rat PC12 pheochromocytoma cells, melittin, a phospholipase A2 activator, stimulated the release of arachidonic acid in a dose-dependent manner in the range between 0.1 and 1 microM. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, inhibited the melittin-induced release of arachidonic acid dose-dependently in the range between 0.1 nM and 0.1 microM, whereas 4 alpha-phorbol 12, 13-didecanoate, which is inactive for protein kinase C, was ineffective in this capacity. Staurosporine, a protein kinase C inhibitor, recovered the inhibitory effect of TPA on the melittin-induced release of arachidonic acid. These results suggest that the activation of protein kinase C inhibits phospholipase A2 activity in PC12 pheochromocytoma cells.     

Relationship of cytosolic ion fluxes and protein kinase C activation to platelet-derived growth factor induced competence and growth in BALB/c-3T3 cells

Platelet-derived growth factor (PDGF) and other agents that activate protein kinase C (PKC) rapidly alter cytosolic pH (pHi) and intracellular free calcium ([Ca++]i) in BALB/c-3T3 fibroblasts. To define whether changes in pHi or [Ca++]i are linked to PDGF-stimulated mitogenesis, these parameters were assessed in control and PKC depleted fibroblasts. PDGF addition to BALB/c-3T3 fibroblasts resulted in transient acidification of the cytoplasm followed by prolonged cytosolic alkalinization. Exposure of cells to 12-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester that activates PKC, resulted in cytosolic alkalinization without prior acidification. Overnight incubation with 600 nM TPA decreased the total cell PKC histone phosphorylating activity in BALB/c-3T3 fibroblasts by greater than 90%. In PKC-deficient fibroblasts, TPA, and PDGF-induced alkalinization was abolished. In addition, the transient drop in pHi seen initially in control cells treated with PDGF is sustained to the point where pHi is fully 0.6-0.7 pH units below control cell values for up to 30 minutes. PDGF increased [Ca++]i threefold; this transient rise in [Ca++]i was only minimally affected (less than 15%) by lowering of the extracellular calcium level with ethylene glycol bis(b-aminoethyl ether)0 N,N,N' tetraacetic acid (EGTA) or blocking calcium influx with CoCl2. In contrast, 8-(diethylamine)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an agent thought to inhibit calcium release from intracellular stores, substantially inhibited the rise in [Ca++]i caused by PDGF. TPA and 1-oleoyl-2-acetylglycerol (OAG) increased [Ca++]i but in contrast to PDGF this effect was blocked by pretreatment of cells with EGTA or CoCl2. In PKC-deficient fibroblasts, PDGF still increased [Ca++]i and stimulated DNA synthesis as effectively as in controls. TPA and OAG however, no longer increased [Ca++]i. The continued ability of PDGF to stimulate DNA synthesis in the face of sustained acidification and the absence of PKC activity suggests that cytosolic alkalinization and PKC activation are not essential for PDGF-induced competence in BALB/c-3T3 fibroblasts.     

Induction of circular membrane ruffling on human fibroblasts by platelet-derived growth factor

One of the earliest effects of platelet-derived growth factor (PDGF) on human fibroblasts in culture is an induction of membrane ruffling. The morphology of the ruffles induced by PDGF is unique in that they form circular arrangements on the dorsal side of the cells. Here we report that the induction of circular ruffle arrangements is an effect specific for PDGF, dose-dependent and inhibitable by anti-PDGF antibodies. We have attempted to utilize this effect to design a rapid and sensitive bioassay for PDGF. The "membrane ruffling assay" is compared with other methods to measure PDGF and its specificity with regard to the different dimeric forms of PDGF is discussed. Introduction of Ca2+ into the cells via the Ca2+ ionophore A23187 or the addition of the tumor-promor 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a stimulator of protein kinase C, does not induce circular ruffle formations on human fibroblasts, neither does the addition of the combination of these two agents. However, addition of TPA almost completely inhibits PDGF-induced circular ruffle formations. Further, we find a shift in the time-course of the PDGF-induced circular ruffle formations by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases. This may indicate the involvement of protein phosphorylation in the regulation of PDGF-induced membrane ruffling.     

Stimulatory and inhibitory effects of TMB-8 on pancreatic enzyme secretion

The putative intracellular calcium antagonist 3,4,5-trimethoxybenzoate 8-(diethylamino)-octyl ester (TMB-8) affects carbachol-induced enzyme secretion from rabbit pancreatic acini in a different way than it does that induced by either the C-terminal octapeptide of cholecystokinin (CCK-8), the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) or the calcium ionophore, A23187. In the presence of TMB-8 the dose-response curve for carbachol-induced amylase release shifts to the right, suggesting competitive antagonism at the muscarinic receptor. The hypothesis that TMB-8 acts as a muscarinic receptor antagonist is supported by the observation that TMB-8 dose-dependently inhibits the carbachol-, but not CCK-8-induced increases in cytosolic free calcium, measured in acinar cells by means of the fluorescent calcium indicator quin2. At a concentration of 100 microM, TMB-8 maximally potentiates the secretory response to suboptimal, but not (supra)optimal, concentrations of CCK-8. At the same concentration the drug also potentiates TPA- and A23187-induced enzyme secretion. Cytosolic free calcium levels and CCK-8-induced increases in cytosolic free calcium remain unaffected by 100 microM TMB-8. The above results strongly suggest that potentiation occurs at or beyond the site of interaction between the diacylglycerol- and the Ca2+-activated pathways. At concentrations beyond 100 microM the potentiating effect of TMB-8 declines and, finally, at a concentration of 500 microM the drug completely abolishes the secretory response to CCK-8 and TPA. Basal enzyme secretion, however, remains unaffected. At 500 microM severe side effects are observed as is shown by Trypan blue uptake, lactic dehydrogenase release and release of trapped quin2. It is concluded that at lower concentrations TMB-8 does not act as a specific intracellular calcium antagonist in pancreatic enzyme secretion and that inhibitory effects obtained with rather high concentrations of this drug should be treated with caution.     

Mitogen and co-mitogen stimulation of lymphocytes inhibited by three Ca++ antagonists

The Ca++ requirement for in vitro lymphocyte stimulation by lectins is well known and can be demonstrated by the use of Ca++ chelators. In this study, three Ca++ antagonists were examined for their effects on lymphocyte proliferation. [3H]-thymidine incorporation was employed to measure DNA synthesis in several systems. Stimulation and proliferation were achieved by the addition of one of the following: the mitogenic lectin concanavalin A (ConA); the combination of two co-mitogens, the calcium ionophore A23187 and the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA), neither of which is mitogenic alone; or the non-mitogenic lectin, wheat germ agglutinin (WGA) with TPA. These mitogenic systems were tested for their sensitivity to the Ca++ channel blockers verapamil and nicardipine and the intracellular Ca++ antagonist TMB-8. We found that the ConA and WGA plus TPA treated cells were inhibited approximately 50% by 10 microM verapamil, nicardipine or TMB-8. The stimulation caused by A23187 and TPA was only inhibited by TMB-8 and nicardipine. The inhibitory effects caused by the Ca++ antagonists could not be reversed by the addition of exogenous Ca++ (0.1-1.5 mM), but were reversed by repeated washings in antagonist free media. Using TMB-8 we saw an apparent intracellular Ca++ dependence throughout the G1 phase. Previous studies using Ca++ chelators or Ca++ antagonists suggested an endpoint at about halfway through this period.     

The role of calcium in macrophage chemotaxis to the phorbol ester tumor promoter TPA

The significance of the macrophage in the inflammatory response that occurs concurrently with phorbol ester induced tumor promotion has not yet been determined. Biologically active phorbol ester tumor promoters modify several functional responses of macrophages including chemotaxis, cytotoxicity, secretion and prostaglandin synthesis and release. The present study examines calcium metabolism as a possible underlying biochemical mechanism through which 12-0-tetradecanoyl-phorbol-13-acetate (TPA) exerts its effects on macrophage chemotaxis. The chemotaxis of mouse resident peritoneal macrophages was evaluated in the presence of pharmacological agents known to alter cellular calcium metabolism. The calcium ionophore A23187 in microM concentrations enhanced macrophage chemotaxis to TPA by approximately 41%. This enhancement was dependent on the presence of extracellular calcium. TPA-induced chemotaxis was also enhanced by the histological dye ruthenium red (RR), an agent known to modify mitochondrial calcium fluxes and calcium-dependent neuronal transmission. Ruthenium red (0.1 and 1.0 microM) produced a maximal stimulation of macrophage chemotaxis to TPA of approximately 62%. An intracellular calcium antagonist, 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) inhibited macrophage chemotaxis to TPA in a dose related fashion (1.0 to 100 microM). Varying extracellular calcium concentrations (0-3.6 mM) had no effect on macrophage chemotaxis in response to TPA. In drug combination studies neither A23187 nor RR was able to overcome the inhibitory effects of TMB-8 on macrophage chemotaxis to TPA. These results indicate that intracellular calcium metabolism may be playing a significant role in modulating TPA's effect on macrophage chemotaxis, while extracellular calcium may be of little import. A possible mode of TPA's effect on the macrophage via mobilization of calcium from cellular storage sites is discussed.     

Trifluoperazine,a calmodulin antagonist,inhibits muscle cell fusion

We investigated the effect of trifluoperazine (TFP), a calmodulin antagonist, on the fusion of chick skeletal myoblasts in culture. TFP was found to inhibit myoblast fusion. This effect occurs at concentrations that have been reported to inhibit Ca2+-calmodulin in vitro, and is reversed upon removal of TFP. In addition, other calmodulin antagonists, including chlorpromazine, N-(6-aminohexyl)-5- chloro-1-naphthalene-sulfonamide (W7), and N-(6-aminohexyl)-1- naphthalene-sulfonamide (W5), inhibit fusion at doses that correspond closely to the antagonistic effects of these drugs on calmodulin. The expression of surface acetylcholine receptor, a characteristic aspect of muscle differentiation, is not impaired in TFP-arrested myoblasts. Myoblasts inhibited from fusion by 10 microM TFP display impaired alignment. In the presence of the Ca2+ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, the fusion block by 10 microM TFP is partially reversed and myoblast alignment is restored. The presence and distribution of calmodulin in both prefusional myoblasts and fused muscle cells was established by immunofluorescence. We observed an apparent redistribution of calmodulin staining that is temporally correlated with the onset of myoblast fusion. Our findings suggest a possible role for calmodulin in the regulation of myoblast fusion.     

Schistosoma mansoni: possible involvement of protein kinase C in linoleic acid-induced proteolytic enzyme release from cercariae

The possible involvement of protein kinase C and Ca2+ metabolism in the proteolytic enzyme release from schistosome cercariae was studied. Cercariae were placed in dechlorinated tap water containing 0.37 mM calcium in the small glass petri dish and exposed to the stimuli (linoleic acid, phorbol esters, and Ca2+ ionophore) with or without inhibitors of protein kinase C or Ca2+ metabolism. The proteolytic activity of incubation medium of cercariae thus treated was measured by the azocoll assay. The penetration response of cercariae induced by linoleic acid, a physiological stimulus, was mimicked by phorbol esters. When exposed to phorbol esters, 0.02 to 2 microM of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 to 2 microM of phorbol-12,13-dibutyrate (PDBu), cercariae ceased the swimming movement, began a rhythmic thrusting of the anterior tip of the parasite, and released the proteolytic enzyme, but they did not shed the tails. Lowering Ca2+ in water by addition of 5 mM ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), phorbol ester-induced release of enzyme was completely inhibited. Phorbol ester-induced release of enzyme was partially inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, at a concentration of 100 microM. H-7 alone, at a concentration of 100 microM, did not affect the swimming movement of cercariae. The cercariae were stimulated to release the enzyme by high concentrations (10 and 100 microM) of the Ca2+ ionophore, A23187, but enzyme was not released by low concentrations (0.5 and 1 microM) of this drug. Cercariae exposed to A23187 behaved differently from those exposed to phorbol esters. They ceased swimming, showed strong muscle contraction, and shed their tail. A23187 stimulated cercariae to release the enzyme in the water containing 5 mM EGTA. A23187-induced enzyme release was not inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, trifluoperazine (TFP), a better calmodulin antagonist on schistosome, or by verapamil, a Ca2+ channel blocker. Linoleic acid-induced release of enzyme was partially inhibited by 0.5 and 5 mM of EGTA and by 1 to 100 microM of H-7. While it was not inhibited by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), inhibitors of cyclic nucleotide-dependent protein kinase which were used as negative controls of H-7, W-7, TFP, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an intracellular Ca2+ antagonist, and verapamil.(ABSTRACT TRUNCATED AT 400 WORDS)     

Platelet-derived growth factor-induced c-myc RNA expression. Analysis of an inducible pathway independent of protein kinase C

Platelet-derived growth factor (PDGF) is generally considered to stimulate phosphoinositide turnover resulting in activation of protein kinase C and increased cytoplasmic [Ca2+]. We have examined the role of these secondary effects in regulation of c-myc mRNA accumulation in the MG-63 human osteogenic sarcoma line. Treatment of quiescent cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA) to down-regulate protein kinase C inhibited TPA-stimulated c-myc expression but did not affect the PDGF-modulated process. When cytoplasmic [Ca2+] was increased by addition of a Ca2+ ionophore (A23187 or ionomycin), no stimulation of c-myc RNA was seen; furthermore, these agents did not enhance the PDGF-modulated c-myc expression. Addition of EGTA to cultures treated with both PDGF and a Ca2+ ionophore did not inhibit c-myc induction but rather caused a superinduction of c-myc RNA accumulation. Superinduction occurred only if the [EGTA] was greater than [Ca2+] in the medium. This superinduction was distinct from the increased induction caused by inhibition of protein synthesis. Because PDGF-induced c-myc expression is independent of protein kinase C and increased cytoplasmic [Ca2+], the evidence suggests that PDGF modulates c-myc RNA accumulation in MG-63 cells via a novel pathway, seemingly uncoupled from the classic action of increased phosphoinositide metabolism.     

Protein kinase C mediates platelet-derived growth factor-induced tyrosine phosphorylation of p42

One of the early events after stimulation of Swiss 3T3 cells with either platelet-derived growth factor (PDGF), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), diacylglycerol, or several other mitogens is the near stoichiometric phosphorylation at tyrosine and serine of a scarce cytoplasmic protein (p42). TPA and diacylglycerol are known to directly stimulate the activity of a protein-serine/threonine kinase, protein kinase C (PKC). PDGF and several other mitogens stimulate tyrosine kinases directly and PKC indirectly. We have therefore examined the involvement of PKC in p42 tyrosine phosphorylation in Swiss 3T3 cells. Firstly, six agents which stimulated phosphorylation of p42 also stimulated phosphorylation of a known PKC substrate, an 80,000-Mr protein (p80). Secondly, in PKC-deficient cells (cells in which PKC activity was reduced to undetectable levels by prolonged exposure to TPA), PDGF-induced p42 phosphorylation was reduced three- to fourfold. Phosphoamino acid analysis of phosphorylated p42 from PDGF-stimulated PKC-deficient cells revealed primarily phosphoserine and only a trace of phosphotyrosine, suggesting that the reduction in PDGF-stimulated tyrosine phosphorylation of p42 resulting from PKC deficiency is greater than three- to fourfold. Finally, comparison of antiphosphotyrosine immunoprecipitates of PKC-deficient versus naive cells revealed that most other PDGF-induced tyrosine phosphorylation events were quite similar. These data suggest that mitogens such as PDGF, which directly stimulate phosphorylation of some proteins at tyrosine, induce p42 tyrosine phosphorylation via a cascade of events involving PKC.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 mediated calcium uptake in ATP depleted human red cells

The A23187 induced calcium uptake in ATP depleted cells was determined at pH 6.9 in the presence of trifluoperazine (TFP, 0.30 mM), compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). Apart from verapamil the drugs all increased the maximum rate of ionophore-mediated calcium flux by 50-60 per cent. After the ionophore addition some time elapsed before the calcium flux attained the maximum value, and this time dependence could be interpreted as a slow uptake of A23187 into the membrane: five seconds after the addition of A23187 half of the added ionophore was able to transport calcium through the membrane. The effect of pH on the ionophore-mediated calcium uptake was determined in the absence and presence of TFP. At pH 7.4 the maximum rate of calcium flux in the absence of TFP was two to three times higher than that at pH 6.9 and TFP increased the uptake rate by 98 per cent.     

Synergistic stimulation of prostaglandin E2 production by calcium ionophore and protein kinase C activator in rat granulosa cells

The effects of luteinizing hormone-releasing hormone (LHRH) and its putative intracellular mediators on progesterone (P) and prostaglandin E2 (PGE2) formation were studied in rat granulosa cells. A calcium ionophore (A23187), 12-0-tetradecanoylphorbol-13-acetate (TPA), and melittin (a phospholipase A2-stimulator) were used to later intracellular calcium, protein kinase C, and arachidonic acid levels, respectively. During a 5-h incubation, LHRH increased basal P levels but failed to affect the formation of P induced by cholera toxin (CT). On the other hand, both basal and CT-stimulated PGE2 formation were increased by LHRH. Treatment of the cells with A23187 or TPA attenuated the formation of P induced by CT or FSH. By contrast, A23187 or TPA significantly augmented CT- or FSH-stimulated PGE2 formation. Interestingly, the effects of A23187 and TPA on PGE2 were synergistic, whether or not FSH or CT was present during the incubation. This synergy was not observed with regard to P formation. Melittin also increased basal P and PGE2 levels, and enhanced the stimulation of PGE2 by A23187 or TPA. However, in the combined presence of A23187 and TPA, melittin failed to further enhance the high levels of PGE2 accumulated. These findings further support a role for the intracellular calcium, protein kinase C, and arachidonic acid metabolic pathways in the multiple actions of LHRH in the ovary.     

Reorganization of alpha-actinin and vinculin in living cells following ATP depletion and replenishment

Although it is known that the depletion of cellular ATP induces a dramatic, reversible disruption of microfilament structures, the morphological pathway remains obscure. I have studied this process by following directly the dynamic redistribution of fluorescently labeled alpha-actinin and vinculin which had been microinjected into living mouse 3T3 fibroblasts. Before treatment, microinjected alpha-actinin displayed characteristic distribution along stress fibers, whereas vinculin was localized predominantly at adhesion plaques. The first response after adding NaN3 and 2-deoxyglucose was the retraction of lamellipodia, followed, over a period of 2 h, by a dramatic contraction of stress fibers and loosening of focal contacts. Vinculin plaques shrank from an elongated shape to small aggregates. During recovery, which was initiated by removing NaN3 and 2-deoxyglucose from the medium, lamellipodia appeared rapidly and alpha-actinin dispersed from contracted aggregates. Some partially dispersed aggregates later served as initiation sites for the formation of stress fibers. The recovery of vinculin plaques occurred predominantly through direct elongation, and focal contacts developed concomitantly. A small fraction of vinculin aggregates, however, moved into the perinuclear region without developing into adhesion plaques, and some new vinculin plaques formed de novo. Possible mechanisms involved and relationships to disruptions induced by other agents are discussed.     

Stimulation by melittin of adrenocorticotropin and beta-endorphin release from rat adenohypophysis in vitro

The effect of melittin on the release of adrenocorticotropin (ACTH) and β-endorphin from the corticotropic cells of the rat adenohypophysis was examined in vitro. Anterior pituitary quarters were perifused or incubated in vitro and ACTH- (ACTH-IR) or β-endorphin-like immunoreactivity (β-End-IR) in the medium was measured by radioimmunoassays. Melittin stimulated ACTH-IR and β-End-IR release. This effect was rapid in onset, reversible, and concentration-related (50–5000 ng/ml) and dependend on the presence of calcium ions in the incubation medium. Melittin also elevated the tissue content of unesterified ³H-arachidonic acid that had previously been incorporated into lipids. Purported phospholipase A₂ inhibitors, mepacrine (up to 1 mM), dexamethasone (0.5 mg/kg in vivo, 50 nM in vitro), or p-bromophenacylbromide (100 μM), did not decrease the Melittin (500 ng/ml) — induced β-End-IR release, although mepacrine and dexamethasone may have inhibited phospholipase A₂ activity as indicated by an inhibition of melittin-evoked prostaglandin E₂ formation. After stimulation by melittin (500 ng/ml), β-End-IR release was not affected by the cyclooxygenase inhibitor inddomethacin (up to 140 μM), whereas nordihydroguaiaretic acid (100 μM), a lipoxygenase inhibitor, or BW755C (250 μM), an inhibitor of both cyclooxygenase and lipoxygenase, abolished melittin-induced hormone secretion. We conclude that melittin generates a signal in the corticotropic cells of the rat adenohypophysis which induces hormone secretion by exocytosis. This signal may be unrelated to the activation by melittin of phospholipase A₂.