Effects of phorbol ester on mitogen and orthovanadate stimulated responses of cultured human fibroblasts

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with serum or a mixture of growth factors (consisting of vasopressin, bradykinin, EGF, and insulin) stimulates the release of inositol phosphates, mobilization of intracellular Ca, activation of Na/H exchange and subsequent incorporation of [3H]-thymidine. We have determined previously that pretreatment with the tumor-promoting phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) inhibits mitogen-stimulated Na influx in HSWP cells. We report herein that TPA pretreatment also substantially inhibits the mitogen-stimulated release of inositol phosphates in HSWP cells. Half maximal inhibition of mitogen-stimulated inositol phosphate release occurs at 1-2 nM TPA. Treatment of cells with TPA alone has no effect on inositol phosphate release. The effect of TPA pretreatment on inositol phosphate release induced by individual growth factors has also been determined. Orthovanadate, reported by Cassel et al. (1984) to increase Na/H exchange in A431 cells, has been demonstrated to stimulate both Na influx and inositol phosphate release in HSWP cells. TPA pretreatment also inhibits both orthovanadate-stimulated inositol phosphate release and Na influx. In addition, orthovanadate was determined to increase intracellular Ca activity by mobilizing intracellular calcium stores, as determined with the fluorescent intracellular calcium probe fura-2. TPA pretreatment blocks orthovanadate stimulated mobilization of intracellular Ca stores. It appears clear that in HSWP cells pretreatment of cells with phorbol ester is capable of artificially desensitizing the early cellular responses to mitogenic stimuli (growth factors, orthovanadate) by blocking the signal transduction mechanism involved at a point prior to the release of inositol phosphates. We hypothesize that in HSWP cells the normal desensitization of both inositol phosphate release and Na/H exchange is mediated via activation of protein kinase C subsequent to the stimulus-mediated activation of phospholipase C and release of protein kinase C activator diacylglycerol. However it is interesting to note that TPA-mediated inhibition of these early responses in HSWP cells does not inhibit their ability to be stimulated to incorporate [3H]-thymidine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Na+ influx and cell growth in cultured human fibroblasts. Effect of indomethacin

The effect of indomethacin on Na+ influx and cell growth in human diploid fibroblasts (HSWP) has been investigated. It was found that both indomethacin and aspirin block serum-stimulated Na+ influx in a dose-dependent manner (Ki = 0.34 +/- 0.04 mM and 11 +/- 1 mM respectively) while having no effect on influx of Na+ in the absence of serum. The Ki for inhibition of [3H]thymidine incorporation into HSWP cells (0.28 +/- 0.02 mM) closely correlated with the Ki for inhibition of Na+ influx. The onset of action of indomethacin is rapid (within 2 min) and inhibition of Na+ flux is readily reversed (within 5 min). Other workers have reported that indomethacin is cytostatic for human fibroblasts presumably via a slowly developing inhibition of "A" system amino acid transport [6]; however, present results indicate that inhibition of Na+ influx in HSWP cells occurs much more rapidly than the inhibition of amino acid transport observed in other human foreskin fibroblasts and therefore may be more closely related to the primary cellular locus of indomethacin action.     

Ionic responses and growth stimulation in rat astroglial cells: differential mechanisms of gangliosides and serum

Rat astroglial cells respond to fetal calf serum (FCS) and gangliosides, including GM1, by undergoing proliferation. Here, we show that addition of FCS but not GM1 causes an increase in Na+, K+-pump activity, as measured by ouabain-sensitive 86Rb+ influx. The increase of Na+, K+-pump activity by FCS was due to increased Na+ influx (measured with 22Na+). This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+/H+ exchange. Amiloride also blocked the FCS-stimulated incorporation of [3H]thymidine into DNA. Two defined polypeptide growth factors, epidermal growth factor and fibroblast growth factor were also able to elicit an amiloride-sensitive Na+ influx and an ouabain-sensitive K+ uptake in these astroglial cells, in the presence of FCS or insulin. Thus, GM1 differs from serum and growth factors in the mechanisms by which these agents stimulate the proliferation of the astroglial cells used here.     

Initiation of DNA synthesis by human thrombin: relationships between receptor binding, enzymic activity, and stimulation of 86Rb+ influx

Stimulation of amiloride-sensitive sodium (Na+) influx and the subsequent activation of NA+, K+-ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human alpha-thrombin was converted to gamma-thrombin, nitro-alpha-thrombin, and diisopropylphospho (DIP)-alpha-thrombin. These derivatives retain either the capacity to bind cell surface alpha-thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of alpha-thrombin or the various thrombin derivatives, only alpha-thrombin stimulates 86Rb+ influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP-alpha-thrombin that saturate the alpha-thrombin receptors (up to 2 micrograms/ml) do not stimulate either the early or late influx of 86Rb+, indicating that DIP-alpha-thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either gamma-thrombin or nitro-alpha-thrombin, however, stimulate both early and late 86RB+ uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86Rb+ influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.     

Dihydropyridine-sensitive L-type Ca2+ channels in human foreskin fibroblast cells. Characterization of activation with the growth factor Lys-bradykinin.

We have previously characterized the calcium response of cultured human fibroblasts (HSWP cells) to stimulation by the mitogen Lys-bradykinin (BK). We have reported a biphasic response which includes a rapid rise to a peak that appears to result from mobilization of internal calcium, and a plateau phase, which is due to influx of external calcium (Byron, K., Babnigg, G., Villereal, M. L. (1992) J. Biol. Chem. 267, 108-118). In this paper we examine participation of L-type voltage operated calcium channels in the calcium entry phase of BK-stimulated HSWP cells. We show that there is an increase in 45Ca2+ uptake and an increase in intracellular free calcium concentration ([Ca2+]i) as measured by fura-2, when HSWP cells are stimulated with the L-channel agonist Bay K 8644 under depolarizing conditions. Furthermore, both of these effects are inhibited by low doses of the dihydropyridine antagonist nitrendipine. We also report that BK stimulation of 45Ca2+ uptake can be significantly inhibited by low doses of nitrendipine, while nitrendipine treatment has no effect on the BK-induced rise in [Ca2+]i, as measured by fura-2. These results suggest that under normal conditions the portion of the BK-stimulated Ca2+ influx which is mediated by a nitrendipine-sensitive entry pathway is invisible to the fura-2 technique used to measure [Ca2+]i. This suggest that the nitrendipine-sensitive influx pathway admits calcium preferentially into an intracellular store that is isolated from fura-2. This idea is supported by the observation that in media where calcium has been replaced by 2 mM Ba2+ nitrendipine inhibits most of the BK-stimulated Ba2+ influx.     

Desensitization of the serum effect on Na+ influx in cultured human fibroblasts

Stimulation of an amiloride-sensitive Na+ influx pathway, which mediates Na+/H+ exchange, has been postulated to be an important step in the initiation of DNA synthesis in quiescent human fibroblasts. If the elevation of intracellular Na+ or the alkalinization of intracellular pH resulting from the activation of this system is a trigger for subsequent mitogenic events, then its inactivation may also be important to cellular functions. We investigated the duration of the activation of Na+ influx by serum in human foreskin fibroblasts (HSWP). It was found that activation of Na+ influx by 10% serum was transient, declining with a t 1/2 = 15 min. Similarly, the Na+ content of the cells rose rapidly following serum addition and decreased with a t 1/2 = 15 min. In addition, both the lys-bradykinin- and the vasopressin-stimulated Na+ influx and Na+ content declined with a t 1/2 of approximately 15 min. Similar results were obtained using both Tris-buffered and Hepes-buffered, amino-acid-free EMEM. Finally, the above experiments were repeated under conditions normally used to assess the mitogenic response of cells. It was found that in cells arrested in G0 by serum deprivation in CO2-buffered EMEM, the serum activated Na+ flux was also transient with a t 1/2 of approximately 20 min. The desensitization of cells to serum could be readily (t 1/2 = 20') reversed by a subsequent incubation of cells in serum-free medium. Stimulation of Na+ influx by both the divalent cation ionophore A23187 and the phospholipase activator melittin in also desensitized rapidly, suggesting the process is independent of receptor downregulation. The desensitization during serum preincubation occurred in both low Na+ and low pH medium suggesting that the process is not due to negative feedback on the transport system via a rise in cellular Na+ concentration or a rise in intracellular pH. Although the mechanism of desensitization is at present not known, it is likely to be a physiologically important event.     

Rapid ionic events and the initiation of growth in serum-stimulated neuroblastoma cells

Rapid effects of serum stimulation on electrical and ionic membrane properties and their relationship to the initiation of DNA synthesis and cell division have been investigated in mouse N1E-115 neuroblastoma cells. Addition of 10% fetal calf serum to serum-deprived N1E-115 cells results in the initiation of DNA synthesis after a lag of approximately 10 hr. The earliest events following serum addition include: transient membrane potential and resistance changes, detectable within seconds and lasting 5--10 min; a persistent increase in the initial rate of 22Na+ influx, the major part of which is not of electrodiffusional origin, and which is potentiated by weak acid anions; and an external Na+-dependent increase in the rate of the Na+, K+ pump. In the absence of serum the stimulation of the Na+, K+ pump can be mimicked by increasing net Na+ influx with monensin or neurotoxins. Growth-depleted serum fails to induce any of the electrical and ionic events. The diuretic amiloride (0.4 mM) inhibits serum-induced Na+ influx, Na+, K+ pump stimulation and DNA synthesis, but does not affect the electrical response or the basal influx rates. The results suggest that serum growth factors act, at least in part, by stimulating an electroneutral, amiloride-sensitive Na+/H+ exchange mechanism. The enhanced Na+ influx then results in the observed stimulation of the Na+, K+ pump, while the simultaneous efflux of protons may raise the intracellular pH.     

Increased ouabain-sensitive 86Rubidium uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA), a protein which stimulates DNA synthesis and growth of chicken embryo fibroblasts, was purified from serum-free medium conditioned by the growth of a rat liver cell line. Purified MSA was shown to rapidly stimulate ouabain-sensitive Na+, K+-ATPase activity as measured by both enzyme assay and rate of 86Rubidium uptake. Labeled ouabain binding was also shown to increase after stimulation of quiescent cells by serum or purified MSA. Conditions which interfere with the ability of the cells to accumulate potassium, such as the presence of the specific inhibitor, ouabain; incubation in potassium-free medium; or the presence of the potassium ionophore, valinomycin, were all demonstrated to inhibit the stimulation of DNA synthesis by serum or purified MSA. These results suggest that an early event in the stimulation of DNA synthesis by purified MSA is an activation of membrane Na+, K+-ATPase with a resulting accumulation of potassium ions inside the cell.     

Ionic responses rapidly elicited by porcine platelet-derived growth factor in Swiss 3T3 cells.

Addition of porcine platelet-derived growth factor (PDGF) to quiescent cultures of Swiss 3T3 cells caused a marked, dose-dependent stimulation of Na+ influx and Na-K pump-mediated 86Rb+ uptake. Porcine PDGF (a single component in SDS polyacrylamide gels) stimulated ion fluxes to the same maximal extent as partially purified preparations, and exhibited half-maximal effect at 6 ng/ml (2 X 10(-10) M). Maximal effect was achieved at 30 ng/ml (10(-9) M). In the presence of insulin, PDGF elicited mitogenesis at comparable concentrations. PDGF stimulated ion uptake in a time-dependent fashion; maximal effect was obtained after 5 min of exposure to the growth factor. PDGF stimulates Na+ influx via an amiloride-sensitive pathway, suggesting that PDGF enhances the activity of a Na+/H+ antiport system. In accordance with this possibility, the mitogen caused an increase of intracellular pH by 0.15 pH units, as judged by the steady-state distribution of labelled 5,5-dimethyloxazolidine-2,4-dione (DMO). Porcine PDGF stimulated E-type prostaglandin synthesis and cAMP accumulation but these events could be dissociated from the stimulation of the ionic fluxes, which was detected within minutes and was not blocked by indomethacin. It is suggested that PDGF elicits multiple signals to stimulate cell proliferation in 3T3 cells.     

Mitogenesis of 3T3 fibroblasts induced by endogenous ganglioside is not mediated by cAMP, protein kinase C, or phosphoinositides turnover

The B subunit of cholera toxin, which binds specifically to ganglioside GM1, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts grown in chemically defined medium. The mitogenic response to the B subunit was potentiated by insulin and other growth factors. To elucidate the mechanism by which the B subunit stimulates cell growth , its effects on several transmembrane signaling systems which have been suggested to play a vital role in cell growth regulation were examined. The B subunit did not increase cAMP levels nor activate adenylate cyclase. The B subunit induced a rapid and profound increase in intracellular free Ca2+ as measured with the fluorescent Ca2+-sensitive dye quin 2/AM. Removal of external Ca2+ completely inhibited the signal, thus suggesting that the B subunit elevates intracellular Ca2+ through a net influx of extracellular Ca2+ rather than by causing the release of Ca2+ from intracellular stores. These findings are consistent with the observations that the B subunit induced reinitiation of DNA synthesis without activation of phospholipase C. There was no increase in the formation of inositol trisphosphate, the second messenger that mediates release of Ca2+ from intracellular stores. In addition, the B subunit still stimulated DNA synthesis in Swiss 3T3 cells pretreated with phorbol ester to down-regulate protein kinase C. These results suggest that the mitogenic effects of the B subunit are mediated mainly by facilitation of Ca2+ influx and that activations of adenylate cyclase, phospholipase C, or protein kinase C are not obligatory steps in the initiation of cell growth by the B subunit. Furthermore, the observation that Ca2+ ionophores, such as ionomycin and A23187, are not mitogenic implies that additional undefined growth signaling pathways may exist in this system.     

Relationship of muscle growth in vitro to sodium pump activity and transmembrane potential

Serum stimulates embryonic avian skeletal muscle growth in vitro and the growth-related processes of amino acid transport and protein synthesis. Serum also stimulates myotube Na pump activity (measured as ouabain-sensitive rubidium-86 uptake) for at least 2 h after serum addition. Serum-stimulated growth depends on this Na pump activity since ouabain added at the same time as serum totally inhibits the growth responses. The relationship of myotube growth, Na pump activity, and transmembrane potential was studied to determine whether serum-stimulated Na pump activation and growth are coupled by long-term membrane hyperpolarization. When myotube amino acid transport and protein synthesis are prestimulated by serum, ouabain was found to have little inhibitory effect, indicating that the already stimulated growth-related processes are not tightly coupled to continued Na pump activity. Serum-stimulated protein synthesis is tightly coupled to Na pump activity, but only during the first 5-10 min after serum addition. When myotube transmembrane potentials were measured using the lipophilic cation tetraphenylphosphonium, serum at concentrations that stimulate myotube growth and Na pump activity was found to have little effect on the cell's transmembrane potential. Furthermore, partial depolarization of the myotubes with 12- to 55-mM extracellular potassium does not prevent serum stimulation of myotube growth. Monensin was found to hyperpolarize the myotubes, but causes myotube atrophy. These results indicate that although Na pump activity is associated with initiation of serum-stimulated myotube growth, continued Na pump activity is not essential, and there is little relationship between myotube growth and the myotube's transmembrane potential.     

Fibroblast growth factor induces a transient net K+ influx carried by the bumetanide-sensitive transporter in quiescent BALB/c 3T3 fibroblasts

The bumetanide-sensitive transport system performed a net efflux of K+ in serum deprived quiescent cells. The addition of partially purified fibroblast growth factor (FGF) to G0/G1 phase 3T3 fibroblasts induced a transient net influx of K+, carried out by the bumetanide-sensitive transport system for 2-6 minutes. The stimulation of the bumetanide-sensitive K+ influx by FGF was followed by stimulation of the ouabain-sensitive K+ influx. In addition, both the bumetanide-sensitive and the ouabain-sensitive K+ influxes were found to be similarly stimulated when the G0/G1 3T3 cells were treated with insulin. These results suggest that growth factors such as FGF and insulin induce a change in the action of the bumetanide-sensitive transporter from performing net K+ efflux along its concentration gradient to an uphill transport pumping of K+ into the cell. We propose, therefore, that the bumetanide-sensitive transporter contributes to the increase in the intracellular K+ (and probable Na+) stimulated by growth factors such as FGF and insulin in early G1 phase of the cell cycle.     

Na/K/Cl cotransport in cultured human fibroblasts

The transport characteristics and regulation of the Na/K/Cl cotransport system were investigated in cultured human fibroblasts (HSWP cells). The existence of the system was documented by the finding that digitoxin-insensitive K+ influx was dependent upon the presence of both Na+ and Cl- in the extracellular milieu. It was found that only Br- could partially substitute for Cl-, with SCN-, I-, acetate, and gluconate being ineffective. Li+ could partially substitute for Na+; however, choline was without effect. The shape of the titration curves for K+ influx versus extracellular Cl- concentration was dependent upon the substituted anion. Furthermore, the apparent Km for Cl- at saturating [K+]o and [Na+]o, was also dependent upon the substituted anion and ranged from 30 mM (gluconate substitution) to 100 mM (acetate substitution). The titration curves for K+ influx versus extracellular Na+ concentration displayed hyperbolic kinetics and the apparent Km = 15 mM at saturating [K+]o. The curve for K+ influx versus extracellular K+ concentration was a hyperbola and the apparent Km for K+ = 3 mM at saturating [Na+]o. The digitoxin-insensitive K+ flux was found to be sensitive to related 5-sulfamoylbenzoic acid derivatives, commonly known as "loop" diuretics and to be insensitive to both: amiloride (3,5-diamino-N-(aminoiminomethyl)-6-chloropyrazinecarboxamide++ +) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. The Na/K/Cl cotransport system was not stimulated by serum, but was slightly stimulated by two peptide mitogens. Furthermore, agents which cause an elevation in cellular cyclic AMP levels were found to be potent inhibitors of cotransport.     

Polarized distribution of the Na+/H+ exchange system in a renal cell line (LLC-PK1) with characteristics of proximal tubular cells

Studies of Na+ and H+ transport by confluent monolayers of the epithelial cell line LLC-PK1 were performed to verify the presence of a Na+/H+ exchange system. The presence of an outwardly directed H+ gradient produced a large stimulation of Na+ influx measured under net flux conditions. Amiloride (10(-3) M) completely inhibited Na+ influx stimulated by the H+ gradient and part of the Na+ influx measured in the absence of a pH gradient. Half-maximal inhibition of the Na+ influx stimulated by a pH gradient at 143 mM Na was observed at 5 microM amiloride. The presence of an inwardly oriented proton gradient also stimulated Na+ efflux from Na+-loaded cells. The stimulation was completely inhibited by the presence of 10(-3) M amiloride in the washout medium. These results indicate that this system could operate in the opposite direction depending on the orientation of the Na+ and H+ gradient. Incubation in Na+-free medium or in the presence of 10(-3) M ouabain resulted in a dramatic decrease of H+ release from LLC-PK1 cells. This H+ release was largely, although not completely, inhibited by 10(-4) M amiloride. Neither chloride substitution by the impermeable anion isethionate nor incubation in the presence of the ionophore valinomycin in high K+ medium affected Na+ influx by stimulated by a pH gradient. Inhibition of the Na+ influx by amiloride occurred only from the apical side of the monolayer. These results indicate that the Na+/H+ exchange system in LLC-PK1 monolayers is specifically localized in the apical membrane of the epithelial cells.     

Amiloride added together with bumetanide completely blocks mouse 3T3-cell exit from G0/G1-phase and entry into S-phase.

In this study we tested the hypothesis that stimulation of univalent-cation fluxes which follow the addition of growth factors are required for cell transition through the G1-phase of the cell cycle. The effect of two drugs, amiloride and bumetanide, were tested on exit of BALB/c 3T3 cells from G0/G1-phase and entry into S-phase (DNA synthesis). Amiloride, an inhibitor of the Na+/H+ antiport, only partially inhibited DNA synthesis induced by serum. Bumetanide, an inhibitor of the Na+/K+ co-transport, only slightly suppressed DNA synthesis by itself, but when added together with amiloride completely blocked cell transition through G1 and entry into S-phase. Similar inhibitory effects of the two drugs were found on the induction of ornithine decarboxylase (ODC) (a marker of mid-G1-phase) in synchronized cells stimulated by either partially purified fibroblast growth factor (FGF) or serum. To test this hypothesis further, cells arrested in G0/G1 were stimulated by serum, insulin or FGF. All induced similar elevations of cellular K+ content during the early G1-phase of the cell cycle. However, serum and FGF, but not insulin, released the cells from the G0/G1 arrest, as measured by ODC enzyme induction. This result implies that the increase in cellular K+ content may be necessary but not sufficient for induction of early events during the G1-phase. The synergistic inhibitory effects of amiloride and bumetanide on the two activities stimulated by serum growth factors, namely ODC induction (mid-G1) and thymidine incorporation into DNA (S-phase), suggested that the amiloride-sensitive Na+/H+ antiport system together with the bumetanide-sensitive Na+/K+ transporter play a role in the mitogenic signal.     

Ionic responses and growth stimulation induced by nerve growth factor and epidermal growth factor in rat pheochromocytoma (PC12) cells

Rat pheochromocytoma cells (clone PC12) respond to nerve growth factor (NGF) by the acquirement of a phenotype resembling neuronal cells. In an earlier study we showed that NGF causes an increase in Na+,K+ pump activity, as monitored by ouabain-sensitive Rb+ influx. Here we show that addition of epidermal growth factor (EGF) to PC12 cells resulted in a stimulation of Na+,K+ pump activity as well. The increase of Na+,K+ pump activity by NGF or EGF was due to increased Na+ influx. This increased Na+ influx was sensitive to amiloride, an inhibitor of Na+,H+ exchange. Furthermore, no changes in membrane potential were observed upon addition of NGF or EGF. Amiloride-sensitive Na+,H+ exchange in PC12 cells was demonstrated by H+ efflux measurements and the effects of weak acids on Na+ influx. These observations suggest that both NGF and EGF activate an amiloride-sensitive, electroneutral Na+,H+ exchange mechanism in PC12 cells. These findings were surprising in view of the opposite ultimate biological effects of NGF and EGF, e.g., growth arrest vs. growth stimulation. However, within 24 h after addition, NGF was found to stimulate growth of PC12 cells, comparable to EGF. In the presence of amiloride, this stimulated growth by NGF and EGF was abolished. In contrast, amiloride did not affect NGF-induced neurite outgrowth of PC12 cells. From these observations it is concluded that in PC12 cells: (a) NGF has an initial growth stimulating effect; (b) neurite outgrowth is independent of increased amiloride-sensitive Na+ influx; and (c) growth stimulation by NGF and EGF is associated with increased amiloride-sensitive Na+ influx.     

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.     

The relative effects of different types of growth factors on DNA replication, mitosis, and cellular enlargement

It was recently demonstrated that growth in cell size can be dissociated from DNA synthesis and mitosis. 3T3 cells starved to quiescence in low serum concentration can be stimulated to undergo DNA synthesis and one cell division without growing in size (unbalanced growth) (42-44). We report here that in cells stimulated to undergo unbalanced growth, the cell nucleus undergoes balanced growth, i.e., nearly doubles in size prior to mitosis. The reduced ability to grow in cell size under unbalanced growth conditions is thus mainly ascribable to the cytoplasm. Furthermore, the extent to which cells grow in size prior to mitosis is dependent on the serum concentration in the tissue culture medium (44). This data suggests that some macromolecular factor or factors in serum are required for growth in cell size prior to mitosis. We report in this study that epidermal growth factor (EGF) alone exerts a small but significant stimulatory influence on DNA synthesis and mitosis but does not affect cellular enlargement. In contrast, insulin added at supraphysiological concentrations does not stimulate quiescent cells to enter S phase but instead stimulates growth in cell size in the small fraction of dividing cells. Furthermore, cells stimulated to proliferate by EGF could be induced to undergo balanced growth when insulin was added concomitantly. Finally, platelet-derived growth factor (PDGF) stimulates quiescent sparse 3T3 cells to undergo DNA synthesis and mitosis. PDGF also exerts a limited but significant effect on cellular enlargement. However, PDGF alone could not induce a complete balanced growth, i.e., a doubling in cell size prior to mitosis.     

Early and late changes in potassium transport during the start of proliferation in CHO cell cultures. The action of serum, isoproterenol, propranolol and theophylline

The stimulation of DNA synthesis by serum is accompanied by early (30 minutes) and late (2-8 hours) increase in ouabain-sensitive rubidium (potassium) influx and the elevation of intracellular potassium content from 0.5-0.6 to 0.7-0.8 mmole per gram protein in CHO-K1 cells. Isoproterenol alone induces the transient increase both in potassium influx via Na,K-ATPase and in potassium efflux without any effect on intracellular potassium content and cell proliferation. Isoproterenol acts synergistically with serum in eliciting the early and late changes in potassium transport and in stimulating G1----S transition. The combination of serum and theophylline produces a rapid increase in potassium influx, however, it does not stimulate DNA synthesis and does not induce any later increase in intracellular potassium content. It is concluded that early and late activation of Na,K-ATPase by mitogens can be dissociated; the Na,K-ATPase activation is involved in mitogenic response when producing the sustained potassium influx and the elevation of intracellular potassium content during G1----S transition.     

Hypertrophy and hyperplasia cause differing effects on vascular smooth muscle cell Na+/H+ exchange and intracellular pH

Mitogens and vasoconstrictors stimulate many of the same early intracellular signals (e.g. phospholipase C and protein kinase C activation) in vascular smooth muscle cells (VSMC). Despite these shared signals, angiotensin II is not mitogenic for cultured VSMC. The nonmitogenic effect of angiotensin II suggests that other intracellular signals associated with growth should differ between mitogens and vasoconstrictors. Because of the importance of intracellular pH (pHi) in growth, we compared the effects of 10% calf serum, 10 ng/ml platelet-derived growth factor, and 100 nM angiotensin II on pHi and Na+/H+ exchange. All agonists stimulated a rapid (less than 1 min) rise in pHi mediated by Na+/H+ exchange. However, exposure of growth-arrested VSMC to these agonists for 24 h caused significant differences in pHi: 7.18 (10% serum), 7.16 (platelet-derived growth factor), 6.99 (angiotensin II), and 7.08 (0.4% serum). Na+/H+ exchange activity was measured in acid-loaded cells by the ethyl isopropyl amiloride-sensitive influx of Na+ and efflux of H+. Both techniques showed that exposure to 10% serum caused approximately 45% decrease in Na+/H+ exchange activity without significant change in angiotensin II-treated cells. Thus, although the rapid changes in pHi and Na+/H+ exchange function are the same for angiotensin II and mitogens, the long term effects differ. The data suggest that differences in pHi regulatory mechanisms are important in determining whether an agonist causes VSMC hypertrophy or hyperplasia.