Regulation of skeletal muscle satellite cell proliferation by bovine pituitary fibroblast growth factor

Satellite cells in skeletal muscle have been implicated in muscle growth processes and regeneration. However, very little is known about the regulation of their proliferation and differentiation. The effect of fibroblast growth factor (FGF) on the proliferation of myogenic cells from adult rat skeletal muscle, presumably satellite cells, has been examined, and FGF has been found to be a potent mitogen for these cells. The mitogenic properties of serum were also documented and studied in conjunction with FGF. Even under conditions of maximal stimulation by serum, the addition of FGF caused a substantial increase in proliferation of satellite cells. The additive nature of the FGF and serum-stimulatory activity suggests that FGF-like molecules are not the active agents in serum and that more than one pathway may be involved in stimulating satellite cell proliferation.     

Selective effects of ascorbic acid on acetylcholine receptor number and distribution

Ascorbic acid in soluble extracts of neural tissue can account for the increase in surface acetylcholine receptors (AChR's) seen on L5 myogenic cells treated with crude brain extract (Knaack, D., and T. R. Podleski, 1985, Proc. Natl. Acad. Sci. USA., 82:575-579). The present study further elucidates the nature of the response of L5 cells to ascorbic acid. Light autoradiography showed that ascorbic acid treatment affects both the number and distribution of surface AChR's. Ascorbic acid, like crude brain extracts, caused a three- to fourfold increase in average AChR site density. However, the number of AChR clusters induced by ascorbic acid was only one-fifth that observed with crude brain extract. The rate constant for degradation of AChR in ascorbic acid-treated cells of 0.037 +/- 0.006 h-1 (t1/2 = 19 h) was not significantly different from that in untreated controls of 0.050 +/- 0.001 h-1 (t1/2 = 14 h). The increase in AChR site density is primarily due to a 2.8-fold increase in the average rate of AChR incorporation. Ascorbic acid also stimulates thymidine incorporation and increases the total number of nuclei per culture. However, cellular proliferation is not responsible for the increase in AChR's since 10 microM cytosine arabinofuranoside blocks the mitogenic effect without affecting the AChR increase. The specificity of ascorbic acid on AChR expression was established by showing that (a) ascorbic acid produced only a slight increase in total protein, which can be accounted for by the mitogenic effect, and (b) the normal increase seen in creatine kinase activity during muscle differentiation was not altered by the addition of ascorbic acid. We conclude that the action of ascorbic acid on AChR number cannot be explained by changes in cell growth, survival, differentiation, or protein synthesis. Therefore, in addition to a minor stimulation of AChR clustering, ascorbic acid specifically affects some aspect of the AChR biosynthetic pathway.     

Role of chain termini in selective steroidogenic effect of ACTH/MSH(4-10) on isolated adrenocortical cells

To investigate the role of charged chain ends in the corticosteroidogenic effect of ACTH/MSH(4-10), acetyl and amide derivatives of ACTH/MSH(4-10) were synthesized and tested in isolated zona glomerulosa and zona fasciculata cells. ACTH/MSH(4-10)-NH2, Ac-ACTH/MSH(4-10) and Ac-ACTH/MSH(4-10)-NH2 (10 microM to 1 mM) stimulated the aldosterone production of zona glomerulosa cells, whereas these peptides did not stimulate the corticosterone production of zona fasciculata cells, even at 1 mM concentration. As ACTH/MSH(4-10) has been shown to have a steroidogenic effect on both types of adrenocortical cells, both charged chain termini seem to be essential for triggering of the corticosterone production of zona fasciculata cells, but for aldosterone production their presence appears not to be important.     

Formaldehyde-ozone-induced fluorescence in pituitary ACTH cells: Effect of adrenalectomy

Summary ACTH and MSH cells of the pituitary are rich in peptides with NH₂-terminal tryptophan, as revealed by fluorescence histochemistry. Adrenalectomy stimulates the ACTH cells but not the MSH cells. As a result, ACTH as well as tryptophyl-peptides disappear from the ACTH cells but not from the MSH cells. It is concluded that the tryptophyl-peptides are stored together with the respective hormone in the ACTH and MSH cells and that tryptophyl-peptides in the ACTH cells are released together with the hormone.     

Differential Regulation by Butyrate and Dibutyryl Cyclic AMP of δ-Opioid, α2-Adrenergic, and Muscarinic Cholinergic Receptors in NCB-20 Cells

Long-term treatment of NCB-20 cells with sodium butyrate resulted in a marked increase in the specific binding of [3H]D-Ala2,D-Leu5 enkephalin. This increase was concentration and time dependent, with an EC50 of about 480 microM and a maximal effect detected after 3-day treatment. At saturating concentration of butyrate (1 mM) the increase was three- to fourfold of the untreated control. Scatchard analysis revealed that the butyrate effect was due to an increase in the density of the opioid receptor binding sites. Butyrate also induced a smaller (about twofold) increase in the density of muscarinic cholinergic receptor binding assessed by using [3H]quinuclidinyl benzilate, whereas alpha 2-adrenergic receptor binding assessed by using [3H]clonidine was not significantly affected. The butyrate-induced opioid receptor binding could be totally abolished by the presence of cycloheximide, suggesting that the butyrate effect involves synthesis of the receptor protein. Butyrate treatment did not affect basal and prostaglandin E1-stimulated cyclic AMP levels but caused a three- to fourfold decrease in the IC50 of D-Ala2,D-Leu5 enkephalin for attenuating these cyclic AMP levels and approximately 25% increase in the maximal extent of attenuation. In contrast to the butyrate effect, long-term treatment of NCB-20 cells with 1 mM dibutyryl cyclic AMP induced an 80% decrease in the opioid and alpha 2-adrenergic receptor bindings and a 57% loss of muscarinic cholinergic receptor binding. This down-regulation of muscarinic cholinergic receptor binding sites was associated with a 35% decrease of carbachol-induced phosphoinositide breakdown, whereas the receptor up-regulation induced by butyrate was found to increase the carbachol response by about threefold. The differential regulation by butyrate and dibutyryl cyclic AMP suggests that the butyrate effect is mediated by a mechanism independent of intracellular cyclic AMP. The induction by butyrate of opioid-receptors and muscarinic cholinergic receptors in NCB-20 cells may provide a useful system for studying the regulation of gene expression of these receptor proteins.     

Stimulation of proliferation of HL60 cells by low concentrations of 12-O-tetradecanoylphorbol-13-acetate and its relationship to the mitogenic effects of insulin.

The effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth and differentiation of cultured human acute promyelocytic leukemia (HL60) cells have been studied using cells growing in a fully defined medium consisting of RPMI 1640 supplemented with selenium dioxide, insulin, and either transferrin or ferric citrate. High concentrations of TPA (greater than 1 nM) cause the expected inhibition of proliferation and induction of macrophage-like differentiation. In contrast, in cells deprived of insulin, which continue to grow at a slow rate, lower concentrations of TPA stimulate proliferation without inducing differentiation. A TPA concentration between 0.03 and 0.3 nM will approximately double the long-term rate of thymidine incorporation into DNA and the rate of increase in cell density. Low-TPA becomes progressively less able to stimulate further proliferation as the insulin concentration is increased and is virtually without effect on cells stimulated by an optimal insulin concentration (5 micrograms ml-1). Insulin itself stimulates proliferation to a greater extent than low-TPA, increasing the long-term rate of thymidine incorporation and the rate of increase in cell density by three- to fourfold. The ability of higher concentrations of TPA to induce differentiation is independent of the presence of insulin. Low-TPA also stimulates the short-term incorporation of thymidine (during a 1-h pulse after 1 or 2 days incubation) by three- to fourfold, as compared to a sevenfold stimulation by insulin. The proliferation response to low TPA concentrations provides a useful model for dissecting the signalling pathways that control cell proliferation following stimulation by insulin and activators of protein kinase C.     

ACTH-like peptides in postimplantation mouse embryos: a possible role in myoblast proliferation and muscle histogenesis.

ACTH and related peptides are mitogens for certain mesodermal cell types such as adrenocortical cells, T-lymphocytes, and skeletal myoblasts. In order to postulate a possible physiological role for these peptides in skeletal muscle histogenesis, it is necessary to establish whether they are present in muscle forming anlagens of postimplantation mouse embryos. By radioimmunoassay and immunofluorescence with antibodies specific for ACTH, we have detected these peptides in many areas of mouse embryos including neural tube, limb buds, eye lens, and myotomal muscles. During fetal development, immunoreactivity decreased in muscle tissue and appeared in visceral ganglia. Furthermore, primary myotubes or C2C12 myotubes, but not muscle or 3T3 fibroblasts, release significant levels of ACTH immunoreactive peptides into the culture medium. Using a microassay for mitogen production, primary myotubes or C2C12 myotubes, but not other mesodermal cells (with the exception of dermal fibroblasts) were shown to release factors into the medium which support myoblast proliferation. Neutralizing antibodies against ACTH inhibit myoblast but not fibroblast proliferation in a dose-dependent fashion. Based on these results, we propose that myotube-derived mitogens (including ACTH-like peptides) promote the proliferation of surrounding myoblast during muscle histogenesis in vivo.     

Factor Xa is a fibroblast mitogen via binding to effector-cell protease receptor-1 and autocrine release of PDGF

The coagulationcascade protease thrombin is a fibroblast mitogen, but theproliferative potential of other coagulation proteases is not known. Inthis study we show that factor Xa stimulated human fetal lungfibroblast DNA synthesis in a concentration-dependent manner from 1 nMonward with a fourfold increase at 200 nM. The mitogenic effect offactor Xa was confirmed using a colorimetric proliferation assay anddirect cell counting. Factor Xa and thrombin had equivalent potencies,and their stimulatory effects followed a similar time course.Comparable results were also obtained with primary human adultfibroblasts derived from lung, kidney, heart, skin, and liver. FactorVIIa also stimulated fibroblast proliferation, but only atconcentrations >10 nM, whereas factor IXa had no effect. To begin toaddress the mechanism by which factor Xa is acting, we show that humanfibroblasts express effector-cell protease receptor-1 and that blockingantibodies to this receptor and the catalytic site of factor Xainhibited its mitogenic effect. Furthermore, factor Xa upregulatedplatelet-derived growth factor-A (PDGF-A) mRNA expression, whereasPDGF-B could not be detected, and a blocking antibody to PDGF inhibitedthe mitogenic effect of factor Xa. We conclude that factor Xa acts as afibroblast mitogen via binding to effector-cell protease receptor-1 andthe autocrine release of PDGF.

     

The effect of MSH/ACTH 4-10 on delayed response performance and post-test locomotor activity in rats

Delayed response performance was measured in male, Long-Evans rats 1 hr after IP administration of various doses of MSH/ACTH 4-10 or control in a Hunter delayed reaction apparatus. Additional treatments consisting of naloxone 500 micrograms/kg (IP) and naloxone 500 micrograms/kg in conjunction with MSH/ACTH 4-10 95 micrograms/kg were also administered. Directly after delayed response performance was assessed, gross locomotor activity was determined. MSH/ACTH 4-10, at a dose of 95 micrograms/kg, significantly enhanced retention of a visual stimulus, while MSH/ACTH 4-10, at doses of 195 and 285 micrograms/kg, significantly impaired delayed response performance. Naloxone treatment resulted in significantly impaired delayed response performance when compared to control. However, naloxone plus MSH/ACTH 4-10 treatment failed to produce a significant difference from control in the delayed response performance paradigm. In post-test locomotor activity determination, an apparent dose-response existed for MSH/ACTH 4-10 with the two highest doses (190 and 285 micrograms/kg) resulting in significantly increased locomotor activity. The observed delayed response performance data support theories implicating MSH/ACTH peptides in attentional processes involving visual stimuli. The fact that large doses of MSH/ACTH 4-10 disrupt delayed response performance while increasing post-test activity suggest that an optimum level of effect caused by the MSH/ACTH peptide exists in this paradigm.     

In situ melanin assay for MSH using mouse B16 melanoma cells in culture

A sensitive in situ melanin assay using cultured mouse B16 melanoma cells is described for structure-activity studies with melanocyte-stimulating hormone (MSH) peptides. B16 Cells were seeded at a density of 2500 cells per well in 96-well microtest tissue culture plates; after 24 h the cells were incubated in the presence of serial dilutions of MSH peptides for 3 to 5 days. The melanin released into the medium of each well was then determined spectrophotometrically at a wavelength of 405 nm using an automatic microplate reader calibrated against synthetic melanin. Studies with alpha-MSH, [Nle4, D-Phe7]-alpha-MSH, [3'-iodo-Tyr2]-alpha-MSH, adrenocorticotropin (ACTH)(1-24), and ACTH(1-39) showed that the peptides had identical intrinsic activities and that the relative potencies were similar to those obtained with a tyrosinase assay. The EC50 of alpha-MSH was 27 pM, i.e., about five- to sevenfold lower than that in the assays for tyrosinase or intracellular melanin. Thus, the new assay represents the most sensitive melanoma cell assay for MSH available to date.     

Effect of insulin-like growth factor (IGF)-I and Des (1-3) IGF-I on the level of IGF binding protein-3 and IGF binding protein-3 mRNA in cultured porcine embryonic muscle cells

Insulin-like growth factor binding protein (IGFBP)-3 effects proliferation and differentiation of numerous cell types by binding to insulin-like growth factors (IGF) and attenuating their activity or by directly affecting cells in an IGF-independent manner. Consequently, IGFBPs produced by specific cells may affect their differentiation and proliferation. In this study we show that embryonic porcine myogenic cells, unlike murine muscle cell lines, produce significant quantities of a binding protein immunologically identified as IGFBP-3. Nonfusing cells subcultured from highly fused porcine myogenic cell cultures do not produce detectable IGFBP-3 protein or mRNA, thus suggesting the IGFBP-3 is produced by muscle cells in the porcine myogenic cell cultures. Treatment of porcine myogenic cultures with 20 ng of IGF-I or 20 ng of Des (1-3) IGF-I/ml serum-free media for 24 h results in a threefold reduction in the level of IGFBP-3 in conditioned media. This reduction is not affected by cell density over a sixfold range. Additionally, treatment for 24 h with 20 ng of IGF-I/ml media results in a sevenfold decrease in the steady-state level of IGFBP-3 mRNA. This IGF-I-induced decrease in IGFBP-3 mRNA level appears to be relatively unique to myogenic cells. IGF-I treatment also causes a fourfold increase in the steady-state level of myogenin mRNA. This increase in myogenin mRNA suggests that, as expected, IGF-I treatment accelerates differentiation of myogenic cells. The simultaneous decrease in IGFBP-3 mRNA and protein that accompanies IGF-I-induced myogenin expression suggests that differentiation of myogenic cells may be preceded or accompanied by decreased production of IGFBP-3.     

The seminiferous growth factor induces proliferation of TM4 cells in serum-free medium

The seminiferous growth factor (SGF) of the mammalian testes induces DNA synthesis and cell proliferation of Balb/c 3T3 cells (Bellvé and Feig, 1984; Rec Prog Hormone Res 40:531-567). In this study, SGF was purified 80,000- to 100,000-fold from calf testes and used to examine the growth of TM4 cells in a chemically defined medium. Cells were seeded sparsely in Dulbecco's Modified Eagles/Ham's F12 medium (1:1;v:v) (DME/F12 degrees), containing epidermal growth factor (EGF; 1 ng/ml), insulin (1; 10 micrograms/ml), and transferrin (Tr; 5 micrograms/ml) (DME/F12). After 24 h, the medium was replaced with DME/F12 degrees supplemented with SGF, EGF, 1, or Tr, in two-, three- or four-way combinations. Cell numbers were quantified after another 48 h of culture. EGF, I, and Tr, alone or in two-way combinations, were not mitogenic for TM4 cells. By contrast, SGF (1 U) alone, or with any two of these factors, stimulated TM4 cell proliferation to commensurate levels, and to twofold greater numbers than occurred with the combination of EGF, I, and Tr. Synergisms or inhibitions were not measurable. Follicle-stimulating hormone, luteinizing hormone, prolactin, acidic fibroblast growth factor, or basic fibroblast growth factor was weakly or not mitogenic for TM4 cells. The effect of SGF on cell proliferation was inhibited by 1 microM - 1 nM retinoic acid, but not by retinol or retinyl acetate. SGF was mitogenic for bovine adrenal capillary endothelial cells, an effect that was potentiated by 10 micrograms heparin/ml. Thus, SGF can induce proliferation of TM4 cells and capillary endothelial cells. The former provides a sensitive, and selective, serum-free, bioassay system for SGF activity.     

Comparative effect of FGF2, synthetic peptides 1-28 N-POMC and ACTH on proliferation in rat adrenal cell primary cultures

There is evidence that pro-opiomelanocortin (POMC)-derived peptides other than adrenocorticotropic hormone (ACTH) have a role in adrenal cell proliferation. We compared the activity of synthetic rat N-terminal POMC fragment 1-28 with disulfide bridges (N-POMC^w) and without disulfide bridges (N-POMC^w/o), with the activity of fibroblast growth factor (FGF2), a widely studied adrenal growth factor, and ACTH, in well-characterized pure cultures of both isolated adrenal Glomerulosa (G) and Fasciculata/Reticularis (F/R) cells. Three days of FGF2-treatment had a proliferative effect similar to serum, and synthetic peptide N-POMC^w induced proliferation more efficiently than N-POMC^w/o. Moreover, both induced proliferation via the ERK1/2 pathway. In contrast, sustained ACTH treatment decreased proliferation and viability through apoptosis induction, but not necrosis, and independently of PKA and PKC pathways. Further elucidation of 1-28 POMC signal transduction is of interest, and primary cultures of adrenal cells were found to be useful for examining the trophic activity of this peptide.     

Myelin Basic Protein and Myelin Basic Protein Peptides Induce the Proliferation of Schwann Cells Via Ganglioside GM1 and the FGF Receptor

Myelin basic protein (MBP) and two peptides derived from MBP (MBP_1–44 and MBP_152–167) stimulated Schwann cell (SC) proliferation in a cAMP-mediated process. The two mitogenic regions of MBP did not compete with one another for binding to SC suggesting a distinctive SC receptor for each mitogenic peptide. Neutralizing antibodies to the fibroblast growth factor receptor blocked the mitogenic effect of the myelin-related SC mitogen found in the supernatant of myelin-fed macrophages. The binding of ¹²⁵I-MBP to Schwann cells was specifically inhibited by basic fibroblast growth factor (bFGF) and conversely the binding of ¹²⁵I-bFGF was competitively inhibited by MBP. These data suggested that the mitogenic effect of one MBP peptide was mediated by a bFGF receptor. The binding of MBP to ganglioside GM1 and the ability of MBP peptides containing homology to the B subunit of cholera toxin (which binds ganglioside GM1) to compete for the binding of a mitogenic peptide (MBP_1–44) to SC, identified ganglioside GM1 as a second SC receptor. Based on these results, we conclude that MBP_1–44 and MBP_152–167 associate with ganglioside GM1 and the bFGF receptor respectively to stimulate SC mitosis.     

Desensitization of melanotropin receptors in COS-7 cells

Non-transfected COS-7 cells have been found to possess functional melanotropin receptors on their cell surface. These receptors, and the properties of the melanocyte stimulating hormone (MSH) peptides can be characterized by measuring melanotropin stimulation of cAMP accumulation in the cells. In these cells we studied the ultra-long lasting super agonist [Nle⁴-D-Phe⁷]-α-MSH (NDP-α-MSH), and compared it with the endogenous MSH peptides with respect to potency, maximal activity, duration of action, and rate of desensitization. Surprisingly, NDP- α-MSH did not act as a full agonist in COS-7 cells. In multiple experiments, it could stimulate cAMP accumulation to approximately 50% of the level of α-MSH, β-MSH and adrenocorticotropic hormone (ACTH). The MSH receptor mediating this activity is unknown. The time course of cAMP accumulation, and the duration of receptor activation was also investigated. In contrast to other systems, NDP-α-MSH did not induce prolonged activity, with respect to cAMP accumulation, in COS-7 cells. The MSH receptors present in COS-7 were found to desensitize rapidly subsequent to pretreatment by any of the MSH peptides. As expected for a partial agonist, the activity of NDP-α-MSH desensitized more rapidly than any of the full agonists. Surprisingly, desensitization induced by pretreatment with NDP-α-MSH also occurred more rapidly than desensitization induced by the other MSH analogs.     

Corticotropin Peptides and Melanotropins Elevate the Level of Adenosine 3'': 5''-Cyclic Monophosphate in Cultured Murine Brain Cells

Cell cultures derived from mouse and rat brain and consisting mainly of astroblasts are known to respond to several hormones by increasing or decreasing their intracellular concentration of cyclic AMP. In the present study these cultures were analyzed for their susceptibility to various additional hormonal and other neuroactive compounds. Only the peptides of the corticotropin (ACTH)/melanotropin (MSH) family were found active. Their potency for elevating the intracellular level of cyclic AMP decreases in the sequence (values for the half-maximally stimulating concentrations, EC50, in parentheses) ACTH-(1-24) (10 m) greater than alpha-,beta-MSH (30 nm) greater than ACTH (greater than or equal to 100 nm) gamma-MSH, ACTH-(1-10), -(4-10), -(4-11) (greater than or equal to 0.5 microM). The lack of additivity of the maximal effects of the peptides suggests that they all act at the same receptor. The stimulation exerted by these peptides is partially suppressed by hormones known to inhibit cyclic AMP formation in that culture, i.e., noradrenaline (acting via an alpha-adrenergic receptor), adenosine (acting via an A1 receptor), and somatostatin. It is concluded that the receptors for the ACTH/MSH peptides and the inhibitory hormones are located on the same cells, presumably the astroblasts. The maximal response to ACTH and alpha- and beta-MSH depends strongly on the age of culture. The results are discussed in view of the facts that (1) peptides of the ACTH/MSH family affect behavior and learning in animals, and (2) ACTH and alpha-MSH occur in brain.     

Expression of annexins as a function of cellular growth state

Annexins are a structurally related family of Ca2+ binding proteins of undertermined biological function. Annexin I (also called lipocortin 1) is a substrate for the EGF-stimulated tyrosine kinase and is postulated to be involved in mitogenic signal transduction. To investigate further the involvement of lipocortin 1 in cell proliferation, we measured lipocortin 1 levels in normal diploid human foreskin fibroblasts (HFF) to determine whether its expression changed as a function of growth status. For comparison, the expression of annexin V (also called endonexin II) was measured in HFF cells. Endonexin II is a protein with similar Ca2+ and phospholipid binding properties as lipocortin 1, but it is not a substrate for tyrosine kinases. Quiescent HFF cell cultures were induced to proliferate by either subculture to lower cell density, EGF stimulation, or serum stimulation. In all three protocols, proliferating HFF cells contained three- to fourfold higher levels of lipocortin 1 and three- to fourfold lower levels of endonexin II than quiescent HFF cells. In contrast, the expression of annexin II (also called calpactin I) and annexin IV (also called endonexin I) remained relatively unchanged in growing and quiescent HFF cells. Lipocortin 1 synthesis rate was eightfold higher and its turnover rate was 1.5-fold slower in proliferating compared to quiescent HFF cells. Endonexin II synthesis rate remained constant but its turnover rate was 2.2-fold faster in proliferating compared to quiescent HFF cells. In a separate set of experiments, annexin expression levels were measured in cultures of rat PC-12 cells, a pheochromocytoma that ceases proliferation and undergoes reversible differentiation into nondividing neuronlike cells in response to nerve growth factor (NGF). After NGF treatment, PC-12 cells expressed fivefold higher levels of endonexin II and 32-fold higher levels of calpactin 1. Lipocortin 1 and endonexin I were not expressed in PC-12 cells. In summary, lipocortin 1 expression exhibited a positive correlation with cell proliferation in HFF cells. The increased expression of endonexin II in quiescent HFF cells and differentiating PC-12 cells implies that this protein may play a more prominent role in nondividing cells.     

Biologically active synthetic fragments of human basic fibroblast growth factor (bFGF): identification of two Asp-Gly-Arg-containing domains involved in the mitogenic activity of bFGF in endothelial cells.

Synthetic peptides derived from the amino acid sequence of human basic fibroblast growth factor (bFGF) have been assayed for the capacity to exert bFGF agonist and antagonist activities in cultured endothelial cells. bFGF fragments A and C, which correspond to the sequences bFGF (38-61) and bFGF (82-101), induce a limited but statistically significant increase in cell number when administered to cultures of fetal bovine aortic endothelial GM 7373 cells and adult bovine aortic endothelial cells. The two peptides also exert a partial antagonist activity when GM 7373 cells are stimulated to proliferate by bFGF, but they do not affect cell proliferation induced by serum, epidermal growth factor (EGF), phorbol ester (TPA), or 1,2-diacylglycerol (diC8). Moreover, antibodies raised against peptides A and C specifically quench the mitogenic activity of bFGF. Peptides A and C contain the amino acid sequence Asp-Gly-Arg (DGR), which is the inverse of the cell adhesion signal sequence RGD recognized by integrins. DGR- and RGD-containing tetra- and heptapeptides inhibit the mitogenic activity exerted by bFGF and by the two active bFGF fragments. They do not affect cell proliferation induced by acidic FGF, EGF, serum, TPA, and diC8. However, neither peptides A and C, their corresponding antibodies, nor DGR-and RGD-containing peptides inhibit the binding of 125I-bFGF to its low and high affinity binding sites. The data suggest that amino acid residues 38-61 and 82-101, both containing a core DGR sequence, represent two "activation" domains of bFGF. Both domains are involved in the modulation of the mitogenic activity of bFGF without interacting directly with the bFGF receptor.     

Effects of human and ovine pancreatic secretory trypsin inhibitors on the proliferation of normal human fibroblasts

Human pancreatic secretory trypsin inhibitor inhibited cell-surface proteolytic activity in human fibroblasts. In the range of concentrations which caused proteinase inhibition, fibroblast proliferation was also inhibited by this reagent and by the ovine equivalent. At lower concentrations, there was some evidence for a mitogenic effect, and this was confirmed by obvious stimulation of DNA synthesis at these concentrations. Human alpha 1-proteinase inhibitor, previously demonstrated to be an inhibitor of fibroblast proliferation, was also mitogenic at concentrations lower than those which inhibited proteolytic activity and cell proliferation. Human pancreatic secretory trypsin inhibitor and epidermal growth factor apparently work through independent mechanisms, since their mitogenic effects are additive.     

Pressor and cardioaccelerator effects of gamma MSH and related peptides

We have recently demonstrated that the hypertensinogenic and natriuretic actions of ACTHI-39 can be found in a non-steroidogenic fragment of ACTH, ACTH4-10. These effects of ACTH or ACTH4-10 may be due to their ability to act as weak agonists of gamma MSH. gamma MSH is found in the 16K N-terminus of pro-opiocortin, and contains a sequence analogous to ACTH4-10, gamma MSH3-9. We investigated the cardiovascular effects of gamma 2MSH, gamma MSH3-9, and sterically restricted analogs of ACTH4-10. The results indicate that gamma MSH3-9, had essentially the same activities as ACTH4-10. The addition of five other amino acid residues to gamma MSH3-9 (gamma 2MSH) resulted in significant enhancement of pressor and cardioaccelerator activity. Steric restriction of the ACTH4-10 sequence by the substitution of a D-Phe in place of an L-Phe residue in position #7, or cyclization of the peptide by a half-Cys4, half Cys10 intramolecular disulfide-bridge derivatization, resulted in increased cardiovascular activities. Based on these data, the cardiovascular actions of ACTH4-10, gamma MSH3-9, and gamma 2MSH are predicted to be due to the assumption of a reverse-turn three-dimensional structure. The additional residues in gamma 2MSH appear to specifically enhance the cardiovascular activities of gamma MSH3-9. The results suggest the existence of a new class of hypophyseal peptides with cardiovascular activities, which require the assumption of a defined three-dimensional structure.