Mitogenesis by serum and PDGF is independent of PI degradation and PKC in VSMC

1989 ◽  
Vol 256 (4) ◽  
pp. C886-C892 ◽  
Author(s):  
M. Kihara ◽  
P. J. Robinson ◽  
S. H. Buck ◽  
R. C. Dage
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Growth Control ◽  
Calcium Ionophore ◽  
Rat Aorta ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Pi Turnover ◽  
Fetal Bovine ◽  
20 Nm

Capacities of serum, platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) on phosphatidylinositol (PI) degradation and cell growth were compared in cultured vascular smooth muscle cells (VSMC) from rat aorta. The role of protein kinase C (PKC) in growth control was also evaluated using polymixin B, a selective inhibitor of PKC. Both dialyzed and nondialyzed fetal bovine serum (FBS) in concentrations from 2 to 20% stimulated [3H]thymidine incorporation into DNA and cell growth without producing corresponding increases in PI turnover. Moreover, both PDGF (40-160 ng/ml) and FGF (6.25-150 ng/ml) also stimulated mitogenesis, but PDGF was more effective although less potent. Mitogenic amounts of PDGF did not stimulate PI turnover, whereas a maximally mitogenic amount of FGF (50 ng/ml) did produce a slight increase. Polymixin B inhibited PKC activity (IC50, 32 microM) from these cells but failed to suppress DNA synthesis produced by 10% FBS or PDGF (50 ng/ml). However, it did suppress that by FGF (50 ng/ml). Angiotensin II (10(-11)-10(-7) M) and phorbol 12,13-dibutyrate (PDB, 1-20 nM) were not mitogenic in the presence or absence of insulin (10 micrograms/ml) or the calcium ionophore A23187 (0.25-4 microM), under serum-free conditions. Instead, PDB inhibited mitogenesis of cells maintained under 0.2% FBS or stimulated with insulin (10 micrograms/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The NGFI-B protein, an inducible member of the thyroid/steroid receptor family, is rapidly modified posttranslationally.

1990 ◽  
Vol 10 (12) ◽  
pp. 6454-6459 ◽  
Author(s):  
T J Fahrner ◽  
S L Carroll ◽  
J Milbrandt
Keyword(s):  
Growth Factor ◽  
Molecular Mass ◽  
Pc12 Cells ◽  
Receptor Gene ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Carboxy Terminal

The NGFI-B gene is rapidly activated by a variety of stimuli that induce cells to differentiate or proliferate. It encodes a protein with a predicted molecular mass of congruent to 61 kDa and is a member of the thyroid/steroid hormone receptor gene family. To characterize this protein, monoclonal antibodies were raised against a bacterial TrpE-NGFI-B fusion protein that encompasses a large portion (Glu-410 to Leu-527) of the carboxy-terminal domain of NGFI-B. These antibodies detected a protein that was rapidly synthesized in response to nerve growth factor (NGF) and migrated as a broad band on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular mass that ranged from 63 to 88 kDa. Pulse-chase analysis demonstrated that NGFI-B was rapidly posttranslationally modified and was a short-lived protein. NGFI-B was found to be a phosphorylated protein, and the multiple NGFI-B species coalesced into a single, more rapidly migrating species when treated with alkaline phosphatase. PC12 cells grown in the absence of NGF contained low levels of NGFI-B that was underphosphorylated. Epidermal growth factor, phorbol ester, and the calcium ionophore A23187 stimulated the synthesis of NGFI-B that was composed largely of underphosphorylated, rapidly migrating species. In contrast, basic fibroblast growth factor, which promotes differentiation of PC12 cells, induced the synthesis of NGFI-B species similar to those synthesized in response to NGF treatment. The underphosphorylated NGFI-B found in uninduced PC12 cells was found only in the nucleus, whereas NGFI-B in NGF-stimulated PC12 cells was present in approximately equal quantities in the cytoplasm and nucleus. Consistent with the cellular distribution observed in nonstimulated PC12 cells, the highly phosphorylated species were predominantly cytoplasmic whereas the more rapidly migrating forms were nuclear.

Effect of glucose in the culture medium on development of horse oocytes matured and microfertilized in vitro

1995 ◽  
Vol 7 (5) ◽  
pp. 1067 ◽  
Author(s):  
T Azuma ◽  
YH Choi ◽  
S Hochi ◽  
N Oguri
Keyword(s):  
Calcium Ionophore ◽  
Fertilization Rate ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cell Stage ◽  
Morula Stage ◽  
Fetal Bovine ◽  
Oviduct Fluid ◽  
Effect Of Glucose

The development of in-vitro matured and microfertilized horse oocytes was examined in vitro. Fertilized oocytes were produced by 20-h insemination of in-vitro matured and partially zona-removed oocytes with frozen spermatozoa that had been treated with caffeine/calcium ionophore A23187 (fertilization rate 34.2%, monospermy rate 76.9%). Embryonic development was assessed by the number of nuclei stained with Giemsa solution. In Experiment 1, a continuous 8-day culture of the microfertilized oocytes in TCM199 or modified synthetic oviduct fluid (m-SOF) supplemented with 10% fetal bovine serum or 0.1% polyvinyl alcohol (PVA) in 5% O2, 5% CO2 and 90% N2 resulted in very few embryos developing beyond the 8-cell stage. In Experiment 2, the effects of different glucose concentrations (0, 0.5, 5.5 mM) in m-SOF/PVA during Days 1-4 and Days 5-8 of culture were examined. Proportions of oocytes having more than one nucleus ranged from 17.7% to 44.7% among the combinations of glucose concentrations. Supplementation with glucose at 0.5 mM during Days 1-4 followed by 5.5 mM during Days 5-8 resulted in the best embryo development; 12/55 (21.8%) nuclei-positive oocytes developed to the 8-16-cell stage, 11 (20.0%) developed to the 17-50-cell stage, and 5 (9.1%) comprised more than 50 cells and were assumed to be at the morula stage.

Inhibitory Effect of Fentanyl on Acetylcholine-induced Relaxation in Rat Aorta

2004 ◽  
Vol 101 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Ju-Tae Sohn ◽  
Seong-Ho Ok ◽  
Hee-Jin Kim ◽  
Seon-Hak Moon ◽  
Il-Woo Shin ◽  
...  
Keyword(s):  
Muscarinic Receptor ◽  
Response Curve ◽  
Calcium Ionophore ◽  
Receptor Activation ◽  
Inhibitory Effect ◽  
Rat Aorta ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Acetylcholine Concentration ◽  
Concentration Response Curve

Background Previous study has shown that fentanyl attenuates acetylcholine-induced vasorelaxation. The goal of the current in vitro study was to identify the muscarinic receptor subtype that is mainly involved in the fentanyl-induced attenuation of endothelium-dependent relaxation elicited by acetylcholine. Methods The effects of fentanyl and muscarinic receptor antagonists on the acetylcholine concentration-response curve were assessed in aortic vascular smooth muscle ring preparations precontracted with phenylephrine. In the rings pretreated independently with pirenzepine, 4-diphenylacetoxyl-N-methylpiperidine methiodide, and naloxone, acetylcholine concentration-response curves were generated in the presence and absence of fentanyl. The effect of fentanyl on the concentration-response curve for calcium ionophore A23187 was assessed. Results Fentanyl (0.297 x 10 0.785 x 10 m) attenuated acetylcholine-induced vasorelaxation in ring preparations with or without 10 m naloxone. Pirenzepine (10 to 10 m) and 4-diphenylacetoxyl-N-methylpiperidine methiodide (10 to 10 m) produced a parallel rightward shift in the acetylcholine concentration-response curve. The concentrations (-log M) of pirenzepine and 4-diphenylacetoxyl-N-methylpiperidine methiodide necessary to displace the concentration-response curve of an acetylcholine by twofold were estimated to be 6.886 +/- 0.070 and 9.256 +/- 0.087, respectively. Methoctramine, 10 m, did not alter the acetylcholine concentration-response curve. Fentanyl, 0.785 x 10 m, attenuated acetylcholine-induced vasorelaxation in the rings pretreated with 10 m pirenzepine but had no effect on vasorelaxation in the rings pretreated with 10 m 4-diphenylacetoxyl-N-methylpiperidine methiodide. Fentanyl, 0.785 x 10 m, did not significantly alter calcium ionophore A23187-induced vasorelaxation. Conclusions These results indicate that fentanyl attenuates acetylcholine-induced vasorelaxation via an inhibitory effect at a level proximal to nitric oxide synthase activation on the pathway involving endothelial M3 muscarinic receptor activation in rat aorta.

The NGFI-B protein, an inducible member of the thyroid/steroid receptor family, is rapidly modified posttranslationally

1990 ◽  
Vol 10 (12) ◽  
pp. 6454-6459
Author(s):  
T J Fahrner ◽  
S L Carroll ◽  
J Milbrandt
Keyword(s):  
Growth Factor ◽  
Molecular Mass ◽  
Pc12 Cells ◽  
Receptor Gene ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Carboxy Terminal

The NGFI-B gene is rapidly activated by a variety of stimuli that induce cells to differentiate or proliferate. It encodes a protein with a predicted molecular mass of congruent to 61 kDa and is a member of the thyroid/steroid hormone receptor gene family. To characterize this protein, monoclonal antibodies were raised against a bacterial TrpE-NGFI-B fusion protein that encompasses a large portion (Glu-410 to Leu-527) of the carboxy-terminal domain of NGFI-B. These antibodies detected a protein that was rapidly synthesized in response to nerve growth factor (NGF) and migrated as a broad band on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular mass that ranged from 63 to 88 kDa. Pulse-chase analysis demonstrated that NGFI-B was rapidly posttranslationally modified and was a short-lived protein. NGFI-B was found to be a phosphorylated protein, and the multiple NGFI-B species coalesced into a single, more rapidly migrating species when treated with alkaline phosphatase. PC12 cells grown in the absence of NGF contained low levels of NGFI-B that was underphosphorylated. Epidermal growth factor, phorbol ester, and the calcium ionophore A23187 stimulated the synthesis of NGFI-B that was composed largely of underphosphorylated, rapidly migrating species. In contrast, basic fibroblast growth factor, which promotes differentiation of PC12 cells, induced the synthesis of NGFI-B species similar to those synthesized in response to NGF treatment. The underphosphorylated NGFI-B found in uninduced PC12 cells was found only in the nucleus, whereas NGFI-B in NGF-stimulated PC12 cells was present in approximately equal quantities in the cytoplasm and nucleus. Consistent with the cellular distribution observed in nonstimulated PC12 cells, the highly phosphorylated species were predominantly cytoplasmic whereas the more rapidly migrating forms were nuclear.

scholarly journals Glycogenolytic effects of the calcium ionophore A23187, but not of vasopressin or angiotensin, in foetal-rat hepatocytes

1984 ◽  
Vol 220 (2) ◽  
pp. 441-445 ◽  
Author(s):  
M Freemark ◽  
S Handwerger
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Glycogen Synthesis ◽  
Calcium Ionophore ◽  
Rat Hepatocytes ◽  
Glycogen Metabolism ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Foetal Rat ◽  
20 Nm

Vasopressin, angiotensin and phenylephrine stimulate glycogenolysis in postnatal rat liver by a Ca2+-mediated mechanism not involving cyclic AMP. To determine whether these hormones promote glycogenolysis in foetal liver, we have examined their effects, and those of the Ca2+ ionophore A23187, on glycogen metabolism in cultured foetal-rat hepatocytes. Vasopressin and angiotensin (0.1 nM-0.1 microM) had no effects on either glycogen synthesis (as assessed by [14C]glucose incorporation into glycogen) or phosphorylase a activity. However, A23187 at 1 and 10 microM inhibited glycogen synthesis by 31.3 and 89.1% respectively (both P less than 0.001) and stimulated phosphorylase a activity by 66.9 and 184.1% respectively (both P less than 0.01). Incubation of cells in Ca2+-deficient medium attenuated the effects of 10 microM-A23187 on glycogen synthesis and abolished the effects of 1 microM-A23187. As in postnatal liver, glucagon (1 and 20 nM) and isoprenaline (1 and 10 microM), which activate adenylate cyclase, inhibited glycogen synthesis and stimulated phosphorylase a activity in foetal hepatocytes. The minimal effective concentration of phenylephrine was 10 times that of isoprenaline. These results indicate striking differences in the ontogeny of cyclic AMP-mediated and Ca2+-mediated processes which regulate hepatic glycogenolysis. Since increases in cytosolic Ca2+ induce glycogenolysis in foetal-rat liver, the weak or absent responses to vasopressin, angiotensin and the alpha-adrenergic agonists may result from defects in hormone-receptor binding or in post-receptor events leading to the mobilization of intracellular Ca2+ stores.

Platelet-derived growth factor-stimulated c-myc RNA accumulation in MG-63 human osteosarcoma cells is independent of both protein kinase A and protein kinase C

1990 ◽  
Vol 10 (1) ◽  
pp. 184-192
Author(s):  
K K Frick ◽  
C D Scher
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Phosphodiesterase Inhibitor ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Platelet Derived Growth Factor ◽  
Ionophore A23187 ◽  
Simultaneous Treatment ◽  
Human Osteosarcoma Cells

Treatment of quiescent MG-63 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) or platelet-derived growth factor (PDGF) stimulates the rapid accumulation of c-myc RNA. We have now determined that a similar effect can be induced by cAMP. Treatment with forskolin (an activator of adenylate cyclase), IBMX (a phosphodiesterase inhibitor), PGE1, and isoproterenol stimulated accumulation of both cAMP and c-myc RNA, but no increase in either cAMP or c-myc RNA was seen with the inactive forskolin analog 1,9-dideoxyforskolin. Forskolin and IBMX acted synergistically in stimulating accumulation of both cAMP and c-myc RNA. However, three lines of evidence indicated that PDGF action is not mediated by cAMP. First, PDGF treatment caused no elevation of cAMP within 1 h, even in the presence of IBMX. Second, the kinetics of c-myc RNA elevation after treatment with PDGF or forskolin were similar, ruling out delayed onset of cAMP stimulation. Finally, simultaneous treatment with forskolin and the calcium ionophore A23187 enhanced the elevation of c-myc RNA levels; no such effect was seen with PDGF. We had previously shown that PDGF action is not affected by prior treatment of MG-63 cells with TPA, a treatment which desensitizes the c-myc response to TPA. Similarly, TPA pretreatment had minimal effect on forskolin or IBMX-induced c-myc expression. These data suggest that cAMP, phorbol esters, and PDGF act independently to stimulate c-myc RNA expression in MG-63 cells. However, nuclear runoff experiments and RNA half-life measurements demonstrated that PDGF, phorbol ester, and cAMP all act to increase the transcription of the MYC gene.

scholarly journals Platelet-derived growth factor-stimulated c-myc RNA accumulation in MG-63 human osteosarcoma cells is independent of both protein kinase A and protein kinase C.

1990 ◽  
Vol 10 (1) ◽  
pp. 184-192 ◽  
Author(s):  
K K Frick ◽  
C D Scher
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Phosphodiesterase Inhibitor ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Platelet Derived Growth Factor ◽  
Ionophore A23187 ◽  
Simultaneous Treatment ◽  
Human Osteosarcoma Cells

Treatment of quiescent MG-63 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) or platelet-derived growth factor (PDGF) stimulates the rapid accumulation of c-myc RNA. We have now determined that a similar effect can be induced by cAMP. Treatment with forskolin (an activator of adenylate cyclase), IBMX (a phosphodiesterase inhibitor), PGE1, and isoproterenol stimulated accumulation of both cAMP and c-myc RNA, but no increase in either cAMP or c-myc RNA was seen with the inactive forskolin analog 1,9-dideoxyforskolin. Forskolin and IBMX acted synergistically in stimulating accumulation of both cAMP and c-myc RNA. However, three lines of evidence indicated that PDGF action is not mediated by cAMP. First, PDGF treatment caused no elevation of cAMP within 1 h, even in the presence of IBMX. Second, the kinetics of c-myc RNA elevation after treatment with PDGF or forskolin were similar, ruling out delayed onset of cAMP stimulation. Finally, simultaneous treatment with forskolin and the calcium ionophore A23187 enhanced the elevation of c-myc RNA levels; no such effect was seen with PDGF. We had previously shown that PDGF action is not affected by prior treatment of MG-63 cells with TPA, a treatment which desensitizes the c-myc response to TPA. Similarly, TPA pretreatment had minimal effect on forskolin or IBMX-induced c-myc expression. These data suggest that cAMP, phorbol esters, and PDGF act independently to stimulate c-myc RNA expression in MG-63 cells. However, nuclear runoff experiments and RNA half-life measurements demonstrated that PDGF, phorbol ester, and cAMP all act to increase the transcription of the MYC gene.

Sign in / Sign up

Export Citation Format

Share Document