Biomedical subjects

R R Padua

Publications and source records attributed to R R Padua.

7 recordsLinked to original sources

PubMed1999

Fibroblast growth factor-2 stimulates phospholipase Cbeta in adult cardiomyocytes.

Although fibroblast growth factor-2 (FGF-2) plays an important role in cardioprotection and growth, little is known about the signals triggered by it in the adult heart. We therefore examined FGF-2-induced effects on phosphoinositide-specific phospholipase C (PI-PLC) isozymes, which produce second messengers linked to the inotropic and hypertrophic response of the myocardium. FGF-2, administered by retrograde perfusion to the isolated heart, induced an increase in inositol-1,4,5-trisphosphate levels in the cytosol, as well as an increase in total PI-PLC activity associated with sarcolemmal and cytosolic fractions. Furthermore FGF-2 induced a time-dependent elevation in cardiomyocyte membrane-associated PLC gamma1 and PLC beta1 activities, assayed in immunoprecipitated fractions, and moreover, increased the membrane levels of PLC beta1 and PLC beta3. Activation of PLC beta is suggestive of FGF-2-induced cross-talk between FGF-receptor tyrosine kinase and G-protein-coupled signaling in adult cardiomyocytes and underscores the importance of FGF-2 in cardiac physiology.

Animals↗

PubMed1998

FGF-2-induced negative inotropism and cardioprotection are inhibited by chelerythrine: involvement of sarcolemmal calcium-independent protein kinase C.

Fibroblast growth factor-2 (FGF-2), administered to the isolated rat heart by perfusion and under constant pressure, is protective against ischemia-reperfusion (I-R). Here we have investigated whether FGF-2 cardioprotection: (a) is dependent on flow modulation; (b) is linked to effects on contractility; (c) is mediated by protein kinase C (PKC); and (d) is linked to PKC and/or mitogen activated protein kinase (MAPK) associated with the sarcolemma. The isolated rat heart was used as a model. Under conditions of constant flow FGF-2 induced significant improvement in recovery of contractile function during I-R. Under constant perfusion pressure, FGF-2 induced a negative inotropic effect (15% decrease in developed pressure). Chelerythrine, a specific PKC inhibitor, prevented both the FGF-2-induced negative inotropic effect before ischemia, and cardioprotection during I-R. FGF-2 induced a chelerythrine-preventable, five-fold increase in sarcolemmal calcium-independent PKC activity. It also increased the association of PKC subtypes -epsilon and -delta with sarcolemmal membranes, detected by Western blotting, as well as, for PKC delta, by immunolocalization. FGF-2 increased the association of PKC epsilon with the membrane fraction of adult cardiomyocyte in culture, confirming that it can affect PKC signaling in cardiomyocytes directly and in a manner similar to its effects in situ. Finally, FGF-2 induced increased active MAPK at sarcolemmal as well as cytosolic sites. Active sarcolemmal MAPK remained elevated when the FGF-2-induced protection was prevented by chelerythrine. In conclusion, we have provided evidence that cardioprotection by FGF-2 is independent of flow modulation. PKC activation mediates both the FGF-2-induced negative inotropic effect before ischemia and the cardioprotective effect assessed during reperfusion, suggesting a cause and effect relationship. Furthermore, FGF-2 cardioprotection is linked to targeting of sarcolemmal sites by calcium-independent PKC.

Alkaloids↗

PubMed1995

Basic fibroblast growth factor is cardioprotective in ischemia-reperfusion injury.

To examine whether basic fibroblast growth factor (bFGF) administered to the heart by perfusion can improve cardiac resistance to injury we employed an isolated rat heart model of ischemia-reperfusion injury and determined the extent of functional recovery in bFGF-treated and control hearts. Global ischemia was simulated by interruption of flow for 60 min. Recovery of developed force of contraction (DF), recorded after reestablishment of flow for 30 min, reached 63.8 +/- 1.5% and 96.5 +/- 3.5% of preischemic levels in control and bFGF-treated hearts (10 micrograms/heart), respectively, indicating that bFGF induced significantly improved recovery of mechanical function. Recoveries of the rates of contraction or relaxation were also significantly improved in bFGF-treated hearts. Extent of myocardial injury, assessed by determination of phosphocreatine kinase in the effluent, was reduced as a result of bFGF treatment. As a first step towards understanding the mechanism and direct cellular target(s) of bFGF-induced cardioprotection, we investigated its fate after perfusion. Perfusion of 10 micrograms bFGF/heart resulted in a 4-fold increase in bFGF associated with the heart compared to control levels, as estimated by biochemical fractionation and immunoblotting. Immunofluorescent staining of the bFGF-perfused hearts revealed intense anti-bFGF staining in association with blood vessels as well as the periphery of cardiomyocytes, suggesting that the latter may be a target for direct bFGF action. In conclusion, our findings of bFGF-induced increases in cardiac resistance to, and improved functional recovery from, ischemia-reperfusion injury indicate that bFGF may have clinical applications in the treatment of ischemic heart disease.

Animals↗

PubMed1995

Adult cardiomyocytes express functional high-affinity receptors for basic fibroblast growth factor.

As a first step in addressing the question of function for basic fibroblast growth factor (bFGF) in the adult myocardium, expression of bFGF receptors by adult rat myocytes was investigated. Cross-linking of 125I-labeled bFGF to purified sarcolemmal vesicles from adult hearts indicated specific binding to 90- to 104-kDa proteins, whereas equilibrium binding studies revealed the presence of "low"-affinity (1 nM) and "high"-affinity (115 pM) sites. Adult myocytes were found to express short and long variants of bFGF receptor 1 (FGFR-1, tyrosine kinase) mRNA. Adult heart overall levels of FGFR-1 mRNA were decreased by about one-third of corresponding fetal values. Several lines of evidence indicated that bFGF receptors in adult cardiomyocytes in situ and/or in isolation are functional. Isolated adult myocytes were found to be capable of heparin-resistant internalization of 125I-labeled bFGF, to lose their viability after interaction with bFGF-saporin (a mitotoxin known to kill cells after entry via the bFGF receptor), and to respond to bFGF by activation of mitogen-activated protein kinase. In addition, introduction of exogenous bFGF into the myocardium by Langendorff perfusion resulted in stimulation of tyrosine phosphorylation in association with cardiomyocyte intercalated disks, as assessed by immunofluorescence. It is concluded that adult cardiomyocytes express functionally coupled high-affinity bFGF receptors and that they are capable of a biologic response to bFGF in vivo.

Animals↗

PubMed1993

Increased basic fibroblast growth factor (bFGF) accumulation and distinct patterns of localization in isoproterenol-induced cardiomyocyte injury.

Basic FGF is a multifunctional protein which promotes regeneration in several tissues. To investigate involvement in cardiac injury-repair, bFGF accumulation and localization was examined in hearts of rats injected with a single high dose of isoproterenol. The bFGF content of cardiac extracts was analyzed at 6 and 24 hours as well as 1, 4, and 6 weeks by western blotting of heparin-sepharose-bound fractions. The 18 kilodalton bFGF species showed an approximately 2-fold increase in extracts from treated animals compared to non-treated controls. A transient rise in a 21-23 kilodalton bFGF species was seen at 24 hours after treatment. An induction of bFGF mRNA was also observed in treated animals. To localize bFGF in vivo, immunofluorescent labelling with specific antibodies was used at 4-24 hours and 1-4 weeks after treatment. Simultaneous labelling for the cytoskeletal proteins vinculin or vimentin was employed to identify viable myocytes or non-muscle interstitial cells, respectively. Necrotic myocytes, identified by loss of vinculin, displayed a pronounced increase in cytoplasmic anti-bFGF staining compared to adjacent normal myocytes. This increase occurred prior to and may play a role in promoting mobile cell migration and proliferation in areas of necrosis. Viable cardiomyocytes adjacent to fibrotic regions displayed strong pericellular anti-bFGF staining and, occasionally, were also stained by anti-vimentin antibodies, suggesting reexpression of an embryonic phenotype and thus an attempt for regeneration. These data showing increased accumulation and distinct patterns of localization of bFGF in the hearts of isoproterenol-treated animals suggest that this growth factor plays a role in short-term as well as long term response of the myocardium to injury.

Animals↗

PubMed1990

Calcium protects pituitary basic fibroblast growth factors from limited proteolysis by co-purifying proteases.

Extracts from bovine pituitaries and other tissues contained basic fibroblast growth factor-like peptides of 22-26 kda, co-fractionating with smaller, 16-20 kda bFGFs. Heparin-bound, 22-26 kda bFGFs were converted to smaller, heparin-binding forms by tryptic proteolysis. In solution, 22-26 kda bFGFs were converted to smaller, heparin-binding forms by an activity present in pituitary extracts. Calcium protected higher molecular weight pituitary bFGFs from truncation by the endogenous activity, which was not acid-activated, co-purified with bFGF during heparin-sepharose chromatography, remained operant at high salt concentrations and was inhibited by phenylmethan-sulfonyl fluoride.

Animals↗

PubMed1990

Characterization of two preparations of antibodies to basic fibroblast growth factor which exhibit distinct patterns of immunolocalization.

Immunoglobulins reactive against basic fibroblast growth factor (bFGF) were obtained from the serum of a single rabbit immunized against residues [1-24] of bFGF conjugated to keyhole limpet hemocyanin (KLH). Pure immunoglobulin preparations no. 1 and no. 2 were prepared using different affinity chromatography columns and preabsorption to KLH-coupled Sepharose for preparation no. 1. Both preparations no. 1 and no. 2 were specific for bFGF in in vitro assays. Competition with synthetic peptides suggests that preparations no. 1 and no. 2 recognize predominantly epitope(s) within residues [16-24]bFGF or residues [1-10]bFGF, respectively, in situ. Furthermore, no. 2 (but not no. 1) antibodies can react with tissue-(heparin-)-bound antigen. When used in indirect immunofluorescence for bFGF in frozen heart sections, preparation no. 1 stained predominantly muscle intercalated discs (IcDs); muscle nuclei were also stained, in an overall punctate fashion. Preparation no. 2 stained muscle nuclei strongly, in association with the nuclear envelope; it also stained basement-membrane associated bFGF. Differences in immunostaining were also observed in uterine smooth muscle and kidney sections but not in skeletal muscle. It is plausible that accessibility of various epitopes within the amino-terminal region depends strongly on the local interactions of bFGF. Our data illustrate the importance of using several different antibodies to localize bFGF in a tissue.

Absorption↗