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Biomedical subjects

H M Piper

Publications and source records attributed to H M Piper.

At least 55 records · Page 3Linked to original sources
PubMed1999

Prime causes of rapid cardiomyocyte death during reperfusion.

In ischemic-reperfused myocardium, necrosis of cardiomyocytes may develop not only due to the ischemic conditions but also the specific circumstances of reperfusion. The existence of reperfusion injury becomes apparent when modifications of the conditions of reperfusion can prevent cell death otherwise occurring. Three prime causes of rapidly developing reperfusion injury are here discussed, ie, reenergization of cells at increased cytosolic Ca2+ contents, rapid normalization of tissue pH, and rapid normalization of tissue osmolality. All three causes lead to severe mechanical stress of cardiomyocytes which can cause their rapid deterioration. Propagation of cell injury among adjacent cells can cause a spreading of necrosis throughout myocardial tissue. The understanding of these initial causes of rapidly developing lethal reperfusion injury leads to new concepts for specific protection of reperfused myocardium.

Acid-Base Equilibrium↗
PubMed1999

Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat.

OBJECTIVES: Apoptosis is one feature of myocardial damage after ischemia-reperfusion, but the causes for its induction are unclear. The present study was undertaken to investigate whether apoptosis in cardiomyocytes is directly initiated by their sub-lethal injury that results from ischemia-reperfusion. METHODS: Ischemia was simulated on isolated ventricular cardiomyocytes of adult rats by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected by (1) DNA laddering of genomic DNA, (2) TUNEL positive cells (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelling) and (3) annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide. Necrotic cells were identified by (1) staining with both annexinV-FITC and propidium iodide, (2) unspecific DNA degradation and (3) enzyme release. RESULTS: Simulated ischemia caused a > 75% loss of high-energy phosphates within 2 h, which was reversible upon reoxygenation at pH 7.4. Even after 18 h of simulated ischemia, creatine phosphate contents recovered to 55.2 +/- 7.3% of control within 1 h. Apoptosis could be induced by UV irradiation (80 J/m2), H2O2 and the NO-donor N2-acetyl-S-nitroso-D,L-penicillinaminamide. In contrast to this, simulated ischemia and reoxygenation could not induce apoptosis in the cells, but with prolonged ischemia more cells became necrotic. After 18 hours of simulated ischemia and 4 h of reoxygenation 41.2 +/- 10.2% myocytes were necrotic (vs. 6.3 +/- 4.4% of control) and only 1.7 +/- 0.5% (vs. 8.7 +/- 4.6% of control) were apoptotic. The percentage of necrotic cells correlated with an increase in lactate dehydrogenase release from 9.9 +/- 0.6% (of total activity) of normoxic controls to 37.9 +/- 5.1% after 18 h of simulated ischemia and 12 h of reoxygenation. CONCLUSIONS: Simulated ischemia-reoxygenation causes necrosis of isolated cardiomyocytes but is not sufficient for induction of apoptosis.

Analysis of Variance↗
PubMed1999

Regulation of growth in the adult cardiomyocytes.

Cardiomyocytes of adult myocardium increase their cellular mass in response to growth stimuli. They undergo hypertrophic growth but they do not proliferate in contrast to immature cardiomyocytes. Growth stimuli of the adult cardiomyocytes include classical growth hormones, various neuroendocrine factors, and the increase in mechanical load. The signal transduction of alpha1-adrenoceptor stimulation has been investigated in greatest detail and may therefore be taken as a reference for other humoral stimuli. It involves the activation of protein kinase C (PKC) and, downstream of PKC activation, of two separate signaling pathways, one including the mitogen-activated protein kinase and another including PI3-kinase and p70(s6k) as key steps. Activation of the first pathway leads to re-expression of fetal genes, activation of the second pathway to a general activation of protein synthesis, and cellular growth. In neonatal cardiomyocytes, mechanical stretch causes growth by an activation of an autocrine mechanism including angiotensin II and endothelin. This mechanism does not operate, however, in adult cardiomyocytes. A mechanism of mechanotransduction has not yet been identified on adult cardiomyocytes but integrins may play a part. In microgravity, the scenario of myocardial growth stimulation is altered. On the systemic level, there are changes in hemodynamic and neuroendocrine regulation that exert indirect effects on the myocardium. Microgravity may also exert a direct cellular effect by the absence of a constant gravitational load component.

Adrenergic alpha-1 Receptor Agonists↗
PubMed1999

Early response kinase and PI 3-kinase activation in adult cardiomyocytes and their role in hypertrophy.

The present study investigated the role of early response kinase (ERK) and phosphatidylinositol 3 (PI 3)-kinase in ventricular cardiomyocytes from adult rat for the hypertrophic response to alpha-adrenoceptor stimulation. Parameters of the hypertrophic response were stimulation of protein synthesis and induction of creatine kinase BB. The alpha-adrenoceptor agonist phenylephrine (10 micromol/l) activated ERK2 and PI 3-kinase. The protein kinase C inhibitor bisindolylmaleimide (5 micromol/l) and the mitogen-activated protein kinase kinase inhibitor PD-98059 (10 micromol/l) but not the tyrosine kinase inhibitor genistein (100 micromol/l) blocked ERK2 activation. Inhibition of ERK2 activation abolished induction of creatine kinase BB by phenylephrine but not the increase in protein synthesis. The PI 3-kinase inhibitor wortmannin (100 nmol/l) blocked protein synthesis under alpha-adrenoceptor stimulation but did not interfere with ERK2 activation. Inhibition of the ERK2 pathway with PD-98059 did not affect PI 3-kinase activation. We conclude that ERK2- and PI 3-kinase-dependent pathways represent two mutually exclusive ways of signaling that lead to different aspects of the hypertrophic response to alpha-adrenoceptor stimulation.

Androstadienes↗
PubMed1999

Cardioprotective effects of KB-R7943: a novel inhibitor of the reverse mode of Na+/Ca2+ exchanger.

The novel inhibitor of the reverse mode of the Na+/Ca2+ exchanger (NCE) KB-R7943 (KB) was tested in isolated rat cardiomyocytes exposed to 80 min of simulated ischemia [substrate-free anoxia, extracellular pH (pHo) of 6.4] and 15 min of reoxygenation (pHo 7.4). At pHo 6.4, 20 micromol/l KB was required for complete inhibition of the reverse mode of NCE. Treatment with 20 micromol/l KB only during anoxia did not influence the onset of rigor contracture and intracellular pH (pHi) (monitored with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) but significantly reduced the cytosolic accumulation of Ca2+ (monitored with fura 2) and Na+ (monitored with sodium-binding benzofuran isophthalate). During reoxygenation, cardiomyocytes developed hypercontracture. This was significantly reduced by anoxic KB treatment. To investigate this protection against reoxygenation-induced injury in the whole heart, we exposed Langendorff-perfused rat hearts to 110 min of anoxia (pHo 6.4) and 50 min of reoxygenation (pHo 7.4). Application of 20 micromol/l KB during anoxia significantly reduced the reoxygenation-induced enzyme release. We conclude that KB offers significant protection of cardiomyocytes against Ca2+ and Na+ overload during anoxia and hypercontracture or enzyme release on reoxygenation.

Animals↗
PubMed1999

ATP reduces macromolecule permeability of endothelial monolayers despite increasing [Ca2+]i.

We investigated the relationship between the ATP-evoked rise of cytosolic Ca2+ concentration ([Ca2+]i) and barrier function in porcine aortic endothelial monolayers. ATP (0.01-100 microM) induced a transient rise of [Ca2+]i and reduced permeability in a concentration-dependent manner. In contrast, the Ca2+ ionophore ionomycin (1 microM) elicited a rise in [Ca2+]i comparable to that induced by ATP (10 microM), but it increased permeability. For the reduction of permeability, nucleotides were found to be in the following order of potency: ATP = ATPgammaS > ADP = UTP. Blockade of adenosine receptors by 8-phenyltheophylline (10 microM) did not affect ATP (10 microM)-induced reduction of permeability. ATP reduced permeability even in endothelial monolayers that had been loaded with the Ca2+ chelator BAPTA to prevent the rise in [Ca2+]i. U-73122 (1 microM), an inhibitor of phospholipase C (PLC), completely abolished the effect of ATP (10 microM) on permeability. It also abolished the translocation of protein kinase C (PKC) in response to ATP, which could also be achieved by the PKC inhibitors Gö-6976 (100 nM) or bisindolylmaleimide I (1 microM). In the presence of PKC inhibitors, however, the permeability effect of ATP was not affected. The presence of inhibitors of adenylate or guanylate cyclase (50 microM SQ-22536 or 20 microM ODQ) prevented changes in cyclic nucleotides but did not affect the permeability effects of ATP. The study shows that ATP reduces macromolecule permeability via a PLC-mediated mechanism that is independent of the concomitant effects of ATP on cytosolic Ca2+, cyclic nucleotides, or PKC.

Adenosine Triphosphate↗
PubMed1999

The protein phosphatase inhibitor cantharidin alters vascular endothelial cell permeability.

In this study, we characterized the effects of the protein phosphatases type 1 (PP 1) and type 2A (PP 2A) inhibitor cantharidin in endothelial cells. We identified catalytic subunits of PP 1alpha, PP 2Aalpha, and PP 2Abeta immunologically in bovine aortic endothelial cells. Moreover, we detected mRNAs coding for catalytic subunits of PP 1alpha, PP 1beta, and PP 2Aalpha by hybridization with specific DNA probes in total RNA from these cells. Okadaic acid and cantharidin inhibited the activities of catalytic subunits of PP 1 (okadaic acid, 0.01-1 microM; cantharidin, 1-100 microM) and PP 2A (okadaic acid, 0.1 nM to 1 microM; cantharidin, 0.1-100 microM) separated by column chromatography in a concentration-dependent manner. Moreover, cantharidin (1 microM to 1 mM) increased the phosphorylation state of endothelial proteins including the regulatory light chains of myosin without affecting cytosolic calcium concentrations. Cantharidin (5-100 microM) increased the permeability of cultured endothelial cells in a time- and concentration-dependent manner. We suggest that inhibition of PP 1 and PP 2A activities by cantharidin increases endothelial permeability by enhancing the phosphorylation state of endothelial regulatory proteins. Thus, cantharidin might be a useful tool to study the function of protein phosphatases in endothelial barrier function.

Animals↗
PubMed1998

Protection of rat cardiomyocytes against simulated ischemia and reoxygenation by treatment with protein kinase C activator.

The aim of this study was to investigate whether treatment with the protein kinase C (PKC) agonist 1,2-dioctanoyl-sn-glycerol (1,2DOG) can protect isolated adult Wistar rat cardiomyocytes against simulated ischemia and reoxygenation. Cytosolic Ca2+ (assessed by fura 2 fluorescence), pHi (assessed by BCECF fluorescence), and cell length were measured during 80 minutes of simulated ischemia (anoxia, pHo 6.4) and 20 minutes of reoxygenation (pHo 7.4) and compared between control cells and cells treated with 20 micromol/L 1,2DOG before anoxia (10-minute treatment and 10-minute washout), before and during anoxia (two-step treatment), or only during anoxia. Treatment before anoxia attenuated rigor contracture but did not influence anoxic Ca2+ overload. In contrast, two-step treatment before and during anoxia accelerated rigor contracture but reduced the rate of anoxic Ca2+ accumulation. During reoxygenation, control cells developed irreversible hypercontracture (reduction of cell length to 43+/-2% of the initial cell length, n=62), which was accompanied by spontaneous oscillations of cytosolic Ca2+ (19.6+/-1.6 per minute). Two-step treatment with 1,2DOG before and during anoxia significantly reduced hypercontracture (reduction of cell length to 60+/-2%, P<.01 versus control, n=41) and suppressed spontaneous Ca2+ oscillations (2.8+/-0.9 per minute, P<.01 versus control). These effects could not be reproduced by treatment with 1,2DOG before anoxia or during anoxia or by a two-step treatment with the PKC-inactive 1,3-dioctanoyl-sn-glycerol and were fully abolished with 1 micromol/L bisindolylmaleimide (PKC inhibitor). We conclude that a two-step activation of PKC before and during anoxia is required for effective protection of cardiomyocytes against anoxic Ca2+ overload and reoxygenation-induced hypercontracture.

Animals↗
PubMed1998

Wegener's granulomatosis: anti-proteinase 3 antibodies are potent inductors of human endothelial cell signaling and leakage response.

Anti-neutrophil cytoplasmic antibodies (ANCAs) targeting proteinase 3 (PR3) have a high specifity for Wegener's granulomatosis (WG), and their role in activating leukocytes is well appreciated. In this study, we investigated the influence of PR3-ANCA and murine monoclonal antibodies on human umbilical vascular endothelial cells (HUVECs). Priming of HUVECs with tumor necrosis factor alpha induced endothelial upregulation of PR3 message and surface expression of this antigen, as measured by Cyto-ELISA, with a maximum occurrence after 2 h. Primed cells responded to low concentrations of both antibodies (25 ng-2.5 microg/ml), but not to control immunoglobulins, with pronounced, dose-dependent phosphoinositide hydrolysis, as assessed by accumulation of inositol phosphates. The signaling response peaked after 20 min, in parallel with the appearance of marked prostacyclin and platelet-activating factor synthesis. The F(ab)2 fragment of ANCA was equally potent as ANCA itself. Disrupture of the endothelial F-actin content by botulinum C2 toxin to avoid antigen-antibody internalization did not affect the response. In addition to the metabolic events, anti-PR3 challenge, in the absence of plasma components, provoked delayed, dose-dependent increase in transendothelial protein leakage. We conclude that anti-PR3 antibodies are potent inductors of the preformed phosphoinositide hydrolysis-related signal tranduction pathway in human endothelial cells. Associated metabolic events and the loss of endothelial barrier properties suggest that anti-PR3-induced activation of endothelial cells may contribute to the pathogenetic sequelae of autoimmune vasculitis characterizing WG.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1998

Influence of pHi and creatine phosphate on alpha-adrenoceptor-mediated cardiac hypertrophy.

Stimulation of alpha-adrenoceptors on ventricular cardiomyocytes isolated from adult rat hearts leads to cellular alkalization, increases of creatine phosphate concentration, RNA mass, and protein synthesis. This study investigated whether the increase of creatine phosphate concentrations is causally linked to the hypertrophic response of cardiomyocytes under alpha-adrenoceptor stimulation. Cellular alkalization achieved with phenylephrine (10 microM), an alpha-adrenoceptor agonist, was abolished in the presence of the sodium-proton-exchange (NHE)-inhibitor HOE 694 (1 microM). HOE 694 inhibited also the alpha-adrenoceptor-mediated increase in cellular creatine phosphate and the increase in cellular RNA mass. The phenylephrine-induced stimulation of protein synthesis (determined by incorporation of 14C-phenylalanine) was reduced by one-third when HOE 694 was present. beta-Guanidinopropionic acid was added to cardiomyocytes to reduce cellular creatine phosphate concentrations. In these cultures, alpha-adrenoceptor stimulation activated NHE, but creatine phosphate concentrations were not increased. Protein synthesis was augmented to the same extent as in control cultures, but total RNA mass did not increase. From these results we conclude that alpha-adrenoceptor stimulation causes the increase in protein synthesis via activation of NHE, but independent of the concomitant increase in creatine phosphate contents. The effect of alpha-adrenoceptor stimulation on total RNA mass (translational capacity) is also caused by NHE activation, but depends on the changes in creatine phosphate contents as well.

Adrenergic alpha-Agonists↗
PubMed1998

Activation of p70(S6) kinase by beta-adrenoceptor agonists on adult cardiomyocytes.

Cardiac cellular hypertrophy plays an important role in cardiovascular diseases. Up until now, little has been known about the regulation of cellular growth on the level of intracellular signalling. Here, the implication of the p70(S6)-kinase (p70(S6K)) in the hypertrophic response after beta-adrenergic stimulation of cardiac myocytes from adult rats was investigated. Isoproterenol stimulation can activate p70(S6K) in adult cardiomyocytes analysed by direct kinase assays and retarded gel mobility. This signalling of beta-adrenoceptor stimulation is found only under conditions where the cardiomyocytes exhibit also a hypertrophic response to beta-adrenoceptor stimulation as measured by increase in protein content, RNA content and incorporation of radiolabelled amino acids. Rapamycin, a specific inhibitor of this kinase, reduces the trophic responses to control levels, suggesting an involvement of the p70(S6)-kinase in the development of cellular hypertrophy. An engagement of the MAP-kinase (ERK-1/2) pathway in the beta-adrenergic induced growth of cardiac myocytes from adult rats was excluded.

Adrenergic beta-Agonists↗
PubMed1998

Metabolic recovery of isolated adult rat cardiomyocytes after energy depletion: existence of an ATP threshold?

The question was investigated whether cardiomyocytes can be resuscitated after extreme energy depletion, i.e. after loss of ATP >70%. Isolated ventricular cardiomyocytes of the adult rat were exposed to metabolic inhibition with dinitrophenol and cyanide (DNP 0.2 mm; KCN 2 mm). After rapid energy depletion, cells were "reoxygenated" by wash-out of DNP and KCN. Intracellular calcium, cell length, ATP and creatine phosphate (CrP) of the cardiomyocytes were monitored. Metabolic inhibition resulted in a depletion of the stores of ATP and CrP by more than 95% of the normoxic values and caused a cytosolic Ca2+ overload. Parameters of metabolic recovery were: (i) resynthesis of CrP; (ii) recovery of a normal cytosolic Ca2+ control; and (iii) the elicitation of energy-dependent hypercontracture. "Reoxygenation", i.e. wash-out of metabolic inhibitors, reactivated oxidative phosphorylation. Consecutively, CrP levels recovered to 76.0+/-7.3%, ATP levels recovered to 10. 4+/-2.3% (means+/-s.dn=10) of the initial normoxic values, a normoxic intracellular calcium level was re-established and hypercontracture was elicited. Prolongation of metabolic inhibition with KCN (2 mm) or inhibition of the Na+/K+ pump with ouabain (0.5 mm) disabled the cardiomyocytes to recover from cytosolic Ca2+ overload and prevented hypercontracture. It is concluded that even after extensive energy depletion metabolic resuscitation of the myocardial cell remains possible and a critical range of ATP for recovery, i.e. a "threshold" of a 70% loss of ATP, does not exist.

Acidosis↗
PubMed1998

Protection of isolated cardiomyocytes against reoxygenation-induced hypercontracture by SIN-1C.

Previous studies have shown that SIN-1C (N-morpholinoiminoacetonitrile) can protect ischemic-reperfused myocardium. The aim of the present study was to analyse on the cellular level the mechanism by which SIN-1C may exert this effect. To simulate ischemia-reperfusion, isolated adult rat cardiomyocytes were incubated at pH 6.4 under anoxia and reoxygenated at pH 7.4 in presence or absence of SIN-1C. Reoxygenation was started when intracellular Ca2+ (measured with fura-2) had increased to > or = 10(-5) mol/L and pHi (BCECF) decreased to 6.6. Development of hypercontracture was determined microscopically. In the control group reoxygenation provoked oscillations of cytosolic Ca2+ (60.9 +/- 9.6 min-1 at 5 min of reoxygenation) accompanied by development of hypercontracture (to 77.2 +/- 3.8% of end-ischemic cell length). When SIN-1C was added upon reoxygenation, Ca2+ oscillations were markedly reduced (27.0 +/- 4.5 min-1, p < 0.001) and hypercontracture virtually abolished (90.6 +/- 2.0% of end-ischemic cell length, p < 0.001). SIN-1C did not influence the recovery of pHi during reoxygenation. The results indicate that SIN-1C protects cardiomyocytes against reoxygenation-induced hypercontracture by its ability to suppress oscillations of intracellular Ca2+ during the early phase of reoxygenation.

Acetonitriles↗
PubMed1998

Cardiovascular actions of parathyroid hormone and parathyroid hormone-related peptide.

Cardiovascular cells (cardiomyocytes and smooth muscle cells) are target cells for parathyroid hormone (PTH) and the structurally related peptide parathyroid hormone-related peptide (PTH-rP). PTH activates protein kinase C (PKC) of cardiomyocytes via a PKC activating domain previously identified on chondrocytes. Activation of PKC leads to hypertrophic growth and re-expression of fetal type proteins in cardiomyocytes. This hypertrophic effect of PTH might contribute to left ventricular hypertrophy in hemodialysis patients with secondary hyperparathyroidism. PTH-rP is expressed in cardiovascular cells (endothelial cells and smooth muscle cells). It does not mimic the above described actions of PTH but exerts effects of its own on cardiomyocytes. These effects involve activation of protein kinase A, via a N-terminal domain distinct from that identified on PTH, and activation of PKC, via a C-terminally located domain distinct from that found on PTH. On smooth muscle cells PTH and PTH-rP reduce the influence of extracellular calcium, through cAMP-dependent mechanisms. These inhibitory effects on voltage-dependent L-type calcium channels of smooth muscle cells cause vasorelaxation. Present studies concerning cardiovascular actions of either PTH and PTH-rP suggest that increased plasma levels of PTH and PTH-rP influence cardiomyocyte and smooth muscle cell physiology. It can be assumed that PTH-rP acts as a paracrine or autocrine modulator in heart and vessels.

Blood Pressure↗
PubMed1998

Role of phosphatidylinositol 3-kinase activation in the hypertrophic growth of adult ventricular cardiomyocytes.

OBJECTIVE: The present study investigated whether activation of phosphatidylinositol 3-kinase (PI3-kinase) is involved in the stimulation of hypertrophic growth of adult ventricular cardiomyocytes under alpha- or beta-adrenoceptor stimulation. METHODS: Adult ventricular rat cardiomyocytes were used either directly after isolation (day 1 culture) or after cultivation for 6 days in presence of 20% fetal calf serum (day 7 culture). PI3-kinase activity was determined in extracts of cardiomyocytes after immunoprecipitation with an antibody against the p85 subunit of PI3-kinase. The influence of PI3-kinase inhibition on myocardial growth was determined using the specific PI3-kinase inhibitors wortmannin and LY294002. RESULTS: In day 1 cultures alpha-adrenoceptor stimulation, but not beta-adrenoceptor stimulation caused activation of PI3-kinase. In response to alpha-adrenoceptor stimulation but not beta-adrenoceptor stimulation an acceleration of protein synthesis (incorporation of 14C-phenylalanine) and an increase in the total masses of cellular protein and RNA was observed. In these cultures inhibition of PI3-kinase attenuated the acceleration of protein synthesis and the increase in cellular masses of protein or RNA in response to alpha-adrenoceptor stimulation. In day 7 cultures alpha- and beta-adrenoceptor stimulation caused activation of PI3-kinase and increased protein synthesis. In these cultures inhibition of PI3-kinase attenuated the growth response to alpha- and beta-adrenoceptor stimulation. CONCLUSIONS: PI3-kinase activation via protein kinase C-dependent or cAMP-dependent pathways is required for hypertrophic growth of adult cardiomyocytes.

Adrenergic alpha-Agonists↗
PubMed1998

Intracellular signaling leads to the hypertrophic effect of neuropeptide Y.

Signal transduction pathways involved in the hypertrophic effect of neuropeptide Y (NPY) were investigated in adult cardiomyocytes. Reduction of transforming growth factor-beta activity in serum-supplemented media abolished the induction of hypertrophic responsiveness to NPY. In responsive cells, NPY (100 nM) increased protein synthesis, determined as incorporation of [14C]phenylalanine, by 35 +/- 15% (P < 0.05, n = 16 cultures). In these cells, NPY activated pertussis toxin (PTx)-sensitive G proteins and phosphatidylinositol (PI) 3-kinase. PTx and inhibition of PI 3-kinase abolished the hypertrophic effect of NPY. NPY also activated protein kinase C (PKC) and mitogen-activated protein (MAP) kinase. Inhibition of these two kinases attenuated the induction of creatine kinase (CK)-BB but not the growth response to NPY. In conclusion, NPY stimulates protein synthesis in adult cardiomyocytes via activation of PTx-sensitive G proteins and PI 3-kinase and it induces the fetal-type CK-BB via activation of PKC and MAP kinase.

Androstadienes↗