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Biomedical subjects

H M Piper

Publications and source records attributed to H M Piper.

At least 91 records · Page 5Linked to original sources
PubMed1995

Parathyroid hormone induces protein kinase C but not adenylate cyclase in adult cardiomyocytes and regulates cyclic AMP levels via protein kinase C-dependent phosphodiesterase activity.

Adult ventricular cardiomyocytes have been identified as target cells for parathyroid hormone (PTH) but little is known about its signal transduction in these cells. In the present study the influence of PTH on cyclic AMP accumulation and the activity of protein kinase C (PKC) in cardiomyocytes was evaluated. A mid-regional synthetic fragment of PTH, PTH-(28-48), which exerts a hypertrophic effect on cardiomyocytes, increased the activity of membrane-associated PKC in a dose-dependent manner (1-100 nM). Activated membranous PKC was dependent on Ca2+ and sensitive to an inhibitor of Ca(2+)-dependent isoforms of PKC. When adenylate cyclase was stimulated by the addition of isoprenaline, a beta-adrenoceptor agonist, PTH-(28-48) antagonized cyclic AMP accumulation. This antagonistic effect of PTH-(28-48) could be mimicked by activation of PKC with a phorbol ester and inhibited by isobutylmethylxanthine, a phosphodiesterase inhibitor. An N-terminal synthetic fragment, PTH-(1-34), which includes an adenylate cyclase-activating domain, did not stimulate the accumulation of cyclic AMP in cardiomyocytes. The results demonstrate that in adult cardiomyocytes PTH (1) is able to stimulate PKC, (2) is not able to cause accumulation of cyclic AMP and (3) functionally antagonizes the effect of beta-adrenoceptor stimulation to increase cellular cyclic AMP concentrations via PKC-dependent phosphodiesterase activity.

Adenylyl Cyclases↗
PubMed1995

Hypertrophic effects of calcitonin gene-related peptide (CGRP) and amylin on adult mammalian ventricular cardiomyocytes.

Calcitonin gene-related peptide (CGRP), a neuropeptide localized in the cardiac autonomic nervous supply, shares 46% similarity in sequence of amino acids with amylin, a peptide synthesized in pancreatic beta-cells. In the present study, the question was addressed whether these peptides could exert hypertrophic effects in cardiomyocytes isolated from the ventricles of adult rats and maintained in short-term, serum-free primary culture. FCS (10% v/v), employed as a positive control, increased the incorporation of l-[14C]phenylalanine into cellular protein, total content of cellular RNA and total mass of cellular protein significantly. CGRP and amylin also increased each of these parameters significantly and in a concentration-dependent manner; maximum responses occurred at 100 pM and 10 nM for CGRP and amylin, respectively. The selective antagonist at CGRP1-receptors, CGRP8-37(100 nM), inhibited significantly the incorporation of l-[14C] phenylalanine into cellular protein in response to CGRP and amylin. The selective inhibitor of protein kinase C (PKC), bisindolylmalemide (BIM) (5 microM), reduced significantly the incorporation of l-[14C] phenylalanine into cellular protein in response to phenylephrine (1 microM), employed as a positive control, but did not inhibit the response to insulin (1 unit/ml), employed as a negative control. BIM (5 microM) reduced significantly the responses to FCS (10% v/v), amylin (10 nM) and CGRP (10 pM), but did not inhibit the response to CGRP (100 pM). The activity of protein kinase C in membranes prepared from intact myocytes pre-treated for 10 min with the phorbol ester, phorbol 12-myristate 13-acetate (PMA) (100 nM), employed as a positive control, and CGRP (10 pM) was significantly greater than in membranes prepared from cardiomyocytes not subjected to agonist stimulation. Phenylephrine (1 microM) increased significantly the specific activity of creatine kinase but not of lactate dehydrogenase in day 1 cultures of freshly isolated cardiomyocytes. Significant induction of creatine kinase, but not lactate dehydrogenase, was also stimulated by CGRP and amylin; the maximum responses occurred at 100 pM and 100 nM CGRP and amylin, respectively. In conclusion, CGRP and amylin exert hypertrophic effects directly on ventricular cardiomyocytes from the hearts of adult rats in vitro. These effects are: (1) due to de novo protein synthesis since total content of cellular RNA and incorporation of l-[14C]phenylalanine into cellular protein were also increased; (2) mediated by a common population of CGRP1-preferring receptors at which amylin binds with lower potency: (3) mediated, at least partly, by the activation of PKC; (4) may be associated with a fetal shift in gene expression, characterized by selective induction of creatine kinase.

Age Factors↗
PubMed1995

Intracoronary SIN-1C during reperfusion reduces infarct size in dog.

Reperfusion of ischemic myocardium may aggravate the ischemic state of injury and thus augment infarct size (reperfusion injury). The aim of this study was to reduce infarct size by an intervention at the time of reperfusion that acts only on a reperfusion-specific pathomechanism. It was investigated whether SIN-1C, a metabolite of molsidomine, can protect against reperfusion injury in canine hearts in vivo. Ten anesthetized open chest dogs underwent 1 h of left anterior descendent artery (LAD) occlusion and were randomly assigned to receive either intracoronary SIN-1C or vehicle infusion as a placebo during the first hour of reperfusion. The infusion was adjusted to LAD flow to achieve a regional blood concentration of 5 x 10(-3) M. Infarct size was assessed by triphenyltetrazolium staining after 6 h of reperfusion. Left ventricular pressure (LVP) was similar in both groups (SIN-1C: 101 +/- 6, placebo: 89 +/- 6 mm Hg, mean +/- SEM, n = 5) at the beginning of the experiment and did not change significantly thereafter from baseline values in both groups. During SIN-1C infusion, the LAD flow was increased (SIN-1C: 195 +/- 38, control: 86 +/- 17 ml/min/100 g at 30 min of reperfusion, p < 0.05), while systemic hemodynamics remained unaltered. A reduction in infarct size (percent of area at risk) was seen in the SIN-1C group (11.4 +/- 2.8%) compared with the placebo group (24.4 +/- 3.9%, p < 0.05). Infusion of papaverin (5 x 10(-5) M) following an identical protocol caused a similar vasodilation as SIN-IC, but did not reduce infarct size in five additional dox experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetonitriles↗
PubMed1995

Induction of hypertrophic responsiveness to isoproterenol by TGF-beta in adult rat cardiomyocytes.

In a previous publication we reported that hypertrophic responsiveness to beta-adrenoceptor stimulation can be induced in isolated cardiomyocytes when these are cultured for 6 days in presence of fetal calf serum (FCS; Pinson et al., J. Mol. Cell. Cardiol.. 25: 477-490, 1993). The role of transforming growth factor-beta (TGF-beta) in this induction process has now been investigated. Isolated cardiomyocytes from adult rats were cultured for 6 days in presence of 20% FCS. It was found that induction of hypertrophic responsiveness to beta-adrenoceptor stimulation was abolished when a neutralizing anti-TGF-beta 1 antibody was added to FCS-containing culture medium. In culture media with FCS contents (5%) too low to induce hypertrophic responsiveness to beta-adrenoceptor stimulation, addition of 1 ng/ml TGF-beta 1,2 induces this responsiveness. It was demonstrated that cardiomyocytes already release TGF-beta into culture media on day 1 of culture and that they continue to do so in presence of FCS supplements of > 5%. The results demonstrate that hypertrophic responsiveness to beta-adrenoceptor stimulation is induced in cardiomyocytes by an autocrine mechanism involving TGF-beta 1 as mediator.

Adrenergic beta-Agonists↗
PubMed1995

Regulation of protein synthesis and degradation in adult ventricular cardiomyocytes.

For studies on the regulation of myocardial protein metabolism, isolated adult cardiomyocytes were introduced as an experimental model about a decade ago. When used shortly after isolation, this model represents a tool for studying the properties of normal and diseased myocardium on the cellular level. The influence of various peptide hormones, neurotransmitters, and mechanical stimulation on protein synthesis and degradation in isolated cardiomyocytes has been studied. It has been demonstrated, for example, that alpha 1-adrenoceptor stimulation increases protein synthesis in newly isolated cardiomyocytes, independently of any mechanical effects. Other potential growth stimuli require appropriate conditions to induce cellular responsiveness. Neuropeptide Y, for example, does not stimulate cellular protein synthesis in newly isolated cells, whereas it does so in cells that have been cultured for a week in the presence of serum. Mechanical stretch also represents a growth stimulant. It seems that its signal transduction involves an autocrine loop. Thus different mechanisms, by which exogenous influences can modify cellular protein synthesis and degradation, have been identified on the cellular level, with the use of isolated adult cardiomyocytes.

Adrenergic alpha-Agonists↗
PubMed1995

Initiation of hyperpermeability in energy-depleted coronary endothelial monolayers.

How the initiation of energy depletion affects macromolecule permeability of a barrier of coronary endothelial cells was investigated. Cultured monolayers of adult rat coronary endothelial cells were exposed to 5 mM KCN and 5 mM 2-deoxy-D-glucose (2-DG). Transendothelial flux of albumin, cellular ATP content, and cytosolic Ca2+ concentration were monitored. Within the first minute, a merely partial loss (28%) of ATP reserves provoked a distinct increase (41%) in albumin flux. Rise of permeability was dependent on Ca2+ release from a thapsigargin- and ATP-sensitive endogenous store, and hyperpermeability was greatly attenuated when energy depletion was extremely rapid, as under sequential addition of 20 mM 2-DG and 5 mM KCN. Attenuation of hyperpermeability could also be achieved by use of 5-20 mM 2,3-butanedione monoxime, an inhibitor of actin-myosin interaction. This finding, together with dependence on Ca2+ and availability of residual energy, indicates that the rapid initiation of hyperpermeability is caused by a contractile mechanism.

Adenosine Triphosphate↗
PubMed1995

Protection of reoxygenated cardiomyocytes against hypercontracture by inhibition of Na+/H+ exchange.

Effects of Na+/H+ exchange inhibition and cytosolic acidosis on reoxygenated adult rat ventricular cardiomyocytes were investigated. Cells were incubated in anoxic media at pH 6.4 until pCa of < or = 5, intracellular pH (pHi) of 6.5, and cytosolic [Na+] of 50 mM were reached. On reoxygenation, medium pH was changed to 7.4 to activate Na+/H+ exchange. In one group, 20 microM HOE-694, an inhibitor of Na+/H+ exchange, was added. With or without HOE-694, cytosolic Ca2+ and Na+ returned to control levels within 10 min of reoxygenation. In the absence of HOE-694, the pHi renormalized (to 7.2) within 8 min, but irreversible hypercontracture and transient Ca2+ oscillations were observed. In the presence of HOE-694, pHi stayed acidotic (at 6.5), hypercontracture was prevented, and Ca2+ oscillations were attenuated. When the Na+ pump was inhibited with 0.1 mM ouabain, even partial recovery of Ca2+ control became impossible unless HOE-694 was added. Our conclusions are 1) activation of Na+/H+ exchange does not impair recovery of cytosolic Na+ and Ca2+ control unless activity of the sarcolemmal Na+ pump is critically reduced, and 2) due to prolongation of cytosolic acidosis, inhibition of Na+/H+ exchange protects against reoxygenation-induced hypercontracture and cytosolic Ca2+ oscillations.

Animals↗
PubMed1994

Endothelial cell toxicity of preservation solutions: comparison of endothelial cells of different origin and dependence on growth state.

Previously, we have shown that cultured liver endothelial cells are affected by an energy-dependent injury when incubated in cold University of Wisconsin (UW) or histidine-tryptophan-ketoglutarate solution. Here, we studied the susceptibility of other endothelial cells to this type of injury. Aortic endothelial cells in early-confluent, i.e., still proliferating, monolayer cultures were damaged more quickly during cold incubation in UW solution than during cold incubation in Krebs-Henseleit buffer. At this stage the addition of KCN did not alter the loss of viability in UW solution, but when the culture period was prolonged, cells were protected by the addition of cyanide. A paradoxical, protective effect of KCN could also be observed in late-confluent, i.e., nonproliferating, cultures of coronary endothelial cells incubated in UW solution. Similarly, liver endothelial cells in subconfluent, growing cultures were damaged by the addition of cyanide (loss of viability after 48 h, 3 +/- 1% in UW, 65 +/- 19% in UW + KCN), whereas in late-confluent cultures the addition of cyanide to UW solution was protective (loss of viability after 48 h, 100 +/- 0% in UW, 31 +/- 15% in UW + KCN). Variations of culture period and seeding density and the use of inhibitors of cell proliferation demonstrated that liver endothelial cells acquire their susceptibility to energy-dependent injury along with confluence. Subcultured cells retained this susceptibility for some hours. These results suggest that the energy-dependent injury described previously is not confined to liver endothelial cells and that the occurrence of energy-dependent injury requires a capacity of the cells that develops only after cultures have grown to confluence.

Adenosine↗
PubMed1994

Regional contractile blockade at the onset of reperfusion reduces infarct size in the dog heart.

An important mechanism of lethal myocardial reperfusion injury is the development of cellular hypercontracture at the onset of reperfusion. Hypercontracture can lead to cytolysis by mutual mechanical disruption of myocardial cells. 2,3-Butanedione monoxime (BDM) inhibits myofibrillar cross-bridge cycling and may therefore reduce infarct size in ischaemic reperfused myocardium. This study investigated whether a temporary presence of BDM protects against myocardial reperfusion injury in an intact-animal preparation. Anaesthetized open-chest dogs (n = 10) underwent 1 h of left anterior descendent artery (LAD) occlusion and received intracoronary BDM (25 mM, n = 5) or vehicle (n = 5) for 65 min starting with an anoxic local infusion 5 min before reperfusion. Infarct size was assessed by triphenyltetrazolium staining after 6 h reperfusion. The infusion of BDM was accompanied by a transient reduction of left ventricular systolic pressure from 84.3 +/- 11.2 mm Hg during occlusion to 66.4 +/- 9.9 mm Hg at 30 min reperfusion (mean +/- SD, P < 0.01 vs. control). LAD-flow and regional wall motion in the area at risk showed no difference between groups. Infarct size (% of area at risk) was reduced from 24.4 +/- 8.7 (control) to 6.6 +/- 2.0% (BDM) (P < 0.01). The results demonstrate that development of necrosis in reperfused myocardium can be greatly reduced by temporary presence of the contractile inhibitor BDM at the onset of reperfusion.

Animals↗
PubMed1994

Neuropeptide Y stimulates hypertrophy of adult ventricular cardiomyocytes.

It was investigated whether neuropeptide Y (NPY) could exert a trophic effect on ventricular myocytes isolated from the adult rat heart. Two different culture models were used: day 1 and 7 cultures of cardiomyocytes. In day 1 and 7 cultures, NPY caused an increase in cellular protein mass. In day 1 cultures, NPY (10 nM) increased the protein-to-DNA ratio within 24 h by 10.1 +/- 2.8% (P < 0.01), but did not stimulate the incorporation of [14C]phenylalanine into cell proteins. The degradation of proteins was retarded in presence of NPY, revealed by pulse-chase experiments. In day 7 cultures, NPY (10 nM) increased the protein-to-DNA ratio within 24 h by 33.9 +/- 5.0% (P < 0.01), increased the RNA-to-DNA ratio by 19.2 +/- 6.4%, and stimulated the incorporation of [14C]phenylalanine by 45.5 +/- 4.5% (P < 0.01). As in day 1 cultures, protein degradation was retarded. The specific activities of cytosolic creatine kinase and lactate dehydrogenase were increased in presence of NPY. This study demonstrates for the first time that NPY is a trophic factor for cardiomyocytes. NPY can cause an increase in cellular mass of protein, i.e., hypertrophy, by two mechanisms: 1) reduction of degradation of protein, found in day 1 and 7 cultures, and 2) stimulation of protein synthesis, observed only in day 7 cultures. The responsiveness of protein synthesis to NPY stimulation is induced during prolonged incubation in culture.

Animals↗
PubMed1994

Importance of sodium for recovery of calcium control in reoxygenated cardiomyocytes.

The role of Na+ in the recovery from severe anoxic Ca2+ overload was investigated in isolated quiescent ventricular cardiomyocytes from adult rat. Changes of cytosolic Ca2+ and Na+ concentrations were followed by the fura 2 and Na(+)-binding benzofuran isophthalate techniques, respectively. When the fura 2 ratio (340/380 nm) reached saturation in anoxic cells, indicating a severe cytosolic Ca2+ overload, the cells were reoxygenated. This caused a rapid initial drop of cytosolic Ca2+ to a lower but still elevated level (phase I), followed by oscillatory Ca2+ transients at this level (phase II) and, within 10 min, the reestablishment of a stable cytosolic Ca2+ concentration at the normal resting level (phase III). As previously shown [B. Siegmund, R. Zude, and H. M. Piper. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1262-H1269, 1992], Ca2+ shifts in phase I and II are mainly due to uptake and release of Ca2+ by the sarcoplasmic reticulum. Phase I was unchanged, and phase II was much prolonged (> 60 min) in cells reoxygenated under Na+ pump inhibition (0.2 mM ouabain) or Na+ depletion. Phase III could only be reestablished (< 10 min) when ouabain was eluted or external Na+ replenished, respectively. The results show that full recovery of cytosolic Ca2+ control (phase III) requires an active sarcolemmal Na+ pump and the availability of external Na+. This indicates that phase III is determined by the transsarcolemmal extrusion of Ca2+ by a tandem mechanism consisting of 1) the Na+ pump, generating an extracellular-to-intracellular Na+ gradient, and 2) the sarcolemmal Na+/Ca2+ exchange, driven by that gradient to extrude Ca2+.

Animals↗
PubMed1994

NO donor SIN-1 protects against reoxygenation-induced cardiomyocyte injury by a dual action.

It was investigated whether morpholinosydnonimine (SIN-1), which spontaneously decomposes into NO and 3-morpholinoiminoacetonitrile (SIN-1C), can be used for protection of cardiomyocytes against reoxygenation-induced hypercontracture. Isolated ventricular cardiomyocytes (from adult rats) were used as the experimental model. SIN-1 [concentration with half-maximal effect (EC50) 2.5 x 10(-4) M] and SIN-1C (EC50 8.3 x 10(-3) M) inhibited the contractile response of electrically paced cardiomyocytes. When the cells were submitted to substrate-free anoxia (135 min) and subsequent reoxygenation (30 min), the onset of reoxygenation provoked their hypercontracture. It was studied whether the temporary presence of the test agents during the last 15 min of anoxia and the first 15 min of reoxygenation prevented hypercontracture. At 10 nM, SIN-1 prevented hypercontracture in 96% of the cells and SIN-1C in 72% of the cells. The protective effect of SIN-1 was reduced to that of SIN-1C by simultaneous presence of methylene blue (50 microM). Methylene blue had no influence on the protective action of SIN-1C. SIN-1C (10 mM) plus sodium nitroprusside (another NO donor, 250 microM) provided the same degree of protection as SIN-1 (10 mM). The results show that reoxygenation-induced hypercontracture can be prevented or attenuated by the temporary presence of high concentrations of SIN-1 or SIN-1C. SIN-1 acts through a dual mechanism, protecting through the generation of NO and SIN-1C.

Adenosine Diphosphate↗
PubMed1993

Alpha- and beta-adrenergic stimulation of protein synthesis in cultured adult ventricular cardiomyocytes.

The effect of the alpha 1-adrenoceptor agonist phenylephrine (PE, 1-10 microM) and the beta-adrenoceptor agonist isoprenaline (ISO, 1-10 microM) on protein synthesis and ultrastructure of ventricular cardiomyocytes from adult rat in culture (6 days in medium 199 plus 20% fetal calf serum) was studied. In these cultures cardiomyocytes were spread, but not spontaneously contractile. ISO and PE significantly increased total cell protein and incorporation of (14C)-phenylalanine within 24 h of exposure. These effects were inhibited by the antagonists propranolol and prazosin, respectively. The incorporation of (14C)-uridine was stimulated only by PE but not ISO. Induction of fetal BB-isoform of cytosolic creatine kinase was also caused only by PE but not ISO. The ultrastructure of PE-treated cardiomyocytes was altered as compared to controls, by a greater number of Golgi complexes, denser myofibrillar structures and the appearance of paracrystalline bands in mitochondrial matrices. In conclusion, in this culture model the protein synthesis of cardiomyocytes can be stimulated, independently of the contractility, by either alpha 1- or beta-adrenoceptor agonists. Catecholamines differ, however, in their effects on specific cellular proteins and structures. Only alpha 1-adrenergic stimulation leads to a "fetal shift" in the expression of CK-isoforms.

Animals↗
PubMed1993

Calcium and the oxygen paradox.

When myocardial cells are reoxygenated after a prolonged period of energy depletion, they rapidly hypercontract. In tissue, hypercontracture induced by reoxygenation is accompanied by cytolysis ("oxygen paradox"). Recent studies have indicated that severe cytosolic Ca2+ overload and reactivation of energy production represent the causal key factors for the deleterious hypercontracture, through the following mechanism: prolonged energy depletion leads to a progressive cytosolic Ca2+ overload in cardiomyocytes; when oxidative phosphorylation is then resumed with the resupply of oxygen, activation of the myofibrils at (still) increased cytosolic Ca2+ concentrations provokes a sustained maximal force development and consecutive mechanical cell injury. This injury can largely be prevented when the contractile machinery is inhibited during the initial phase of reoxygenation. In the model of isolated cells it has been shown that a normal cytosolic Ca2+ control can be reestablished upon reoxygenation. This seems to explain why contractile blockade is needed only temporarily for the prevention of reoxygenation induced hypercontracture and cellular deterioration. Temporary contractile blockade at the onset of reperfusion has also been shown to protect the heart in vivo against lethal reperfusion injury.

Animals↗
PubMed1993

Disintegration of cytoskeletal structure of actin filaments in energy-depleted endothelial cells.

In a previous study [Watanabe, H., W. Kuhne, R. Spahr, P. Schwartz, and H. M. Piper. Am. J. Physiol. 260 (Heart Circ. Physiol. 29): H1344-H1352, 1991] metabolic inhibition (5 mM KCN + 5 mM 2-deoxy-D-glucose, for 2 h) was found to cause disintegration of F-actin filaments, cell retraction, and augmented paracellular macromolecule permeability in monolayer cultures of porcine aortic endothelial cells after a rapid depletion of ATP stores (90% in 5 min). These changes were reversible. In the present study, the nature of this cytoskeletal disintegration was investigated. 1) Disintegration of F-actin filaments within 2-h incubation under metabolic inhibition was accompanied by appearance of F-actin clumps in the cells, but total contents of F-actin remained unaltered. 2) Cytosolic Ca2+ levels rapidly rose in metabolically inhibited cells; after 2 h a 10-fold increase was observed. 3) Presence of the Ca2+ ionophore A23187 (10 microM) mimicked the reversible effect of metabolic inhibition on F-actin filaments and monolayer permeability but not the extensive depletion of ATP stores. 4) Existence of the Ca(2+)-activatable actin-severing protein gelsolin in endothelial cells was demonstrated. The results show that during the reversible phase of endothelial energy depletion disintegration of F-actin filaments is only partial, since it is based on their fragmentation and not depolymerization. Increase in cytosolic Ca2+ levels seems to be the primary cause for the fragmentation, possibly through the activation of gelsolin.

Actins↗