Biomedical subjects
Publications and source records attributed to H M Piper.
The mechanism of action of NPY to produce contractions of adult rat ventricular myocytes was investigated. Positive effects were mediated through an L-type Ca2+ channel. Negative effects, linked through an inhibitory G-protein to a transient outward K+ channel, could be antagonised by NPY-(18-36).
The question was investigated whether mild acidosis can provide energetic protection on the hypoxic myocardial cell by mechanisms other than negative inotropy. The experimental model used was quiescent ventricular cardiomyocytes isolated from the adult rat. The extracellular pH (pHe) in modified hypoxic Tyrode's solution was varied by changing the concentration of bicarbonate at constant PCO2. The intracellular pH (pHi) was determined with 2',7'-bis-(carboxyethyl)5-(6')-carboxyfluorescein. When the pHe was varied between 6.6 and 7.4, the pHi varied only between 6.91 and 7.14. At pH 7.0, pHi and pHe were identical. Around pHe 7.0, the rate of hypoxic energy loss was lowest. Energetic differences were most prominent after 90 min hypoxia. At this time loss of ATP was 57.1 +/- 2.9% at pHe 6.95 vs. 83.5 +/- 4.1% at pHe 7.4 and 73.9 +/- 2.8% at pHe 6.6; the free energy change of ATP hydrolysis was reduced from 58.8 +/- 0.4 kJ/mol under control conditions to 52.0 +/- 1.1 kJ/mol at pHe 6.95 vs. 45.8 +/- 1.2 and 47.3 +/- 1.4 kJ/mol at pHe 7.4 and 6.6, respectively (means +/- S.D.). Anaerobic lactate production was not significantly altered in the investigated pHe range, indicating that glycolytic energy production remained constant. The results of the present study demonstrate that mild acidosis (pHe around 7.0) has a prominent energy-saving effect on the hypoxic cardiomyocyte due to a reduction in the demand of energy.
It has been hypothesized that, in oxygen-depleted myocardial cells, mitochondria are depolarized and the F1,F0-proton adenosinetriphosphatase (ATPase) catalyzes net ATP hydrolysis when the cells exhibit the signs of an aerobic-anaerobic metabolic transition, which are increased lactate formation and decline in high-energy phosphate reserves [W. Rouslin, C. W. Broge, and I. L. Grupp. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1759-H1766, 1990]. This hypothesis was tested by incubating isolated cardiomyocytes from the adult rat in substrate-free Tyrode solution (37 degrees C, pH 7.4) at a PO2 less than or equal to 0.1 Torr, i.e., 1,000-fold below the normal arterial level. At this deep hypoxia, the following results were found. 1) Lactate production was activated to maximal rates and high-energy phosphate contents decreased (aerobic-anaerobic metabolic transition). The inhibitor of the mitochondrial F1,F0-proton ATPase oligomycin, however, added upon establishment of hypoxia, did not slow down, as in the case of depolarized mitochondria, but moderately accelerated energy depletion. 2) Activation of mitochondrial ATP hydrolysis could be provoked in these hypoxic cells by addition of cyanide, antimycin A, and rotenone, i.e., specific inhibitors of certain sites of the respiratory chain. The enhancement of loss of ATP could be inhibited by oligomycin. The results demonstrate that states of deep hypoxia of the cardiomyocyte are possible in which it undergoes an aerobic-anaerobic metabolic transition, indicated by increased lactate formation and progressive loss of cellular energy reserves, and yet mitochondrial ATPase hydrolytic activity is not activated.
The effect of the A2-adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) on macromolecule permeability (PM; indicator fluorescein isothiocyanate-labeled albumin) of endothelial cells was investigated using confluent monolayers of rat coronary microvascular endothelial cells (CEC) and porcine aortic macrovascular endothelial cells (AEC). In CEC, NECA (10(-7) M) increased PM by 39%. Similar results were obtained by isoproterenol (10(-6) M) and forskolin (10(-5) M). The effect of NECA could be antagonized by 8-phenyltheophylline (8-PT; 10(-5) M). In AEC, NECA (10(-7) M) caused an opposite effect in that it decreased PM by 26% as did isoproterenol (10(-6) M) and forskolin (10(-5) M). The response to NECA was abolished in the presence of 8-PT (10(-5) M). In AEC but not CEC, NECA could reduce the rise in PM caused by endothelial energy depletion (in the presence of 5 mM KCN and 5 mM 2-deoxy-D-glucose). It was common to AEC and CEC that NECA (10(-7) M), isoproterenol (10(-6) M), and forskolin (10(-5) M) stimulated production of adenosine 3',5'-cyclic monophosphate (cAMP). The stimulatory effect of NECA on production of cAMP could be antagonized by 8-PT (10(-5) M). In summary, the results indicate that in AEC and CEC PM is modulated by an A2-adenosine receptor-mediated stimulation of adenylate cyclase. The secondary effects of stimulation of adenylate cyclase are different in CEC and AEC, however, since it caused a reduction of PM in AEC, but an increase in CEC.
The ability of hypoxic-reoxygenated cardiomyocytes to recover from severe cytosolic Ca2+ overload was investigated using the fluorescent Ca2+ indicator fura-2 in ventricular cardiomyocytes from adult rats. When the fura-2 ratio (340/380 nm) reached saturation in hypoxic cardiomyocytes, indicating severe Ca2+ overload, they were reoxygenated. The cell then suddenly hypercontracted but reestablished, after a phase of Ca2+ oscillations, a normal Ca2+ control. Because these oscillations could be abolished by ryanodine (50 nM), they seem to depend on the function of the sarcoplasmic reticulum (SR). In the presence of caffeine (5 mM) and thapsigargin (100 nM), i.e., agents impairing Ca2+ sequestration in the SR, reoxygenation did not lead to Ca2+ oscillations or to a stable recovery of cytosolic Ca2+ control. The additional presence of ruthenium red (5 microM), an inhibitor of mitochondrial Ca2+ uptake, restored the ability of cells treated with caffeine or thapsigargin to reestablish a normal cytosolic Ca2+ control. The results show that cardiomyocytes are able to recover from severe hypoxic Ca2+ overload if, first, a closed sarcolemma is retained (as in isolated cardiomyocytes) and, second, the SR is available for rapid Ca2+ storage (impaired by caffeine and thapsigargin). The results also suggest that, in the case of an impairment of SR function, the inhibition of mitochondrial Ca2+ uptake (as by ruthenium red) has a protective effect.
Trophic effects of isoproterenol (Iso), norepinephrine (NE), phenylephrine (PE), and biologically active fragments of parathyroid hormone (PTH), PTH-(1-34) and PTH-(28-48), were investigated in mechanically quiescent, isolated ventricular cardiomyocytes from adult rat. In 24-h incubations in modified serum-free medium 199 incorporation of [14C]phenylalanine, changes in total protein and specific activities of cytosolic enzymes, creatine kinase (CK) and lactate dehydrogenase (LDH) were monitored. NE and PE (10 microM), but not Iso, distinctly increased phenylalanine incorporation, total cell protein, and specific activity of CK but not LDH. Induction of CK, but not LDH, was also produced by phorbol 12-myristate 13-acetate (10 nM) but not dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 1 mM). It was abolished by copresence of cycloheximide (35 microM) or actinomycin D (5 microM). CK-BB was the only induced isoform of CK, as shown for PE incubations. PTH-(1-34) and PTH-(28-48) (30-300 nM) had effects comparable to NE and PE. They increased phenylalanine incorporation and total protein content and induced CK but not LDH. In summary, distinct trophic effects on adult cardiomyocytes were found with alpha 1-adrenergic agonists, fragments of PTH containing the midregional amino acids 28-34, and direct activation of protein kinase C but neither beta-adrenergic agonists nor DBcAMP.
It was investigated how cytosolic Ca2+ overload affects the cytoskeletal structure and macromolecule permeability (for albumin) of monolayers of endothelial cells (from porcine aorta). States of cytosolic Ca2+ overload were produced either 1. by metabolic inhibition (5 mM KCN plus 5 mM 2-deoxyglucose) or 2. by increasing membrane permeability with the use of a Ca2+ ionophore (10 microM A 23187). The effects of cytosolic Ca2+ overload on the structure of F-actin filaments and monolayer permeability were monitored. ATP stores were rapidly degraded (> 90% in 15 minutes) in the presence of metabolic inhibitors, but only partially reduced in the presence of A 23187 (30%) in two hours). Concomitantly with ATP loss, cytosolic Ca2+ levels were increased in metabolically inhibited cells. Two-hour exposure to the Ca2+ ionophore A 23187 mimicked the effect of two-hour metabolic inhibition on F-actin filaments and monolayer permeability, in spite of the divergence in energy metabolism. Disintegration of F-actin filaments in presence of metabolic blockers or ionophore was accompanied by appearance of F-actin clumps in the cells, but total contents of F-actin remained unaltered. Within three hours after removal of these agents, a normal F-actin structure and normal macromolecule permeability were re-established in the monolayers. The results show that cytosolic Ca2+ overload causes disintegration of F-actin filaments and a subsequent increase in macromolecule permeability. These changes are readily reversible as long as the dis-integration is based on fragmentation and not depolymerization of F-actin filaments.
It was investigated in hypoxic-reoxygenated cardiomyocytes and isolated perfused hearts from rat whether temporary contractile blockade by 2,3-butanedionemonoxime (20 mM: BDM) during the initial phase of reoxygenation could prevent severe reoxygenation-induced cell injury. In isolated rat cardiomyocytes, reoxygenation after 120 minutes substrate-free anoxia caused sudden hypercontracture but not cytolysis. Within 15 minutes, a nearly normal free energy change of ATP hydrolysis and a normal cytosolic Ca2+ control were reestablished, in spite of irreversible hypercontracture. When BDM was present during the initial 15 minutes reoxygenation and then eluted, hypercontracture remained absent. In the isolated perfused heart, reoxygenation after 60 minutes substrate-free hypoxic perfusion provoked rapid hypercontracture and a sudden massive loss of creatine kinase ("oxygen paradox"). When BDM was present for the first 60 minutes reoxygenation and then eluted, these characteristics of the "oxygen paradox" remained virtually absent. The results demonstrate that the reoxygenation-induced hypercontracture and severe cell injury characteristic for the "oxygen paradox" can be prevented in the hypoxic-reoxygenated heart muscle cell when the contractile apparatus is temporarily paralyzed during the initial phase of reoxygenation. This time seems to be needed for the recovery of cytosolic Ca2+ control.
The requirements and influence of thiols on the production of nitric oxide (NO) were examined in cultured porcine aortic endothelial cells. NO production was diminished when cells were pretreated with thiol-depleting agents (IC50: N-ethylmaleimide, 30 microM; 1-chloro-2,4-dinitrobenzene, 200 microM; diamide, 1.5 mM; diethyl maleate, 20 mM). The depletion of glutathione (45-99% loss at the various IC50 values) and protein thiols (3-25% loss at IC50) showed no consistent relationship to decreased NO production. The effects of the agents on NO production were not linked to altered sensitivity to the stimulant (calcium ionophore A23187; maximal effect at 10 microM), but roughly paralleled the appearance of cell damage (17-44% lactate dehydrogenase release at IC50). The decrease in NO production due to 1-chloro-2,4-dinitrobenzene was partially reversed by cysteine, dithioerythritol, and dihydrolipoate, whereas cystine partially reversed the decrease due to diamide or diethyl maleate. On the other hand, several thiols diminished NO production in control cells. Overall, alterations of NO production did not parallel the depletion or replenishment of either glutathione, protein thiol, or soluble thiol pools, and so the results argue against hypotheses that cellular thiols are either substrates or necessary cofactors in the pathway of NO synthesis in endothelial cells.
The study had two aims: first, to improve the longevity of isolated adult cardiomyocytes in serum-free culture, and, second, to investigate whether catecholamines which promote hypertrophy in vivo can prolong survival of isolated adult rat cardiomyocytes in serum-free culture. The basic cell culture medium consists of serum-free medium 199 with 10(-7) M insulin. In this medium 50% of the initially plated cardiomyocytes survive in elongated form for 2 days. Omission of glutamine and supplementation of the basic medium with 5 mM creatine, 2 mM carnitine and 5 mM taurine extends survival of elongated cells to 14 days. In supplemented medium, normal cell ATP content is maintained (27 nmol/mg protein after 15 days), but cells gradually atrophy and reduce their protein mass. The trophic effects of catecholamines (epinephrine, norepinephrine, phenylephrine; 10 microM, added on day 3 of culture) were investigated. After addition of catecholamines the cells spread. Spreading can be prevented by prazosin (10 microM) and phentolamine (10 microM) but not by propranolol (10 microM), indicating that spreading is stimulated via the alpha 1-adrenoreceptor. Cells also spread in the presence of the phorbol ester phorbol myristate acetate (10 microM). Catecholamines reduce the progressive cell atrophy and protein loss. With 10 microM phenylephrine, cellular ATP content remained constant at 27 nmol/mg protein until day 15. The results indicate that agents which stimulate protein kinase C (alpha 1-agonists, phorbol esters) stimulate cell spreading, protein synthesis and long-term survival of cardiomyocytes in vitro.
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Reoxygenation after 120-min substrate-free anoxia causes sudden hypercontracture in isolated rat cardiomyocytes. Reoxygenated-hypercontracted cardiomyocytes maintain their sarcolemmal integrity as indicated by the absence of enzyme release and reestablish a nearly normal free energy change of ATP hydrolysis within 15 min [Siegmund, B., A. Koop, T. Klietz, P. Schwartz, and H. M. Piper.Am J. Physiol. 258 (Heart Circ. Physiol. 27): H285-H291, 1990]. In the same model, it was now investigated whether a temporary contractile blockade by 20 mM 2,3-butanedione monoxime (BDM) can prevent reoxygenation-induced hypercontracture. When BDM was present during 120-min anoxia and the subsequent 15-min reoxygenation, hypercontracture could be prevented. The anoxic changes of high-energy phosphate contents, the free energy change of ATP hydrolysis, and the ultrastructure of the cells remained unaffected by the presence of BDM. When BDM was applied anoxically immediately before reoxygenation, it also prevented hypercontracture. Contracture still remained absent when BDM was washed out after the first 15 min of reoxygenation. These results demonstrate that a temporary contractile blockade (15 min) at the onset of reoxygenation prevents hypercontracture in anoxic-reoxygenated cardiomyocytes. This result, the energetic recovery, and the sarcolemmal integrity of cardiomyocytes in anoxia-reoxygenation demonstrate that reoxygenation-induced hypercontracture is not based on an already irreversible cell damage.
The dependence of macromolecule permeability (MP; indicator fluorescein isothiocyanate-labeled albumin) of endothelial cells on their energetic state was investigated using confluent monolayers of rat coronary microvascular endothelial cells and porcine aortic macrovascular endothelial cells. When oxidative and glycolytic energy productions were inhibited (5 mM KCN plus 5 mM 2-deoxy-D-glucose) 90% of the endothelial ATP contents were lost within 15 min, followed by a progressive increase of MP, disintegration of the actin cytoskeleton, and the opening of intercellular gaps. Elution of the blocker and a subsequent 3-h incubation in complete culture medium reversed the effects of 2-h metabolic blockade, completely for MP and partially for ATP levels. Coronary microvascular and aortic macrovascular endothelial cells responded similarly to energy depletion and repletion, the microvascular cells being more sensitive. The results demonstrate that 1) energetic inhibition augments endothelial macromolecule permeability when both oxidative and glycolytic energy production are inhibited, 2) increased macromolecule permeability in energy-depleted endothelial monolayers is caused by the opening of intercellular gaps, and 3) endothelial cells reversibly tolerate up to 2 h of almost complete ATP depletion.
Reoxygenation after 60 min substrate-free hypoxic perfusion (modified Tyrode solution, 37 degrees C) caused isolated Langendorff hearts (from rats) to rapidly develop hypercontracture and sarcolemmal disruptions indicated by massive and sudden loss of enzymes ("oxygen paradox"). Reoxygenation (30 min) caused an augmented loss of creatine kinase by 25.8% (lactate dehydrogenase by 40.1%) of the initial total tissue activity. It was investigated whether a temporary contractile blockade by 2,3-butanedione monoxime (BDM; 20 mM) can prevent reoxygenation-induced injury. In the presence of BDM, reoxygenation no longer caused hypercontracture or increased enzyme release. Instead, ultrastructure recovered, and contents of creatine phosphate (CrP) were partially restored (60 min hypoxia: 0.4 mumol CrP/g dry wt; after subsequent 60 min reoxygenation in presence of BDM: 7.8 mumol CrP/g dry wt). When BDM was eluted after first 20 min of reoxygenation, an attenuated but distinct increase in enzyme release was still observed. When BDM was eluted after 60 min of reoxygenation, ultrastructure did not deteriorate and increase of enzyme release remained virtually absent. During first 30 min after removal of BDM, the increased loss of creatine kinase amounted to only 5.7% (lactate dehydrogenase to 6.9%) of the initial total tissue activity. The results demonstrate that the oxygen paradox can be prevented in the hypoxic-reoxygenated heart when the contractile apparatus is temporarily paralyzed during the initial phase of reoxygenation.
The potency of neuropeptide Y (NPY) to cause negative and positive contractile responses in rat ventricular cardiomyocytes was investigated. In these cells, NPY was found to activate the transient outward K+ current (Ito) and the slow inward Ca2+ current (Isi). As reported before (H. M. Piper, B. C. Millar, and J. R. McDermott, Naunyn Schmiedeberg's Arch. Pharmacol. 340: 333-337, 1989), NPY attenuated the increase in the contractile response induced by isoprenaline (10(-7) M). This effect of NPY could be abolished by 1) the presence of the inhibitor of Ito, 4-aminopyridine (4-AP, 0.5 mM); 2) pretreatment of the cells with pertussis toxin (1 microgram/ml for 6 h); and 3) the presence of the 19-amino acid COOH-terminal fragment of NPY, NPY-(18-36) (10(-6) M). In the absence of isoprenaline, but in the presence of 4-AP, NPY exerted a stimulatory effect on the cardiomyocytes. This effect could be abolished 1) by using the inhibitor of the Isi, verapamil (10(-8) M), but not 2) by pretreatment with pertussis toxin, nor 3) by coincubation with NPY-(18-36). The results indicate that in the rat the antiadrenergic negative contractile effect of NPY results from its action on the Ito. Blockade of this current by 4-AP unmasks a positive contractile effect of NPY that is related to activation of the Isi.
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Energy metabolism of bovine cultured lens epithelial cells (CLEC) was compared to that of fresh bovine lens. CLEC contained high levels of ATP (44 nmol mg protein-1) and creatine phosphate (13 nmol mg protein-1). An ATP/ADP ratio of ten and a creatine phosphate/creatine ratio of two indicated the cells were in a well-energized state. ATP concentration in fresh epithelium was comparable to that of CLEC; however in the anterior cortex it was tenfold lower. In contrast to fresh lenses, CLEC were able to oxidize glucose, lactate and palmitic acid. Lactate was oxidized at the highest rate. In CLEC, 42% of the ATP generated by catabolizing glucose resulted from oxidative phosphorylation. Glucose (5 mM) was degraded to lactate and CO2 at a 2:1 ratio. The hexose monophosphate pathway accounted for two thirds of the CO2 produced. In the fresh whole bovine lens palmitate was not oxidized and lactate was oxidized to a lesser degree than in CLEC. Only one-tenth of the ATP generated by glucose catabolism in the fresh whole lens was derived from oxidative phosphorylation. This was also the case for a preparation of fresh epithelium, maintained in air and 100% oxygen, demonstrating that the preferential glycolytic catabolism of glucose in lens is not caused by limited oxygen diffusion. In the fresh bovine lens the epithelium accounted for one third of the glucose catabolism of the whole lens, even though it had only about 0.1% of its protein mass. In fresh human lenses, conversion of glucose into lactate was even more pronounced--the lactate/CO2 ratio was 73:1.(ABSTRACT TRUNCATED AT 250 WORDS)
The ability of coronary endothelial cells in 14 day confluent cultures to metabolize glucose, palmitate, lactate and various amino acids was investigated. Under aerobic conditions, 99% of glucose, (5 mM) was degraded to lactate and only 0.04% was oxidized in the Krebs cycle. One percent of the glucose catabolized was directed into the hexose monophosphate pathway, but this fraction could be increased by 81% by 0.4 mM methylene blue. Glucose oxidation in the Krebs cycle was increased at glucose concentrations lower than 1 mM, or by the uncoupler 2,4-dinitrophenol. Oxidation to CO2 of palmitate (300 microM), lactate (1 mM), and glutamine (0.5 mM) was diminished in the presence of glucose (5 mM) by 80, 66, and 48%, respectively. These results demonstrate that coronary endothelial cells utilize exogenous glucose, at physiological concentration, predominantly for glycolytic energy production. The metabolic pattern is characteristic of the Crabtree effect. In these cells, glucose not only effectively suppresses the oxidation of the substrates lactate and palmitate, i.e. of substrates preferred by the whole heart, but also of glutamine, which is a major oxidative substrate for coronary endothelial cells. Absolute rates of substrate catabolism are low as compared to those of the beating heart indicating a low energy demand of coronary endothelial cells.