Simulated Ischemic Conditions Research Articles

Diltiazem protects human NT-2 neurons against excitotoxic damage in a model of simulated ischemia

In vitro models are often used to investigate pathophysiological mechanisms of brain cell injury as they occur for instance during cerebral ischemia. To analyze the efficacy of potential neuroprotective compounds, cell physiological experiments were performed in a recently improved culture system of human model neurons. The postmitotic neurons were generated from the human NT-2 teratocarcinoma cell line, using a cell sphere culture method to facilitate rapid terminal differentiation. We simulated ischemic conditions in cultures of purified NT-2 neurons and found that low doses of the antihypertensive drug diltiazem protected against excitotoxic neuronal damage in vitro. Experiments with primary cortical mouse neuron cultures demonstrated a similar response to simulated ischemia and confirmed the neuroprotective effect of diltiazem. Calcium imaging experiments showed that diltiazem reduced both NMDA- and glutamate-induced calcium influxes in NT-2 neurons suggesting that its neuroprotective effect is based on the inhibition of voltage-gated calcium channels. These results indicate that diltiazem is an effective blocker of glutamate-induced excitotoxicity. Moreover, we suggest that cell cultures of human model neurons can provide an important initial test system for drug development in stroke therapy.

NMDA Receptors, Glial Cells, and Clinical Medicine

Recent reports have overturned a series of dogmas that have been well entrenched in the neuroscience literature concerning NMDA-type glutamate receptors (NMDARs). The new data show that NMDARs exist on the myelin sheath formed by oligodendrocytes, that an uncompetitive NMDAR antagonist has successfully passed human clinical trials, and that NMDARs trigger multiple deleterious cascades to inflict cellular damage on both neurons and glia during cerebral ischemia (stroke). These recent findings bode well for clinical intervention with NMDAR antagonists in more neurological disorders than previously thought, including multiple sclerosis, cerebral palsy (periventricular leukomalacia), and spinal cord injury.

Phospholipase C-δ1 rescues intracellular Ca 2+ overload in ischemic heart and hypoxic neonatal cardiomyocytes

Ischemia and simulated ischemic conditions cause intracellular Ca 2+ overload in the myocardium. The relationship between ischemia injury and Ca 2+ overload has not been fully characterized. The aim of the present study was to investigate the expression and characteristics of PLC isozymes in myocardial infarction-induced cardiac remodeling and heart failure. In normal rat heart tissue, PLC-δ1 (about 44 ng/mg of heart tissue) was most abundant isozymes compared to PLC-γ1 (6.8 ng/mg) and PLC-β1 (0.4 ng/mg). In ischemic heart and hypoxic neonatal cardiomyocytes, PLC-δ1, but not PLC-β1 and PLC-γ1, was selectively degraded, a response that could be inhibited by the calpain inhibitor, calpastatin, and by the caspase inhibitor, zVAD-fmk. Overexpression of the PLC-δ1 in hypoxic neonatal cardiomyocytes rescued intracellular Ca 2+ overload by ischemic conditions. In the border zone and scar region of infarcted myocardium, and in hypoxic neonatal cardiomyocytes, the selective degradation of PLC-δ1 by the calcium sensitive proteases may play important roles in intracellular Ca 2+ regulations under the ischemic conditions. It is suggested that PLC isozyme-changes may contribute to the alterations in calcium homeostasis in myocardial ischemia.

Pharmacology of ischemia-induced glutamate efflux from rat cerebral cortex in vitro

Simulated ischemic conditions (hypoxia–hypoglycaemia) in vitro enhanced glutamate efflux from rat cerebrocortical prisms. Here we characterised efflux mechanisms using pharmacological tools. The Na + channel blocker TTX (1 μM) did not affect ischemia-induced efflux, while sipatrigine (100 μM), a Na +/Ca 2+ channel blocker and ω-conotoxin MVIIC (2 μM), an N/P/Q type Ca 2+ channel blocker, inhibited efflux by fractions of 0.53 and 0.46, respectively (1.00 corresponding to total inhibition). Omission of extracellular Ca 2+ and addition of EGTA (2 mM) inhibited ischemia-induced efflux only during the first 25 min of incubation. A similar result was observed on omission of extracellular Ca 2+ together with addition of La 3+ (10 μM) and Mg 2+ (6 mM). TTX, sipatrigine and La 3+/Mg 2+ all inhibited control efflux. Ischemia-induced efflux was sensitive to the volume activated anion channel inhibitor NPPB (100 μM) only after the first 25 min of incubation, with the maximal fraction inhibited being 0.54. The glutamate transporter inhibitor d,l-TBOA reduced ischemia-induced efflux throughout a 45-min incubation period, and enhanced efflux from control tissue. d,l-TBOA inhibited efflux at 30 min by a maximum fraction of 0.49, at 50 μM. These data indicate that the early phase of ischemia-induced glutamate efflux is in part Ca 2+ dependent, while the later phase involves volume activated anion currents and both phases involve excitatory amino acid transporters.

The mechanism of the force enhancement by MgADP under simulated ischaemic conditions in rat cardiac myocytes.

In this study, the effects of MgADP and/or MgATP on the Ca2+ -dependent and Ca2+ -independent contractile force restoration were determined in order to identify the origin of the tonic force increase (i.e. ischaemic contracture) which develops during advanced stages of ischaemia. Experiments were performed at 15 degrees C during simulated ischaemic conditions in Triton-skinned right ventricular myocytes from rats. In the presence of 5 mM MgATP the maximal Ca2+ -dependent force (P(o)) of 39 +/- 2 kN m(-2) (mean +/- S.E.M.) under control conditions (pH 7.0, 15 mM phosphocreatine (CP)) decreased to 8 +/- 1 % during simulated ischaemia (pH 6.2, 30 mM inorganic phosphate (P(i)), without CP). This change was accompanied by a major reduction in Ca2+ sensitivity (pCa(50) 4.10 vs. 5.62). Substitution of MgADP for MgATP restored isometric force production and its Ca2+ sensitivity (pCa(50) 4.74 at 4 mM MgADP and 1 mM MgATP). In addition, it shifted the MgATP threshold concentration of Ca2+ -independent force development to higher levels in a concentration-dependent manner. However, Ca2+ -independent force was facilitated less by MgADP than Ca2+ -dependent force. The MgADP-induced increase in force was accompanied by marked reductions in the velocity of unloaded shortening and the rate of tension redevelopment. These data and simulations using a model of cross-bridge kinetics suggest that the ischaemic force is not a consequence of a reduction in intracellular MgATP concentration, but identify MgADP as a key modulator of the cross-bridge cycle under simulated ischaemic conditions in cardiac muscle, with a much lower inhibition constant (0.012 +/- 0.003 mM) than in skeletal muscle. Therefore, MgADP has a high potential to stabilize the force-generating cross-bridge state and to facilitate the development of ischaemic contracture, possibly involving a Ca2+ activation process in the ischaemic myocardium.

Effects of MgATP on ATP utilization and force under normal and simulated ischaemic conditions in rat cardiac trabeculae.

The dependency of ATP utilization and isometric force on [MgATP] was studied in skinned rat trabeculae under normal (pH 7.0) and simulated ischaemic (pH 6.2, 30 mM added Pi) conditions at 20+/-1 degrees C. At saturating [Ca2+], mean (+/-SEM) ATP utilization at 5 mM MgATP (A0) was 0.48+/-0.03 mM/s and force (F0) was 37+/-2 kN/m2. At 10 microM MgATP under normal conditions ATP utilization decreased gradually to 66+/-3% of A0, and force increased to 169+/-7% of F0. Under ischaemic conditions at 10 microM MgATP, ATP utilization decreased from 30+/-5% to 11+/-2% of A0 whereas force increased eightfold from 12+/-4% to 97+/-7% of F0. The [MgATP] at half-maximal ATP utilization (Km) under ischaemic conditions was 21+/-3 microM. At pH 7.0, Km was estimated to be less than 10 microM. These results show that tension cost decreases markedly with decreasing MgATP. Under ischaemic conditions parallel changes in Ca2+ sensitivity of force and ATP utilization were observed, corresponding to 1.3 pCa units. Reducing [MgATP] from 0.5 to 0.05 mM caused a modest reversal of this change in Ca2+ sensitivity. These changes in Ca2+ sensitivity are consistent with a marked reduction in active force and force-related ATP utilization during ischaemia but are insufficient to explain the ischaemic contracture on the basis of active force development.

Protective Effect of Benidipine Against the Abnormal Electrical Activity in Single Ventricular Myocytes of the Guinea Pig Under Simulated Ischemic Conditions and Reperfusion

Induction of electrical abnormalities (EAs) under simulated ischemic conditions and after reperfusion was measured from single cardiac myocytes isolated from guinea pig ventricle using whole-cell voltage or current clamp with perforated patch variation. Conditions of simulated ischemia were produced by the exchange of medium from the standard one oxygenated with 95% 02-5% C02 gas (pH 7.4) to the modified one, which contained no glucose, 8 mM K+ and 30 mM sodium-D,L-lactate and was gassed with 90% argon -10% C02 (pH 6.6). Under the simulated ischemia for 20 min, EAs such as delayed after depolarization, early afterdepolarization, automatic activity or transient inward current were observed in about 37% of myocytes driven electrically at 1 Hz. Irreversible hypercontracture occurred in myocytes of 10% or less. Upon reperfusion with the standard solution, EAs and hypercontracture were observed in about 43% and 22% of cells, respectively. Glibenclamide-sensitive current was detected during ischemia, but tended to be enhanced during reperfusion. Amplitude of Ca2+ current and ATP-sensitive K+ current after reperfusion varied widely with time and from cell to cell. When myocytes were pretreated for 10 min with 10 nM benidipine, a 1,4-dihydropyridine derivative Ca2+ blocker, the incidence of EAs and hypercontracture was markedly reduced, suggesting the protective effect of benidipine against cardiac cell injury during ischemia and reperfusion.

Effects of simulated myocardial ischemic conditions on the responses of isolated human umbilical artery strips to 5-hydroxytryptamine and prostaglandin F 2α

1. 1. The effects of simulated myocardial ischemic conditions on the contractile response of isolated human umbilical artery (HUA) strips to 5-hydroxytryptamine (5-HT) and prostaglandin F 2α(PGF 2α) were studied. 2. 2. During simulated myocardial ischemic conditions the contractile response of HUA strips to 5-HT was lower than the response to the monoamine under oxygenated conditions. Under simulated ischemic conditions the response to 5-HT was further depressed by the cyclooxygenase inhibitor indomethacin (10 μM) and increased by the NO synthase inhibitor L-NAME (100 μM). 3. 3. During simulated ischemic conditions the response of the HUA to a submaximal concentration of PGF 2α (3 μM) was reduced. Indomethacin (10 μM) further reduced the response to the prostanoid whereas L-NAME (100 μM) enhanced the response to PGF 2α. 4. 4. It is concluded that during simulated myocardial ischemic conditions lactate negatively modulates the contractile response of HUA strips to 5-HT. Apparently, during simulated myocardial ischemic conditions in the HUA the production and/or release of EDRF/NO was not affected.

Role of Ca2+ in metabolic inhibition-induced norepinephrine release in rat brain synaptosomes.

Ischemia and simulated ischemic conditions induce enhanced release of norepinephrine (NE) in the brain and the heart. Although studies with neuronal preparations demonstrated a rise in [Ca2+]i under energy-depleted conditions, such release of NE in the heart appears to be predominantly Ca2+ independent. Since Ca2+ overload occurs in ischemia or energy depletion and since a rise in [Ca2+]i triggers exocytosis without membrane depolarization, we tested the possibility, using brain synaptosomes, that increased NE release could be, at least in part, a consequence of raised [Ca2+]i. Brain synaptosomes were incubated with Krebs-Henseleit medium, and ischemia was mimicked by treatment with metabolic inhibitors. NE content in incubation medium (supernatant) and synaptosomes was analyzed chromatographically. Treatment with metabolic inhibitors reduced ATP content by 75% and increased [Ca2+]i by more than fourfold within minutes. Metabolic inhibition elicited NE release, which started within 10 minutes and reached a maximum after 30 minutes, with a corresponding 55% reduction in synaptosomal NE content after 40 minutes. NE release, together with a marked increase in [Ca2+]i, was also induced in energy-depleted synaptosomes by Ca2+ repletion after incubation with the Ca(2+)-free medium. Effects on NE release of various interventions to prevent Ca2+ overload were tested. Omission of Ca2+ from the incubation medium or loading synaptosomes with the Ca2+ chelator BAPTA-AM (20 and 100 mumol/L) prevented NE release, indicating a Ca(2+)-dependent mechanism. Inhibition of Ca2+ channels with omega-conotoxin, cadmium, or nifedipine had no effect on NE release during energy depletion. In contrast, nickel and 3,4-dichlorobenzamil, Na(+)-Ca2+ exchange inhibitors, dose-dependently inhibited NE release. In conclusion, this study provides evidence that under energy-depleted conditions, Ca2+ overload in synaptosomes of noradrenergic neurons from the brain is an important mechanism for the enhanced release of NE and that a reversal of Na(+)-Ca2+ exchange may be the key pathway leading to intraneuronal Ca2+ overload.

Regulation of cardiac Ca2+ release channel (ryanodine receptor) by Ca2+, H+, Mg2+, and adenine nucleotides under normal and simulated ischemic conditions.

In myocardial ischemia, pHi and [ATP] fall, whereas the free [Ca2+] and [Mg2+] rise. The effects of these changes on cardiac Ca2+ release channel (ryanodine receptor) activity were investigated in [3H]ryanodine binding and single-channel measurements, using isolated membrane and purified channel preparations. In the absence of the two channel ligands Mg2+ and ATP, cardiac Ca2+ release channels were half-maximally activated at pH 7.4 by approximately 4 mumol/L cytosolic Ca2+ and half-maximally inhibited by approximately 9 mmol/L cytosolic Ca2+. Regulation of channel activity by Ca2+ was modulated by Mg2+ and ATP. Single-channel activities were more sensitive to a change of cytosolic pH than SR lumenal pH. Reduction in lumenal and/or cytosolic pH from 7.3 to 6.5 and 6.0 resulted in decreased single-channel activities without a change in single-channel conductance. [3H]Ryanodine binding measurements also indicated that acidosis impairs cardiac Ca2+ release channel activity. Mg2+ and adenine nucleotide concentrations regulated the extent of inhibition and the Ca2+ dependence of binding. In the presence of 5 mmol/L Mg2+ and 5 mmol/L beta, gamma-methyleneadenosine 5'-triphosphate (AMPPCP, a nonhydrolyzable ATP analogue), the free [Ca2+] for half-maximal [3H]ryanodine binding was increased from 1.9 mumol/L at pH 7.3 to 36 mumol/L at pH 6.5 and to 89 mumol/L at pH 6.2. These results suggest that ionic and metabolic changes that might be expected to affect sarcoplasmic reticulum Ca2+ release channel activity in ischemic myocardium include an altered Ca2+ sensitivity of the channel, a fall in pH, and a loss of the high-energy adenine nucleotide pool, leading to an increased inhibition by Mg2+.

Effects of dexmedetomidine on hypoxia-evoked glutamate release and glutamate receptor activity in hippocampal slices.

The selective alpha(2) agonist dexmedetomidine may improve neurologic outcome after incomplete ischemia in animals when it is administered either before or after the start of the ischemic insult. To clarify further the mechanisms by which alpha(2) agonists may provide neuroprotective effects, the authors tested the hypotheses that dexmedetomidine decreases synaptic and extrasynaptic glutamate release stimulated by potassium chloride or hypoxia, and decreases postsynaptic glutamate receptor activity during aerobic or hypoxic conditions. Glutamate released from brain slices (300-microns thick) from rat hippocampus was measured in a cuvette during two experimental stresses: (1) potassium chloride-evoked depolarization (30 mM) with and without 10 nM, 100 nM, or 1,000 nM dexmedetomidine; and (2) hypoxia (95% N2 - 5% CO2) with and without 100 nM dexmedetomidine. Glutamate release was quantified by fluorescence assay using 1 mM nicotinamide adenine dinucleotide, and 5 international units per ml glutamate dehydrogenase. The formation of nicotinamide dinucleotide reduced from nicotinamide adenine dinucleotide by glutamate dehydrogenase was measured fluorometrically (excitation light 340 nm, emission intensity 460 nm) in the solution above the slice. Glutamate receptor activity was determined by the change in cytosolic calcium concentration in CA1 neurons in the presence and absence of 100 nM dexmedetomidine during administration of N-methyl-D-aspartate (100 microM) and during simulated ischemic penumbra conditions (PO2 = 20 mmHg, glutamate 3 mM). Calcium concentration was measured using a microscope fluorometer in fura 2-loaded rat hippocampal brain slices. Dexmedetomidine attenuated potassium chloride-evoked glutamate release by 37%, 51%, (P = 0.03) and 27%, respectively, for the 10 nM, 100 nM, and 1,000 nM concentrations, and decreased (at 100 nM) the increase in glutamate release in response to hypoxia by 61% (P < 0.0001). Dexmedetomidine (100 nM) had no effect on N-methyl-D-aspartate or hypoxia plus 3 mM L-glutamate-mediated calcium changes. The selective alpha(2)-adrenergic agonist dexmedetomidine decreases evoked glutamate release from hippocampal rat brain slices during depolarization or hypoxic stress, but does not alter calcium changes mediated by the stimulation of glutamate receptors during aerobic or hypoxic conditions.

Cellular electrophysiological effects of MS‐551, a new class III antiarrhythmic agent

AbstractThe cellular electrophysiological effects of MS‐551, a pure class III antiarrhythmic agent lacking beta‐blocking activity, were compared with those of d‐sotalol and dofetilide in canine Purkinje fibers and ventricular muscles and in guinea pig atrial muscles using the standard microelectrode technique. MS‐551 prolonged the action potential duration (APD) of these cardiac tissues to the same extent in a concentration‐dependent manner (10−6−10−4 M). MS‐551 did not affect other action potential parameters of these tissues except for the Vmax, which was depressed approximately 10% at 10−4 M MS‐551. The extents of prolongation after 10−5 M of MS‐551 were 36, 27, and 35% for canine Purkinje and ventricular muscle and guinea pig atrial muscle, respectively. d‐Stalol at a concentration 10 times higher than MS‐551 produced a similar prolongation of the APD of canine cardiac tissues but only a slight prolongation of the APD of guinea pig atrial muscles. Dofetilide at a concentration 30 times lower than MS‐551 produced a prolongation of the APD of canine Purkinje fibers but a relatively slight prolongation of the APD of both canine ventricular muscles and guinea pig atrial muscles. In canine Purkinje fibers, MS‐551 did not prevent the APD shortening induced by high extracellular potassium (8 and 12 mM), but the APD under these conditions was lengthened compared with controls. Under simulated ischemic conditions (pO2 25–30 mmHg, pH 6.5, extracellular K 8 mM and no glucose) for 30 min, the APD was abruptly shortened during the first 5 min, then gradually shortened further up until 30 min. MS‐551 did not prevent the abrupt APD shortening, but slowed the later gradual shortening. The APD prolonging action of MS‐551 was enhanced at low frequency. These data indicate that (1) MS‐551 is a pure class III antiarrhythmic agent with electrophysiological characteristics basically similar to d‐sotalol and dofetilide; (2) MS‐551 has a relatively greater potency in guinea pig atrial muscle than d‐sotalol and dofetilide; (3) the APD prolonging action of MS‐551 is preserved under high K+ or simulated ischemic conditions; and (4) MS‐551 exhibits a reverse use‐dependence in prolonging APD. © 1995 Wiley‐Liss, Inc.

Simultaneous blockade of non‐NMDA ionotropic receptors and NMDA receptor‐associated ionophore partially protects hippocampal slices from protein synthesis impairment due to simulated ischemia

A large body of evidence exists to demonstrate that excitatory amino acids (EAA) and their receptors are involved in the pathophysiological mechanisms linking several acute brain insults, such as cerebral ischemia, to neuronal degeneration and death. Accordingly, the use of EAA receptor antagonists can be beneficial in attenuating or preventing the neuronal irreversible damage subsequent to various neuropathological syndromes. We have investigated the effect of 15 min of simulated ischemic conditions, i.e., oxygen/glucose deprivation, on hippocampal slices preparation measuring, as neurotoxicity indexes, both the amino acids efflux in the incubation medium, detected by HPLC, and the inhibition of protein synthesis, evaluated as 3H-Leucine incorporation into proteins. Accumulation of neurotransmitter amino acids was measured in the medium during the "ischemic" period. Glutamate increased 30-fold over the basal level while aspartate was sevenfold and GABA 12-fold higher than in normal conditions. After a reoxygenation period of 30 min, the rate of protein synthesis of hippocampal slices subjected to "ischemia" was reduced to 35-50% of controls. The non-competitive NMDA antagonist MK-801 (100 microM) and the competitive NMDA antagonist CGP 39551 (100-250 microM) as well as the non-NMDA receptor antagonists NBQX (100 microM) and AP3 (300 microM) were unable to counteract the metabolic impairment when they were present alone in the incubation fluid during simulated "ischemia." An incomplete, but highly significant (p < 0.001), protection from protein synthesis impairment was achieved in the presence of an equimolar concentration (100 microM) of MK-801 and NBQX.(ABSTRACT TRUNCATED AT 250 WORDS)

Multivariate prediction of spontaneous repetitive responses in ventricular myocardium exposed in vitro to simulated ischemic conditions

Guinea-pig ventricular myocardium was partly exposed to normal Tyrode's superfusion and partly to altered conditions (using modified Tyrode's solution) set to simulate acute myocardial ischemia (P o 2 80 ± 10 mmHg; no glucose; pH 7.00 ± 0.05; K + 12 mM). Using a double-chamber tissue bath and standard microelectrode technique, the occurrence of spontaneous repetitive responses was investigated during simulated ischemia (occlusion) and after reperfusing the previously ischemic superfused tissue with normal Tyrode's solution (reperfusion). In 62 experiments (42 animals) the effects of:(1) duration of simulated ischemia (1321 ± 435 s),(2) stimulation rate (1002 ± 549 ms) and (3) number of successive simulated ischemic periods (occlusions) (1.58 ± 0.92) on: (1) resting membrane potential, (2) action potential amplitude, (3) duration of 50 and 90% action potentials and (4) maximal upstroke velocity of action potential were studied. All variables were considered as gradients (delta) between normal and ischemic tissue. Both during occlusion and upon reperfusion, spontaneous repetitive responses were coded as single, couplets, salvos (three to nine and > 10) or total spontaneous repetitive responses (coded present when at least one of the above-mentioned types was seen). The incidence of total spontaneous repetitive responses was 31% ( 19 62 ) on occlusion and 85% ( 53 62 ) upon reperfusion. Cox's models (forced and stepwise) were used to predict multivariately the occurrence of arrhythmic events considered as both total spontaneous repetitive responses and as separate entities. These models were applicable since continuous monitoring of the experiments enabled exact timing of spontaneous repetitive response onset during both occlusion and reperfusion. In predicting reperfusion spontaneous repetitive responses, total spontaneous repetitive responses and blocks observed during the occlusion period were also considered. Total occlusion spontaneous repetitive responses were predicted by: (1) longer delta 50% action potential duration ( t = 2.68), (2) shorter delta 90% action potential duration ( t = −2.17) and (3) fewer occlusive periods ( t = −2.46). Total reperfusion spontaneous repetitive responses were predicted by a longer delta action potential amplitude ( t = 2.18). Due to few events during occlusion, prediction of individual arrhythmic entities was not possible. Upon reperfusion single spontaneous repetitive responses were predicted by longer delta maximal upstroke velocity of action potential ( t = 2.59) and shorter delta 90% action potential duration ( t = −2.55); couplets were predicted by longer delta 50% action potential duration ( t = 3.26); longer delta action potential amplitude predicted salvos (> 10) ( t = 3.26). This study demonstrates that different electrophysiological variables, simultaneously measured in normal and simulated ischemic tissue, predict in vitro arrhythmic events seen during occlusion and reperfusion. While occlusion spontaneous repetitive responses are mainly related to the difference in action potential duration between normal and ischemic tissue, reperfusion spontaneous repetitive responses are associated with lower action potential amplitudes in the ischemic zone. Differences also exist among various arrhythmic entities upon reperfusion. Drug interventions might be tested in this preparation to see whether prevention of spontaneous repetitive responses is achieved. Based on the evidence presented here, class III antiarrhythmics and ATP potassium-channel openers and/or blockers are potential candidates.

Modification by hypoxia, hyperkalaemia and acidosis of the cardiac electrophysiological effects of a range of antiarrhythmic drugs

1. The electrophysiological effects of a series of drugs with Class I antiarrhythmic activity were examined in sheep Purkinje fibres, superfused in vitro with either a normal or hypoxic, hyperkalaemic and acidotic physiological salt solution (PSS). 2. In normal sheep Purkinje fibres, lignocaine, disopyramide, nicainoprol and propranolol all significantly reduced action potential height and the maximum rate of depolarization of phase zero (MRD) and abbreviated the action potential, without modifying resting membrane potential (RMP). 3. Verapamil at the highest concentration studied, 8 microM, significantly reduced MRD with an associated slight membrane depolarization and abbreviated action potential duration measured at 50% repolarization (APD50). 4. Superfusion of sheep Purkinje fibres with a hypoxic, hyperkalaemic and acidotic PSS resulted in marked reductions in resting membrane potential, upstroke and duration of the action potential. 5. In the presence of modified PSS, lignocaine, propranolol and verapamil all reduced MRD to a greater extent than in normal PSS. The effects of nicainoprol on MRD were not affected whereas those of disopyramide were significantly attenuated. 6. Under simulated ischaemic conditions, lignocaine, propranolol and nicainoprol did not produce a concentration-dependent reduction in action potential duration whereas disopyramide and verapamil, respectively, prolonged and abbreviated both APD50 and APD90. 7. The Na+ channel blocking actions of the different subtypes of Class I antiarrhythmic agents studied, as well as their effects on action potential duration, were modified differently by simulated ischaemia.

Ischemia does not induce the release of excitotoxic amino acids from the hippocampus of newborn rats

Simulated ischemic conditions or a source of oxygen-derived free radicals, such as xanthine plus xanthine oxidase, released a significant amount of the excitotoxic amino acids Asp and Glu from adult rat hippocampal slices incubated in vitro. The concentrations of Asp and Glu in the incubation medium increased by 20 and 30 times respectively when such slices were exposed to simulated ischemic for a 10-min period. However, preparations obtained from 4- to 9-day-old rats did not release Asp or Glu either when exposed to ischemia or after K + depolarization. This release appeared 10–15 days after birth and progressively increased up to 13 months of age. No further increase was observed in 25-month-old animals. The exposure of the slices to a source of oxygen-derived free radicals induced a release of excitotoxic amino acids independently from the age of the rats. The massive excitotoxic amino acid release from adult hippocampal slices and the formation of free radicals induced by ischemic insults has been previously associated with degeneration of hippocampal neurons. The lack of ischemia-induced excitotoxic amino acid release from the newborn hippocampus may help to explain why the newborn hippocampus is more resistant than the adult to hypoxic/ischemic insults.

Effects of neuropeptide Y and calcitonin gene‐related peptide on sheep coronary artery rings under oxygenated, hypoxic and simulated myocardial ischaemic conditions

1. The effects of calcitonin gene-related peptide (CGRP) and neuropeptide Y (NPY) were examined on sheep circumflex (2-2.5 mm o.d.) coronary artery rings, with and without endothelium, under oxygenated, hypoxic and simulated ischaemic conditions. The interaction between the vasoconstrictor effects of NPY and the thromboxane mimetic, U46619, was also studied. 2. Ischaemia was simulated by modification of the composition of the physiological salt solution by increasing potassium and H+, including lactate and reducing glucose and PO2. 3. Hypoxia alone and simulated ischaemia increased the maximum vasodilatation produced by CGRP. CGRP (30 nM) abolished and markedly reduced the contraction that was induced by hypoxia and simulated ischaemia respectively. 4. Hypoxia increased and simulated ischaemia reduced the contractile response to NPY in endothelium intact rings. When the endothelium was removed, NPY caused a contraction under ischaemic conditions only. The hypoxic and ischaemic-induced contractions were augmented by NPY (30 nM). 5. In the rings containing endothelium, NPY enhanced the contraction caused by U46619 during hypoxia only. In endothelium-denuded preparations, NPY increased or partially restored the contraction caused by U46619. 6. These results show that the responsiveness of coronary artery rings isolated from sheep to either CGRP or NPY is modified by hypoxia or simulated myocardial ischaemia.

SELECTIVE DEPRESSION OF MAXIMUM RATE OF DEPOLARIZATION OF GUINEA‐PIG VENTRICULAR ACTION POTENTIALS BY AMIODARONE AND LIGNOCAINE IN SIMULATED ISCHAEMIA: COMPARISON WITH ENCAINIDE

1. Standard microelectrode techniques were used to record intracellular action potentials from guinea-pig ventricular myocardium superfused with either control physiological saline (pH 7.5; pO2 500 mmHg; [K+] 5.6 mmol/L) or 'simulated ischaemic' solution (pH 6.4; pO2 90 mmHg; [K+] 11.2 mmol/L). 2. The effects on action potential parameters of therapeutic concentrations of lignocaine, amiodarone and encainide were studied under both conditions. 3. Simulated ischaemia, in the absence of drugs, produced marked reductions in resting potential (-86.6 +/- 2.3 to -64.7 +/- 3.5 mV), maximum rate of depolarization (Vmax; 263 +/- 66 to 106 +/- 36 V/s) and action potential duration (164 +/- 24 to 97 +/- 26 ms). No drug produced any additional effect on resting potential. 4. All three drugs produced enhanced depression of Vmax in ischaemia compared to control conditions (class I effect). This was much more marked for lignocaine and amiodarone (inactivated channel blockers) than for encainide (open channel blocker). 5. In addition the prolongation of action potential duration seen with acute exposure to amiodarone (174 +/- 12 to 192 +/- 17 ms; class III effect) was abolished under simulated ischaemic conditions. 6. It is concluded that lignocaine and amiodarone exert greater selectivity for ischaemic tissue than does encainide and that amiodarone may function primarily as a class I agent under ischaemic conditions.

The effect of halothane, isoflurane, and verapamil on ischemic-isolated rabbit renal tubules.

The effects of the volatile anesthetics halothane and isoflurane, and the calcium entry blocker verapamil, were studied in isolated rabbit renal tubules under nonischemic and simulated ischemic conditions. Isolated rabbit renal tubules were subjected to zero (control), 30 (I-30), or 60 (I-60) minutes of simulated ischemia following the method of Weinberg. Following the ischemic period, tubules were reoxygenated in a Gilson respirometer (simulated reperfusion) and treated with either halothane (1%) or isoflurane (1%) in the controls and at I-30, or halothane (1%, 2%, 4%) or verapamil (5 microM, 15 microM, 30 microM) at I-60. Tubules were analyzed for lactate dehydrogenase (LDH) release (measuring cell membrane integrity), intracellular potassium and adenosine triphosphate (ATP), and oxygen consumption (cellular respiratory rate). In nonischemic tubules, exposure to 1% isoflurane caused significantly reduced LDH release compared with that released by controls, indicating cell membrane protection, whereas 1% halothane had no effect on these cells. With 30 min of ischemia, 1% isoflurane was associated with significantly higher cellular LDH release and lower ATP concentration, suggesting increased cellular damage. Halothane (1%) was associated with only an increased ATP concentration in tubules exposed to 30 min of ischemia. Following 60 min of ischemia, halothane (4%) decreased LDH release by 45% (29.2 +/- 2.3% vs. 47.0 +/- 9.6% without halothane). Tubules exposed to halothane also had higher intracellular potassium and ATP concentrations, and increased respiratory rates. Halothane (2%) was less protective and only increased the ATP concentration. The release of LDH was not statistically different with or without 2% halothane.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological effects of adenosine and adenosine triphosphate on sheep Purkinje fibres under normal and simulated ischaemic conditions.

1. The electrophysiological effects of adenosine and adenosine triphosphate (ATP) were examined in sheep Purkinje fibres, superfused in vitro with either a normal or a hypoxic, hyperkalaemic and acidotic physiological salt solution (PSS). The ability of adenosine to modify the effects of noradrenaline on action potential characteristics was also investigated. 2. The only statistically significant effects of adenosine (10(-6)-10(4) M) and of ATP (10(-6)-10(-4) M) on normal action potential characteristics were a slight dose-dependent shortening of the action potential by adenosine and a depolarization by ATP, 10(-4) M. 3. Superfusion with a hypoxic, hyperkalaemic and acidotic PSS caused marked reductions in resting membrane potential, upstroke and duration of the action potential. 4. Both adenosine and ATP attenuated the reduction in the rate of rise of the upstroke and the amplitude of the action potential caused by the modified PSS. 5. Adenosine did not alter the noradrenaline-induced effects on automaticity or on action potentials of normal or depressed Purkinje fibres. 6. Adenosine and ATP had electrophysiological effects on Purkinje fibres, exposed to conditions in vitro that mimic mild myocardial ischaemia, that were different from those observed on normally polarized fibres.