Protective effects of manganese, cobalt, nickel, and barium against a calcium paradox in the isolated frog heart

The effect of inorganic slow channel blockers on the calcium paradox in the frog heart was examined. Addition of the divalent cations of manganese, cobalt, nickel, or barium during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that in the control paradox in the absence of divalent cations, there is an efflux of calcium from myocardial cells during calcium depletion and a massive influx of calcium during the following reperfusion, leading to a calcium overload. Divalent cations protected frog myocardial cells, when present in the calcium-free perfusion medium, by reducing both calcium efflux during calcium depletion and the massive calcium influx during reperfusion. The effectiveness of the added divalent cations showed a strong dependence upon their ionic radius. The most potent inhibitors of the calcium paradox in the frog heart were the divalent cations having an ionic radius closer to the ionic radius of calcium. These results are discussed in terms of the possible mechanism involved in the protective effect of manganese, cobalt, nickel, and barium.     

Drinking rates and Na+ effluxes in response to temperature change in two species of marine flatfish: dab,Limanda limanda and plaice,Pleuronectes platessa

The relationship between fish mass and drinking rate in two species of flatfish, dab and plaice, weighing between 1 and 150 g was investigated. Both plaice and dab showed increased drinking rates with increasing fish mass, although — when calculated on a weight-specific basis — the increase was negligible. Fish were acclimated to winter and summer temperatures of 9 and 14°C, respectively. In winter both species were acutely transferred to 5, 14 and 21°C and in summer to 5, 9, 21 and 25°C. Drinking rates, Na⁺ efflux and body ion content were measured. Dab showed lower drinking rates than plaice (e.g. the weight-specific drinking rates of summer-and winter-acclimated dab were 0.12±0.01 and 0.06±0.006 ml·h^-1·100 g^-1, respectively, compared to that of plaice which were 0.25±0.02 ml·h^-1·100 g^-1 in summer and 0.17±0.02 ml·h^-1·100g^-1 in winter). Summer dab exhibited decreased weight-specific drinking rates at 5, 9 and 25°C, while winter dab increased drinking at 21°C. Winter plaice also showed increased drinking at 21°C and a decrease at 5°C, but in contrast summer plaice did not increase drinking at either 21 or 25°C but showed a decrease at 5 and 9°C. Winter dab and plaice showed similar Na⁺ efflux rates but summer dab showed higher efflux at all temperatures except 5°C. The data indicates that (a) the osmoregulatory function of plaice is much weaker than that of dab at higher temperatures (>20°C) and (b) mass has a greater effect on drinking and Na⁺ efflux rates than temperature; although when calculated on a weight-specific basis neither drinking nor efflux showed any variation with fish mass suggesting that these functions occur at similar intensities across the entire weight range.Abbreviations ANOVA analysis of variance - ⁵¹Cr-EDTA ⁵¹chromium ethylenediaminetetra-acetic acid - SW sea water     

The Influence of Temperature on Metabolic and Cellular Protection of the Heart during Long-Term Ischemia: A Study Using P-31 Magnetic Resonance Spectroscopy and Biochemical Analyses

We have compared the influence of two different cold temperatures (below 10°C) for cardiac ischemia by measuring a large variety of hemodynamic and metabolic parameters during ischemia and reflow. Isolated isovolumic rat hearts were arrested with a preservation solution which was developed in our laboratory and then submitted to 5 h of cold storage (4°C, group I; and 7.5°C, group II) in the same solution. After an additional period of 50 min of ischemia at 15°C with intermittent cardioplegic infusion, hearts were reperfused for 60 min at 37°C. Function was assessed during the control period and reflow. High-energy phosphates and intracellular pH were followed by³¹P magnetic resonance spectroscopy. Analyses of metabolites and enzymes were performed by biochemical assays and HPLC in coronary effluents and in freeze-clamped hearts to assess cellular integrity. The energetic pool was better preserved at 4°C during ischemia (ATP at the end of 4°C ischemia, 59 ± 7% in group I vs 31 ± 5% in group II,P< 0.01) and reflow (P< 0.05) but membrane protection was higher when increasing the temperature to 7.5°C (reduction of creatine kinase leakage, 89 ± 16 IU/min in group I vs 51 ± 5 IU/min in group II,P< 0.05). As a result, functional recovery, represented by the rate pressure product, was higher in hearts preserved at 7.5°C (52 ± 6% recovery in group I vs 77 ± 7% in group II at the end of reflow,P< 0.05). Altogether, cold storage at 7.5°C provides a better protection than storage at 4°C.     

Calcium Dependence of Muscarinic Receptor-Mediated Catecholamine Secretion from the Perfused Rat Adrenal Medulla

It had previously been thought that muscarinic cholinergic receptors utilize an influx of extracellular calcium for activation of adrenomedullary catecholamine secretion. However, it has recently been demonstrated that muscarinic receptors on isolated adrenal chromaffin cells can elevate cytosolic free calcium levels in a manner independent of extracellular calcium, presumably by mobilizing intracellular calcium stores. We now demonstrate that muscarinic receptor-mediated catecholamine secretion from perfused rat adrenal glands can occur under conditions of extracellular calcium deprivation that are sufficient to block both nicotine- and electrically stimulated release. Three independent conditions of extracellular calcium deprivation were used: nominally calcium-free perfusion solution (no calcium added), EGTA-containing calcium-free perfusion solution, and perfusion solution containing the calcium channel blocker verapamil. Secretion was evoked from the perfused glands by either transmural electrical stimulation or injection of nicotine or muscarine into the perfusion stream. Each condition of calcium deprivation was able to block nicotine- and electrically stimulated catecholamine release in an interval that left muscarine-evoked release largely unaffected. The above results demonstrate that muscarine-evoked catecholamine secretion from perfused rat adrenal glands can occur in the absence of extracellular calcium, presumably by mobilization of intracellular calcium. The latter may be due to muscarinic receptor-mediated generation of inositol trisphosphate.     

Calcium exposure required for full expression of injury in the calcium paradox

Isolated hearts repleted with calcium after a short period of perfusion with a calcium-free medium undergo the injury of the calcium paradox and release cellular protein. In the present experiments, 15 min perfusion with a calcium containing HEPES solution subsequent to 10 min calcium-free perfusion resulted in the loss of 42.7 +/- 3.9 mg of protein/g heart. If hearts were repleted with calcium for 30 s only, then returned to calcium-free perfusion, resultant protein loss was the same. When calcium repletion was further decreased to 20 s, 23.9 +/- 1.3 mg/g of protein was lost. This critical period coincided with the onset of contracture but was prior to major ion movements. It is concluded that the transition to irreversible injury occurs within 30 s of calcium repletion and that subsequent removal of extra-cellular calcium does not alter the course of events leading to cell death.     

Protective effect of adenosine against a calcium paradox in the isolated frog heart.

The effect of adenosine on the calcium paradox in the isolated frog heart was studied. Addition of adenosine during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein and creatine kinase release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that adenosine protected frog myocardial cells by reducing the massive calcium influx during reperfusion possibly through an action on calcium channels. Adenosine exerted its action in a dose-dependent manner; a concentration of 10 microM adenosine provided maximum protection of myocardial cells against the calcium paradox damage. Higher concentrations of adenosine produced side effects on both electrical and mechanical activity. These results are discussed in terms of the possible mechanism involved in the protective effect of adenosine.     

Potassium transport in the rabbit renal proximal tubule: Effects of barium,ouabain, valinomycin,and other ionophores

Summary Potassium fluxes in a suspension of rabbit proximal tubules were monitored using a potassium-sensitive extracellular electrode. Ouabain (10^–4 m) and barium (5mm) were used to selectively quantitate the potassium efflux pathway (105±5 nmol K⁺·mg protein^–1·min^–1) and the sodium pump-related potassium influx (108±7), respectively. These equal and opposite fluxes suggest that potassium accumulation in the cell occurs mainly through the sodium pump and that potassium efflux occurs mainly through barium-sensitive potassium channels. Thus the activity of the sodium pump (Na, K-ATPase) in the basolateral membrane of the proximal tubule is balanced by the efflux of potassium, presumably across the basolateral membrane, which has a high potassium permeability. In addition, the effect of valinomycin and other ionophores was examined on potassium fluxes and several metabolic parameters [oxygen consumption (QO₂), ATP content]. The addition of valinomycin to the tubules produced a net efflux of potassium which was quantitatively equivalent to the efflux produced by the addition of ouabain. The valinomycin-induced efflux was mainly due to the activity of valinomycin as a mitochondrial uncoupler, which indirectly inhibited the sodium pump by allowing a rapid reduction of the intracellular ATP. Amphotericin, nystatin, and monensin all produced large net releases of intracellular potassium. The action of the ionophores could be localized to the plasma or mitochondrial membrane and classified into three groups, as follows: (a) those which demonstrated full mitochondrial uncoupler activity (FCCP, valinomycin), (b) those which had no uncoupler activity (amphotericin B, nystatin); and (c) those which displayed partial uncoupler activity (monensin, nigericin).     

Reduced free radical generation during reperfusion of hypothermically arrested hearts

Several studies indicate the presence of hydroxyl radical (OH·) as well as its involvement in the myocardial reperfusion injury. A transition metal-like iron is necessary for the conversion of superoxide anion (O₂ ^–) to a highly reactive and cytotoxic hydroxyl radical (OH·). In the present study, we have examined the generation of OH· and free iron in reperfused hearts following either normothermic (37°C) or hypothermic ischemia (5°C). Employing the Langendorff technique, isolated rat hearts were subjected to global ischemia for 30 min at 37°C or 5°C and were then reperfused for 15 min at 37°C. The results of the study suggest that both the OH· generation in myocardium and free iron release into perfusate were significantly lower in hearts made ischemic at 5°C as compared to 37°C. Release of myoglobin and lactic acid dehydrogenase into perfusate also followed a similar pattern. Furthermore, in in vitro studies, chemically generated O₂ ^– at 5°C caused a significantly lower rate of oxidation of oxymyoglobin as well as generation of OH° and free iron as compared to 37°C. These results suggest that (1) reperfusion of hypothermic ischemic heart is associated with a reduction in the generation of OH· and cellular damage compared to that of normothermic ischemic heart, and (2) myoglobin, an intracellular protein, is a source of free iron and plays a role in the reperfusion injury mediated by free radicals.Abbreviations OH· hydroxyl radical - O₂ ^– superoxide anion - ODFR oxygen-derived free radicals - KHB Krebs-Henseleit buffer - LDH lactate hydrogenase - SOD superoxide dismutase     

Energy dependence of enzyme release from hypoxic isolated perfused rat heart tissue

There is a sudden release of intracellular constituents upon reoxygenation of isolated perfused hypoxic heart tissue (O2 paradox) or on perfusion with calcium-free medium after a period of hypoxia. Rat hearts were perfused by the method of Langendorff (Pfluegers Arch. 61: 291-332, 1895) with Krebs-Henseleit medium containing 10 mM glucose. Hearts were equilibrated for 30 min, followed by 90 min of hypoxia or 60 min of hypoxia and 30 min of reoxygenation. The massive enzyme release observed upon reoxygenation after 60 min of hypoxia was prevented by infusing 0.5 or 5 mM cyanide 5 min before reoxygenation. Lactate dehydrogenase (LDH) release commenced immediately upon withdrawal of cyanide. Hearts perfused with calcium-free medium throughout hypoxia did not release increased amounts of LDH at reoxygenation. Perfusing heart tissue with medium containing 0 or 25 microM calcium, but not 0.25 or 2.5 mM, after 50 min of hypoxia initiated a release of cardiac LDH, which was not further enhanced by reoxygenation. Enzyme release was significantly inhibited when the calcium-free perfusion medium included 10 mM 2-deoxyglucose (replacing glucose), 0.5 mM dinitrophenol, or 2.5 mM cyanide. Histologically, hearts perfused with calcium-free medium after 50 min of hypoxia showed areas of severe necrosis and contracture without any evidence of the contraction bands that were seen in hearts reoxygenated in the presence of calcium. Cardiac ATP and creatine phosphate (PCr) levels were significantly decreased after 50-60 min of hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovalent cation ionophores

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca²⁺-activated ATPase activity could remain unaltered.Gramicidin stimulated calcium uptake irrespective of the presence of a K⁺ gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K⁺ gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates.Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux were inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore.After cleavage of the 100 000 dalton ATPase to 50 000 dalton fragments, which was not associated with changes in Ca²⁺-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca²⁺-activated ATPase protein.     

Ca2+-Dependent and Ca2+-Independent [3H]GABA Release from Rat Brain Synaptosomes: the Effect of Temperature

The main inhibitory neurotransmitter GABA in the mammalian brain is distributed in the nerve terminals between two pools, vesicular (synaptic vesicles) and cytosolic. GABA is released from these pools by different mechanisms; there are calcium-activated exocytotic release and calcium-independent sodium-dependent release from the cytosolic pool (resulting from the membrane GABA transporter reversal). We investigated the influence of temperature on [³H]GABA release from rat brain synaptosomes, which was induced by stimulation of both these processes. In addition, we used -latrotoxin as a stimulant of [³H]GABA release. Synaptosomes from the rat brain were used in the experiments. 4-Aminopyridine (4-AP) and high [KCl] were applied to stimulate calcium-activated and calcium-independent [³H]GABA release, respectively. 4-AP-evoked [³H]GABA release was of the same intensity at 37 and 25°C (10.1 ± 1.2 and 10.1 ± 0.8% of total [³H]GABA incorporated into the synaptosomes, respectively). The effect of 4-AP on the ⁴⁵Ca²⁺ influx into synaptosomes was also temperature-independent: 0.775 ± 0.075 and 0.725 ± 0.100 nmol/min/mg of protein at 37 and 25°C, respectively. A drop in the effect of 4-AP was observed only at 15°C. When synaptosomes were depolarized with 50 mM KCl, a temperature decrease from 37°C to 25°C resulted in a twofold drop in the [³H]GABA release, from 20.5 ± 1.4 to 10.3 ± 0.7%; at 15°C [³H]GABA release dropped to less than one-third of the norm (6.0 ± 0.5%). -Latrotoxin-stimulated [³H]GABA release was diminished from 32.5 ± 2.5 at 37°C to 17.2 ± 1.3 at 25°C and 5.9 ± 0.4% at 15°C and was not affected by the presence or absence of calcium in the medium. It seems likely that the observed effect of temperature can be interpreted as based on the temperature dependence of the -latrotoxin insertion into the membrane. It is suggested that the pattern of the temperature sensitivity of GABA release from the synaptosomes can be used as a criterion for identification of the mode of neurotransmitter release.     

Measurement of calcium fluxes in plants using 45Ca

This paper deals with the effect of calcium binding in the cell wall on the measured ⁴⁵Ca influx in Chara corallina Klein ex Will. esk. R.D. Wood. Calcium in the cell wall was in the range 687–1197 (mol · m^–2 compared to the sap which contained only 144–256 mol · m^–2. In dilute culture solutions the calcium content of the cell wall was relatively independent of external calcium at concentrations above about 0.1 mol · m^–3. The half-times for exchange of calcium from ⁴⁵Ca-labelled cell walls varied from 45 min at 0.05 mol · m^–3 to less than 2 min at 2 mol · m^–3. The effectiveness of other cations in displacing calcium from cell walls was in the order La > Zn > Co > Ni > Mg. Rinsing of ⁴⁵Ca-labelled cell walls in 2 mol · m^–3 LaCl₃ for 20 min removed more than 99% of the bound ⁴⁵Ca. However, the residual ⁴⁵Ca activity in isolated cell walls following La³⁺ rinsing was similar to that in whole cells. It is concluded that in whole cells ⁴⁵Ca influx cannot normally be distinguished from extracellular binding of calcium. Methods are described for the measurement of ⁴⁵Ca fluxes in charophyte cells by isolation of intracellular ⁴⁵Ca after the uptake period using techniques which avoid contamination from the large amount of tracer bound in the cell wall. At an external calcium concentration of 1 mol · m^–3, the plasmalemma influx was approx. 0.2 nmol · m^–2 · s^–1 of which about half entered the vacuole and half was effluxed back into the external solution. The cytoplasm filled with calcium with a half-time of 40–50 min with an apparent pool size of 50 mmol · m^–3. After 2 h the net flux to the cell was almost the same as the vacuolar flux. The fluxes reported are an order of magnitude lower than previously reported calcium fluxes in plants.Abbreviations APW artificial pond water This work was supported by the Australian Research Council. The authors wish to thank Patrick Kee for his skilful technical assistance and Professor E.A.C. MacRobbie, University of Cambridge, UK, and Dr. M. Tester for helpful discussions.     

Effect of cooling rate on the survival of frozen wood frogs,Rana sylvatica

Summary Wood frogs (Rana sylvatica) were frozen to-2.5°C under five distinct cooling regimes to investigate the effect of cooling rate on survival. Frogs survived freezing when cooled at -0.16°C · h^-1 or -0.18°C · h^-1, but mortality resulted at higher rates (-0.30°C · h^-1,-1.03°C · h^-1, and -1.17°C · h^-1). Surviving frogs in the latter groups required longer periods to recover, and transient injury to the neuromuscular system was evident. Some of the frogs that died had patches of discolored, apparently necrotic skin; vascular damage, as indicated by hematoma, also occurred. It is concluded that slow cooling may be critical to the freeze tolerance of wood frogs. Additional studies examined the effect of cooling rate on physiological responses promoting freeze tolerance. Mean glucose concentrations measured in plasma (15–16 mol · ml^-1) and liver (42–45 mol · g^-1) following a 2-h thaw did not differ between slowly- and rapidly-cooled frogs but in both groups were elevated relative to unfrozen controls. Thus freezing injury to rapidly-cooled frogs apparently was not mitigated by the presence of elevated glucose. Water contents of liver tissue, measured 2 h post-thawing, did not differ between slowly-cooled (mean = 77.6%) and rapidly-cooled (mean = 78.5%) frogs. However, the mean hematocrit of slowly-cooled frogs (48%) was significantly higher than that (37%) of frogs cooled rapidly, possibly owing to differences in the dynamics of tissue water during freezing.     

Response of chloride efflux from skeletal muscle ofRana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl^– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl^– efflux. The pH-dependent Cl^– efflux can be described by the relationu=1/(1+10n(pK _a -pH)), whereu is the Cl^– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK _a is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK _a at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK _a suggests that the critical pH-sensitive moiety producing the pH-dependent Cl^– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl^– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl^– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k ^–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q₁₀ of 2.5. The Cl^– efflux inhibitor SCN^– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl^– efflux by DEPC. The findings that the apparent pK _a is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl^– efflux, and that this action is retarded by SCN^– supports the notion that protonation of histidine groups associated with Cl^– channels is the controlling reaction for the pH-dependent Cl^– efflux.     

Seasonal thermogenic capacity in a hibernator,Spermophilus richardsonii

Summary The maximum thermogenic capacity (HPmax) and the maximum capacity for non-shivering thermogenesis (NSTmax) were assessed in a hibernator, the Richardson's ground squirrel at different times of year. The HPmax was elicited by exposing animals to He–O₂ (21% oxygen, balance helium) at –10 to –25°C. The NSTmax was estimated by i.v. infusion of isoproterenol in anesthetized animals at thermoneutrality. Non-hibernating phase adults were collected and tested in April and June, and youngs in June and August for effects of seasonal acclimatization; animals were also tested after acclimation to cold (5°C) or warm (20°C). Hibernating phase animals were tested both shortly after the onset of hibernation season and after several months into the hibernation season. Although HPmax differed significantly between the lowest [101 cal (wt^0.73·h)^–1 in the June-Young group] and the highest [142 cal (wt^0.73·h)^–1 in the June-Adult group], it was not significantly different between other groups regardless of hibernation status or temperature acclimation (Fig. 4). The NSTmax, however, increased from 40–50 cal (wt^0.73·h)^–1 in the Warm-Acclimated, August-Young, June-Adult, and April-Adult to 66.5 and 79.2 cal (wt^0.73·h)^–1 in the two hibernating groups (Fig. 3). No significant difference in NSTmax was observed between Cold- and Warm-Acclimated groups. Since HPmax was maintained essentially constant at different times of year or after temperature acclimation, the increase of NSTmax during the hibernating phase can best be viewed as an adjustment for facilitation of periodic rewarmings from depressed body temperature during hibernation rather than to counter cold.Abbreviations HP heat production - HPmax maximum heat production - NST non-shivering thermogenesis - NSTmax maximum non-shivering thermogenesis - ST shivering thermogenesis - T _a ambient temperature - T _b body temperature     

Calcium uptake and survival of Bacillus stearothermophilus

The calcium transport in resting vegetative cells of Bacillus stearothermophilus was studied by determining the retention of ⁴⁵Ca in a membrane filter assay. The kinetics of death by vegetative cells, when suspended in buffer at 55°C, was also investigated. The calcium influx required the presence of an energy source, e.g. glucose-1-phosphate and the system exhibited saturation kinetics. The requirements for survival of the thermophilic cells reflected those of the calcium transport system. Thus, cells treated with nitrogen gas showed an increased thermal stability and a decreased efflux of calcium. The initial velocity of calcium influx correlated linearly with the survival of the cells after 1 min heating at 55° C. Lanthanum inhibited calcium influx and reduced survival. Magnesium did not inhibit calcium influx but could replace calcium as a stabilizing agent. The results suggest that the thermophilic cells are not intrinsically heat stable but survive due to a high cellular concentration of divalent ions.Abbreviations CFU colony forming units - CPM counts per min - NCA National canners association - CCCP carbonyl cyanide m-chlorophenylhydrazone - PMS phenazine methosulfate     

Pulmonary diffusing capacity in reptiles (Relations to temperature and O2-uptake)

Summary Pulmonary CO-diffusing capacity (D l _CO), lung volume, pulmonary perfusion and O₂-uptake were measured by non-invasive techniques in the lizardsVaranus exanthematicus andTupinambis teguixin (mean body weight 2.2 kg for both species).The CO-diffusing capacity was at 25–27°C 0.059 mlstpd·kg^–1·min^–1·Torr^–1 inVaranus, which is 47% greater than the value of 0.040 mlstpd·kg^–1·min^–1·Torr^–1 inTupinambis. The lung volume ofVaranus was 36 ml·kg^–1 and that ofTupinambis 20 ml·kg^–1. At 35–37°C the diffusing capacity of lizard lungs are about 25% of those for mammals of comparable size.InVaranus pulmonary CO-diffusing capacity increased with temperature from 0.027 mlstpd·kg^–1·min^–1·Torr^–1 at 17–19 °C to 0.075 mlstpd·kg^–1·min^–1·Torr^–1 at 35–37 °C. This change closely matched a concomitant increase of O₂-uptake. Pulmonary perfusion increased from 27 ml·kg^–1·min^–1 to 55 ml·kg^–1·min^–1 within this temperature range.The study emphasizes that pulmonary diffusing capacity cannot be fully evaluated without information on pulmonary perfusion and O₂-uptake. In reptiles and other ectotherms diffusing capacity must be reported at specified body temperature.     

Temperature insensitive O2 in blood of the tree frogChiromantis petersi

Summary Respiratory gas exchange and blood respiratory properties have been studied in the East-African tree frogChiromantis petersi. This frog is unusually xerophilous, occupies dry habitats and prefers body temperatures near 40°C and direct solar exposure. Total O₂ uptake was low at 81 l O₂·g^–1·h^–1±19.0 (SD) at 25°C increasing to 253.5 l O₂·g^–1·h^–1±94.8 (SD) at 40°C giving aQ ₁₀ value of 2.1. Skin O₂ uptake at 25°C was 38.5% of total. The gas exchange ratio was 0.71 for whole body gas exchange, 0.61 for the lungs and 1.02 for the skin at 25°C.Blood O₂ affinity was low with aP ₅₀ of 47.5 mmHg at 25°C and pH 7.65. Then _H-value at 25°C increased from 2.7 aroundP ₅₀ to 5.0 at O₂ saturations exceeding 70–80%. Surprisingly, blood O₂ affinity was nearly insensitive to temperature expressed by a H value of ±1.0 kcal·mole between 25 and 40°C.The adaptive significance of the low O₂ affinity, the increase ofn _H with O₂ saturation and the temperature insensitive O₂-Hb binding is discussed in relation to the high and fluctuating body temperatures ofChiromantis.     

Mitochondrial and cytoplasmic aspartate aminotransferase enzyme release in the calcium paradox

The release of mitochondrial and cytoplasmic aspartate aminotransferase (AST) enzymes from the myocardium was studied in the isolated rabbit heart under conditions of the calcium paradox. Four different periods of calcium-free perfusion for 10, 15, 20, and 25 min were selected to produce different degrees of the calcium paradox and the associated myocardial damage which was indicated by impairment in the left ventricular contractile function. Calcium-free perfusion periods of less than 20 min were associated with partial recovery of ventricular function, while periods of 20 min or greater were associated with little or no recovery of contraction after reperfusion with calcium. Mitochondrial (ASTm) and cytoplasmic (ASTc) aspartate aminotransferase were released from the heart beginning within 1 min of reintroduction of Ca2+. The cumulative amount of ASTm release was about one-tenth the amount of ASTc release. The cumulative amount of ASTm and ASTc released were significantly (p less than 0.05) related to the duration of calcium-free perfusion. The time to 90% of maximum AST release was slightly longer for ASTm compared with ASTc (6.8 +/- 0.6 vs. 5.7 +/- 0.5 min, 0.10 greater than p greater than 0.05). ASTc but not ASTm correlated significantly (p less than 0.05) with total protein release from the myocardium, while ASTm was not as consistently related to protein loss. The cumulative amount of ASTm and ASTc were inversely related to the extent of recovery of left ventricular contractile function. Disparities did occur as the longest duration of the calcium-free period, which did not produce any further damage to left ventricular function, was nonetheless associated with more enzyme release from the myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ischemia,calcium depletion and repletion,acidosis and hypoxia on cellular taurine content

Summary.  Occlusion of the left main coronary artery led to a time-dependent release of taurine from the heart. Upon reperfusion, there was a second phase of taurine release, which exceeded the amount of taurine that exited the heart during the 45 min ischemic insult. To obtain information on the mechanism underlying the release of taurine, three variables were examined, acidosis, hypoxia and calcium overload. It was found that large amounts of taurine also leave the cell during the calcium paradox, a condition induced by perfusing the heart with calcium containing buffer following a period of calcium free perfusion. However, little taurine effluxes the hearts exposed to buffer whose pH was lowered to 6.6. Isolated neonatal cardiomyocytes subjected to chemical hypoxia also lost large amounts of taurine. However, the amount of taurine leaving the cells appeared to be correlated with the intracellular sodium concentration, [Na⁺]_i. The data suggest that taurine efflux is regulated by [Na⁺]_i and cellular osmolality, but not by cellular pH. Received November 15, 2001 Accepted January 15, 2002 Published online October 3, 2002 Acknowledgements This study was supported with a grant from the Taisho Pharmaceutical Company. Authors' address: Dr. Stephen W. Schaffer, Department of Pharmacology, University of South Alabama, School of Medicine, Mobile, Alabama, U.S.A., E-mail: sschaffe@jaguarl.usouthal.edu