AndréB. Borle

Source of oxygen free radicals produced by rat hepatocytes during postanoxic reoxygenation

The aim of this study was to determine the cellular source of oxygen free radicals generated by isolated hepatocytes during post-anoxic reoxygenation. Superoxide anions (O2.-) were detected by lucigenin chemiluminescence. Cell damage was assessed by LDH release. During anoxia, the chemiluminescence decreased to background levels while LDH release increased 8-fold. During reoxygenation, O2.- formation increased 15-fold within 15 min then declined towards control levels. LDH release increased from 161 to 285 mU/min in the first 30 min of reoxygenation, then declined toward the control rate. Allopurinol, an inhibitor of the xanthine-xanthine oxidase system, did not inhibit O2.- formation nor LDH release. Antimycin, a mitochondrial complex III inhibitor that does not block O2.- formation, increased both O2.- generation and LDH release 82 and 133% respectively. Diphenyleneiodonium (DPI), a mitochondrial and microsomal NADPH oxidase inhibitor, reduced O2.- and LDH release 60-70%. SOD, which catalyzes the dismutation of O2.- to H2O2, was without effect on O2.- and LDH release, but TEMPO, a stable nitroxide which mimics SOD and easily penetrates the cell membrane, decreased O2.-86% without affecting LDH. These results suggest that mitochondria or microsomes are the principal sites of O2.- production during reoxygenation of isolated hepatocytes, whereas the cytosolic xanthine/xanthine oxidase system is apparently not involved.

Pitfalls of the 45Ca uptake method

Abstract The interpretation of the data derived from 45Ca uptake measurements by cells and tissues can be difficult. Several of these difficulties and possible misinterpretations are described: 1) 45Ca uptake is not equivalent to calcium influx; 2) interpretations based on the sole visual examination of 45Ca uptake curves can be misleading because an increased tracer uptake can coexist with a decreased calcium transport and vice-versa; 3) drugs and hormones can have diametrically opposite effects when they are tested at steady state on in nonsteady state conditions. It is concluded that 45Ca uptake curves must be kinetically analyzed and that the steady or non-steady state of the system must be known for a valid interpretation of such data.

Fasting enhances the effects of anoxia on ATP, Ca2+i and cell injury in isolated rat hepatocytes

The effect of fasting and anoxia on the intracellular concentration of ATP, Na+, Ca2+, Mg2+, and H+ was studied in isolated perfused rat hepatocytes. ATP and intracellular Mg2+ were measured by 31P-NMR spectroscopy, cytosolic free calcium was measured with aequorin, intracellular Na+ with SBFI, intracellular pH with BCECF, lactic dehydrogenase by NADH absorbance. In hepatocytes from fasted rats, intracellular ATP was depressed 52% (P < 0.001), Nai+ was increased 70% from 16.9 to 27.7 mM (P < 0.02), and Cai2+ was increased 79% from 137 to 245 nM (P < 0.05) when compared to fed rats. Mgi2+ and pHi were unchanged. During anoxia, ATP and the cell phosphorylation potential decreased 90% to practically the same low levels in both fed and fasted groups. On the other hand, in hepatocytes from fasted animals, Cai2+ increased faster and to significantly higher levels than in hepatocytes from fed rats: Cai2+ reached 2.19 microM in 10 min compared to 1.45 microM in 1 h, respectively (P < 0.05). Cell injury assessed by LDH release and trypan blue exclusion also occurred earlier and was more severe in hepatocytes from fasted rats. Fructose and Ca(2+)-free perfusion reduced the rise in Cai2+, abolished LDH release and significantly improved the cell viability measured by Trypan blue exclusion. The data demonstrate that fasting decreases the hepatocytes energy potential and increases Nai+ and Cai2+ which are inversely related to the cell energy potential. Consequently, in hepatocytes isolated from fasted rats, the increase in Cai2+ and the resulting cell injury evoked by anoxia occur earlier and are more severe than in fed rats. These results suggest that Ca2+ plays a crucial role in the development of anoxic cell injury.

Sex difference in cellular calcium metabolism of rat hepatocytes and in α-adrenergic activation of glycogen phosphorylase

Abstract Three topics were the subject of these investigations: (i) the difference between males and females in the basal calcium metabolism of hepatocytes; (ii) the source of the calcium which triggers the phosphorylase a stimulation induced by epinephrine through alpha-adrenergic receptors; (iii) the time relation between the rise in phosphorylase activity and the increase in calcium efflux. We found that there was no difference between males and females in total or exchangeable cell calcium. However, there were significant differences in the mitochondrial calcium pool and fluxes measured by steady-state kinetic analyses: they were smaller and the rate constants of mitochondrial calcium influx and efflux were lower in males than in females. The 45Ca content of isolated mitochondria and microsomes was also significantly lower in males than in females. In both males and females, epinephrine stimulated phosphorylase activity and calcium efflux even in the absence of extracellular calcium, indicating that the principal source of calcium which triggers the enzyme stimulation is intracellular. During the first 10 min following stimulation by 10−6 M epinephrine, the total cell calcium, 45Ca and the mitochondrial calcium were significantly depressed in male hepatocytes. After 10 min, these changes were reversed and the cell or mitochondrial calcium content was greater than in controls. In females, on the other hand, changes could only be detected if the cells were transferred to calcium-free media before the stimulation. In both males and females, there was a good temporal relationship between the stimulation of calcium efflux and the rise in phosphorylase a activity when hepatocytes were exposed to increasing concentrations of epinephrine: both rose at least 75% in less than 15 s. We conclude that there are important differences in cellular calcium metabolism between males and females. The rise in cytosolic calcium induced by alpha-adrenergic activation is principally due to a mobilization of calcium from an intracellular pool, probably the mitochondria.

Ca2+ antagonists do not protect isolated perfused rat hepatocytes from anoxic injury

Ca2+ antagonists were studied during anoxia in perfused isolated rat hepatocytes. Cytosolic free calcium (Ca2+i) was measured with aequorin. Anoxia was induced for 2 h by saturating the perfusate with 95% N2/5+ CO2. Anoxia increased Ca2+i in two distinct phases reaching a maximum of 1.5 microM. The increase in Ca2+i was caused by Ca2+ influx from the extracellular fluids because the main Ca2+i surge was totally abolished in Ca(2+)-free media. LDH release increased 6-fold during the second hour of anoxia, but when Ca2+ was removed from the perfusate during the anoxic period, LDH rose only 2.7-fold. Ca2+ antagonists (10(-7) to 10(-5) M) did not prevent the increase in Ca2+i and the rise in LDH release. On the contrary, high concentrations (10(-6) and 10(-5) M) of the blockers nifedipine and diltiazem significantly increased anoxic cell injury. The observation that the increase in LDH and the rise in Ca2+i were not suppressed by Ca2+ antagonists suggests that (i) Ca2+ antagonists protect the whole liver from anoxic injury by acting on cells other than parenchymal cells; (ii) the influx of Ca2+ responsible for the massive increase in hepatocyte Ca2+i evoked by anoxia did not take place through voltage-sensitive Ca2+ channels but must have occurred via the Na(+)-Ca2+ antiporter operating in the reverse mode (Ca2+ influx vs. Na+ efflux), and (iii) high concentrations of Ca2+ antagonists may be deleterious to the parenchymal cells of the liver.

The effects of hydrogen ions on the kinetics of calcium transport by rat kidney mitochondria

Abstract The influence of pH on the kinetics of the initial rate of calcium uptake by isolated kidney mitochondria was studied using the ruthenium red-ethylene glycol bis(β-aminoethyl ether) N,N -tetraacetic acid quench method (K. Reed and F. Bygrave, 1975, Eur. J. Biochem. 55 , 497–504). In the absence of phosphate, the K m is increased 50% and the V is decreased 57% when the pH is decreased from pH 7.4 to 7.0. Conversely, when the pH is increased to 7.8, the K m is decreased 25% while the V is not affected. The presence of 0.1 or 0.4 m m phosphate in the incubation medium abolishes the change in K m at a low pH while the V remains depressed by 36 and 25%, respectively. The presence of phosphate does not affect the decrease in the K m seen with an increased medium pH. Mitochondria incubated in steady-state conditions with a medium free calcium of 0.7 μ m also show significant changes in calcium exchange and distribution with pH. Two kinetic calcium pools are found in isolated mitochondria. Decreasing pH from 7.4 to 7.1 decreases mitochondria total calcium 32%, decreases the rapidly exchanging pool 28%, and depresses both the mitochondrial membrane and an intramitochondrial calcium exchange by 54 and 22%, respectively. Raising the pH to 7.7 increases both exchangeable pools (63 and 46%), and increases the mitochondrial membrane calcium exchange 44%. These results are consistent with previous studies on the influence of intracellular pH on calcium metabolism of kidney cells in which the mitochondrial pool was markedly affected by pH ( R. Studer, and A. Borle, 1979, J. Membrane Biol. 48 , 325–341 ). Alterations in cellular pH may modify mitochondrial calcium transport and cellular calcium metabolism and thus affect cell functions which are calcium dependent.

Effects of high and low pH on Ca2+i and on cell injury evoked by anoxia in perfused rat hepatocytes.

The effect of high and low pH on anoxic cell injury was studied in freshly isolated rat hepatocytes cast in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB) saturated with 95% O2 and 5% CO2. Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular pH (pHi) with BCECF, and lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate. A 2 h period of anoxia was induced by perfusing the cells with KHB saturated with 95% N2 and 5% CO2. The extracellular pH (pHo) was maintained at 7.4, 6.8 or 8.0 by varying the bicarbonate concentration. The substrate was either 5 mM glucose, 15 mM glucose or 15 mM fructose. In some experiments, anoxia was performed in Ca(2+)-free media by perfusing the cells with KHB without Ca2+ but with 0.1 mM EGTA. Reducing pHo to 6.8 during anoxia did not reduce the increase in Ca2+i, but but completely abolished LDH release. Under these conditions, pHi decreased to 6.56 +/- 0.3 when glucose was the substrate and to 6.18 +/- 0.25 with 15 mM fructose. Apparently, protection against anoxic injury caused by a low pHo is associated with a low pHi but not with a reduced elevation in Ca2+i. Increasing pHo to 8.0 during anoxia increased pHi above 8.0 +/- 0.01 and doubled LDH release without significantly altering the rise in Ca2+i. When 15 mM fructose was present with a pHo of 8.0, pHi was still 8.0, but there was practically no rise in Ca2+i, and LDH release was again completely abolished. On the other hand, a Ca(2+)-free perfusate with a pHo of 8.0 kept the rise in Ca2+i below 400 nM but did not abolish the massive release of LDH caused by high pH. Since cell injury is caused by the activation of Ca(2+)-sensitive hydrolytic enzymes such as phospholipase A2, these experiments suggest that a low pH (< 6.5) prevents their activation even in the presence of a high Ca2+i. Conversely, a high pH (> 8.0) can activate hydrolytic enzymes and cause injury even in the absence of an elevated Ca2+i. The precise mechanism by which fructose protects hepatocytes against cell injury at pHi 8.0 is unclear.

Effect of adrenalectomy on cellular calcium metabolism and on the response to adrenergic stimulation of hepatocytes isolated from male and female rats

The effects of adrenalectomy on cell calcium metabolism and on the effects of epinephrine on cAMP, phosphorylase a activity, and calcium efflux were studied in hepatocytes isolated from adult male and female rats. Adrenalectomy increased the total calcium of hepatocytes, all exchangeable calcium pools, and all calcium fluxes between the cellular pools in both sexes. After adrenalectomy, basal cAMP was elevated, phosphorylase a + b was decreased, but basal phosphorylase a activity was not changed. In adrenalectomized males and at all concentrations of epinephrine studied (1.10(-8)-1.10(-5)M) stimulation of calcium efflux was decreased and cAMP accumulation was enhanced, while the resulting phosphorylase a activation was depressed. In hepatocytes from adrenalectomized females there was a similar increase in cAMP accumulation induced by epinephrine, and a decrease in the stimulation of calcium efflux; however, the depression in phosphorylase a activation was much less and was significant only at 1.8(-8) and 1.10(-5)M epinephrine. In the male, while activation of phosphorylase a shifted from a pure alpha-adrenergic response mediated by calcium to one also involving a cAMP-mediated beta-adrenergic response, the contribution of the attenuated calcium signal was still significant. Hepatocytes from female rats did not show a comparable alpha- to beta-shift, since the relative contribution of calcium and cAMP to phosphorylase activation was similar in sham-operated and adrenalectomized animals.

Solutions for the kinetic analysis of 45calcium uptake curves

Abstract The classic solutions based on specific activity curves for the kinetic analysis of 45Ca movements in three compartment cellular systems cannot be used when the extracellular compartment is one to two orders of magnitude larger than the cellular or tissue compartments. However if the relative radioactivity curve (tracer uptake curve) is analyzed it is possible to calculate all the relevant kinetic parameters. This paper offers the solutions based on relative radioactivity measurements for the calculation of exchange rates, rate constants and compartment sizes of three compartment systems, for series and parallel cases, for closed and open systems.

Biochemical and 31P-NMR spectroscopic evaluation of immobilized perfused rat Sertoli cells.

Cell immobilization and perfusion are used for physiologic studies of Sertoli cells with phosphorus 31 nuclear magnetic resonance (NMR) spectroscopy and biochemical methods. In this study the 31P NMR spectra of Sertoli cells isolated from 18-to 21-day-old rats and immobilized in agarose threads continuously perfused with oxygenated Dulbeccos modifed Eagle medium were obtained at 81 MHz on an NMR system. Cytosolic Ca2+, intracellular Mg2+, lactate and pyruvate, and oxygen consumption were measured with standard biochemical methods. Perfused Sertoli cells maintain a stable intracellular adenosine triphosphate concentration for more than 10 hours. Sertoli cells placed in cold storage overnight and then subjected to perfusion partially regenerate cellular adenosine triphosphate levels. Sertoli cells consume an average of 4.8 +/- 0.4 nmol O2/min/10(6) cells and maintain average ambient lactate and pyruvate levels of 7.1 +/- 0.8 mg/dl and 0.65 +/- 0.05 mg/dl, respectively, with a lactate/pyruvate ratio in the range 8 to 12. The basal Ca2+(i) of Sertoli cells is 98 +/- 0.7 nmol/L (n = 58), which declines to a level less than 10 nmol/L when the Sertoli cells are perfused with a calcium-free medium. Perfusion of Sertoli cells with a sodium-free medium, with 10(-6) mol/L carbonyl cyanide P-trifluoromethoxy-thenylhydrozone, or with Ca2+ ionophore A23187 at a concentration of 10(-6) mol/L increases the Ca2+(i) to a level of 426 +/- 107 nmol/L, 274 +/- 29 nmol/L, or 282 +/- 57 nmol/L, respectively. A bioreactor for physiologic studies of Sertoli cells in real time with NMR spectroscopy has been developed. These data demonstrate that isolated, immobilized, and perfused Sertoli cells are stable for prolonged periods. In addition, these data suggest that Sertoli cells possess a functional Na+-Ca2+ antiporter and that they sequester extracellular Ca2+ in one or more intracellular compartments.