Oscillatory activity of P-type membrane adenosine triphosphatases: a kinetic model.

A kinetic model for membrane P-type adenosine triphosphatases is considered, the main application being to the erythrocyte Ca2+-ATPase. It is shown that a simple modification of the known catalytic mechanism of the ATPase by addition of a self-inhibition step and the steady calcium influx leads to damped oscillations in the system discussed. In this way, the model can explain the kinetic experimental results obtained for the purified enzyme in solution as well as for the enzyme incorporated into liposome membranes. The estimated kinetic parameters are close to the experimental ones. Alternative changes in time, demonstrated by the kinetic model for the conformational enzyme states, E(1 )and E(2), confirm the model of two alternatively functioning gates in the ion pumping Ca2+-ATPase.

Influence of Ca2+ oscillatory influx on membrane Ca2+-ATPase activity: a kinetic model.

A kinetic model for the membrane Ca2+-ATPase is considered. The catalytic cycle in the model is extended by enzyme auto-inhibition and by oscillatory calcium influx. It is shown that the conductive enzyme activity can be registered as damped or sustained Ca2+ pulses similar to observed experimentally. It is shown that frequency variations in Ca2+ oscillatory influx induce changes of pulsating enzyme activity. Encoding is observed for the signal frequency into a number of fixed levels of sustained pulses in the enzyme activity. At certain calcium signal frequencies, the calculated Ca2+-ATPase conductivity demonstrates chaotic multi-level pulses, similar to those observed experimentally.

The evaluation of brain CBF and mitochondrial function by a fiber optic tissue spectroscope in neurosurgical patients.

The brain of neurosurgical patients are exposed to various manipulations in the ICU or during surgery. Under such conditions brain O2 balance may become negative and as a result brain vitality and function will deteriorate. In order to evaluate brain vitality in real time it is important to measure more than one parameter. The multiparametric monitoring system used in our previous study to monitor comatose patients (Mayevsky et al., Brain Res. 740: 268-274, 1996) was changed into a "simplified" tissue spectroscope for real time monitoring of brain O2 balance. Mitochondrial function was evaluated by monitoring the NADH redox state by surface fluorometry. Microcirculatory blood flow was assessed by laser Doppler flowmetry. The combined optical probe was located on the surface of the brain during various neurosurgical procedures and the responses were recorded and presented in real time to the surgeon. A total of 32 patients were monitored during various procedures. The results could be summarized as follows: 1. Hypercapnia led to 3 different types of responses. In two patients the 'stealing' like event was recorded. In the other 7 patients the responses to high CO2 was not detectable. In the last group of 6 patients a clear CBF elevation was recorded with variable response of mitochondrial NADH. 2. Our monitoring device was able to evaluate the efficacy of the STA-MCA anastomosis during aneurysm surgery. 3. A significant correlation was recorded between CBF and NADH redox state during changes in blood pressure, papaverine injection, spontaneous drop in blood supply to the brain or during releasing of high ICP levels. We conclude that in order to evaluate the metabolic state of the brain during neurosurgical procedures it is necessary to monitor both CBF and mitochondrial NADH by using the tissue spectroscope.

Brain multiparametric responses to carbon monoxide exposure in the aging rat.

The multiparametric monitoring system was applied to study the effects of 2000 ppm carbon monoxide (CO) on brain functions in vivo in the aging rat. The vasodilatory (non hypoxic) effects of CO on CBF in normal adult rats, which were shown in concentrations of 1000-2000 ppm involved the effect of nitric oxide (NO). Energy metabolism was evaluated by optical monitoring of CBF and mitochondrial function by fluorometry of NADH. Ionic homeostasis was evaluated by monitoring the extracellular level of K(+) and H(+) and the DC steady potential. Seven aging rats (24 months) were exposed to 2000 ppm for 60 min and 120 min of recovery, while five control rats were exposed to air under the same conditions. A comparison between the CO group and the control group showed that the changes in CBF, NADH and light reflectance were not statistically significant while extracellular K(+) was elevated and tissue pH became more acidic. Thus, the typical CO induced increase in CBF, was not recorded in the aging rats. We concluded that the brain vasodilatory response to CO was not active in the aging rat, while the ionic homeostasis responses were similar to those found in the adult rat.

Thiopental induced cerebral protection during ischemia in gerbils.

Temporary interruption or reduction of cerebral blood flow during cerebrovascular surgery may rapidly result in ischemia or cerebral infarction. Thiopental has been shown to have cerebroprotective effects. However, the cerebroprotective dose of thiopental causes burst suppression of the EEG, thus this parameter cannot be used continuously for the detection of metabolic changes in the brain during thiopental anaesthesia. This study was performed in order to examine whether the multiparametric assembly (MPA), which measures energy metabolism CBF and mitochondrial (NADH) as well as extracellular ion concentrations (K+), can shed light on the mechanism of the cerebroprotective effects of thiopental. The MPA was placed on the brain of Mongolian gerbils and burst suppression of the ECoG was induced by thiopental. Cerebral ischemia was induced by occlusion of carotid arteries after burst suppression. Burst suppression of the ECoG was accompanied by a significant decrease in cerebral blood flow. In animals that received thiopental prior to ischemia, NADH increased to a lesser degree and extracellular potassium ion concentration increased to a lesser degree than in the control animals, indicating that thiopental affords protection of the brain under ischemic conditions due to improved energy metabolism. This study also demonstrates that the MPA can monitor changes occurring in the cerebral cortex even after the ECoG can no longer be used. Those findings have a significant value in the development of a new clinical monitoring device.

Multiparametric evaluation of brain functions in the Mongolian gerbil in vivo.

We have developed the multiprobe assembly (MPA) by which metabolic, ionic and electrical activities can be monitored from the surface of the brain. In the present study we included optical fibers for the monitoring of intracapillary hemoglobin oxygenation by use of the Erlangen Microlight Guide Spectrophotometer (EMPHO-I) from the surface of the gerbil brain. The newly developed MPA provides simultaneous information about oxygen delivery (oxydeoxy Hb), tissue pO2 level, as well as the intracellular oxygen balance (intramitochondrial redox state). The ionic homeostasis was evaluated by monitoring extracellular K+ and Ca2+ activities reflecting the permeability changes of cation channels as well as the activities of Na+,K(+)-ATPase and other ion linked transport processes. The electrical activities were monitored by a bipolar electrocortical surface probe and DC steady potential. The subjects of the present study were Mongolian gerbils (Meriones unguiculatus) anesthetized and operated according to our routine techniques. After 30 min of recovery from the operation each gerbil was exposed to a short anoxia, graded hypoxia, ischemia as well as spreading depression. The results can be summarized as follows: 1. A clear correlation was recorded between the changes in oxydeoxy Hb spectra, tissue pO2 level and oxidation-reduction state of intramitochondrial NADH under oxygen deficiency situations (hypoxia, ischemia). 2. Blood volume changes under various perturbations monitored by various probes (366 reflectance and EMPHO-I) correlated very well with each other. 3. The degree of inhibition of Na+,K(+)-ATPase induced by oxygen deficiency could be interpreted by changes in extracellular levels of K+ measured by the surface mini-electrode. 4. Brain stimulation induced by spreading depression mechanism led to transient changes in ionic homeostasis and increase in energy requirements. The major HbO2 response was an increase in oxygenation due to the large CBF increase as monitored by the laser Doppler flowmeter. 5. Changes in oxy-deoxy Hb under fast scanning of 500-600 nm during 2-3 seconds of bilateral carotid arterial occlusion provided an indirect index for tissue O2 consumption.

Age dependence of steady state mitochondrial oxidative metabolism in the in vivo hypoxic dog brain.

Mitochondrial bioenergetics were investigated in newborn, neonatal and adult dog brains during normoxia and hypoxia. The ratio of the rate of ATP synthesis to the maximum synthesis rate (V/Vmax), phosphorylation potential, [ADP] and PCr/Pi, were used to evaluate age related mitochondrial hypoxic tolerance. These indicators were calculated from the phosphorus compounds measured by in vivo 31P MRS quantitatively using ATP as an internal reference. Indicators and substrates of mitochondrial function, V/Vmax, ADP, and Pi reached a peak value during the neonatal (3-21 days) period of development, suggesting that the oxidative metabolism of the neonate is more vulnerable to stress when compared to newborns and adults. Distinction among newborns and neonates became apparent during hypoxia. Newborns (0-2 days old) showed substantial tolerance by maintaining V/Vmax until exposure to severe hypoxia. Older neonates (3-21 days old) showed increases in V/Vmax, [Pi] and [ADP] under less than severe conditions of hypoxia. Adults exhibited low V/Vmax values even during exposure to severe hypoxia, further indicating that mitochondrial oxidative processes are more stable in adults than in newborns and neonates. This study provides evidence that newborns and adults are more capable of maintaining mitochondrial function under conditions of minimal to moderate hypoxia than 3-21 day old neonates.

In vivo mechanisms of myocardial functional stability during physiological interventions.

Metabolic regulatory mechanisms are designed to maintain stable myocardial function during extremes in physiological insult; they can now be studied in vivo and may provide insight into mechanisms of altered myocardial functional decompensation during disease processes. To determine mechanisms of myocardial stability during hypoxia and acute pressure loading, creatine kinase (CK) kinetics (forward rate constant, Kf, and flux of phosphocreatine, PCr, to adenosine triphosphate, ATP), and nicotinamide adenine dinucleotide (NADH) redox state were determined with 31P nuclear magnetic resonance (NMR) and NADH fluorometry, respectively, and correlated with heart work (heart rate x systolic blood pressure, HR x SBP), cardiac output (CO) and O2 consumption (MVO2) in 15 anesthetized open chest dogs. Hypoxia (PaO2 of 30-35 mm Hg) was produced in 6 dogs with an inspired O2/N2 of 200/3,000. Cardiac loading was produced in 9 dogs by administration of norepinephrine (NE, 1 micrograms/kg/min). Each dog acted as its own control. Baseline NADH fluorometry, 31P-NMR saturation transfer and cardiac function measurements were performed simultaneously in each dog, after which the experimental interventions were made. Similar increases in HR x SBP, CO, and MVO2 which occurred during both interventions were associated with different bioenergetic responses. During NE infusion, the Kf of CK increased from control; during hypoxia, the Kf decreased from control (p less than 0.05). Flux of PCr----ATP was significantly lower during hypoxia than during NE infusion (p less than 0.05). PCr was decreased significantly during NE infusion (p less than 0.05). In addition, NADH redox state increased (from baseline of 100%) during hypoxia (140 +/- 10%) and decreased during NE infusion (78 +/- 6%).(ABSTRACT TRUNCATED AT 250 WORDS)

Brain oxidative metabolism of the newborn dog: correlation between 31P NMR spectroscopy and pyridine nucleotide redox state.

The effects of both anoxia and short- and long-term hypoxia on brain oxidative metabolism were studied in newborn dogs. Oxidative metabolism was evaluated by two independent measures: in vivo continuous monitoring of mitochondrial NADH redox state and energy stores as calculated from the phosphocreatine (PCr)/Pi levels measured by 31P nuclear magnetic resonance (NMR) spectroscopy. The hemodynamic response to low oxygen supply was further evaluated by measuring the changes in the reflected light intensity at 366 nm (the excitation wavelength for NADH). The animal underwent surgery and was prepared for monitoring of the two signals (NADH and PCr/Pi). It was then placed inside a Phosphoenergetics 260-80 NMR spectrometer magnet with a 31-cm bore. Each animal (1-21 days old) was exposed to short-term anoxia or hypoxia as well as to long-term hypoxia (1-2 h). The results can be summarized as follow: (a) In the normoxic brain, the ratio between PCr and Pi was greater than 1 (1.2-1.4), while under hypoxia or asphyxia a significant decrease that was correlated to the FiO2 levels was recorded. (b) A clear correlation was found between the decrease in PCr/Pi values and the increased NADH redox state developed under decreased O2 supply to the brain. (c) Exposing the animal to moderately long-term hypoxia led to a stabilized low-energy state of the brain with a good recovery after rebreathing normal air. (d) Under long-term and severe hypoxia, the microcirculatory autoregulatory mechanism was damaged and massive vasoconstriction was optically recorded simultaneously with a significant decrease in PCr/Pi values.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dibromochloropropane and ethylene dibromide on biochemical events and ultramorphology of ejaculated ram spermatozoa in vitro.

The effects of dibromochloropropane (DBCP) and ethylene dibromide (EDB) on the function and ultrastructure of freshly ejaculated washed ram spermatozoa were assessed. These two compounds inhibited the collective motility of the sperm in a dose-dependent fashion when the sperm cells generated their energy either by mitochondrial respiration (2-deoxyglucose-treated sperm cells) or by the intact energy production system. DBCP and EDB inhibited the oxygen uptake by the sperm in a dose-dependent manner. No change in lactic acid accumulation and glucose utilization by the sperm cells was noted following DBCP and EDB addition. No change in the collective motility of the sperm was noted when DBCP or EDB were added to spermatozoa treated with the electron transfer inhibitor, antimycin A. Electron microscopy studies of sperm cells treated with DBCP revealed lesions in the plasma membrane adjacent to the acrosome and in the acrosomal membrane forming vesiculations. The inner membrane and the matrix space of the mitochondria were condensed following DBCP treatment, leaving a large mitochondrial peripheral space, compared with the control. EDB, at the concentration studied, caused no change in the ultramorphological structure of the sperm. DBCP was more potent, at least 4-fold, compared with EDB. An in vitro direct effect of DBCP and EDB on ram spermatozoa was established. It is suggested that quantitative measurements of sperm collective motility derived by different metabolic pathways can be used as an in vitro toxicological model for evaluation of toxicological and environmental factors affecting biological systems.

Metabolic, ionic, and electrical responses of gerbil brain to ischemia.

The effects of short- or long-term complete cerebral ischemia were studied in the gerbil brain using a multi-parameter monitoring system. Metabolic (NADH redox state) and hemodynamic responses were monitored by surface fluorometry-reflectometry. Ionic activities (K+ and pH) were measured by surface macroelectrodes. Electrical activity was evaluated by monitoring the general electrocorticogram (ECoG) as well as local DC steady potential (two sites). Two groups of gerbils were studied to compare the effects of 4-5 min occlusions with those of 30 min complete ischemia. During bilateral carotid artery occlusion the cortex is exposed to complete ischemia resulting in the complete depletion of O2 with attendant maximal reduction of NADH. Extracellular K+ began to increase as soon as energy reserves were decreased with a time course suggesting two different kinetic areas. Surface pH decreased very shortly after the occlusion. During the recovery phase, NADH was reoxidized soon after recirculation, whereas the pH and K+ recovery showed a short delay. ECoG did not recover even when all other parameters reached base-line levels. The recovery of all the measured parameters was correlated to the duration of the ischemic insult; i.e., the recovery from 30 min of ischemia took significantly longer than after 5 min of ischemia. We conclude that pH recovery depends on recirculation and adequate O2 supply to the tissue, whereas K+ recovery required not only an adequate O2 supply but also the integrity of the adenosine triphosphatase system.

Metabolic changes induced in rat hippocampal slices by norepinephrine.

The oxidative metabolic activity of restricted regions of hippocampal slices was assessed by a continuous measurement of the fluorescence of intramitochondrial nicotinamide-adenine dinucleotide (NADH). A large increase in NADH fluorescence was triggered by substituting the oxygen supply to the slice by nitrogen gas. A large and transient increase in NADH fluorescence was also produced by superfusion of the the slice with a high (50 mM) potassium-containing medium. Addition of norepinephrine (NE) to the superfusion medium caused a propranolol-inhibited increase in NADH fluorescence. Furthermore, ouabain, which inhibits the Na-K pump, blocked the effects of NE. An analog of cyclic adenosine monophosphate (cAMP), 8-bromo cAMP, mimicked the effect of NE. Finally, effects of NE could still be produced in a kainic acid-treated hippocampus, where most neurons were previously destroyed by the drug. It is suggested that NE activates a Na-K-ATPase, that this effect might be mediated by cAMP, and that these interrelations may underly the physiological action of NE in the brain.