The effect of medium tonicity on the rate of respiration and oxidative phosphorylation in liver mitochondria of active and hibernating ground squirrels

The rate of respiration and ATP synthesis in liver mitochondria (M) isolated from hibernating ground squirrels and incubated in the medium with normal tonicity (250 mosm) was shown to be considerably lower than the rate of respiration and ATP synthesis in liver M from active animals. The increase of the medium tonicity to 600 mosm simulated the state of M from hibernating animals, resulting in a decrease of the respiration rate of M from active ground squirrels. On the contrary, the decrease of the tonicity to 60 mosm caused the activation of the respiration and increase of the ATP synthesis in M from hibernating ground squirrels. Bromophenacylbromide (BPhB), an inhibitor of phospholipase A2, prevented the activation of the respiration of M from hibernating animals incubated in the medium with low tonicity. BPhB had practically no effect on the respiration of M from both hibernating and active ground squirrels as well as on the swelling of M in hypotonic medium. It was concluded that the activation of the respiration and increase of the ATP synthesis rate in M from hibernating ground squirrels incubated in the medium with low tonicity is related to the activation of phospholipase A2. It was assumed that decrease of phospholipase A2 activity and change in the lipid composition of mitochondrial membrane may be one of the reasons for inhibition of the respiration rate in M from hibernating ground squirrels.     

Regulation of the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels, Citellus undulatus

1. The rates of oxidation of various substrates (beta-hydroxybutyrate, succinate, ascorbate + TMPD) and the rate of ATP synthesis in liver mitochondria from active and hibernating ground squirrels were measured. 2. It was shown that the rate of mitochondrial respiration is significantly lower in hibernating animals than in active animals. 3. The degree of inhibition of mitochondrial respiration in hibernating ground squirrels was found to correlate with the length of the respiratory chain fragment involved in the oxidation of a given substrate. 4. The inhibition of mitochondrial respiration in hibernating animals was accompanied by a decrease in the rate of ATP synthesis. 5. The activity of phospholipase A2 in liver mitochondria from hibernating ground squirrels was found to be decreased. The activation of phospholipase A2 by Ca2+ ions eliminated the inhibition of respiration almost completely. 6. It was assumed that the inhibition of mitochondrial respiration during hibernation is (a) related to the suppression of phospholipase A2 activity and (b) caused by the reduced rates of electron transport through the respiratory chain and/or of substrate transport across the mitochondrial membrane.     

Biosynthesis of dolichyl pyrophosphate N-acetylglucosaminyl intermediates in liver microsomes from hibernating ground squirrels (Citellus citellus L.)

Dolichyl pyrophosphate N-acetyl[14C]glucosamine was synthesized after incubation of liver microsomes from hibernating ground squirrels with UDP-N-acetyl[14C )glucosamine. The radioactivity of glycolipid formed by liver microsomes from hibernating ground squirrels was about 2-fold greater than by liver microsomes from active animals. Addition of exogenous dolichyl phosphate to the incubation mixture increased the formation of dolichyl pyrophosphate N-acetyl[14C]glucosamine by microsomes from both active and hibernating ground squirrels about 6 times. Liver microsomes from hibernating ground squirrels converted dolichyl pyrophosphate N-acetyl[14C]glucosamine into dolichyl pyrophosphate N,N'-diacetyl[14C]chitobiose in the presence of unlabelled UDP-N-acetylglucosamine. This conversion was maximal at 1.0 M concentration of unlabelled UDP-N-acetylglucosamine. The level of dolichyl phosphate assessed by the level of dolichyl pyrophosphate N-acetylglucosamine formation was nearly 2 times greater in liver microsomes from hibernating ground squirrels than from active animals.     

Biochemical bases of the inhibition and activation of liver mitochondrial respiration in hibernating susliks

The possible mechanisms in regulation of the respiration rate of mitochondria from liver of hibernating ground squirrels have been investigated. The inhibition of respiration has been shown to be mainly due to the inhibition of electron transfer to the respiratory chain from flavoproteins to cytochrome c. Calcium ions evoke the gradual increase in the respiration rate of mitochondria from liver of hibernating ground squirrels which is abolished by adding albumin, ruthenium red and sovcaine. The lower content of free fatty acids and decreased rate of the oxidation of exogenic NADH in the external pathway indicate the decrease in the activity of phospholipase A2 in mitochondria from liver of hibernating ground squirrels. The decreased calcium capacity of mitochondria indicates the higher sensitivity to calcium ions. A conclusion concerning the leading role of phospholipase A2 in Ca-induced activation of respiration of mitochondria from liver of hibernating ground squirrels is made.     

Mitochondrial alpha-glycerophosphate dehydrogenase activity in active and torpid ground squirrels, Spermophilus richardsoni.

1. Mitochondrial alpha-glycerophosphate dehydrogenase (GPD) activity was assayed in liver homogenates from active and torpid ground squirrels. 2. Arrhenius plots of GPD activity were linear in non-hibernating animals and discontinuous in hibernators. Compared with non-hibernators, the energy of activation in hibernators was reduced between 37 and 25 degrees C, but increased between 25 and 6 degrees C. 3. A dose-response relation between GPD activity and injected thyroxine was determined in active animals. No correlation was found between enzyme activity at 37 or 6 degrees C and circulating titres of thyroid hormones, in ground squirrels sampled during the preparative and hibernating phases.     

The intensity of oxidative phosphorylation and the function of the adenylate system in the liver mitochondria of active and hibernating susliks Citellus undulatus

The state of adenylate system and intensity of oxidative phosphorylation in liver mitochondria of active and hibernating ground squirrels were studied depending on the concentration of extramitochondrial Ca2+ ([Ca2+]ex). It was shown that at [Ca2+]ex.10(-7) M, the content of ATP as well as ATP/ADP ratio are slightly lower in the mitochondria of hibernating ground squirrels than in the mitochondria of active animals. The other parameters of the adenylate system under the same conditions differ insignificantly. [Ca2+]ex increase to 10(-6) M has little effect on the parameters of the adenylate system of active animals. On the contrary, the mitochondria of hibernating ground squirrels are strongly affected: the level of ATP is 1.5-fold and the ratio of ATP/ADP is almost 2-fold decreased. At both [Ca2+]ex the intensity of oxidative phosphorylation is essentially higher in the mitochondria of active ground squirrels. With increasing [Ca2+]ex the rate of ATP synthesis decreases, and in the mitochondria of hibernating animals the decrease is more pronounced than in the mitochondria of active animals. Thus, oxidative phosphorylation and adenylate system of mitochondria from hibernating ground squirrels are more sensitive to [Ca2+]ex increase than those of the mitochondria of active animals.     

Seasonal Changes in Microsomal Fraction Enriched with Na,K-ATPase from Kidneys of the Ground Squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

The Na,K-ATPase activity in microsomal fraction isolated from kidneys of winter hibernating ground squirrels was found to be 1.8–2.0-fold lower than that in active animals in summer. This is partially connected with a decrease in Na,K-ATPase protein content in these preparations (by 25%). Using antibodies to different isoforms of Na,K-ATPase α-subunit and analysis of enzyme inhibition by ouabain, it was found that the decrease in Na,K-ATPase activity during hibernation is not connected with change in isoenzyme composition. Seasonal changes of Na,K-ATPase a-subunit phosphory- lation level by endogenous protein kinases were not found. Proteins which could be potential regulators of Na,K-ATPase activity were not found among phosphorylated proteins of the microsomes. Analysis of the composition and properties of the lipid phase of microsomes showed that the total level of unsaturation of fatty acids and the lipid/protein ratio are not changed significantly during hibernation, whereas the cholesterol content in preparations from kidneys of hibernating ground squirrels is approximately twice higher than that in preparations from kidneys of active animals. However, using spin and fluorescent probes it was shown that this difference in cholesterol content does not affect the integral membrane micro-viscosity of microsomes. Using the cross-linking agent cupric phenanthroline, it was shown that Na,K-ATPase in mem- branes of microsomes from kidneys of hibernating ground squirrels is present in more aggregated state in comparison with membranes of microsomes from kidneys of active animals. We suggest that the decrease in Na,K-ATPase activity in kidneys of ground squirrels during hibernation is mainly connected with the aggregation of proteins in plasma membrane.     

The excitability of neurons of the medial septal area in brain slices from hibernating and active Citellus undulatus susliks]

Spontaneous and evoked neuronal activity of the medical septum-diagonal band complex (MS-DB) has been investigated in slices from the brain of hibernating and active ground squirrels, as well as guinea pigs. In all experimental groups, the majority of the MS-DB neurones exhibited high regular of rhythmic burst spontaneous activity which persisted even after synaptic blockade in half of the neuronal population. Under the same conditions, the activity of the surrounding structures was completely suppressed. The density of the spontaneously active neurones in the slices, as well as the mean frequency of discharges in the MS-DB of hibernating ground squirrels, were significantly higher than in active ground squirrels and guinea pigs. Stimulation of the medial forebrain bundle evoked initial suppression of the activity in the majority of MS-DB units; in many of them, the suppression was followed by a burst discharge. Neurones with background rhythmic burst activity always reacted by resetting the spontaneous bursts. In total, 50-60% of the MS-DB neurones in active ground squirrels and guinea pigs reacted by post-inhibitory bursts, whereas in hibernating animals these responses were observed nearly in all neurones. Threshold values of the stimulating current were lower in hibernating animals; the intraburst density of spikes was increased.     

Characteristics of sarcoplasmic reticulum membrane preparations isolated from skeletal muscles of active and hibernating ground squirrel Spermophilus undulatus

The total Ca-ATPase activity in the sarcoplasmic reticulum (SR) membrane fraction isolated from skeletal muscles of winter hibernating ground squirrel Spermophilus undulatus is 2.2-fold lower than in preparations obtained from summer active animals. This is connected in part with 10% decrease of the content of Ca-ATPase protein in SR membranes. However, the enzyme specific activity calculated with correction for its content in SR preparations is still 2-fold lower in hibernating animals. Analysis of the protein composition of SR membranes has shown that in addition to the decrease in Ca-ATPase content in hibernating animals, the amount of SR Ca-release channel (ryanodine receptor) is decreased 2-fold, content of Ca-binding proteins calsequestrin, sarcalumenin, and histidine-rich Ca-binding protein is decreased 3-4-fold, and the amount of proteins with molecular masses 55, 30, and 22 kD is significantly increased. Using the cross-linking agent cupric–phenanthroline, it was shown that in SR membranes of hibernating ground squirrels Ca-ATPase is present in a more aggregated state. The affinity of SR membranes to the hydrophilic fluorescent probe ANS is higher and the degree of excimerization of the hydrophobic probe pyrene is lower (especially for annular lipids) in preparations from hibernating than from summer active animals. The latter indicates an increase in the microviscosity of the lipid environment of Ca-ATPase during hibernation. We suggest that protein aggregation as well as the changes in protein composition and/or in properties of lipid bilayer SR membranes can result in the decrease of enzyme activity during hibernation.     

Production of tumor necrosis factor in cells of hibernating ground squirrels Citellus undulatus during annual cycle

TNF production has been studied in peritoneal macrophages and splenic T cells of Arctic Yakutian ground squirrel (Citellus Undulatus Pallas) in hibernating and awake animals in winter and in prehibernating autumn as well as in active euthermic spring-summer animals. A high level of TNF production in macrophages of ground squirrel is observed over the active period and during arousals in winter. There are no significant season variations in TNF production in splenic T lymphocytes of ground squirrels. This suggests the major role of activated macrophages in the arousals of hibernating animals. T lymphocyte proliferation in ground squirrels in the active period is higher than in winter, and the most significant seasonal variations are found in T cell mitogenic response, which increases in spring-summer period. Evidence is presented that functional activity of macrophages of squirrel in autumn has much in common with that in winter rather than in spring-summer period.     

The behavior of titin and the proteins of its family from skeletal muscles of ground squirrel (Citellus undulatus) during hibernation and rats under conditions of simulated microgravity

By the use of SDS PAGE, the behavior of titin and MyBP-C in fast (m. psoas) as well as titin and MyBP-X in slow (m. soleus) muscles of ground squirrels (Citellus undulatus) during hibernation was compared with the behavior of titin and MyBP-X in rat m. soleus under conditions of simulated microgravity. A decrease in the amount of titin 1 and MyBP-C relative to that of myosin heavy chains by approximately 30% and approximately 40%, correspondingly, in muscles of hibernating and arousing ground squirrels was revealed in comparison with active animals. No differences in the relative amount of MyBP-X in m. soleus of hibernating, arousing and active ground squirrels were found. Under conditions of simulated microgravity, a decrease in the amount of titin 1 by approximately 2 times and MyBP-X by approximately1.5 times relative to that of myosin heavy chains in rat m. soleus was observed. By the method of SDS PAGE modified by us, an almost twofold decrease in the amount of short isovariants of the titin N2A isoform relative to that of myosin heavy chains was shown in muscles of hibernating and arousing ground squirrels, whereas no changes were found in the amount of long titin isovariants. The conditions of simulated microgravity resulted in a twofold decrease in the relative amount of both short and long titin isovariants in rat m. soleus. The results indicate that hibernating ground squirrels have an evolutionarily determined adaptive mechanism of selective degradation of fast muscle fibers and preservation or increase of slow fibers, as the most economic and energetically advantageous, with proteins typical of them. The microgravitation of nonhibernating animals (rats) leads to a non-selective degradation of MyBP-X and titin isovariants, which contributes to considerable atrophy of soleus fibers.     

The inhibition of succinate, beta-oxybutyrate and glutamate transport in the liver mitochondria of hibernating susliks

Studies have been made on the permeability of the inner membrane of the liver mitochondria from hibernating and active ground squirrels for succinate, glutamate, hydroxybutyrate and inorganic phosphate. The permeability was calculated from the rate of mitochondrial swelling in 100 mM ammonium salts of the substrates and phosphate. It was shown that the rate of mitochondrial swelling in hibernating animals is 2--3 times lower than in active ones, being essentially identical in a solution of ammonium phosphate. It was concluded that the permeability of the inner mitochondrial membrane for the substrates decreases in hibernating animals, remaining unaffected for phosphate. Calcium-induced activation of membrane phospholipase A2 facilitates the transport of oxidative substrates into the mitochondria of hibernating ground squirrels, significant increase in the mitochondrial respiration being simultaneously observed. The data obtained suggest that inhibition of transport of oxidative substrates is one of the main factors which account for a low respiration rate in the mitochondria of hibernating animals.     

The rate and capacity of calcium uptake by sarcoplasmic reticulum in fast, slow, and cardiac muscle: effects of ryanodine and ruthenium red

The rate and capacity of oxalate-supported calcium uptake was measured in homogenates of rat fast, slow, and cardiac muscle. The contribution of the releasing fraction of the sarcoplasmic reticulum (SR) to the calcium uptake abilities was estimated using ruthenium red or ryanodine to block the release channel. A relatively small fraction (12-20%) of the calcium pumping activity was associated with the release channel in skeletal muscle compared to 50% or more in cardiac muscle. The total capacity of the SR in the muscle types was in the ratio 1:0.75:1.5 for cardiac, slow, and fast muscle, respectively, while the rates of uptake were in the ratio 1:3.8:14.4. The major difference in the muscle types appears to be the density of pumping activity in the SR rather than the volume of the SR. The difference in the density of pumping activity is due to intrinsic differences in the kinetics of the calcium pump units and in their surface density.     

Evaluation of the role of AMP-activated protein kinase and its downstream targets in mammalian hibernation

Mammalian hibernation requires an extensive reorganization of metabolism that typically includes a greater than 95% reduction in metabolic rate, selective inhibition of many ATP-consuming metabolic activities and a change in fuel use to a primary dependence on the oxidation of lipid reserves. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in this reorganization. AMPK activity and the phosphorylation state of multiple downstream targets were assessed in five organs of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) comparing euthermic animals with squirrels in deep torpor. AMPK activity was increased 3-fold in white adipose tissue from hibernating ground squirrels compared with euthermic controls, but activation was not seen in liver, skeletal muscle, brown adipose tissue or brain. Immunoblotting with phospho-specific antibodies revealed an increase in phosphorylation of eukaryotic elongation factor-2 at the inactivating Thr56 site in white adipose tissue, liver and brain of hibernators, but not in other tissues. Acetyl-CoA carboxylase phosphorylation at the inactivating Ser79 site was markedly increased in brown adipose tissue from hibernators, but no change was seen in white adipose tissue. No change was seen in the level of phosphorylation of the Ser565 AMPK site of hormone-sensitive lipase in adipose tissues of hibernating animals. In conclusion, AMPK does not appear to participate in the metabolic re-organization and/or the metabolic rate depression that occurs during ground squirrel hibernation.     

Effects of exercise of varying duration on sarcoplasmic reticulum function

Sarcoplasmic reticulum (SR) Ca2+ uptake and Ca2+-Mg2+-ATPase activity were examined in muscle homogenates and the purified SR fraction of the superficial and deep fibers of the gastrocnemius and vastus muscles of the rat after treadmill runs of 20 or 45 min or to exhaustion (avg time to exhaustion 140 min). Vesicle intactness and cross-contamination of isolated SR were estimated using a calcium ionophore and mitochondrial and sarcolemmal marker enzymes, respectively. Present findings confirm previously reported fiber-type specific depression in the initial rate and maximum capacity of Ca2+ uptake and altered ATPase activity after exercise. Depression of the Ca2+-stimulated ATPase activity of the enzyme was evident after greater than or equal to 20 min of exercise in SR isolated from the deep fibers of these muscles. The lowered ATPase activity was followed by a depression in the initial rate of Ca2+ uptake in both muscle homogenates and isolated SR fractions after greater than or equal to 45 min of exercise. Maximum Ca2+ uptake capacity was lower in isolated SR only after exhaustive exercise. Ca2+ uptake and Ca2+-sensitive ATPase activity were not affected at any duration of exercise in SR isolated from superficial fibers of these muscles; however, the Mg2+-dependent ATPase activity was increased after 45 min and exhaustive exercise bouts. The alterations in SR function could not be attributed to disrupted vesicles or differential contamination in the SR from exercise groups and were reinforced by similar changes in Ca2+ uptake in crude muscle homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Undirectional calcium and nucleotide fluxes in cardiac sarcoplasmic reticulum. II. Experimental results.

Unidirectional calcium influx and efflux were evaluated in cardiac sarcoplasmic reticulum (SR) by 45Ca-40Ca exchange at steady state calcium uptake in the absence of calcium precipitating anions. Calcium efflux was partitioned into a pump-mediated efflux and a parallel passive efflux by separately measuring passive efflux referable to the steady state. Unidirectional and net ATP-ADP fluxes were measured using [3H]-ATP----ADP and [3H]-ADP----ATP exchanges. Methods are presented that take into account changing specific activities and sizes of the nucleotide pools during the measurement of nucleotide fluxes. The contribution of competent and incompetent vesicles to the unidirectional and net nucleotide fluxes was evaluated from the specific activity of these fluxes in incompetent vesicles and from the fraction of vesicles that were incompetent. The results indicate that, in cardiac SR, unidirectional calcium fluxes are larger than the unidirectional nucleotide fluxes contributed by competent vesicles. Because the net ATPase rate of competent vesicles is similar to the parallel passive efflux, it appears that cardiac SR Ca-ATPase tightly couples ATP hydrolysis to calcium transport even at static head, with a coupling ratio near 1.0.     

Fatty acids and cholesterol in the liver cell nuclei of hibernating Yakutian ground squirrels

The content of neutral lipids in tissue homogenates and liver cell nuclei of hibernating Yakutian ground squirrels was studied. In homogenates, hibernation increases the content of fatty acids and reduces the content of glycerides and cholesterol. When studying the liver cell nuclei of torpid winter ground squirrels, we detected a twofold increase in the content of fatty acids, cholesterol, and monoglycerides as compared to the “summer” ground squirrels. In the active “winter” ground squirrels, as compared to the torpid winter ones, the content of cholesterol did not change, whereas the content of fatty acids, monoglycerides, and diglycerides decreased but remained higher than in the “summer” ground squirrels.     

Analysis of sarcoplasmic reticulum Ca2+ transport and Ca2+ ATPase enzymatic properties using mouse cardiac tissue homogenates

Recent studies have focused on developing transgenic mouse models to explore the physiological roles of sarcoplasmic reticulum (SR) calcium handling proteins. The goal of this study was to develop methodology to measure SR Ca2+ transport function and enzymatic properties of SR Ca2+ ATPase (SERCA) in individual mouse hearts. We describe here the procedures to specifically measure SR Ca2+ uptake, the formation and decomposition of SERCA phosphoenzyme intermediate (E-P) in mouse cardiac homogenates. The specificity of SERCA enzymatic activity in cardiac homogenates was established by (a) the selective inhibition of SERCA enzyme by inhibitor-thapsigargin, and (b) comparison of the kinetic parameters of SERCA activity between homogenates and isolated microsomes. Here we show that the apparent affinity of SERCA for Ca2+ and ATP, the time to reach steady-state levels of E-P, and the rate of E-P decomposition (turnover rate of SERCA enzyme) are similar in homogenates and microsomes. These studies demonstrate that SERCA Ca2+ transport and enzymatic properties can be accurately measured in mouse cardiac tissue homogenates. Additionally, we show that frozen cardiac homogenates can be used without significant loss of enzymatic activity. In conclusion, we have developed and established the methods to employ tissue homogenates to study SR Ca2+ transport function in individual mouse hearts.     

The paradoxically high reactivity of septal neurons in hibernating ground squirrels to endogenous neuropeptides is lost after chronic deafferentation of the septum from the preopticohypothalamic areas

The effect of neuropeptides (TSKYR, TSKY and DY) and neurotransmitters (serotonin and noradrenaline) on the activity of medial septum (MS) neurons from the brain of summer wakening ground squirrels (WGS), hibernating ground squirrels (HGS), and hibernating ground squirrels with the undercut septum (UHGS) was studied. It was shown that in HGS, the neuropeptides were substantially more effective in modulating the spontaneous activity of MS neurons than in WGS. The undercutting of MS led to the disappearance of the increased responsiveness to the neuropeptides: in UHGS, neuropeptide-induced changes in the spontaneous activity became nearly identical to those in WGS. The decrease in MS responsiveness in UHGS is due mainly to pacemaker neurons, which cease to respond to the peptides. It was shown that the neuropeptides have a dual effect: they change the level of spontaneous activity through direct modulation of pacemaker potential and control responses to electrical stimulation by modulating the synaptic transmission. Contrary to neuropeptides, neurotransmitters were highly effective in neurons of all groups of animals. Presumably, the enhanced excitability of MS during hibernation, which is necessary for performing the 'sentry post' function, is formed under the influence of the preopticohypothalamic area, and this influence is mediated by peptides.     

Phospholipase A activity determines the rate of respiration of the mitochondria in hibernating animals

The mechanisms for regulating the rate of respiration and oxidative phosphorylation in liver mitochondria from hibernating ground squirrels were studied. The microviscosity of the mitochondrial membrane in hibernating squirrels was found to be higher than that in active animals. Probably, a high microviscosity of the membrane causes a decreases in the rate of the transport of oxidation substrates into the mitochondrial matrix, which in turn may be one of the main reasons for the inhibition of mitochondrial respiration in hibernating squirrels. The activation of phospholipase A2 in a hypotonic medium results in the acceleration of the respiration and phosphorylation in the mitochondria from hibernating squirrels and is accompanied by the increase of the transport of substrates across the mitochondrial membrane. The inhibition of phospholipase A2 decreases Ca2+--induced acceleration of the transport of substrates and prevents the activation of the respiration and phosphorylation in a hypotonic medium.