Norepinephrine-stimulated increase in Na+, K+-ATPase activity in the rat brain is mediated through alpha1A-adrenoceptor possibly by dephosphorylation of the enzyme

Rapid eye movement sleep deprivation is reported to increase Na+,K+-ATPase activity. This increase was shown earlier to be stimulated by norepinephrine acting on alpha1-adrenoceptor. The involvement of a subtype of alpha1-adrenoceptor and the possible molecular mechanism of action of norepinephrine in increasing the enzyme activity were investigated using receptor agonists and antagonists, as well as stimulants and blockers of signal transduction pathway. It was observed that incubation of the homogenate with cyclic AMP, forskolin, A23187 (a calcium ionophore), or calmodulin alone did not stimulate the Na+,K+-ATPase activity. However, although the spontaneous activity of the Na+,K+-ATPase was not affected by prazosin, WB4101, heparin, W13, or cyclosporin A alone, each of them could prevent the norepinephrine-stimulated increase in the enzyme activity. Based on these results and our previous findings, it is proposed that norepinephrine acted on alpha1A-adrenoceptor and increased intracellular calcium, which in the presence of calmodulin activated a calmodulin-dependent phosphatase, calcineurin. This calcineurin possibly dephosphorylated Na+,K+-ATPase and increased its activity. The physiological significance especially in relation to rapid eye movement sleep deprivation is discussed.     

快速眼动睡眠产生的神经机制:蓝斑核神经元停止发放是一个必要的条件

两种类型的神经元参与了快速眼动（rapid eye movement,REM）睡眠的调节：快速眼动一发放（REM-ON）神经元和快速眼动-沉寂神经元（REM-OFF）。快速眼动-沉寂神经元属去甲肾上腺素能神经元,正如名字表示的那样——在快速眼动睡眠期间停止发放。已有研究表明,这些神经元放电活动的停止是导致快速眼动睡眠的前提条件,γ-氨基丁酸（γ-aminobutyric acid,GABA）可使它们停止发放。如果这嗤神经元不停止发放,脑中的去甲肾上腺素水平将升高,不出现快速眼动睡眠。剥夺快速眼动睡眠所引起的去甲肾上腺素增加,至少是快速眼动睡眠丧失引起Na^＋-K^＋ATP酶活性增加的原因,而这可能是导致快速眼动睡眠剥夺所引发的各种效应的主要因素。     

Norepinephrine induced alpha-adrenoceptor mediated increase in rat brain Na-K ATPase activity is dependent on calcium ion

It has been reported that norepinephrine increases Na-K ATPase activity by acting on -1 adrenoceptors. The mechanism of such an increase was investigated. The norepinephrine induced increase in synaptosomal Na-K ATPase activity was prevented by pretreating the rat brain homogenate with either EDTA, a divalent cation chelator or prazosin, an -1 adrenoceptor blocker. The norepinephrine and EGTA increased the Na-K ATPase activity in the synaptosome prepared from rat brain homogenate untreated with EDTA. The EGTA was ineffective in stimulating the enzyme activity if the synaptosome was prepared from homogenate treated with norepinephrine. However, the EGTA was effective in increasing the enzyme activity if the synaptosome was prepared from the homogenate treated with norepinephrine in the presence of prazosin.

Thus, norepinephrine did not increase the Na-K ATPase activity in the presence of EDTA or -1 adrenoceptor blocker. Similarly, the Ca⁺⁺ chelator, EGTA, could not increase the enzyme activity if the homogenate was pretreated with norepinephrine alone. However, if norpeinephrine action was blocked by -1 antagonist prazosin, EGTA increased the enzyme activity possibly by chelation of Ca⁺⁺. Further, chlorotetracycline fluorescence study showed that norepinephrine removes membrane bound Ca⁺⁺. Thus, it is likely that norepinephrine acts on adrenoceptors and removes membrane bound Ca⁺⁺ and thereby increases the Na-K ATPase activity in the synaptosome.     

Tyrosine hydroxylase activity in rat brain following "REM" sleep deprivation

I n R ecent years biogenic amines have been implicated in the control mechanism for induction and maintenance of sleep processes (J ouvet , 1969). Investigators have looked for changes in the rate of synthesis of cerebral norepinephrine from [³H]tyrosine after REM sleep deprivation and reported increased rates of synthesis during REM sleep deprivation (M ark , H einer , M andel and G odin , 1969) and REM sleep rebound following 91 h of deprivation (P ujol , M ouret and G lowinski , 1968). Because tyrosine is thought to be the rate-limiting enzyme (U denfriend , 1966) in the synthetic pathways for norepinephrine and since the above-mentioned studies are suggestive of changes in the activity of the enzyme, we decided to measure tyrosine hydroxylase activity following REM sleep deprivation.     

Kinetic characteristics of hamster (Mesocricetus auratus) brown fat (Na+/K+)-ATPase: effects of catecholamines

1. The (Na+/K+)-ATPase activity of brown fat membranes is increased by norepinephrine, the physiological mediator of thermogenesis in this tissue. 2. This increased ATPase activity was inhibited approximately 50% by either propranolol (a beta-adrenergic blocker) or phentolamine (an alpha-blocker). 3. The alpha-agonist, phenylephrine and the beta-agonist, isoproterenol, also stimulated the ATPase activity. 4. That these latter effects were receptor-specific is supported by the finding that: (a) l(-)isoproterenol stimulation was inhibited by propranolol but not by phentolamine; (b) d(+)isoproterenol had no stimulatory effect on the ATPase activity; and (c) the l(-)phenylephrine-induced increase was inhibited by phentolamine but not by propranolol. 5. (-)norepinephrine, l(-)isoproterenol and l(-)phenylephrine all decreased the apparent Km for K+ of the (Na+/K+)-ATPase but did not alter the apparent Km for ATP or the Vmax of the reaction.     

Study of protective effects of exogenous heat shock protein 70 kDa in model of sleep deprivation in pigeon <Emphasis Type="Italic">Columba livia</Emphasis>

Electroencephalographic methods were used to study effects of preparation of the exogenous heat shock protein with molecular mass of 70 kDa (Hsp70i/Hsc70) on time characteristics of sleep and wakefulness, brain temperature, peripheral vasomotor reactions, and thoracic muscle contractile activity after the 5-hour forceful sleep deprivation in the pigeon Columba livia. Administration of Hsp70i/Hsc70 into the third brain ventricle at once after the end of sleep deprivation eliminated disturbances in the sleep-wakefulness cycle organization and decreased the thoracic muscle contractile activity and the brain temperature as early as for the first hour of postdeprivation period. For the subsequent hours, the Hsp70i/Hsc70 action was characterized by an increase of the total time of deep sleep and a decrease of the total time of the rapid eye movement sleep. We suggest that the protective effects of the exogenous Hsp70i/Hsc70 preparation are associated with its ability to decrease activity of the hypothalamo-pituitary-adrenal axis and to enhance the stress-limiting function of the slow eye movement sleep.     

Phosphatidylserine and calmodulin effects on Ca2+-stimulated ATPase activity of dog brain synaptosomal plasma membranes

Phosphatidylserine (PtdSer)-liposomes when incubated with synaptosomal plasma membranes (SPM) of dog brain, evoked a significant increase (approx 80%) of the Ca2+-stimulated ATPase activity with maximal effect achieved at around 0.7 mumol PtdSer/mg SPM protein. Higher concentrations of PtdSer led to inhibition of the enzyme activity with respect to the maximal percentage of stimulation. Treatment of SPM with EGTA, to minimize the presence of bound cytoplasmic activator calmodulin, resulted in a mixed mechanism of inhibition of the enzyme activity (Vmax was decreased and Km increased) as estimated by Lineweaver-Burk plots. Addition of exogenous calmodulin resulted in an increase of Vmax and in a restoration of Km to control value. Ca2+-stimulated ATPase activity, in EGTA-treated SPM, showed the same figure of changes at different concentrations of PtdSer-liposomes as those of the control, but the turning point was now located at higher PtdSer concentrations. The results suggest that Ca2+-stimulated ATPase activity of SPM is modulated by PtdSer and that calmodulin participates in these interactions, probably, by regulating the contact between the enzyme and Ca2+ ions.     

Potentiation of ketamine effects on the spiking activity in the lateral geniculate nucleus by rapid eye movement (REM) sleep deprivation.

In cats prepared for chronic recording of sleep, an investigation was made on the effects of an anaesthetic agent, ketamine [cl-581, 2-(O-chlorophenyl)-2-methylaminocyclohexamine HCl] and rapid eye movement (REM) sleep deprivation on spiking activity recorded from lateral geniculate (LGN) nucleus. In normal cats most of the LGN spikes occurring during sleep are found in REM sleep. Follwoing injection of 10 mg/kg of ketamine a substantial increase of slow wave sleep (SWS) spikes occurred. While selective REM sleep deprivation had the same effects, combined influences of ketamine and REM-sleep deprivation led to a marked potentiation of their individual effects probably by simultaneous stimulation of the neurone system which determines the endogenous electrical activity of LGN cells.     

Effects of selective sleep deprivation on ventilation during recovery sleep in normal humans.

To assess the effects of selective sleep loss on ventilation during recovery sleep, we deprived 10 healthy young adult humans of rapid-eye-movement (REM) sleep for 48 h and compared ventilation measured during the recovery night with that measured during the baseline night. At a later date we repeated the study using awakenings during non-rapid-eye-movement (NREM) sleep at the same frequency as in REM sleep deprivation. Neither intervention produced significant changes in average minute ventilation during presleep wakefulness, NREM sleep, or the first REM sleep period. By contrast, both interventions resulted in an increased frequency of breaths, in which ventilation was reduced below the range for tonic REM sleep, and in an increased number of longer episodes, in which ventilation was reduced during the first REM sleep period on the recovery night. The changes after REM sleep deprivation were largely due to an increase in the duration of the REM sleep period with an increase in the total phasic activity and, to a lesser extent, to changes in the relationship between ventilatory components and phasic eye movements. The changes in ventilation after partial NREM sleep deprivation were associated with more pronounced changes in the relationship between specific ventilatory components and eye movement density, whereas no change was observed in the composition of the first REM sleep period. These findings demonstrate that sleep deprivation leads to changes in ventilation during subsequent REM sleep.     

Noradrenergic β-Adrenoceptor-Mediated Intracellular Molecular Mechanism of Na–K ATPase Subunit Expression in C6 Cells

Rapid eye movement sleep deprivation-associated elevated noradrenaline increases and decreases neuronal and glial Na–K ATPase activity, respectively. In this study, using C6 cell-line as a model, we investigated the possible intracellular molecular mechanism of noradrenaline-induced decreased glial Na–K ATPase activity. The cells were treated with noradrenaline in the presence or absence of adrenoceptor antagonists, modulators of extra- and intracellular Ca⁺⁺ and modulators of intracellular signalling pathways. We observed that noradrenaline acting on β-adrenoceptor decreased Na–K ATPase activity and mRNA expression of the catalytic α2-Na–K ATPase subunit in the C6 cells. Further, cAMP and protein kinase-A mediated release of intracellular Ca⁺⁺ played a critical role in such decreased α2-Na–K ATPase expression. In contrast, noradrenaline acting on β-adrenoceptor up-regulated the expression of regulatory β2-Na–K ATPase subunit, which although was cAMP and Ca⁺⁺ dependent, was independent of protein kinase-A and protein kinase-C. Combining these with previous findings (including ours) we have proposed a working model for noradrenaline-induced suppression of glial Na–K ATPase activity and alteration in its subunit expression. The findings help understanding noradrenaline-associated maintenance of brain excitability during health and altered states, particularly in relation to rapid eye movement sleep and its deprivation when the noradrenaline level is naturally altered.     

Sleep/wake dependent changes in cortical glucose concentrations

Most of the energy in the brain comes from glucose and supports glutamatergic activity. The firing rate of cortical glutamatergic neurons, as well as cortical extracellular glutamate levels, increase with time spent awake and decline throughout non rapid eye movement sleep, raising the question whether glucose levels reflect behavioral state and sleep/wake history. Here chronic (2–3 days) electroencephalographic recordings in the rat cerebral cortex were coupled with fixed‐potential amperometry to monitor the extracellular concentration of glucose ([gluc]) on a second‐by‐second basis across the spontaneous sleep‐wake cycle and in response to 3 h of sleep deprivation. [Gluc] progressively increased during non rapid eye movement sleep and declined during rapid eye movement sleep, while during wake an early decline in [gluc] was followed by an increase 8–15 min after awakening. There was a significant time of day effect during the dark phase, when rats are mostly awake, with [gluc] being significantly lower during the last 3–4 h of the night relative to the first 3–4 h. Moreover, the duration of the early phase of [gluc] decline during wake was longer after prolonged wake than after consolidated sleep. Thus, the sleep/wake history may affect the levels of glucose available to the brain upon awakening.     

Sleep and cortical temperature in the Djungarian hamster under baseline conditions and after sleep deprivation

The Djungarian hamster (Phodopus sungorus) is a markedly photoperiodic rodent which exhibits daily torpor under short photoperiod. Normative data were obtained on vigilance states, electroencephalogram (EEG) power spectra (0.25–25.0 Hz), and cortical temperature (T_CRT) under a 168 h light-dark schedule, in 7 Djungarian hamsters for 2 baseline days, 4 h sleep deprivation (SD) and 20 h recovery.During the baseline days total sleep time amounted to 59% of recording time, 67% in the light period and 43% in the dark period. The 4 h SD induced a small increase in the amount of non-rapid eye movement (NREM) sleep and a marked increase in EEG slow-wave activity (SWA; mean power density 0.75–4.0 Hz) within NREM sleep in the first hours of recovery. T_CRT was lower in the light period than in the dark period. It decreased at transitions from either waking or rapid eye movement (REM) sleep to NREM sleep, and increased at the transition from NREM sleep to waking or REM sleep. After SD, T_CRT was lower in all vigilance states.In conclusion, the sleep-wake pattern, EEG spectrum, and time course of T_CRT in the Djungarian hamster are similar to other nocturnal rodents. Also in the Djungarian hamster the time course of SWA seems to reflect a homeostatically regulated process as was formulated in the two-process model of sleep regulation.Abbreviations EEG electroencephalogram - EMG electromyogram - N NREM sleep - NREM non-rapid eye movement - R REM sleep - REM rapid eye movement - SD sleep deprivation - SWA slow-wave activity - T_CRT cortical temperature - TST total sleep time - VS vigilance state - W waking     

Hormonal responses of glucose-6-phosphatase catalytic unit studied by stopped-flow analysis

To obtain insight regarding the mechanism(s) of response of the catalytic unit of glucose-6-phosphatase (D-glucose-6-P phosphohydrolase; EC 3.1.3.9) to glucocorticoid administration and insulin deprivation, the functional enzyme concentration E0 was estimated from presteady-state kinetics by the stopped-flow technique. The E0 values were compared with Vmax values determined by the steady-state kinetic approach. Studies were carried out with detergent-disrupted microsomes from livers of normal fed, 48-h fasted, streptozotocin-diabetic, and triamcinolone-treated rats. All of the treatments caused an increase in E0, but Vmax values were increased only in fasting and diabetes. Km values were unaffected by all the treatments. The increase in Vmax observed with fasted and diabetic rats was explained by an increase in E0 alone. These results showed that insulin deprivation resulted in an increased formation of fully active glucose-6-phosphatase catalytic unit. In contrast, administration of triamcinolone caused an increase in E0 but not in Vmax. It was concluded that glucocorticoid administration may promote formation of catalytic units of glucose-6-phosphatase which are less active than the enzyme normally present or formed in response to insulin deprivation.     

Salinity adaptation of HCO3--dependent ATPase activity in the gills of blue crab (Callinectes sapidus)

A bicarbonate-dependent ATPase (EC 3.6.1.3) was found in microsomal preparations from blue crab gills. When the crabs were transferred to low salinity (200 mosmolal) from seawater (1000 mosmolal), the HCO3- dependent ATPase increased in all gill pairs, reaching its new steady state in 2 weeks. The greatest increase occurred in the sixth and seventh gill pairs (approx. 2.5-fold). Maximal enzyme activity was observed at an Mg2+ concentration of 2 mM and an optimal pH of 7.8. The apparent Ka for HCO3- was found to be 8.9 mM. Kinetic analysis showed that low-salinity adaptation increased the Vmax without altering the Km for ATP. When the microsomes from high-salinity crab gills were treated with detergent or assayed at different temperatures, the total enzyme activity did not reach the activity levels after adaptation to low salinity. These results suggest that the alteration of HCO3- -ATPase activity may be due to synthesis, rather than modulation of membranes or of the existing enzyme activity.     

Localization and cation dependence of a Ca2+- or Mg2+-ATPase from electrocytes of Electrophorus electricus, L

Electrocyte membranes of Electrophorus electricus exhibit high ATPase activity, as demonstrated by cytochemical and biochemical techniques. This activity is visualized as electron-dense deposits in electron micrographs, and appears to be localized only at the innervated face of the electrocyte. ATP hydrolysis can be detected cytochemically or biochemically only in the presence of calcium or magnesium. The effects of Ca or Mg on ATPase activity can be described by Michaelis-like functions with similar apparent Km values for Ca and Mg (0.41 mM and 0.23 mM, respectively). Vmax, however, is fivefold higher in the presence of Mg. The effects of the two cations are not additive, and pH dependence of ATP hydrolysis is identical in the presence of Ca or Mg (maximal at pH 8-9). Therefore, it can be concluded that Ca and Mg activate the same enzyme, the differences in Vmax being attributable to influences in kcat.     

Selective REM Sleep Deprivation Improves Expectation-Related Placebo Analgesia

The placebo effect is a neurobiological and psychophysiological process known to influence perceived pain relief. Optimization of placebo analgesia may contribute to the clinical efficacy and effectiveness of medication for acute and chronic pain management. We know that the placebo effect operates through two main mechanisms, expectations and learning, which is also influenced by sleep. Moreover, a recent study suggested that rapid eye movement (REM) sleep is associated with modulation of expectation-mediated placebo analgesia. We examined placebo analgesia following pharmacological REM sleep deprivation and we tested the hypothesis that relief expectations and placebo analgesia would be improved by experimental REM sleep deprivation in healthy volunteers. Following an adaptive night in a sleep laboratory, 26 healthy volunteers underwent classical experimental placebo analgesic conditioning in the evening combined with pharmacological REM sleep deprivation (clonidine: 13 volunteers or inert control pill: 13 volunteers). Medication was administered in a double-blind manner at bedtime, and placebo analgesia was tested in the morning. Results revealed that 1) placebo analgesia improved with REM sleep deprivation; 2) pain relief expectations did not differ between REM sleep deprivation and control groups; and 3) REM sleep moderated the relationship between pain relief expectations and placebo analgesia. These results support the putative role of REM sleep in modulating placebo analgesia. The mechanisms involved in these improvements in placebo analgesia and pain relief following selective REM sleep deprivation should be further investigated.     

Cardiovascular effects of partial sleep deprivation in healthy volunteers

Sleep deprivation is common in Western societies and is associated with increased cardiovascular morbidity and mortality in epidemiological studies. However, the effects of partial sleep deprivation on the cardiovascular system are poorly understood. In the present study, we evaluated 13 healthy male volunteers (age: 31 ± 2 yr) monitoring sleep diary and wrist actigraphy during their daily routine for 12 nights. The subjects were randomized and crossover to 5 nights of control sleep (>7 h) or 5 nights of partial sleep deprivation (<5 h), interposed by 2 nights of unrestricted sleep. At the end of control and partial sleep deprivation periods, heart rate variability (HRV), blood pressure variability (BPV), serum norepinephrine, and venous endothelial function (dorsal hand vein technique) were measured at rest in a supine position. The subjects slept 8.0 ± 0.5 and 4.5 ± 0.3 h during control and partial sleep deprivation periods, respectively (P < 0.01). Compared with control, sleep deprivation caused significant increase in sympathetic activity as evidenced by increase in percent low-frequency (50 ± 15 vs. 59 ± 8) and a decrease in percent high-frequency (50 ± 10 vs. 41 ± 8) components of HRV, increase in low-frequency band of BPV, and increase in serum norepinephrine (119 ± 46 vs. 162 ± 58 ng/ml), as well as a reduction in maximum endothelial dependent venodilatation (100 ± 22 vs. 41 ± 20%; P < 0.05 for all comparisons). In conclusion, 5 nights of partial sleep deprivation is sufficient to cause significant increase in sympathetic activity and venous endothelial dysfunction. These results may help to explain the association between short sleep and increased cardiovascular risk in epidemiological studies.     

Analysis of azide-insensitive Ca2+-dependent ATPase activity in atrial specimens from patients with coronary or valvular heart disease

The activity of the azide-insensitive Ca2+-dependent ATPase (highly enriched in myofibrillar ATPase activity) was studied in specimens of both right and left atria which were taken from patients with ischemic and/or valvular heart disease during coronary by pass and/or valvular substitution. A significantly lower enzymatic activity was found in atrial specimens from patients with left ventricular heart failure in comparison to the atrial fragments obtained from the patients with normal heart function. Such an inhibition reflected a significant increase in the Km of the enzyme for ATP and was associated with a concomitant reduction in Vmax, both more evident in the left atrial fragments. Moreover, tissue homogenates of atrial specimens from failing hearts exhibited a lower protein SH group content when compared to the atrial homogenates from the heart with normal left ventricular heart function.     

Renal sodium handling and stimulation of medullary Na-K-ATPase during blockade of prostaglandin synthesis

The effect of suppression of prostaglandin synthesis on renal sodium handling and microsomal Na-K ATPase was studied in control and indomethacin treated intact rats maintained on a normal sodium diet (series A) and chronically salt loaded (series B). Indomethacin administration resulted in a decreased GFR and a significantly depressed urinary excretion and an increased fractional reabsorption of sodium in animals fed the normal sodium diet or chronically salt loaded. In rats maintained on a normal Na diet, the activity of the renal medullary Na-K ATPase after indomethacin was 206.3 +/- 6.4 ug Pi/mg protein, i.e. significantly higher as compared with the enzyme activity in the medullary renal fraction from control animals in which it averaged 148 +/- 7.79 ug Pi/mg protein (p less than 0.001). While after chronic salt load a similar increment in the activity of renal medullary Na-K ATPase was observed, no additional stimulation was elicited by subsequent indomethacin administration. The addition of exogenous PGE2, 0.1 mM to microsomal fractions obtained from kidneys of normal rats, was associated with a moderate suppression of the medullary Na-K-ATPase activity, from a basal level of 170 +/- 16 to 151.3 +/- 13 umol Pi/mg protein/hr (p less than 0.005). In isolated segments of medullary thick ascending limb of Henle's loop (MTAL) addition of PGE2 to the incubation medium resulted in a significant inhibition of Na-K ATPase from 37.2 +/- 2 to 21.25 +/- 1.17 x 10(-11) mol/mm/min (p less than 0.0001). These findings suggest that the increased renal Na reabsorption after inhibition of PG synthesis might be related, at least partly, to stimulation of medullary Na-K ATPase. In parallel, the reported natriuretic effect of prostaglandins might imply a direct inhibitory effect of these mediators on renal Na-K ATPase.