Peripheral effects of thyroid hormones: Alteration of intracellular Na-concentration,ouabain-sensitive Na-transport,and Na-Li countertransport in human red blood cells

Summary To investigate the effect of thyroid hormones on erythrocyte cation transport systems and intracellular electrolyte content we have measured the activity of Na-K ATPase, Na-Li countertransport, as well as red cell sodium and potassium contents in patients with hyperthyroidism and in euthyroid controls. Intracellular Na- and K-concentrations were determined in erythrocytes washed three times in isotonic MgCl₂ solution. Ouabain-sensitive Na-transport was estimated as the increase of Na before and after addition of ouabain in an erythrocyte suspension in isotonic Na-free medium. Na-Li countertransport was measured according to the method described by Canessa et al. [2]. The patients with hyperthyroidism exhibited a significantly elevated intracellular sodium content as well as a highly increased Na-K ATPase activity. Intracellular potassium content was not altered in the hyperthyroid subjects, but Na-Li countertransport was markedly decreased as compared to the controls.The results indicate that different ion transport systems of the erythrocyte membrane are influenced by thyroid hormones. We suggest that the elevation of Na-K ATPase activity might be due to the increased intracellular sodium concentration which is caused by the diminished countertransport pathway. Furthermore, the activity of Na-K ATPase, Na-Li countertransport, and intracellular sodium content in erythrocytes might be a useful peripheral indicator of thyroid hormone excess.Supported by the Bundesministerium für Forschung und Technologie (MMT 27)     

Melatonin modulates the light-induced sympathoexcitation and vagal suppression with participation of the suprachiasmatic nucleus in mice

In mammals, the autonomic nervous system mediates the central circadian clock oscillation from the suprachiasmatic nucleus (SCN) to the peripheral organs, and controls cardiovascular, respiratory and gastrointestinal functions. The present study was conducted in mice to address whether light signals conveyed to the SCN can control peripheral autonomic functions, and further examined the impact of centrally administered melatonin on peripheral autonomic functions via activation of melatonin receptor signalling. In vivo electrophysiological techniques were performed in anaesthetised, open-chest and artificially ventilated mice whilst monitoring the arterial blood pressure and heart rate. Light induced an increase of the renal sympathetic nerve activity, arterial blood pressure and heart rate immediately after lights on. Conversely, light rapidly suppressed the gastric vagal parasympathetic nerve activity, which was affected neither by hepatic vagotomy nor by total subdiaphragmatic vagotomy. These autonomic responses were mediated by the SCN since bilateral SCN lesion totally abolished the light-evoked neuronal and cardiovascular responses. Melatonin administered intracerebroventricularly ( i.c.v .) attenuated the sympathetic and vagal nerve activities in a dose-dependent manner with a threshold of 0.1 ng and these effects were blocked by i.c.v . pre-treatment of the competitive melatonin receptor antagonist luzindole. These results suggest that light induces sympathoexcitation and vagal suppression through the SCN and that melatonin modulates the light-induced autonomic responses via activation of the central melatonin receptor signalling.     

Effect of suprachiasmatic lesions on diurnal heart rate rhythm in the rat

Heart rate and locomotor activity of rats kept under 12L/12D illumination regimen was recorded every six minutes for ten days using implantable radio transmitters. Some of the rats then received bilateral radio frequency lesions into the suprachiasmatic nucleus (SCN). Control sham operations were performed on the rest of the animals. After recovery from surgery, recording of heart rate and locomotor activity was continued for ten days. SCN lesioned rats showed no significant diurnal fluctuation in heart rate, while normal and sham operated rats showed the normal diurnal rhythm in that function. The arrhythmic diurnal heart rate pattern of SCN rats appeared to be correlated to their sparadic activity pattern. The integrity of the suprachiasmatic nucleus therefore is necessary for the generation and/or the expression of diurnal rhythmicity in heart rate in the rat.     

Erythrocyte membrane cation carrier in mania.

Erythrocyte sodium and potassium concentrations, erythrocyte membrane ATPase (Na-K specific and non-specific) and the rate of potassium influx into erythrocytes (ouabain-sensitive and insensitive) were estimated in a group of female patients suffering from mania and repeated on about two thirds of them when they had recovered. With recovery there was a statistically significant increase in the erythrocyte ouabain-sensitive potassium influx. The other parameters showed no significant overall change with recovery but the initial severity correlated significantly and negatively with the change in erythrocyte Na-K ATPase with recovery. The changes that occurred in the erythorcyte sodium concentration and Na-K ATPase activity were not random since they correlated significantly with changes in the active potassium influx.     

Period-amplitude analysis of rat electroencephalogram: effects of sleep deprivation and exercise

Electroencephalogram (EEG) wavelength and amplitude within NREM sleep, paradoxical sleep (PS), and wake were measured by computer in five intact rats and four rats with suprachiasmatic nucleus (SCN) lesions for the first recovery day following 24-h total sleep deprivation (TSD) achieved by keeping them on a rotating cylinder over water. To assess exercise effects, EEG within NREM was also analyzed in four intact rats for 8 h after separate 4-h TSD sessions at low and high rates of cylinder rotation (high rate = 12 times low rate). During recovery from 24-h TSD, EEG changed most dramatically in NREM. The number of slow waves per unit time (1-4 Hz wave incidence) and the amplitude at all wavelengths from 1 to 16 Hz were increased for up to 12 h and then fell below baseline levels for most of the next 12 h. Fast (5-16 Hz) wave incidence changed inversely with slow wave incidence. Wake and PS also showed initially increased amplitude, but shifts in incidence were from slow to fast waves. Relative to baseline, intact and SCN-lesioned rats showed similarly shaped recovery functions, indicating that EEG responses to sleep loss are largely independent of diurnal rhythms. Four-hour TSD at a low rotation rate affected NREM EEG similarly to 24-h TSD, but more mildly. The high rotation rate further increased slow wave incidence during recovery without further increasing slow wave amplitude. The results suggest that both EEG wave incidence and amplitude are responsive to prior wakefulness, but only incidence is responsive to prior exercise.     

Early membrane damage during ischemia in rat heart

Effects of ischemia on cell membrane of rat heart were investigated. The endothelial surface revealed the existence of ruthenium red-positive glycocalyx at the anionic site. Membrane bound enzyme as Na-K ATPase was mostly located in the inner side and pinocytotic vesicles of endothelial cell. The clumping and dispersion in glycocalyx of endothelial cells was observed in an ischemic heart and it may prove the functional disturbance of plasma membrane. A potential and functional defect with reduced activity of Na-K ATPase occurred within 1 hr of vascular ligation. The membrane dysfunction due to these molecular changes has been proved by the membrane permeability alteration as well as the intracytoplasmic localization of horseradish peroxidase as tracer.     

Circadian change in tryptophan hydroxylase protein levels within the rat intergeniculate leaflets and raphe nuclei

Serotonin (5-HT) is involved in the synchronisation of the mammalian circadian clock located in the suprachiasmatic nuclei of the hypothalamus (SCN). This clock is synchronised by light (photic cues) and by non-photic cues. Non-photic cues are notably conveyed to the SCN by a direct 5-HT pathway arising from the mesencephalic median raphe nucleus (MRN). Furthermore, an indirect projection conveys non-photic inputs by 5-HT fibres from the mesencephalic dorsal raphe nucleus (DRN) to the intergeniculate leaflets of the thalamus (IGL) which project to the SCN. In the rat, the quantitative distribution of tryptophan hydroxylase (TpH), used as an index of 5-HT synthesis, was studied by in situ immunoautoradiography in both the serotoninergic cell bodies area of the raphe nuclei and the serotoninergic terminal field of the IGL. Under a 12 h light: 12 h dark (LD 12:12), TpH protein amount exhibited a rhythmic variation within the IGL. The maximum levels were reached at the day/night transition. In both MRN and the lateral groups of the DRN, TpH variations were opposite to those observed in the IGL. Such phase opposition was reported previously in the MRN/SCN pathway and was correlated with a rhythmic release of 5-HT within the SCN [Eur J Neurosci 15 (2002) 833]. Thus, the daily rhythmicity of TpH levels observed in DRN-IGL pathway may be correlated with a rhythmic release of 5-HT in the IGL at the beginning of the night. Under constant darkness, TpH rhythmic variations in the two serotoninergic pathways were maintained and similar to those observed under light/dark cycle. These results demonstrate the existence of a circadian endogenous functioning in the 5-HT neurones projecting to the rat circadian system.     

Circadian changes in arginine vasopressin level in the suprachiasmatic nuclei in the rat

Arginine vasopressin (AVP) neurons were preferentially localized in the dorsomedial part of the suprachiasmatic nucleus (SCN). To know the role of AVP neurons in the SCN, male rats were kept under a normal light-dark cycle (L-D), or under constant darkness (D-D) for 20 days. In L-D condition, AVP levels in the SCN showed the circadian change. In D-D condition, the patterns in AVP levels showed a free-running rhythm, and an about 12-h shift per 20 days. This result suggests that the activity of AVP neurons may be closely associated with the endogenous circadian rhythm of the SCN.     

Rapid eye movement sleep deprivation modulates synapsinI expression in rat brain

Rapid eye movement sleep (REMS) deprivation (REMSD) has been reported to elevate neurotransmitter level in the brain; however, intracellular mechanism of its increased release was not studied. Phosphorylation of synapsinI, a synaptic vesicle-associated protein, is involved in the regulation of neurotransmitter release. In this study, rats were REMS deprived by classical flowerpot method; free moving control (FMC), large platform control (LPC) and recovery control (REC) was carried out. In another set REMS deprived rats were intraperitoneally (i.p.) injected with α1-adrenoceptor antagonist, prazosin (PRZ). Effects of REMSD on Na-K ATPase activity and on the total synapsinI as well as phosphorylated synapsinI levels were estimated in synaptosomes prepared from whole brain. It was observed that REMSD significantly increased synaptosomal Na-K ATPase activity, which was prevented by PRZ. Western blotting of the same samples by anti-synapsinI and anti-synapsinI-phosphoSer603 showed that REMSD increased both the total as well as phospho-form of synapsinI as compared to respective levels in FMC and LPC samples. These findings suggest a functional link between REMSD and synaptic vesicular mobilization at the presynaptic terminal, a process that is essential for neurotransmitter release. The findings help explaining the intracellular mechanism of elevated neurotransmitter release associated to REMSD.     

Basal forebrain neurons modulate the synthesis and expression of neuropeptides in the rat suprachiasmatic nucleus

We tested the hypothesis that efferents from the nucleus basalis magnocellularis (NBM) play a direct role in the regulation of neuropeptide synthesis and expression by neurons of the rat suprachiasmatic nucleus (SCN). Adult male rats in which the NBM was destroyed with quinolinic acid, either unilaterally or bilaterally, were compared with rats injected with physiological saline and with control rats. The estimators used to assess the effects of cholinergic deafferentation on the neuroanatomy and neurochemistry of the SCN were the total number of SCN neurons, the total number and somatic size of SCN neurons producing vasopressin (VP) and vasoactive intestinal polypeptide (VIP), and the respective mRNA levels. Bilateral destruction of the NBM did not produce cell death in the SCN, but caused a marked reduction in the number and somatic size of SCN neurons expressing VP and VIP, and in the mRNA levels of these peptides. The decrease in the number of VP- and VIP-producing neurons provoked by unilateral lesions was less striking than that resulting from bilateral lesions. It was, however, statistically significant in the ipsilateral hemisphere, but not in the contralateral hemisphere. The results show that the reduction of cholinergic inputs to the SCN impairs the synthesis, and thereby decreases the expression of neuropeptides by SCN neurons, and that the extent of the decline correlates with the amount of cholinergic afferents destroyed. This supports the notion that acetylcholine plays an important, and direct role in the regulation of the metabolic activity of SCN neurons.     

Phase preference for the display of activity is associated with the phase of extra-suprachiasmatic nucleus oscillators within and between species

Many features of the suprachiasmatic nucleus (SCN) are the same in diurnal and nocturnal animals, suggesting that differences in phase preference are determined by mechanisms downstream from the SCN. Here, we examined this hypothesis by characterizing rhythmic expression of Period 1 (PER1) and Period 2 (PER2) in several extra-SCN areas in the brains of a diurnal murid rodent, Arvicanthis niloticus (grass rats). In the shell of the nucleus accumbens, dorsal striatum, piriform cortex, and CA1 of the hippocampus, both PER1 and PER2 were rhythmic, with peak expression occurring at ZT10. PER1 in the dentate gyrus also peaked at ZT10, but PER2 was arrhythmic in this region. In general, these patterns are 180° out of phase with those reported for nocturnal species. In a second study, we examined inter-individual differences in the multioscillator system of grass rats. Here, we housed grass rats in cages with running wheels, under which conditions some individuals spontaneously adopt a day active (DA) and others a night active (NA) phase preference. In the majority of the extra-SCN regions sampled, the patterns of PER1 and PER2 expression of NA grass rats resembled those of nocturnal species, while those of DA grass rats were similar to the ones seen in grass without access to running wheels. In contrast, the rhythmic expression of both PER proteins was identical in the SCN and ventral subparaventricular zone (vSPZ) of DA and NA animals. Differences in the phase of oscillators downstream from the SCN, and perhaps the vSPZ, appear to determine the phase preference of particular species, as well as that of members of a diurnal species that show voluntary phase reversals. The latter observation has important implications for the understanding of health problems associated with human shift work.     

Lithium-induced inhibition of Na-K ATPase and Ca ATPase activities in rat brain synaptosome.

To explore the action mechanism of lithium in the brain, the author investigated the effects of lithium on Na-K ATPase and Ca ATPase in rat brain synaptosomes prepared from forebrains by the method of Booth and Clark. The activities of Na-K ATPase and Ca ATPase were assayed by the level of inorganic phosphate liberated from the hydrolysis of ATP. Lithium at the optimum therapeutic concentration of 1 mM decreased the activity of Na-K ATPase from the control value of 19.08 +/- 0.29 to 18.27 +/- 0.10 micromoles Pi/mg protein/h and also reduced the activity of Ca ATPase from 6.38 +/- 0.12 to 5.64 +/- 0.12 micromoles Pi/mg protein/h. The decreased activity of Na-K ATPase will decrease the rate of Ca2+ efflux, probably via an Na-Ca exchange mechanism and will increase the rate of Ca2+ entry by the depolarization of nerve terminals. The reduced activity of Ca ATPase will result in the decreased efflux of Ca2+. As a Conclusion, it can be speculated that lithium elevates the intrasynaptosomal Ca2+ concentration via inhibition of the activities of Na-K ATPase and Ca ATPase, and this increased [Ca2+]i will cause the release of neurotransmitters and neurological effects of lithium.     

The suprachiasmatic nucleus participates in food entrainment: a lesion study

Daily feeding schedules entrain temporal patterns of behavior, metabolism, neuronal activity and clock gene expression in several brain areas and periphery while the suprachiasmatic nucleus (SCN), the biological clock, remains coupled to the light/dark cycle. Because bilateral lesions of the SCN do not abolish food entrained behavioral and hormonal rhythms it is suggested that food entrained and light entrained systems are independent of each other. Special circumstances indicate a possible interaction between the light and the food entrained systems and indicate modulation of SCN activity by restricted feeding. This study explores the influence of the SCN on food entrained rhythms. Food entrained temporal profiles of behavior, core temperature, corticosterone and glucose, as well as Fos and PER1 immunoreactivity in the hypothalamus and corticolimbic structures were explored in rats bearing bilateral SCN lesions (SCNX). In SCNX rats food anticipatory activity and the food entrained temperature and corticosterone increase were expressed with earlier onset and higher values than in intact controls. Glucose levels were lower in SCNX rats in all time points and SCNX rats anticipation to a meal induced higher c-Fos positive neurons in the hypothalamus, while a decreased c-Fos response was observed in corticolimbic structures. SCNX rats also exhibited an upregulation of the PER1 peak in hypothalamic structures, especially in the dorsomedial hypothalamic nucleus (DMH), while in some limbic structures PER1 rhythmicity was dampened. The present results indicate that the SCN participates actively during food entrainment modulating the response of hypothalamic and corticolimbic structures, resulting in an increased anticipatory response.     

The effects of varying sodium diets on haemodynamics and fluid balance in the spontaneously hypertensive rat

Spontaneously hypertensive rats (SHR) were given either 'low' (LNa; 0.5 mmol Na 100 g-1 food), 'control' (CNa; 12 mmol) or 'very high' (vHNa; 120 mmol) sodium diets from 5 to 13-14 weeks of age, to explore how these 240-fold variations in Na intake affected body weight, cardiac, renal and adrenal weights, overall water-electrolyte equilibrium and haemodynamic balance during rest, mental stress and blood loss. Body growth was retarded both in vHNa and LNa SHR presumably reflecting disturbed appetite due to the greatly altered dietary Na contents. Compared with CNa SHR, both cardiac and renal weights 100 g-1 body wt were slightly increased in vHNa and decreased in LNa SHR, with opposite changes of adrenal weights. Total body water, haematocrit and plasma Na-K levels were largely equal in the three groups. Furthermore, cardiac output (CO), stroke volume (SV) and central blood volume (CBV) did not differ significantly between groups; if anything, CO and SV were higher and CBV lower in vHNa and LNa SHR than in CNa SHR. However, while mean arterial pressure (MAP) was only marginally elevated in vHNa compared with CNa SHR, both MAP and total peripheral resistance (TPR) were lowered about 15% in LNa SHR with signs of increased sympathetic activity to the heart also during rest. Despite an apparently normal volume and cardiac output balance in LNa SHR, the latter changes suggest a disturbed neuro-hormonal cardiovascular control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lithium and erythrocyte membrane cation carrier studies in normal and manic depressive subjects.

Changes in the erythrocyte membrane cation carrier following lithium ingestion in normal human subjects were studied; ouabain sensitive potassium influx fell significantly during the lithium treated phase. Lithium was fed to rats and no change in erythrocyte Na-K ATPase was shown. These findings contrast with studies of lithium in manic depressive psychosis. The fluctuations in the erythrocyte membrane cation carrier were studied in 5 normal subjects over 12 weeks and the correlations between the parameters calculated. The erythrocyte sodium concentration correlated positively with the ouabain sensitive potassium influx. This too contrasts with findings in manic depressive psychosis.     

Erythrocyte membrane cation carrier, relapse rate of manic-depressive illness and response to lithium.

Biochemical studies of manic-depressive psychosis usually correlates biochemical findings with current affective state and hence any significant findings could be secondary to mood change. The present study attempts to correlate measures of the erythrocyte membrane cation carrier with clinical events, remote in time from the biochemical assay. Eprythrocyte sodium concentration, ouabain-sensitive potassium influx and Na-K ATPase were estimated in 11 patients before and after the cross-over point in a 2-year double blind clinical trial ratio tended to suffer most episodes of affective illness in the 2 years. Patients who had a low initial Na-K ATPase or a high initial flux sodium ATPase ratio, or in whom this ratio fell most with lithium or whose Na-K ATPase rose most with lithium, clinically responded best to lithium.     

Daily rhythms in PER1 within and beyond the suprachiasmatic nucleus of female grass rats (Arvicanthis niloticus)

Although circadian rhythms of males and females are different in a variety of ways in many species, their mechanisms have been primarily studied in males. Furthermore, rhythms are dramatically different in diurnal and nocturnal animals but have been studied predominantly in nocturnal ones. In the present study, we examined rhythms in one element of the circadian oscillator, the PER1 protein, in a variety of cell populations in brains of diurnal female grass rats. Every 4 h five adult female grass rats kept on a 12-h light/dark (LD) cycle were perfused and their brains were processed for immunohistochemical detection of PER1. Numbers of PER1-labeled cells were rhythmic not only within the suprachiasmatic nucleus (SCN), the locus of the primary circadian clock in mammals, but also in the peri-suprachiasmatic region, the oval nucleus of the bed nucleus of the stria terminalis, the central amygdala, and the nucleus accumbens. In addition, rhythms were detected within populations of neuroendocrine cells that contain tyrosine hydroxylase. The phase of the rhythm within the SCN was advanced compared with that seen previously in male grass rats. Rhythms beyond the SCN were varied and different from those seen in most nocturnal species, suggesting that signals originating in the SCN are modified by its direct and/or indirect targets in different ways in nocturnal and diurnal species.     

The role of calcium ions in circadian rhythm of suprachiasmatic nucleus neuron activity in rat hypothalamic slices

The role of calcium ions in maintaining the circadian rhythm of suprachiasmatic nucleus (SCN) neuron activity was investigated using rat hypothalamic slice preparations. In normal Krebs solution, the firing rate of SCN neurons was higher in the light period than in the dark period. In Ca2+-free Krebs solution, SCN neuron activity was low during all periods and did not show diurnal rhythm. These results suggest that the disappearance of circadian rhythmic change of SCN neuron activity in Ca2+-free Krebs solution may be due to the disappearance of synaptic transmission in the SCN.     

Endogenous ouabain-like compounds in locus coeruleus modulate rapid eye movement sleep in rats

Although the detailed mechanism of spontaneous generation and regulation of rapid eye movement sleep (REMS) is yet unknown, it has been reported that noradrenergic REM-OFF neurons in the locus coeruleus (LC) cease firing during REMS and, if they are kept active, REMS is significantly reduced. On the other hand, the activity as well as expression of Na-K ATPase has been shown to increase in the LC following REMS deprivation. Ouabain is a specific inhibitor of Na-K ATPase, and endogenous ouabain-like compounds are present in the brain. These findings led us to propose that a decrease in the level of ouabain-like compounds spontaneously available in and around the LC would stimulate and increase the REM-OFF neuronal activities in this region and thus would reduce REMS. To test this hypothesis, we generated anti-ouabain antibodies and then microinjected it bilaterally into the LC in freely moving chronically prepared rats and recorded electrophysiological signals for evaluation of sleep−wakefulness states; suitable control experiments were also conducted. Injection of anti-ouabain antibodies into the LC, but not into adjacent brain areas, significantly reduced percent REMS (mean ± SEM) from 7.12 (±0.74) to 3.63 (±0.65). The decrease in REMS was due to reduction in the mean frequency of REMS episode, which is likely due to increased excitation of the LC REM-OFF neurons. Control microinjections of normal IgG did not elicit this effect. These results support our hypothesis that interactions of naturally available endogenous ouabain-like compounds with the Na-K ATPase in the LC modulate spontaneous REMS.