The role of the angiotensin AT2 receptor on the diurnal variations of nociception and motor coordination in rats.

Phasic pain demonstrates significant diurnal variation in rats. Angiotensin II modulates pain transmission and the diurnal variation in nociception in several rodent pain models. The participation of AT2 receptors in the diurnal regulation of nociception is not yet elucidated. In the present study we investigated the effects of selective peptide AT2 agonist CGP 42112A and the nonpeptide AT2 receptor antagonist PD 123319 on the nociception, motor coordination and arterial blood pressure. Male Wistar 12 weeks old rats were used. CGP 42112A was injected at single doses of 1 and 5 µg/rat intracerebroventricularly (ICV) and infused chronically ICV at a dose of 12 µg/rat/day during 14 days by osmotic minipumps. PD123319 was injected at single doses of 1 and 5 µg/rat, ICV and chronically subcutaneously at a dose of 10 mg/kg/day/14 days. Nociception was assessed by an analgesimeter, arterial blood pressure (ABP) was measured by tail cuff method, and motor coordination by Rota-rod method. Single doses of CGP 42112A (1 and 5 µg/rat) provoked a short lasting antinociception. Unlike acute injection, chronic CGP 42112A infusion increased nociception at the beginning and the end of light phase thus attenuating the diurnal variations observed in the controls. Moreover, it produced an increase of ABP and improved motor coordination. Both acute (1 µg/rat) and chronic PD 123319 treatment resulted in a decrease of pain threshold and chronic treatment attenuated its diurnal fluctuation. Our data support a role for Ang II type 2 receptors in the control of diurnal variations of nociception in rats.

Effect of the AT1 receptor antagonist losartan on diurnal variation in pain threshold in spontaneously hypertensive rats.

Angiotensin (AT) II plays a key role in the regulation of blood pressure and water-salt balance and modulates nociception. Peptides based on AT influence central functions through the activation of AT1, AT2 or AT4 receptors. The aim of this study was to elucidate the role of AT1 receptors in diurnal variation in nociception in spontaneously hypertensive rats (SHR). Male Wistar rats (16 weeks old) and SHR were caged individually and exposed to light from 08:00 to 20:00 h. The tail cuff method for noninvasive measurement of arterial blood pressure (ABP), paw pressure test for the determination of pain threshold and rotarod test to study motor coordination were used. Chronic treatment was administered to the SHR with the AT1 receptor antagonist losartan (10 mg/kg/day, s.c.) for 14 days. Spontaneously hypertensive rats showed lower pain threshold and smaller day-night variations of nociception as compared to Wistar rats. Chronic losartan decreased the ABP and produced an inverted diurnal pattern of nociception in SHR, increasing the pain threshold at 03:00 h. Neither strain differences nor changes in motor coordination after losartan treatment were observed in SHR. Our results suggest that SHR have disturbances in diurnal variation in nociception and that the AT1 receptor plays a role in the regulation of the circadian rhythm of mechanical pain threshold in SHR.

Diurnal variation in the effect of angiotensin II on nociception.

The aim of this study was to investigate the effect of angiotensin II (Ang II) on nociception at particular time points of a 24-h cycle using different pain stimuli. A parallel investigation of phasic and tonic pain tests revealed different diurnal patterns of pain responses. Phasic pain test (mechanical paw pressure) in rats was characterized with shortest latencies during the dark phase, when the average of motor activity is greatest. Ang II (0.1 microg/animal) increased the latency of pain responses to mechanical and thermal stimulations mainly during the active dark phase. With regard to tonic pain, regardless of a weak circadian fluctuation of the number of pain responses (writhes) in mice, there was a tendency to attenuate the diurnal pattern of nociception. In contrast to the effect of Ang II on the phasic pain, it exerted an antinociceptive effect in the writhing test during the light phase. In summary, Ang II exerted an antinociceptive effect at the time points that have naturally high pain sensitivity.

Blood pressure and heart rate rhythmicity: differential effects of late pregnancy.

Arterial blood pressure (BP) and heart rate (HR) of 31 hospitalized pregnant women at low risk of hypertension were automatically monitored for 48 h at 15-min intervals. Each of the recorded 56 data series for systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and HR was chronobiologically assessed by linear-nonlinear rhythmometry. The rhythm-adjusted mean (MESOR), circadian amplitude, circadian acrophase, and best-fitting period were grouped by pregnancy trimester and further subjected to analysis of variance. BP MESOR remained unaltered, whereas HR MESOR increased significantly in middle and late pregnancy. Ultradian rhythms, with an amplitude higher than that of the circadian rhythm, were found in 25% of the SAP records in the second and third trimester. Such ultradian rhythms were not detected in the simultaneously recorded HR. Finally, the group BP and HR circadian acrophases coincided in the first trimester, but were significantly apart in mid and late pregnancy. These observations support the notion that the coordination of BP and HR rhythmicity involves different physiological mechanisms. Analysis of the individual variability in the chronobiological end points (based on the records of nine women monitored in each pregnancy trimester) revealed that only the BP MESOR was well reproducible in the course of pregnancy and may be useful in early diagnosis of gestational hypertension.

Circadian rhythms of arterial pressure: basic regulatory mechanisms and clinical value.

The circadian rhythm of arterial pressure (AP) is not a passive consequence of the impact of exogenous factors. Endogenous mechanisms play an important role in the generation and maintenance of AP rhythm. The adaptation of the exogenous components of AP rhythm to the demands of the environment is modulated by the circadian-time-dependent responsiveness of the biologic oscillator. A neuronal network in the rostral hypothalamus including the suprachiasmatic nucleus is implicated in the generation of AP rhythm, in the modification of the rhythm amplitude (possibly due to homeostatic constraints), and in the regulation of its phase. The central sympathoexcitatory pathway to the upper thoracic cord plays a crucial role in the maintenance of normal circadian AP rhythm. The circadian pattern of AP is influenced also by hormonal factors such as the hypothalamic-pituitary-adrenal and the hypothalamic-pituitary-thyroid axes, the renin-angiotensin-aldosterone system, opioids, and various vasoactive peptides. The circadian variations of AP depend on physiological state--sleep and wakefulness, pregnancy, work, and senescence (primary aging). In some essential hypertensive patients and in patients with secondary hypertension the nocturnal fall in AP is reduced or absent (nondippers). Target-organ damage is more advanced in nondippers than in dippers. The occurrence of cardiovascular events exhibits a prominent circadian pattern, with events more frequent in the morning (06:00-12:00 h).

Circadian clocks and hypertension: genetics and interactions.

Recent advances in molecular genetics of circadian rhythms and hypertension led to the discovery of separate groups of genes implicated in their regulation. Importantly, the identification in both mammals and flies of 6 homologous circadian clock genes strongly indicates that the circadian period is controlled by an evolutionary conserved set of genes. Studies in familial and experimental hypertension reveal that elevated blood pressure is due to mutations in genes implicated in the function of the renin-angiotensin-aldosterone system. A chronobiologic approach to experimental hypertension indicates that hypertension can be associated with selectively inverted circadian rhythm of arterial pressure. Several lines of evidence suggest that the rostral hypothalamus is an area of central integration of the endogenous rhythmic and other regulatory influences that modulate the phase and amplitude of circadian arterial pressure rhythmicity. The combination of advanced molecular genetics and continuous blood pressure monitoring with chronobiologic assessment emerges as a fruitful approach in better understanding the pathogenesis of hypertension.

Effect of forward rapidly rotating shift work on circadian rhythms of arterial pressure, heart rate and oral temperature in air traffic controllers.

Twenty-four-hour records of arterial pressure (AP), heart rate (HR), oral temperature (OT) and physical and mental performance were obtained in air traffic controllers during morning (n = 16), afternoon (n = 17) and night (n = 19) shifts. Data were analyzed by the cosinor method. The results obtained during the morning shift were as follows (mesor/amplitude/acrophase): systolic AP (mm Hg)--113.6/10.0/16:03 h; diastolic AP--71.1/8.2/15:19 h; mean AP--85.6/8.8/15:41 h; HR (beats/min)--77.5/8.9/16:00 h; OT (dg C)--36.71/0.21/15:49 h; right-hand grip strength (kg)--49.8/2.0/17:43 h; left-hand grip strength--46.1/2.0/16:08 h; mental performance (calculations/min)--14.9/1.1/16:39 h. During the night shift either no change of the circadian acrophases (HR, right-hand grip strength) or acrophase delays ranging from about 2 h (systolic AP, OT, mental performance) up to 3 h (diastolic and mean AP, left-hand grip strength) were observed. Our data suggest that the shift system studied does not significantly alter the circadian rhythms, and does not induce a desynchronization, particularly as concerns arterial pressure and oral temperature.

Integrative coordination of circadian mammalian diversity: neuronal networks and peripheral clocks.

Diverse circadian rhythms are generated, maintained and/or coordinated by brain structures constituting the circadian timing system. However, the mechanisms underlying the variety in activity types and circadian rhythm phases and amplitudes are currently unknown. We address this problem by comparing rhythms in diurnal and nocturnal mammals, while focusing on alterations not involving the central circadian oscillator. The circadian rhythms are divided into two groups: activity-independent and activity-related. The rhythms in the first group have similar acrophases in all mammals and are anticipated to function as an internal zeitgeber (time giver). Analysis of activity-related circadian rhythms in behavior, blood pressure (BP) and renal excretion suggests separate mechanisms in their regulation in addition to the central suprachiasmatic nuclei-located circadian oscillator. We propose that: (a) a passive hypothalamic oscillator coordinates the phases and underlies the high amplitude of behavioral circadian rhythms; (b) a separate rostral hypothalamic network participates in the regulation of the low-amplitude circadian BP rhythm; and (c) a circadian oscillator in the kidney generates electrolyte excretion rhythms. A model is offered where the overt activity is determined by the phase-relationship between the circadian and the passive hypothalamic oscillator. Specific brain structures or peripheral circadian oscillators integrate circadian and other signals for different activity-related circadian rhythms. The hypothalamic structures implicated in regulation of behavioral and blood pressure rhythms belong to the circadian timing system since they underlie circadian rhythms diversity. The same hypothalamic areas selectively modulate circadian rhythms in response to homeostatic stimuli or stress without engaging the circadian oscillator.

Circadian rhythms of arterial pressure, heart rate and oral temperature in truck drivers.

Circadian rhythms of arterial pressure (AP), heart rate (HR) and oral temperature (OT) were studied in healthy male truck drivers and in a control group of air traffic controllers. Twenty-four-hour records of systolic and diastolic AP and HR and of OT were obtained from 12 truck drivers both during the outward and homeward journey in Europe and Asia, and from 12 air traffic controllers during a morning shift. Data were analyzed by the cosinor method. The results obtained in the control group were as follows (mesor/amplitude/acrophase): systolic AP (mm Hg): 111.1/6.1/16.51 h; diastolic AP: 68.4%5.6/16.58 h; HR (b.p.m.): 77.07/7.6/17.46 h; OT (dg C): 36.74/0.21/17.26 h. Statistically significant acrophase advances were observed for the circadian rhythms of systolic, diastolic and mean AP and of HR in TD during the outward journey in comparison with the control ATC group. These differences were still present during the homeward journey, in combination with an acrophase delay of the circadian rhythm of OT. Our results supply grounds to suppose that extended working time combined with greater load during a long-lasting trip may act to generate an internal desynchronization of circadian rhythms in long-haul truck drivers.

Suprachiasmatic nuclei lesions eliminate the group circadian rhythm of systolic arterial pressure but not of heart rate in rats.

The aim of this study was to evaluate the participation of the suprachiasmatic nuclei (SCN) in the generation and synchronization of cardiovascular rhythms. Seven sham-operated and 11 SCN-lesioned animals maintained under 12/12 hr light/dark cycle were used. Systolic arterial pressure (SAP) and heart rate (HR) were measured indirectly during 24-hour periods at 3-4 hour intervals. The data were analyzed using individual and group cosinor rhythmometry and Fourier analysis. A circadian rhythm of water intake was not detected in animals with successful SCN lesions. A reduction of the double amplitude/MESOR ratio for the 24-hour component of drinking rhythm in the SCN-lesioned rats was observed. After SCN lesions the group 24-hour rhythm of SAP was eliminated while a significant group circadian rhythm for HR was detected. The individual amplitude/MESOR ratios for the 24-, 12-, 8- and 6-hour periodic components of SAP and HR in the lesioned rats showed no marked differences as compared with controls. The generation and entrainment of circadian variations in HR is probably not dependent on the integrity of SCN in rats. The SCN may participate in the entrainment of the circadian rhythm of SAP. The combination of completely abolished (water intake) and persisting (heart rate) rhythms further supports that the circadian regulatory system consists of a network of multiple oscillators.

Lack of effect of suprachiasmatic infusion of a vasopressin antagonist on the circadian rhythm of wheel-running activity in rats.

Circadian patterns of wheel-running activity were studied in male Wistar rats before, during, and after continuous infusion of (Pmp1, Tyr(Me)2)-Arg8-vasopressin, a V1 receptor antagonist, in the suprachiasmatic region by means of Alzet mini-osmotic pumps. The lack of any significant effect on the pattern of wheel-running activity argues against a role for vasopressin in the generation of circadian rhythms by the suprachiasmatic nuclei.

Effect of transplantation of embryonic anterior hypothalamic tissue from spontaneously hypertensive rats to normotensive Wistar rats on circadian rhythms of systolic arterial pressure and heart rate.

The anterior hypothalamus (AH) participates in the regulation of arterial pressure. The suprachiasmatic nuclei (SCN) of the AH are a major circadian oscillator necessary for the generation and/or the entrainment of circadian rhythms. Circadian rhythms of systolic arterial pressure (SAP) and heart rate (HR) were investigated in spontaneously hypertensive rats (SHR) and in normotensive Wistar rats (NWI) with intact SCN, grafted with SHR embryonic AH tissue containing the SCN. Prominent circadian rhythms for SAP and HR in both NWI and SHR with acrophases during dark were found. The elevation of the MESOR (midline-estimated statistic of rhythm) of the SAP in normotensive rats grafted with AH embryonic tissue obtained from SHR was accompanied by disappearance of the circadian rhythm of SAP. This result suggests an interaction between the grafted tissue containing the SCN on the one hand, and the host SCN on the other hand. Our data ascribe a role for the SCN in the entrainment of the circadian rhythm of arterial pressure. The circadian rhythm of HR was not eliminated by the SCN graft in spite of the amplitude decrease and the phase delay observed. It seems that the entrainment of the circadian rhythm of HR is probably not crucially dependent on the SCN in rats. The circadian rhythms of SAP and HR in rats were differently affected by the grafts, thus suggesting a multioscillatory system for circadian regulation in rats.

Metachronanalysis of circannual and circasemiannual characteristics of human suprachiasmatic vasopressin-containing neurons.

We here test for and detect anticipated about-yearly (circannual) changes in the volume and number of vasopressin-containing neurons in the human suprachiasmatic nucleus. We then resolve inferential statistical parameters quantifying the extent and timing (the amplitude and acrophase) of the circannual rhythm previously missed by data inspection and classical biometry. We parametrize about-half-yearly changes previously validated by non-parametric statistical tests. New dynamic circannual and circasemiannual endpoints thus become available for basic investigation and the assessment of disease risk elevation and/or chronoprotopathology. It was earlier demonstrated that the circannual rhythms of prolactin and TSH are prominent classifiers of individuals at high versus low familial and other risk for developing breast or prostate cancer. Any neurocrine or neural mechanisms contributing to this classification are now amenable to study, on a population basis, with the dynamic hypothalamic rhythm characteristics yielded by this metachronanalysis.

Gene expression in suprachiasmatic nucleus and circadian rhythms.

The suprachiasmatic nuclei (SCN) contain a circadian system consisting of circadian oscillator (clock) that is normally synchronized by the light/dark cycle (input) and drives circadian rhythms (output) that are intrinsic to the SCN. Gene expression of immediate-early genes, such as c-fos and jun-B, in the ventrolateral SCN is associated with circadian synchronization by light pulses and subjected to circadian control. Vasopressin and somatostatin gene expression shown distinct circadian rhythms intrinsic to the dorsomedial SCN with higher peptide levels occurring during the day. In addition, embryonic SCN grafted into the brain of an SCN-lesioned arrhythmic host define the period of the restored circadian locomotor rhythm. Taken together, these and other findings support the notion that the expression of genes underlying circadian synchronization, oscillation and output takes place within individual SCN neurons. However, no information regarding the nature and number of those neurons as well as the molecular mechanisms of the single cell-circadian oscillator and output is currently available. Therefore, we propose a simple two-neuron model as a framework for critically discussing the molecular genetic strategies to analyze the circadian system in SCN.

Circadian function of suprachiasmatic nuclei: molecular and cellular biology.

Suprachiasmatic nuclei (SCN) contain a circadian oscillator that is normally synchronized by the light/dark cycle. Embryonic SCN grafted into the brain of an SCN-lesioned arrhythmic host define the period of the restored circadian locomotor rhythm. Gene expression of immediate-early genes, such as c-fos and jun-B, in the ventrolateral SCN is associated with circadian synchronization by light pulses and subjected to circadian control. Vasopressin and somatostatin gene expression in dorsomedial SCN show distinct circadian rhythms with higher peptide levels occurring during the day. It is currently unknown whether the circadian oscillator in SCN resides in a single cell or is a property of cellular network. Briefly presented are some model views about the circadian oscillator in SCN and the molecular and cellular approaches to the circadian function of the nucleus.

Reduced increase in plasma renin activity on water-deprivation in blind hereditary microphthalmic rats.

We compared the plasma renin activity (PRA) before and after 24-h water-deprivation in blind hereditary microphthalmic rats and Donryu rats. In the congenitally blind rats with a morphologically abnormal suprachiasmatic nucleus (SCN), hypovolemia induced significantly less elevation of the PRA and significantly more increase in the hematocrit value than in normal rats. The changes after water-deprivation in the blind rats were quite similar to those reported in rats with SCN lesions. However, the free-running circadian rhythms persisted in these blind rats, whereas those in rats with SCN lesions were completely eliminated. Thus, it is likely that SCN cells are involved in regulation of the PRA, and that if this is the case these cells are different from those containing the circadian pacemaker.

Peripheral p-chloroamphetamine is an unsuitable probe for investigation of central serotoninergic control on renal renin secretion in the rat.

Intraperitoneal application of p-chloroamphetamine (PCA) is considered a suitable probe for investigation of central serotoninergic control on renin release in the rat, although it causes several behavioral and autonomic changes including negative water balance (increased urination and loss of body weight). The possibility that PCA-induced renin release is secondary to the alterations in water balance was investigated 1 hour after intraperitoneal PCA in male Wistar (Wi) (Experiment I). Long-Evans (LE) and diabetes insipidus (DI) (Experiment II), DI rats pretreated by the inhibitor of angiotensin I-converting enzyme captopril (Experiment III), and water-loaded or propranolol-pretreated Wi rats (Experiment IV). PCA treatment induced significant body weight loss, increase in hematocrit, stimulation of renin-aldosterone system (RAS) and elevation of plasma creatinine level. A toxic damage of the kidney and liver was documented histologically 72 h after 5 mg/kg PCA in Wi rats. The blockade of PCA-induced stimulation of RAS (by captopril or propranolol) markedly potentiated the attendant negative water balance, whereas positive water balance (oral water load) abolished PCA-induced renin secretion. In conclusion, intraperitoneal PCA is an unsuitable probe for investigation of central serotoninergic control on renin release in the rat since PCA-induced renin release is caused by the attendant negative water balance.

A reliable technique for midline stereotaxic cannulation of the third ventricle in the rat.

A simple technique is described for chronic or acute cannulation of the third ventricle in the rat slightly displacing the superior sagittal sinus to allow midline implantation of a vertically oriented cannula. The results of an experiment successfully employing the technique for transplantation of embryonic hypothalamic tissue in rats are briefly described.

Suprachiasmatic nuclei lesions eliminate light/dark variations in short-term feeding responses to deprivation or insulin treatment in rats.

Short-term (1-h, 4-h and 12-h) and long-term (24-h) feeding responses to 24-h food deprivation (FD) or insulin treatment (IT) (8 mU/kg IP) were studied in male rats under a 12/12-h light/dark (L/D) cycle. The 24-h FD or the IT began either at onset (Dawn) or offset (Dusk) of the lights. In sham-operated rats (Shams) both protocols elicited greater short-term feeding responses at Dusk (p less than 0.05 or less). In suprachiasmatic nuclei (SCN)-lesioned rats the L/D variations in short-term responses were absent. In both SCN and Shams the 24-h feeding responses did not depend on stimuli time-schedule. We conclude that the regulation of short-term (circadian), but not long-term, feeding responses to metabolic stimuli is dependent on SCN integrity in the rat.

Nephrotoxic effect of the specific brain serotonin depletor para-chlorophenylalanine.

Brain serotonin depletion induced by peripheral parachlorophenylalanine (pCPA) is frequently used to evaluate the role of the central serotoninergic system in the regulation of a number of physiological functions, including the secretion of renin by the kidney. We found that due to the treatments applied in the protocol used for the investigation of pCPA effect on renin and vasopressin secretion in rats (300 mg/kg i.p. 64 and 40 h before sacrifice) renal injury was induced as well. Typical changes indicating acute renal failure were observed--an initial polyuria, natriuresis and body mass loss, succeeded by oliguria, decreased glomerular filtration rate, and salt and creatinine retention. Morphological changes in the glomeruli included a thickening of the basal membranes, a confluence and a reduced number of podocyte pedicles. A slight to moderate granular degeneration was observed in epithelial cells of the proximal convoluted tubule, combined with mitochondrial changes--an increase in number, matrix disorganization, and myelin degeneration. In conclusion, the renal function changes after i.p. pCPA may be due not to brain serotonin depletion-alone, but also to nephrotoxic effect.