Rhythmic supplementation of 17beta-estradiol according to the physiological estrous cycle: effect on blood pressure in female ovariectomized rats.

Female Wistar-Kyoto rats (WKY) show a 4-day estrous cycle. The aim of this study was to examine the impact of 17beta-estradiol supplementation every fourth day to ovariectomized rats - mimicking the physiological estrous cycle - on regulation of blood pressure. We monitored blood pressure telemetrically in intact females, ovariectomized (OVX), and ovariectomized WKY injected subcutaneously with 17beta-estradiol (OVX (E2)) in a 4-day rhythm for 24 weeks. Blood pressure decreased both in intact females and OVX (E2), whereas that of OVX persisted at constant levels. The underlying mechanisms studied include the nitric oxide pathway, the rennin-angiotensin system as well as the endothelin system. Serum and urinary nitrate/nitrite (NOx) as well as aortic eNOS decreased in OVX and were restored to normal in OVX (E2). Conversely, caveolin-1 was higher in OVX than in intact females and OVX (E2) while Hsp90 did not differ among groups. Plasma angiotensin II and aortic AT (1) receptor expression increased in OVX and were normalized in OVX (E2). AT (2) receptor expression was regulated reciprocally. Serum endothelin-1 was significantly elevated in OVX and OVX (E2). There was no difference in aortic ET (A) receptor expression between groups whereas ET (B) receptor expression was higher in intact females and OVX (E2) than in OVX. The study suggests that supplementation of 17beta-estradiol in female WKY according to the natural estrous cycle maintains the physiological blood pressure encompassing vasorelaxing and vasoconstricting pathways. The physiological estrous cycle should be kept in mind when cardiovascular data are to be collected/interpreted under estrogen supplementation.

Effect of short light-dark cycles on young and adult TGR(mREN2)27 rats.

Animals placed under short light-dark (LD) cycles show a dissociation of their circadian rhythms. However, this effect has only been studied in Wistar rats and with the motor activity (MA) rhythm. Thus, in the present experiment, we studied in TGR(mREN2)27 (TGR) rats, a strain of hypertensive rats, the effect of a short LD cycle on the circadian rhythms of MA, heart rate (HR), and blood pressure (BP). Our aim was (1) to investigate whether the exposure of TGR rats to a short LD cycle induced a dissociation of their circadian rhythms, (2) to study the effect of short LD cycles on the development of the circadian rhythms of TGR rats, and (3) to compare the effect of short LD cycles on young and adult TGR rats. One group of TGR rats was maintained under LD cycles of 22h periods (group G22). The progress in time of their rhythms was compared to that of TGR rats of the same age that had been kept under LD cycles of 24h periods (group G24). For the third point, the rhythms of a group of 5-week-old TGR rats kept under LD 22h cycles (young rats) were compared to those of a group of 11-week-old TGR rats (adult rats). Results showed that there is a dissociation of the circadian rhythms of all the variables monitored in TGR rats maintained under LD 22h cycles, independent of age. We have also found that group G22 showed a higher increase in BP with age and a higher mortality due to malignant hypertension compared to group G24. Finally, it seems that it is harder for young rats to entrain to short LD cycles than for adult rats, and young rats have a higher mortality due to malignant hypertension than adult rats. In conclusion, we demonstrated that short LD cycles produce a dissociation in the HR, BP, and MA circadian rhythms. The results of this experiment, compared to those previously obtained in Wistar rats, suggest that the light perception, the responses of the circadian system to light, or both are altered in the TGR rats.

Cardiovascular regulation in TGR(mREN2)27 rats: 24h variation in plasma catecholamines, angiotensin peptides, and telemetric heart rate variability.

Dysfunction of the sympathetic nervous system might play an important role in disturbed 24h blood pressure regulation in transgenic hypertensive TGR (mREN2)27 (TGR) rats. Our study was performed to determine possible differences in activity of the sympathetic nervous system in TGR rats in comparison to their normotensive Sprague-Dawley (SPRD) controls; we measured plasma catecholamine and angiotensin concentrations throughout 24h under synchronized light-dark 12h:12H (LD 12:12) conditions. In the TGR rat strain, rhythms of plasma catecholamines were blunted, and the concentrations were significantly decreased. In addition, TGR rats showed increased plasma angiotensin I and II concentrations without any significant rhythm. An impaired autonomic regulation was confirmed by monitoring heart rate variability in TGR rats. Data showed that the TGR rat strain is characterized by a reduction in plasma catecholamines and an increase in angiotensin peptides. At present, it is not clear whether the reduction in catecholamines represents a decrease in sympathetic tone mediated by baroreflex activation or an increased catecholamine turnover induced by elevated angiotensin II. However, the blunted, but normally phased, rhythms in plasma catecholamines in TGR rats make it unlikely that the sympathetic nervous system is mainly responsible for the inverse circadian blood pressure rhythm in the transgenic strain.

Sickness behaviour, an animal equivalent to human quality of life, is improved in septic rats by G-CSF and antibiotic prophylaxis.

BACKGROUND AND AIM: New biological response modifiers are usually tested in reductionistic, pharmacological animal models by the determination of mechanistic endpoints (mortality rate, cellular/physiological parameters). In the meantime, quality of life had become an important endpoint in clinical trials but adequate animal experiments are very rare. The aim of this study was to demonstrate alterations in the behavioural response of septic rats due to a prophylaxis with cytokine (G-CSF) plus antibiotics. METHODS: Sickness behaviour (locomotor activity, circadian rhythms of blood pressure, heart rate and temperature) was determined by the use of radio telemetry. Complex animal experiments in rats were performed including anaesthesia, antibiotic and G-CSF prophylaxis, volume substitution, laparotomy, contamination and infection with human faecal suspension and postoperative analgesia. RESULTS: Prior to infection, rats showed circadian rhythm in locomotor activity, blood pressure, heart rate and temperature. Sham operation did not alter these parameters significantly. Immediately after abdominal contamination and infection, locomotor activity was strongly reduced and circadian rhythm was lost in all parameters. Body temperature showed a continuous rise, peaking 38 h after infection. Untreated animals died in 63% (8/14) of cases. Antibiotic prophylaxis blunted the febrile response and markedly reduced mortality to 20% (2/10) or 0% (0/10) using G-CSF plus antibiotics. Blood pressure and heart rate were increased in parallel with the rise in temperature. These early physiological changes were not prevented by prophylaxis, but normal behaviour was restored faster with G-CSF plus antibiotic prophylaxis. CONCLUSIONS: In septic rats, sickness behaviour (locomotor activity) is significantly improved in parallel to the mortality rate by a prophylaxis with G-CSF plus antibiotics. Sickness behaviour can be considered as an equivalent to human quality of life.

Loss of 24 h rhythm and light-induced c-fos mRNA expression in the suprachiasmatic nucleus of the transgenic hypertensive TGR(mRen2)27 rat and effects on cardiovascular rhythms.

Immediate early genes, especially c-fos, are thought to play an essential role in photic entrainment of circadian rhythms. A special characteristic of the transgenic hypertensive TGR(mRen2)27 rat strain, expressing an additional mouse renin2 gene, is the inverse blood pressure rhythm in relation to those in heart rate and activity resulting in internal desynchronisation of these physiological rhythms. Assessment of c-fos mRNA expression by microdissection and RT-PCR in the suprachiasmatic nucleus showed, that in contrast to normotensive Sprague-Dawley rats the 24 h and circadian rhythm of c-fos mRNA expression in TGR(mRen2)27 rats is abolished. Moreover, light-induced c-fos expression within the nucleus could be found in the normotensive controls, but was absent in transgenic hypertensive rats. The light pulse applied during the subjective night, at CT 14, significantly phase delayed rhythms in blood pressure, heart rate and activity in the normotensive rats by about 2 h, whereas in the transgenic hypertensive animals rhythms in blood pressure and heart rate were unaffected, only activity showed a slight phase shift. In conclusion, these data suggest that the transgene in TGR leads not only to a disturbance of the cardiovascular system but also influences the light entrainment response, which is accompanied by a suppressed c-fos mRNA expression in the suprachiasmatic nucleus.

Relevance for chronopharmacology in practical medicine.

Nearly all functions of the body, including those influencing pharmacokinetic parameters, such as drug absorption and distribution, drug metabolism, and renal elimination display significant daily variations. Also, the onset and symptoms of diseases such as asthma attacks, coronary infarction, angina pectoris, stroke, and ventricular tachycardia are circadian-phase dependent. Asthma attacks predominantly occur around 4 o'clock at night. Blood pressure and heart rate in normotensives and essential (primary) hypertensive patients display highest values during daytime followed by a nightly drop and an early morning rise. In about 70% of forms of secondary hypertension, however, this rhythmic pattern is abolished or even reversed exhibiting nightly peaks in blood pressure. Similar findings were obtained in children. This form of hypertension is accompanied by increased end organ damages. These observations call for a circadian time-specified drug treatment. In nocturnal asthma unequal dosing of antiasthmatic drugs with a higher/single evening dose is recommended. In secondary hypertension not only the elevated blood pressure must be reduced but the disturbed blood pressure profile should be normalized, too, possibly best achieved by evening dosing. Pharmacokinetics may also not be constant within 24 hours of a day as shown for cardiovascular active drugs, antiasthmatics, anticancer drugs, psychotropics, analgesics and local anesthetics, antibiotics to mention but a few. Far more drugs were shown to display significant daily variations in their effects even after chronic application or constant infusion. Because circadian rhythms undergo maturation with development, drug therapy in children can/may also be modified by circadian time of drug dosing as shown for anticancer drugs. In conclusion, there is clear evidence that the dose/concentration-response relationship of drugs can be significantly dependent on the time of day. Thus, circadian time has to be taken into account as an important variable influencing a drug's pharmacokinetics and/or its effects or side effects.

Basal and stimulated hippocampal adenylate cyclase activity in the experimentally lesioned rat entorhinal cortex.

Early stage development of Alzheimer-related neurofibrillary tangles occurs primarily in neurons of entorhinal cortex layers pre-alpha and pre-beta. These excitatory neurons project into the hippocampus. At this stage ('entorhinal' case), while neurofibrillary tangles are still absent from the hippocampus, a significant reduction in hippocampal adenylate cyclase activity has been detected. To test whether this reduction is a consequence of a deafferentation (and thus not a specifically disease-related alteration), we performed unilateral electrolytic lesions and sham-operations of the rat entorhinal cortex. The animals were killed 2, 12 and 55 days post lesion (dpl) and hippocampal adenylate cyclase activity was assayed. The major results were as follows: (1) both lesioned and unlesioned sides showed higher activity than a sham-operated control; (2) the adenylate cyclase activity of the lesioned side increased to a significantly lesser degree than that of the unlesioned side at 12 dpl; (3) this 'decrease' was attributed to changes in G protein-mediated activation of adenylate cyclase; (4) at no time point post lesion did the pattern of rat adenylate cyclase activity resemble that observed in Alzheimer's disease. Our data suggests that the loss of entorhinal afferents alone cannot explain the reduction in cyclase-activity seen in 'entorhinal' cases.

Signal transduction in cardiac and vascular tissue from normotensive and transgenic hypertensive TGR(mREN2)27 rats.

Adenylyl cyclase and soluble guanylyl cyclase activities were measured in cardiac and aortic tissue from transgenic hypertensive TGR(mREN2)27 and normotensive Sprague-Dawley rats. Cardiac basal and stimulated adenylyl cyclase activity was significantly lower in TGR(mREN2)27 than in Sprague-Dawley rats except after uncoupling of G-proteins by Mn2+-ions. Aortic cAMP formation did not differ between both strains, indicating that the disturbance of cardiac adenylyl cyclase activity was due to local rather than systemic factors. Vascular cGMP formation was significantly reduced in TGR(mREN2)27 aortae under basal conditions and after stimulation with sodium nitroprusside, indicating that there is a subsensitive vasodilating second messenger pathway in the transgenic strain.

Diurnal and seasonal changes in sympathetic signal transduction in cardiac ventricles of European hamsters.

This investigation of the relationship between cardiac beta-adrenoceptors and adenosine 3',5'-cyclic monophosphate (cAMP) formation in cardiac ventricles of the nocturnally active European hamster both during euthermia under a 12:12-h dark-light cycle and during hibernation under constant-darkness conditions showed that neither the densities, affinities, nor distribution of the beta 1- and beta 2-receptor subtypes differed between the dark phase, light phase, and hibernation. Basal formation of cAMP by the cardiac adenylyl cyclase of euthermic hamsters was higher in ventricles obtained at night [core temperature (Tcore) = 37.8 degrees C] than in ventricles obtained during the day (Tcore = 36.4 degrees C). Basal formation of cAMP was also significantly lower in hibernating hamsters (Tcore = 7.0 degrees C) than in euthermic hamsters. When adenylyl cyclase activity was stimulated by isoprenaline, guanylylimidodiphosphate [Gpp(NH)p], or forskolin, the rank order of potency was the same in euthermic hamsters and hibernating hamsters: isoprenaline < Gpp(NH)p < forskolin. Functional competition curves indicated that in the euthermic hamsters beta 1-receptors were responsible for 67% of the response to isoprenaline at night and 62% of the response during the day. In hibernating hamsters, in contrast, most of the response to isoprenaline (58%) was mediated via beta 2-receptors. This shift in the relative importance of the receptor subtypes may facilitate arousal from hibernation by making the heart more sensitive to circulating epinephrine.

Clinical chronopharmacology: the importance of time in drug treatment.

Nearly all functions of the body, including those influencing pharmacokinetic parameters such as drug absorption and distribution, drug metabolism and renal elimination, show significant daily variations: these include liver metabolism, hepatic blood flow and the first-pass effect; glomerular filtration, renal plasma flow and urine volume and pH; blood pressure, heart rate and organ perfusion rates; acid secretion in the gastro-intestinal tract and gastric emptying time. The onset and symptoms of diseases such as asthma attacks, coronary infarction, angina pectoris, stroke and ventricular tachycardia are circadian phase dependent. In humans, variations during the 24 h day in pharmacokinetics (chrono-pharmacokinetics) have been shown for cardiovascularly active drugs (propranolol, nifedipine, verapamil, enalapril, isosorbide 5-mononitrate and digoxin), anti-asthmatics (theophylline and terbutaline), anticancer drugs, psychotropics, analgesics, local anaesthetics and antibiotics, to mention but a few. Even more drugs have been shown to display significant variations in their effects throughout the day (chronopharmacodynamics and chronotoxicology) even after chronic application or constant infusion. Moreover, there is clear evidence that even dose/concentration-response relationships can be significantly modified by the time of day. Thus, circadian time has to be taken into account as an important variable influencing a drug's pharmacokinetics and its effects or side-effects.

Effects of bright light on circadian patterns of cyclic adenosine monophosphate, melatonin and cortisol in healthy subjects.

Bright light is known as a strong zeitgeber on human circadian rhythms and influences several endocrine and neuroendocrine functions. In the present study we examined the influence of a 3-h bright light stimulus, given at different times during the day (morning or evening), on circadian patterns of cyclic adenosine monophosphate (cAMP), melatonin and cortisol. Two groups of synchronized healthy volunteers (lights on: 05.00-23.00 h) were exposed to bright light (2500 lux) for 3 h over 6 days either in the morning (05.00-08.00 h) or in the evening (18.00-21.00 h). The results showed a significant phase advance in the circadian rhythms of melatonin and cortisol when bright light was given in the morning but not when given in the evening. Rhythm in plasma cAMP basically was not affected by either light treatment.

Adenylyl cyclase activity in Alzheimer's disease brain: stimulatory and inhibitory signal transduction pathways are differently affected.

Adenylyl cyclase (AC) activity was studied in post mortem hippocampus and cerebellum from eight patients with Alzheimer's disease/senile dementia of the Alzheimer type (AD/SDAT) and seven non-demented control patients. AC was stimulated via stimulatory guanine nucleotide binding proteins (Gs) using guanosine triphosphate (GTP) and GppNHp (both 10(-4) M) or directly with either forskolin (10(-4) M) or Mn2+ (10(-2) M). Inhibition of AC via A1-receptors was performed with N6-cyclohexyladenosine (CHA) under basal conditions and in the presence of forskolin (10(-5) M). In both brain regions AC activity was significantly reduced in AD/SDAT when compared to controls. Under basal conditions and after stimulation via Gs mean reduction in hippocampus and cerebellum was 47.7% and 58.2%, respectively. The reduction was less pronounced after direct activation of the AC, amounting to 21.8% in hippocampus and 28.1% in cerebellum. CHA inhibited basal and forskolin-stimulated AC concentration-dependently by about 20% (basal) and 30% (forskolin). Inhibition by CHA was similar in hippocampus and cerebellum and tended to be more pronounced in AD/SDAT than in controls. Since the reduction of AC activity in AD/SDAT is greater after stimulation via Gs than after direct activation of the catalytic subunit, we suggest that both Gs and the catalytic subunit seem to be impaired. The fact that CHA-mediated inhibition of AC is not significantly different in AD/SDAT and controls, indicates that in contrast to Gs-, inhibitory G-proteins (Gi) coupling to AC remains intact in Alzheimer's disease.

Circadian rhythm of soluble interleukin-2 receptor in healthy individuals.

The cytokine levels of soluble interleukin-2 receptor (sIL-2R), interleukin-6 (IL-6) and tumor-necrosis-factor alpha (TNF-alpha) were studied in 12 healthy volunteers at 11 different times of day. TNF-alpha levels were below the detection limit, and IL-6 levels were at baseline values of the respective assay used. Interindividual variations were found for the plasma levels of sIL-2R (179-524 U/ml). Shedded IL-2 receptors displayed a pronounced circadian phase-dependency (p less than 0.0001) with a peak value at 12:29 h and a trough at 4:14 h when a complex cosine function (period lengths: 24 h plus 12 h) was fitted to the data. These findings may be of importance when using sIL-2R as a diagnostic tool as well as in controlling efficacy of drug treatment.

Reduced basal and stimulated (isoprenaline, Gpp(NH)p, forskolin) adenylate cyclase activity in Alzheimer's disease correlated with histopathological changes.

Cyclic adenosine monophosphate (cAMP) is an adenylate cyclase borne second messenger involved in basic metabolic events. The beta-adrenoceptor sensitive adenylate cyclase was studied in post-mortem hippocampi of controls and Alzheimer patients. Virtually identical subsets of each hippocampus homogenate were stimulated by 100 mumol isoprenaline, Gpp(NH)p and forskolin, respectively, in presence of an ATP-regenerating system. The determination of cAMP formed was carried out by means of a radioassay. The observed significant 50% reduction in basal as well as in stimulated adenylate cyclase activity in Alzheimer's disease is negatively correlated with semiquantitative evaluations of amyloid plaques (P less than 0.05) but not with neuritic plaques, neurofibrillary tangles or neuropil threads. This reduction in enzyme activity is obviously not due to simple cell loss alone. It is likely that the crucial point of the observed functional disturbance is at the level of the catalytic unit of the adenylate cyclase, since the same degree of reduction is maintained at all steps of the signal cascade.

Reduced cAMP-signal transduction in postmortem hippocampus of demented old people.

The basal as well as the stimulated activity of the adenylate cyclase was determined in postmortem hippocampi. The tissue probes were obtained from 12 demented individuals (10 Alzheimer-type dementia; 1 Down's syndrome; 1 argyrophilic grains syndrome) and from 15 age-matched controls. The diagnoses were done in accordance with histopathological criteria. Adenylate cyclase was stimulated by isoprenaline, Gpp(NH)p, or forskolin. The amount of cAMP formed was determined by the protein binding method using a radioimmuno assay. In tissues of controls as well as of demented patients adenylate cyclase was stimulated in the rank order of isoprenaline less than Gpp (NH) p less than forskolin. In hippocampal tissues of demented individuals a significant reduction (50%, p less than 0.01) in basal as well as stimulated adenylate cyclase activity was found. This reduction in cAMP signal transduction is not caused by simple cell loss.

On the daily variation in the beta-receptor-adenylate cyclase-cAMP-phosphodiesterase system in rat forebrain.

In rat forebrain tissue of single rats beta-adrenoceptor density (Bmax) and affinity (Kd) were determined by saturation isotherms in receptor binding studies with the antagonist ligand (3H)-dihydroalprenolol at 8 different times of day in May. Rats were on a controlled 12L:12D photoperiod. In addition, the cAMP content, the formation of cAMP from ATP by the adenylate cyclase and the hydrolysis of the second messenger by the phosphodiesterase were determined at the same time points. No significant (ANOVA) daily variations were found in the total number of 3H-DHA binding sites (Bmax) nor in the affinity (Kd). In contrast, basal cAMP content as well as basal formation and hydrolysis of cAMP displayed significant rhythms. The peak value in cAMP was at the beginning of light. At that time the daily trough value in cAMP formation was found. Hydrolysis of cAMP by the phosphodiesterase displayed a 12-hr rhythm with trough values occurring at the early light and early dark period. The results demonstrate pronounced rhythmic changes in basal formation, content and hydrolysis of cAMP which are, however, not paralleled by changes in receptor number and/or affinity in the same tissue.

Circadian rhythm and seasonal variations in basal cAMP content of rat heart ventricles.

The cAMP content in rat heart ventricles was studied at 3-hr intervals during 24hr at different times of the year. A significant circadian rhythm in cAMP content was found. Time of the year reproducibly influenced the 24-hr mean, the amplitude as well as the peak value in cAMP in relation to circadian time.