Lubos Molcan

The Presence and Localization of Melatonin Receptors in the Rat Aorta

Melatonin is involved in blood pressure modulation in rats and humans. Some of the effects of melatonin are presumably mediated via two G-protein-coupled receptors (MT1 and MT2), but the distribution of MT1 and MT2 in the cardiovascular system remains to be explored comprehensively. We investigated the expression of both the receptors in the rat aorta on mRNA level by RT-PCR and real time RT-PCR as well as on protein level via western blotting and immunofluorescence microscopy. We verified MT1 mRNA expression in the rat aorta and demonstrated the absence of MT2 mRNA in this vessel type. MT1 receptors were confirmed also at the protein level, and surprisingly they were preferentially localized to the tunica adventitia. Since no daily changes in MT1 mRNA expression were detected, we suppose that the circadian changes in circulating melatonin concentrations are sufficient to mediate circadian effects of melatonin in the aorta. The localization of MT1 in the tunica adventitia suggests an influence of melatonin on vasa vasorum function and signal transduction in the aorta wall.

Prenatal hypoxia in rats increased blood pressure and sympathetic drive of the adult offspring.

Decreased oxygenation during pregnancy and early periods of ontogeny can affect normal body development and result in diseases in adulthood. The aim of this study was to use the model of prenatal intermittent hypoxia (PIH) and evaluate the effects of short-term hypoxia at the end of gestation on blood pressure (BP) control in adulthood. Wistar rats were exposed daily to PIH for 4 h during gestational day 19 and 20. In adult male rats, heart rate (HR), systolic BP and pulse pressure (PP) were acquired by radiotelemetry during 1 week. On the basis of HR variability and BP variability, sympathovagal balance (LF/HF) and spontaneous baroreflex sensitivity (sBRS) were evaluated. Systolic BP and PP were significantly elevated in PIH rats in comparison with control rats during the light and dark phase of the day, while LF/HF increased only during the light phase of the day. In contrast, sBRS tended to decrease only during the dark phase in PIH rats. In all measured and calculated parameters, significant circadian rhythms were present and were not affected by PIH. In conclusion, our data suggest that short intermittent hypoxia at the end of gestation can increase BP and PP via significant changes in LF/HF, which occur especially during the passive phase of the day. Results suggest that minor changes in the autonomous nervous system activity induced by environmental conditions during the perinatal period may contribute to development of hypertension in adulthood.

Increased salt intake during early ontogenesis lead to development of arterial hypertension in salt-resistant Wistar rats

Abstract A direct relationship exists between salt consumption and hypertension. Increased sodium intake does not automatically lead to a rise in blood pressure (BP) because of marked intra-individual variability in salt sensitivity. Wistar rats are a salt-resistant strain and increased salt intake in adults does not induce hypertension. Mechanisms regulating BP develop during early ontogenesis and increased sodium consumption by pregnant females leads to an increase in BP of their offspring, but early postnatal stages have not been sufficiently analyzed in salt-resistant strains of rats. The aim of this work was to study the effects of increased salt during early ontogeny on cardiovascular characteristics of Wistar rats. We used 16 control (C; 8 males + 8 females) rats fed with a standard diet (0.2% sodium) and 16 experimental (S; 8 males + 8 females) rats fed with a diet containing 0.8% sodium. BP was measured weekly and plasma renin activity, aldosterone and testosterone concentrations were assayed by radioimmunoassay after the experiment in 16-week-old animals. In the kidney, AT1 receptors were determined by the western blot. BP was higher in the S as compared with the C rats and did not differ between males and females. The relative left ventricle mass was increased in S as compared with C males and no differences were recorded in females. No significant differences between groups were found in hormonal parameters and AT1 receptors. Results indicate that moderately increased salt intake during postnatal ontogeny results in a BP rise even in salt-resistant rats.

Decreased emotional reactivity of rats exposed to repeated phase shifts of light-dark cycle.

Disturbed light-dark (LD) cycles are associated with circadian disruption of physiological and behavioural rhythms and in turn with an increased risk of disease development. However, direct causal links and underlying mechanisms leading to negative health consequences still need to be revealed. In the present study, we exposed male Wistar rats to repeated phase shifts of LD cycle and analysed their ability to cope with mild emotional stressors. In experiment 1, rats were submitted to either a regular 12:12 LD cycle (CTRL rats) or 8-h phase delay shifts applied every 2days for 5weeks (SHIFT rats). Subsequently, the behaviour was examined in the open-field, black-white box and elevated plus maze tests. In experiment 2, changes in blood pressure (BP), heart rate (HR) as well as the activity of autonomic nervous system were measured in telemeterised rats in response to open-field and black-white box tests before and after 5-week exposure to shifted LD regime. Locomotor activity was consistently higher in SHIFT than CTRL rats in in the open-field and black-white box tests. Interestingly, in the elevated plus maze, SHIFT rats displayed increased risk assessment and decreased grooming compared to CTRL rats. Anxiety measures were affected only in the black-white box, where SHIFT rats displayed reduced anxiety-like behaviour compared to CTRL rats. Differences in behavioural reactivity between SHIFT and CTRL rats did not correspond with BP and HR changes. However, exposure to phase shifts increased the sympathovagal reactivity in the black-white box. Together, our results demonstrated that disturbed LD conditions decreased emotional reactivity of rats and affected their ability to cope with emotional stressors denoting an additional risk mechanism linking disrupted circadian organisation to adverse health effects.

Endocrine and cardiovascular rhythms differentially adapt to chronic phase-delay shifts in rats

ABSTRACT Disturbances in regular circadian oscillations can have negative effects on cardiovascular function, but epidemiological data are inconclusive and new data from animal experiments elucidating critical biological mechanisms are needed. To evaluate the consequences of chronic phase shifts of the light/dark (LD) cycle on hormonal and cardiovascular rhythms, two experiments were performed. In Experiment 1, male rats were exposed to either a regular 12:12 LD cycle (CONT) or rotating 8-h phase-delay shifts of LD every second day (SHIFT) for 10 weeks. During this period, blood pressure (BP) was monitored weekly, and daily rhythms of melatonin, corticosterone, leptin and testosterone were evaluated at the end of the experiment. In Experiment 2, female rats were exposed to the identical shifted LD schedule for 12 weeks, and daily rhythms of BP, heart rate (HR) and locomotor activity were recorded using telemetry. Preserved melatonin rhythms were found in the pineal gland, plasma, heart and kidney of SHIFT rats with damped amplitude in the plasma and heart, suggesting that the central oscillator can adapt to chronic phase-delay shifts. In contrast, daily rhythms of corticosterone, testosterone and leptin were eliminated in SHIFT rats. Exposure to phase shifts did not lead to increased body weight and elevated BP. However, a shifted LD schedule substantially decreased the amplitude and suppressed the circadian power of the daily rhythms of BP and HR, implying weakened circadian control of physiological and behavioural processes. The results demonstrate that endocrine and cardiovascular rhythms can differentially adapt to chronic phase-delay shifts, promoting internal desynchronization between central and peripheral oscillators, which in combination with other negative environmental stimuli may result in negative health effects.

Shifts in the light-dark cycle increase unpredictability of the cardiovascular system

Physiological variables such as heart rate (HR) and blood pressure (BP) exhibit long-term circadian rhythms, which can be disturbed by shift work. On the other hand, short-term oscillations in HR and BP have a high prognostic value. Therefore, we aimed to determine if the short-term variability, complexity and entropy of HR and BP would be affected by a regular light/dark (LD) cycle and phase delay shifts of the LD cycle, leading to chronodisruption. Telemetry-monitored rats were exposed first to the regular LD cycle and then to shifts in LD for 8weeks. On the basis of long-term HR and BP recording and evaluation, we found circadian rhythms in HR and BP variability, complexity and entropy under regular LD cycles. Short-term exposure to shifts disturbed circadian rhythms of HR and BP variability, complexity and entropy, indicating chronodisruption. The power of circadian rhythms was suppressed after 8weeks of phase delay shifts. Long-term exposure to shifts increased variability (p=0.007), complexity (p<0.001) and dark-time entropy (p=0.006) of HR but not BP. This is the first study demonstrating long-term recording and estimation of HR and BP variability, complexity and entropy in conscious rats exposed to irregular lighting conditions. After long-term phase delay shifts, short-term variability of HR was less predictable than in controls. This study suggests that changes in short-term HR and BP oscillations induced by long-term shift work can negatively affect cardiovascular health.