Mary Key

Nocturnal hypertension in mice consuming a high fructose diet

OBJECTIVE To investigate the effect of fructose consumption on the light/dark pattern of blood pressure, heart rate and autonomic neural function in mice. BACKGROUND Insulin resistant diabetes is associated with hypertension and autonomic dysfunction. There is evidence that the increasing incidence of diabetes may be related to dietary changes, including consumption of high levels of fructose. DESIGN/METHODS C57/BL mice, instrumented with radiotelemetric arterial catheters, were fed a control or high fructose diet (60%). Cardiovascular parameters measured were light/dark pattern of mean arterial pressure (MAP), heart rate (HR) and variability (time and frequency domain). We also measured plasma insulin, glucose, lipids and angiotensin II (Ang II) as well as glucose tolerance. In situ hybridization was used to measure brainstem expression of tyrosine hydroxylase (TH) and Ang AT1a mRNA. RESULTS Fructose diet (8 weeks) produced an increase in MAP, variance and low frequency domain (14+/-3 vs. 33+/-4 mm Hg(2), variance and 10+/-2 vs. 26+/-4 mm Hg(2), LF, control vs. fructose, P<0.01). The changes occurred only at night, a period of activity for mice. Glucose tolerance was attenuated in the fructose group. Fructose also increased plasma cholesterol (80+/-1 vs. 126+/-2 mg/dl, control vs. fructose, P<0.05) and plasma Ang II (18+/-5 vs.65+/-12 pg/ml, control vs. fructose, P<0.05). Depressor responses to alpha(1)-adrenergic blockade with prasozin were augmented in fructose-fed mice. Using quantitative in situ hybridization, we found that Ang AT1a receptor and TH mRNA expression were significantly increased in the brainstem locus coeruleus. CONCLUSION A high fructose diet in mice produced nocturnal hypertension and autonomic imbalance which may be related to activation of sympathetic and angiotensin systems.

Stress-induced pressor and corticosterone responses in oxytocin-deficient mice

We used oxytocin knockout (OTKO) mice to investigate the role of oxytocin in regulation of blood pressure, heart rate and stress reactivity (pressure reactivity and plasma corticosterone). Male OTKO and control wild‐type mice with carotid arterial catheters were exposed to intermittent shaker stress for 7 days (2 min stressors, 45 times per day). Mean arterial pressure (MAP) and heart rate (HR) were recorded continuously (24 h) before stress (basal), on stress days 1, 3 and 7 (S1, S3 and S7) and 1 day poststress (recovery). Plasma corticosterone (Cort) was measured before stress and 30 min after the last stress on day 7. Twenty‐four hour averages of MAP and HR were lower in OTKO mice than in controls (P < 0.0001 and P < 0.005, respectively) with a significant diurnal rhythm. Chronic stress (S1 and S3) produced an increase in 24 h average MAP in OTKO mice, but not in controls. There were no stress‐related changes in 24 h average HR values between control and OTKO mice. The immediate pressor responses were analysed during the dark and light periods (19.00 and 08.00 h). During the dark period, stress‐induced pressor responses were observed only in OTKO mice (S1 and S3). In the light period, stress‐induced MAP increases were seen on all days in OTKO mice and on days S1 and S3 in controls. There were no differences in baseline Cort between the groups; however, OTKO mice showed a reduced response to chronic stress (+298 versus+411%, OTKO mice versus controls, P < 0.005). In conclusion, oxytocin deficiency alters the endocrine and pressor responses to chronic stress, suggesting that the endogenous oxytocin system is important in regulating the stress‐induced pressor response.

Circadian Differences in Stress-Induced Pressor Reactivity in Mice

Abstract—The objective of this study was to determine the effect of chronic stress exposure on the circadian pattern of cardiovascular responses in mice. Using male C57BL6 mice with carotid arterial catheters, we tested the effect of 7 days of intermittent shaker stress on body weight, food intake, drinking activity, plasma corticosterone, mean arterial pressure (MAP), and heart rate. The stress was delivered automatically for 2-minute periods (150 cycles/min), 45 times/d for 7 days. Plasma corticosterone was significantly increased in acutely and chronically stressed mice, with a partial attenuation in the chronic condition. Stress increased water intake, produced no change in food intake, and significantly decreased body weight (5% change). MAP and heart rate were measured continuously on stress days 1, 3, and 7 and during the basal and recovery periods. Chronic stress did not produce a sustained increase in MAP; however, there was an increase in MAP during the first stress day and a decrease during the recovery period. There was a circadian pattern in the pressor responses, with greater increases seen during the light period (nonactive phase) than in the dark period (+24% versus +11% on stress day 3, light versus dark). The results suggest that a stress delivered during the nonactive phase represents a higher cardiovascular risk.

Quantitative in situ hybridization for peptide mRNAs in mouse brain

The objective was to determine the feasibility of using a radioactive capture method (Fuji FLA 2000) and image analysis system for the measurement of peptide mRNA levels in specific brain regions in mice. As a test mRNA, we chose vasopressin (VP) and oxytocin (OT) because they are expressed in abundance in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). A comparison was made between free-floating and slide-mounted sections to determine which method yielded better results. Mouse brains were fixed in 4% paraformaldehyde (PFA) and processed for in situ hybridization using 35S-oligonucleotide probes for VP and OT. After overnight hybridization and high stringency washes, 25-microm brain sections and 14C standards were exposed to a BAS-IIIs Fuji imaging film over a range of times (4 h-6 days). Results showed that there was an intense hybridization reaction in the PVN and SON, making it possible to distinguish the specific brain regions. Using Image Gauge Software, the signal was quantified in PVN and SON. A comparison of the different exposure times showed that the signal could be measured after as little as 4 h. The intensity readings increased over time while the calculated radioactivity remained constant. The free-floating method was superior to the slide-based system, providing a lower background and a higher signal. The data illustrates the applicability of the phosphor imaging system for the reproducible measurement of mRNA levels in discrete regions of the mouse brain.

Sarin produces delayed cardiac and central autonomic changes

The aim was to evaluate the acute and delayed effects of low dose sarin exposure on cardiac autonomic and brainstem catecholaminergic function in mice. The rationale was to expand our knowledge of the cardiovascular effects of this neurotoxic, acetylcholinesterase (AChE) inhibitor. C57BL/6 male mice with telemetric arterial catheters were injected with saline or sarin (8 microg/kg, 0.05x LD(50); sc, two injections) with blood pressure (BP) measurements made at 1 and 10 weeks after sarin exposure. BP and pulse interval variability (PI) and low and high frequency spectral oscillations were measured using autoregressive spectral analysis. In situ hybridization (ISH) was used to quantify tyrosine hydroxylase (TH) mRNA expression in brainstem cardiovascular centers. Sarin had no effect on blood AChE activity, heart rate (HR) or BP. There was a biphasic response in PI variance, an early increase (+140%) and a delayed decrease (-62%) at more than 2 months after sarin exposure. There were no changes in BP variance. Assuming that increased PI variance is a positive outcome, the short-term response to sarin should be protective. This is opposite for the delayed decrease in PI variance which is associated with adverse cardiovascular effects. There was an increase in TH mRNA in both locus coeruleus (0.18+/-0.05 vs. 1.4+/-0.2 microCi/g; control vs. sarin) and dorsal vagal complex (0.09+/-0.06 vs. 1.17+/-0.03 microCi/g; control vs. sarin). Results show that a dose of sarin which had no peripheral cholinergic effects caused changes in autonomic modulation, a short-term enhancement followed by a delayed impairment in heart rate variability. Sarin-induced cardiac effects suggest a controversial aspect to the use of pharmacological agents which target AChE for management of cardiovascular risk.

5α-Reduced androgens block estradiol–BSA-stimulated release of oxytocin

In this study we test the postulate that estradiol conjugated to bovine serum albumin (E-BSA) acts via receptors for the steroid-binding protein sex hormone binding globulin (SHBG) by attempting to block E-BSA-stimulated release of oxytocin with two antagonists of SHBG receptor actions: the 5α-reduced androgens dihydrotestosterone (DHT) and 3α-diol. Simultaneous superfusion with either DHT or 3α-diol significantly blocked E-BSA-stimulated release of oxytocin. We also found that a wide range of free 17β-estradiol was unable to stimulate oxytocin release, suggesting that E-BSA stimulates receptors other than those for free estradiol to release oxytocin, perhaps SHBG receptors.

Cardiovascular Interactions Between Losartan and Fructose in Mice

Aim: To determine whether pharmacological blockade of angiotensin (Ang) AT1 receptors alters the cardiovascular, metabolic, and angiotensin-converting enzyme (ACE and ACE2) responses to a fructose diet in mice. Methods: C57BL male mice were fed with a 60% fructose diet for 8 weeks in combination with losartan treatment on week 9 (30 mg/kg per day). Blood pressure (BP), heart rate (HR), and autonomic balance were monitored using radiotelemetry with spectral analysis. Renal ACE and ACE2 activity and protein levels as well as Ang II and Ang 1-7 were measured. Results: Fructose impaired glucose tolerance and increased plasma cholesterol and insulin. These effects were not corrected by losartan treatment. Fructose increased BP and HR but only during the dark period. Short-term losartan treatment decreased BP by 16% in the fructose group but had no effect in controls. This was accompanied by a decrease in BP variance and its low-frequency component. Fructose increased Ang II (plasma and kidney) and ACE 2 (renal activity and protein expression). Losartan alone increased plasma Ang II in plasma and ACE2 in kidney. There were no changes in renal Ang 1-7 levels. Conclusions: Losartan reversed the pressor effect of a high fructose diet, demonstrating that there are prominent interactions between a dietary regimen that produces glucose intolerance and an antihypertensive drug that antagonizes Ang signaling. The mechanism of change may be via renal Ang II rather than the ACE2/Ang 1-7 pathway because the fructose losartan combination resulted in lowered renal Ang II without changes in Ang 1-7.

Rapid neurosecretory and cardiovascular response to osmotic stimulation in conscious mice.

Experiments were performed to evaluate the neuroendocrine and cardiovascular effects of osmotic stimulation in mice. Hypertonic saline (HS) was administered centrally or via the blood stream to conscious mice during measurement of blood pressure (BP), heart rate (HR) and plasma vasopressin (VP) and oxytocin (OT). A test of hypovolemia on VP secretion was also performed. Chronic carotid arterial cannulas were inserted for blood sampling, cardiovascular monitoring and vascular injections. Intracerebroventricular (ICV) cannulas were used for central injections. Vascular injection of HS (30 µl, 3.4 M NaCl) caused rapid and transient increases in plasma VP and OT. Plasma VP increased from 5.6 ± 0.9 to 10.0 ± 1.0 pg/ml, while plasma OT increased from 1.5 ± 0.6 to 8.6 ± 2.4 pg/ml at the earliest time point, immediately after ICV injection. ICV osmotic stimulation produced a rapid and sustained increase in plasma VP, with no change in OT. Plasma VP levels were increased from basal levels of 5.1 ± 1.5 to 13.1 ± 4.6, 11.4 ± 1.5, 12.6 ± 1.7 pg/ml at 0, 1 and 5 min after injection, respectively. ICV HS also increased plasma corticosterone. BP was increased by both vascular and central osmotic stimulation. Vascular HS increased BP immediately (Δ15.3 ± 1.7 mm Hg, 0 min) and transiently (Δ–3.9 ± 4.6 mm Hg, 5 min) while central HS produced a sustained increase in BP (Δ10 ± 1.4 and Δ9.8 ± 1.9 mm Hg, 0 and 5 min). Osmotic stimulation produced no significant changes in HR. Acute hemorrhage (∼10% decrease in blood volume) increased plasma VP (4.9 ± 1.0 vs. 8.4 ± 2.2 pg/ml). These results show the pattern of endocrine and cardiovascular responses to osmotic stimulation in conscious mice. They demonstrate that (1) there are extremely rapid changes in plasma VP and OT; (2) plasma OT is increased only after peripheral vascular hypertonic injection, and (3) central and peripheral osmotic stimulations are associated with pressor responses.