By: Gang Wang
American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 2008
Previous studies have shown that females develop hypertension later in life (and it is usually less severe) than males do. This indicates that sex differences can play a role in determining risk factors for development of high blood pressure. One of the things involved in tonic regulation of arterial pressure is the presence of bulbospinal neurons in the RVLM. When Angiotensin II (ANG II) is injected into the RVLM, an increase in sympathetic nerve activity and blood pressure is observed due to being mediated by angiotensin type I (AT1) receptors. As part of the experiment, the research team analyzed RVLM bulbospinal neurons to determine if there was a sex difference in the following: 1. Immunolabeling for AT1 receptors and the key NADPH-oxidase subunit p47 using dual-label immunoelectron microscopy and 2. Effects of ANG II on ROS production and Ca2+ currents hydroethidine fluoromicroscopy and patch-clamping. The purpose of these analyses were to test the hypothesis that an increase in AT1 receptors in female RVLM neurons is counterbalanced by reduction in p47 levels, so that ANG II-induced production does not differ between males and females.
To test the hypothesis, 4 month and 12 month old male and female Sprague-Dawley rats were used. All the young adult and middle-aged female rats underwent isoflurane anesthesia to receive bilateral ovariectomies. This was done to determine more directly the role of estrogen, since the females were given an estrogen replacement following the surgery. To analyze the data, the following methods were used: retrograde labeling of RVLM neurons, immunolabeling of isolated RVLM neurons, patch-clamp recordings of isolated RVLM neurons, and detection of intracellular reactive oxygen species (ROS) using dihydroethidium as an indicator.
The results showed that female tyrosine hydroxylase (TH) RVLM neurons displayed significantly more AT1 and less p47 than males. In addition, in the population of TH-labeled RVLM neurons, females had a higher level of AT1 receptor-ImG labeling than males. In terms of the L-type Ca2+ channels, currents were used to assess their function. ANG II increased L-type Ca2+ currents of female RVLM bulbospinal neurons through the AT1 receptors and NADPH oxidase. This lead to the discovery that ANG II-induced L-type Ca2+ currents, but not the ROS production, was greater in females than in males.
From the results above, three things can be concluded: 1. Female P23 rats display more AT1 receptor-ImG labeling and less NADPH oxidase subunit p47-ImG labeling in the TH-labeled RVLM neurons compared to males, 2. The elevated levels of AT1 receptor-ImG labeling in females persist into adulthood and midlife and 3. AT1 receptor-ImG labeling was higher in estradiol than in vehicle-treated OVX females.
In summary, female rats contained more AT1-receptors in TH-containing neurons of the RVLM than male rats, and this increase was associated with the reduction of NADPH oxidase subunit p47 in female rats. Overall these findings revealed previously unknown sex differences in ANG II receptor expression and signaling in autonomic neurons, which are critically involved in controlling the cardiovascular system and blood pressure.
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