SEX DIFFERENCES IN THE SUBCELLULAR DISTRIBUTION OF ANGIOTENSIN TYPE 1 RECEPTORS AND NADPH OXIDASE SUBUNITS IN THE DENDRITES OF C1 NEURONS IN THE RAT ROSTRAL VENTROLATERAL MEDULLA

By J. P. PIERCE, J. KIEVITS, B. GRAUSTEIN, R. C. SPETH, C. IADECOLA, and T. A. MILNER 
Neuroscience, 2009

Division of Neurobiology, Department of Neurology and Neuroscience, Weill Cornell Medical College, 407 East 61st Street, New York, NY 
Harold and Margaret Milliken Hatch Laboratory of Neuroendocrinology, The Rockefeller University, 1230 York Avenue, New York, NY 
University of Mississippi School of Pharmacy, Department of Pharmacology, University of Mississippi, University, MS 

There are many studies that document the sex-differences of cardiovascular disorders in humans. Women have a lower risk of developing heart disease compared to men until the onset of menopause—at which point it flips. The sympathetic nervous system has been argued to contribute to the development of hypertension. The C1 neurons in the rostral ventrolateral medulla (RVLM), which express tyrosine hydroxylase (TH), are the main type of neuron that controls the sympathetic nervous system activity (SNA). They are tonically active, working to maintain the sympathetic vasomotor tone of individuals. When the activity of these neurons is increased, the vasoconstriction of the blood vessels increases and causes a similar increase in blood pressure. Aside from expressing TH, the C1 neurons also express angiotensin 1 (AT1) receptors. When angiotensin acts on these receptors, both the SNA and the blood pressure increases. In order to produce its effects, angiotensin binds to the AT1 receptor which stimulates NADPH oxidase after the phosphorylation of p47, an NADPH oxidase subunit. P47 then translocates to the cell membrane. NADPH oxidase then produces reactive oxygen species (ROS) to increase the blood pressure. Aside from the NADPH oxidase subunits that translocate to the cell membrane, there are also transmembrane units that act as an internalized pool, including p22. Thus, both pools in the RVLM C1 neurons need to be measured to understand the influence of NADPH oxidase on the development of hypertension. Additionally, the AT1-NADPH oxidase interaction may be where the sex-differences in hypertension originate.

Male, proestrus (high estrogen) female, and diestrus (low estrogen) female Sprague-Dawley rats were used. Vaginal smear cytology determined the stage of the estrus cycle for the female rats. The expression of p47, p22, and AT1 were measured by tagging them with antibodies (ImG antibody). After being tagged, the brains underwent electron microscopy to image the different densities of receptors and subunits. Furthermore, C1 neurons were labeled with ImP using the “avidin-biotin-peroxidase complex method” previously used in other studies.

The dendrites of the C1 neurons I both male and female rats expressed a high volume of AT1, p47, or p22 ImG labeling. They were expressed less in the presynaptic processes, suggesting that the action of ANG II occurs only at the C1 neurons to increase the sympathetic nerve activity. There were also two ImG populations found also the C1 membranes: 1. Membrane-associated and; 2. Internal particles. The internal pools were suggested to be p47, since it translocates to the membrane to form NADPH oxidase after the AT1 receptor is activated. The membrane-bound pool is theorized to be the p22 and AT1 since both of the receptors are transmembrane proteins. These pools were found to be distributed in a nonrandom fashion, suggesting the ImG antibody was successful in labeling the p47, p22, and AT1 proteins.

To investigate whether or not the AT1 receptor may contribute to the sex differences in hypertension, the levels of the AT1 receptor were measured. Female rats, in general, had 2.1 times more AT1 ImG labeling on the dendrites of their RVLM C1 neurons compared to males. It is important to note that previous studies have found that juvenile rats (23 days-old) and ovariectomized female rats have similar increases in the AT1 expression compared to males. These results suggest that sex differences persist throughout the life of rats. Therefore, although the increase in AT1 receptors may make female rats more sensitive to ANG II signaling, another component may be contributing to the age-related changes seen in menopausal females. However, it would be interesting to see if older rats have a reduced AT1 expression. This future study could help determine what happens at the onset of menopause to increase women’s risk of hypertension.

Females were found to have lower p47 ImG labeling compared their male counterparts. This difference was true for both the protein on the cell membranes and in the cytoplasm. p22 expression was not different between the sexes, which could mean that this pool of the subunit is not as critical in the control of blood pressure in rats. These results are supported by the findings of other studies and suggest that there the lower amount of NADPH oxidase subunit reduces the capacity of female rats to produce ROS in the C1 RVLM neurons. A reduced capacity leads to a lowered capability to increase SNA upon AT1 activation. Therefore, the female rats are unable to tonically increase their blood pressure as much as males. More studies should be done to further investigate the relationship since other pathways may be involved in the control of the expression of both the p47 protein and AT1 receptor.

Interestingly, AT1 and the NADPH oxidase subunits changed depending on the stage of the estrus cycle each rat was in. During proestrus (high estrogen), AT1 was much higher on the cell membrane compared to diestrus (low estrogen). The cytoplasm levels did not differ during the stages, nor did the expression of p22 or p47. This may make females more sensitive to ANG II during proestrus. However, the lower amounts of NADPH oxidase subunit prevent the increase in the blood pressure by reducing the capacity of the females to produce ROS. Without the increase in ROS, the SNA cannot increase with the ANG II signaling. The researchers describe this relationship as "counterbalancing" and may explain why females have an attenuated blood pressure response. 

In summary, the current study suggests that there are sex-differences in the C1 neurons and the ANG II signaling proteins involved in the maintenance of blood pressure. There may also be a direct impact from estrogen on the expression of the receptors.

-LivInLaVida