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Throughout the world, including the United States, men have worse outcomes from COVID-19 than women. SARS-CoV-2, the causative virus of the COVID-19 pandemic, uses angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. ACE2 is a member of the renin-angiotensin system (RAS) and plays an important role in counteracting the harmful effects mediated by the angiotensin type 1 receptor. Therefore, we conducted Ovid MEDLINE and Embase database searches of basic science studies investigating the impact of the biological variable of sex on ACE2 expression and regulation from 2000, the year ACE2 was discovered, through December 31, 2020. Out of 2,131 publications, we identified 853 original research articles on ACE2 conducted in primary cells, tissues, and/or whole mammals excluding humans. The majority (68.7%) of these studies that cited the sex of the animal were conducted in males, while 11.2% were conducted solely in females; 9.26% compared ACE2 between the sexes, while 10.8% did not report the sex of the animals used. General findings are that sex differences are tissue-specific and when present, are dependent upon gonadal state. Renal, cardiac, and adipose ACE2 is increased in both sexes under experimental conditions that model co-morbidities associated with worse COVID-19 outcomes including hypertension, obesity, and renal and cardiovascular diseases; however, ACE2 protein was generally higher in the males. Studies in Ace2 knockout mice indicate ACE2 plays a greater role in protecting the female from developing hypertension than the male. Studying the biological variable of sex in ACE2 research provides an opportunity for discovery in conditions involving RAS dysfunction and will shed light on sex differences in COVID-19 severity.

Estradiol (E2) decreases both water and saline intakes by female rats. The ERα and ERβ subtypes are expressed in areas of the brain that control fluid intake; however, the role that these receptors play in E2's antidipsogenic and antinatriorexigenic effects have not been examined. Accordingly, we tested the hypothesis that activation of ERα and ERβ decreases water and saline intakes by female rats. We found a divergence in E2's inhibitory effect on intake: activation of ERα decreased water intake, whereas activation of ERβ decreased saline intake. E2 decreases expression of the angiotensin II type 1 receptor (AT1R), a receptor with known relevance to water and salt intakes, in multiple areas of the brain where ERα and ERβ are differentially expressed. Therefore, we tested for agonist-induced changes in AT1R mRNA expression by RT-PCR and protein expression by analyzing receptor binding to test the hypothesis that the divergent effects of these ER subtypes are mediated by region-specific changes in AT1R expression. Although we found no changes in AT1R mRNA or binding in areas of the brain known to control fluid intake associated with agonist treatment, the experimental results replicate and extend previous findings that body weight changes mediate alterations in AT1R expression in distinct brain regions. Together, the results reveal selective effects of ER subtypes on ingestive behaviors, advancing our understanding of E2's inhibitory role in the controls of fluid intake by female rats., (Copyright © 2016 the American Physiological Society.)

To investigate age-associated impairments in fluid homeostasis, 4-mo (young) and 32-mo (old) Fischer 344/BN male rats were studied before and after a dietary sodium load. Transferring young rats from a low-sodium (LS) to a high-sodium (HS) diet increased water intake and urine volume by 1.9- and 3.0-fold, respectively, while urine osmolality and plasma aldosterone decreased by 33 and 98%. Concomitantly, adrenocortical angiotensin type 1 receptor (AT1R) density decreased by 35%, and AT1bR mRNA decreased by 39%; no changes were observed in AT1aR mRNA. In contrast, the increase in water intake (1.4-fold) was lower in the old rats, and there was no effect of the HS diet on urine volume or urine osmolality. AT1R densities were 29% less in the old rats before transferring to the HS diet, and AT1R densities were not reduced as rapidly in response to a HS diet compared with the young animals. After 6 days on the HS diet, plasma potassium was lowered by 26% in the old rats, whereas no change was detected in the young rats. Furthermore, while plasma aldosterone was substantially decreased after 2 days on the HS diet in both young and old rats, plasma aldosterone was significantly lower in the old compared with the young animals after 2 wk on the LS diet. These findings suggest that aging attenuates the responsiveness of the adrenocortical AT1R to a sodium load through impaired regulation of AT1bR mRNA, and that this dysregulation contributes to the defects in water and electrolyte homeostasis observed in aging., (Copyright © 2016 the American Physiological Society.)

Previous studies show that the angiotensin type 1 receptor (AT1R) is susceptible to rapid desensitization, but that more chronic treatments that stimulate ANG II lead to sensitization of several responses. It is unclear, however, if the processes of desensitization and sensitization interact. To test for differences in AT1R expression associated with single or repeated injections of ANG II, we measured AT1R mRNA in nuclei that control fluid intake of rats given ANG II either in a single injection or divided into three injections spaced 20 min apart. Rats given a single injection of ANG II had more AT1R mRNA in the subfornical organ (SFO) and the periventricular tissue surrounding the anteroventral third ventricle (AV3V) than did controls. The effect was not observed, however, when the same cumulative dose of ANG II was divided into multiple injections. Behavioral tests found that single daily injections of ANG II sensitized the dipsogenic response to ANG II, but a daily regimen of four injections did not cause sensitization. Analysis of (125)I-Sar(1)-ANG II binding revealed a paradoxical decrease in binding in the caudal AV3V and dorsal median preoptic nucleus after 5 days of single daily injections of ANG II; however, this effect was absent in rats treated for 5 days with four daily ANG II injections. Taken together, these data suggest that a desensitizing treatment regimen prevents behavior- and receptor-level effects of repeated daily ANG II., (Copyright © 2014 the American Physiological Society.)

A seven-amino acid peptide (PEP7) is encoded within a short open reading frame within exon 2 (E2) in the 5'-leader sequence (5'LS) upstream of the rat ANG 1a-receptor (rAT1aR) mRNA. A chemically synthesized PEP7 markedly inhibited ANG II-induced Erk1/2 activation in cell culture by 62% compared with a scrambled PEP7 (sPEP7) [pErk1/2/Erk1/2 (AU): ANG II, 1.000 ± 0.0, ANG II+PEP7, 0.3812 ± 0.086, ANG II+sPEP7, 1.069 ± 0.18; n = 3]. Under these same conditions, PEP7 had no effect on ANG II-stimulated inositol-trisphosphate production. PEP7 also had no effect on epidermal growth factor- and phorbol methyl ester-induced Erk1/2 activation, suggesting PEP7 selectively inhibits AT1aR-mediated Erk1/2 signaling. PEP7 intracerebroventricularly inhibited ANG II-induced saline intake but had no effect on water intake in male and female rats, indicating PEP7 also selectively inhibits the ANG II-Erk1/2 pathway in vivo since saline drinking is Erk1/2-mediated, while water drinking is not. PEP7 inhibition of ANG II-induced saline ingestion was rapidly reversed by a subsequent intracerebroventricular injection of an oxytocin antagonist, suggesting when PEP7 blocks ANG II-stimulated Erk1/2 activation, animals no longer ingest saline to balance the continued water intake, due to the release of oxytocin and its subsequent inhibitory effects on saline drinking. PEP7 also attenuated ANG II-induced increases in arterial pressure by 35% compared with sPEP7 at the same dose. Thus, we have identified a novel peptide encoded within the rAT1aR E2 that selectively inhibits Erk1/2 activation, resulting in physiological consequences for sodium ingestion and arterial pressure that may have implications for treating sodium-sensitive diseases like hypertension and chronic kidney disease.

Sex differences may play a significant role in determining the risk of hypertension. Bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are involved in the tonic regulation of arterial pressure and participate in the central mechanisms of hypertension. Angiotensin II (ANG II) acting on angiotensin type 1 (AT(1)) receptors in RVLM neurons is implicated in the development of hypertension by activating NADPH oxidase and producing reactive oxygen species (ROS). Therefore, we analyzed RVLM bulbospinal neurons to determine whether there are sex differences in: 1) immunolabeling for AT(1) receptors and the key NADPH oxidase subunit p47 using dual-label immunoelectron microscopy, and 2) the effects of ANG II on ROS production and Ca(2+) currents using, respectively, hydroethidine fluoromicrography and patch-clamping. In tyrosine hydroxylase-positive RVLM neurons, female rats displayed significantly more AT(1) receptor immunoreactivity and less p47 immunoreactivity than male rats (P < 0.05). Although ANG II (100 nM) induced comparable ROS production in dissociated RVLM bulbospinal neurons of female and male rats (P > 0.05), an effect mediated by AT(1) receptors and NADPH oxidase, it triggered significantly larger dihydropyridine-sensitive long-lasting (L-type) Ca(2+) currents in female RVLM neurons (P < 0.05). These observations suggest that an increase in AT(1) receptors in female RVLM neurons is counterbalanced by a reduction in p47 levels, such that ANG II-induced ROS production does not differ between females and males. Since the Ca(2+) current activator Bay K 8644 induced larger Ca(2+) currents in females than in male RVLM neurons, increased ANG II-induced L-type Ca(2+) currents in females may result from sex differences in calcium channel densities or dynamics.

Reduced uterine perfusion initiated in late gestation in the rat results in intrauterine growth restriction (IUGR) and development of hypertension by 4 wk of age. We hypothesize that the renin angiotensin system (RAS), a regulatory system important in the long-term control of blood pressure, may be programmed by placental insufficiency and may contribute to the etiology of IUGR hypertension. We previously reported that RAS blockade abolished hypertension in adult IUGR offspring; however, the mechanisms responsible for the early phase of hypertension are unresolved. Therefore, the purpose of this study was to examine RAS involvement in early programmed hypertension and to determine whether temporal changes in RAS expression are observed in IUGR offspring. Renal renin and angiotensinogen mRNA expression were significantly decreased at birth (80 and 60%, respectively); plasma and renal RAS did not differ in conjunction with hypertension (mean increase of 14 mmHg) in young IUGR offspring; however, hypertension (mean increase of 22 mmHg) in adult IUGR offspring was associated with marked increases in renal angiotensin-converting enzyme (ACE) activity (122%) and renal renin and angiotensinogen mRNA (7-fold and 7.4-fold, respectively), but no change in renal ANG II or angiotensin type 1 receptor. ACE inhibition (enalapril, 10 mg x kg(-1) x day(-1), administered from 2 to 4 wk of age) abolished hypertension in IUGR at 4 wk of age (decrease of 15 mmHg, respectively) with no significant depressor effect in control offspring. Therefore, temporal alterations in renal RAS are observed in IUGR offspring and may play a key role in the etiology of IUGR hypertension.

In the present investigation we sought to determine if a perinatal high-salt treatment affects blood pressure at an early age (30 days), and if so, to determine the mechanisms responsible for the hypertension. Pregnant dams were given an 8% NaCl diet [high-salt (HS) rats] during the final one-third of gestation and throughout the suckling period. After weaning, the pups continued to receive the high-salt diet until testing at age 30 days. Control groups received a normal-salt diet (NS rats). In HS rats, mean arterial pressure (MAP) was significantly increased (110 +/- 5 vs. 96 +/- 3 mmHg) compared with NS rats. Blockade of brain AT(1) receptors with intracerebroventricular losartan decreased MAP in HS but not NS rats. Blockade of alpha-adrenergic receptors with intravenous phentolamine or ganglionic transmission with intravenous chlorisondamine produced a greater decrease in MAP in HS rats. Baroreflex control of heart rate was assessed using a four-parameter logistics function. The mid-range MAP (p3) was significantly increased in the HS rats. No other baroreflex parameters were affected. Specific binding of (125)I-[Sa (1),Ile(8)]ANG II to AT(1) receptors was increased in the subfornical organ (SFO) of the HS rats. Expression of AT(1a) receptor mRNA was greater in both SFO and PVN of the HS rats. These data suggest that even at an early age, Sprague-Dawley rats treated with a perinatal high-salt diet are hypertensive. The elevated blood pressure appears to be caused by increased sympathetic nervous activity, resulting, in part, from increased brain AT(1) receptor activation.

The present investigation measured the relative pressor potencies of intracerebroventricularly infused ANG II, ANG III, and the metabolically resistant analogs d-Asp(1)ANG II and d-Arg(1)ANG III in alert freely moving rats. The stability of these analogs was further facilitated by pretreatment with the specific aminopeptidase A inhibitor EC33 or the aminopeptidase N inhibitor PC18. The results indicate that the maximum elevations in mean arterial pressure (MAP) were very similar for each of these compounds across the dose range 1, 10, and 100 pmol/min during a 5-min infusion period. However, d-Asp(1)ANG II revealed significantly extended durations of pressor effects before return to base level MAP. Pretreatment intracerebroventricular infusion with EC33 blocked the pressor activity induced by the subsequent infusion of d-Asp(1)ANG II, whereas EC33 had no effect on the pressor response to subsequent infusion of d-Arg(1)ANG III. In contrast, pretreatment infusion with PC18 extended the duration of the d-Asp(1)ANG II pressor effect by about two to three times and the duration of d-Arg(1)ANG III's effect by approximately 10 to 15 times. Pretreatment with the specific AT(1) receptor antagonist losartan blocked the pressor responses induced by the subsequent infusion of both analogs indicating that they act via the AT(1) receptor subtype. These results suggest that the brain AT(1) receptor may be designed to preferentially respond to ANG III, and ANG III's importance as a centrally active ligand has been underestimated.

The mechanism by which lactating rats increase fluid consumption to meet the demands of milk production is unknown. Because ANG II is the most potent dipsogenic stimulus known, this study examined whether angiotensinergic signaling plays a role in enhanced drinking in lactating rats. ANG II administered intracerebroventricularly caused a significantly greater dipsogenic response in lactating rats than in control rats, suggesting that dipsogenic responsivity to ANG II is enhanced in the brains of lactating rats. The angiotensin type 1 (AT1) ANG II receptor subtype antagonist SKF-108566, also given intracerebroventricularly, caused a significant reduction in water consumption in lactating rats, whereas it did not significantly affect water intake in control rats. In contrast, stimulation of drinking by the muscarinic agonist carbachol, also administered intracerebroventricularly, did not differ between lactating and control rats. Inhibition of drinking by the muscarinic antagonist atropine also did not differ significantly between lactating and control rats. These results suggest that the increased drinking in lactating rats involves an increased responsivity to ANG II in neurons that mediate dipsogenesis, as well as an enhancement in the amount of angiotensinergic input to these ANG II-responsive neurons.

In lactating rats, ANG II receptor binding in the arcuate nucleus (ARH) and median eminence is decreased. To further evaluate brain angiotensinergic activity during lactation, we assessed angiotensinogen (AON) mRNA by in situ hybridization in forebrains of day 10 or 11 postpartum lactating and diestrous rats. AON mRNA was abundantly expressed in the ARH, preoptic, suprachiasmatic, supraoptic, paraventricular, and dorsomedial hypothalamic nuclei, and other regions, similar to that reported in male rat brains. AON mRNA levels were decreased 27% in the midcaudal ARH of lactating rats but did not differ between lactating or diestrous rats in any of the other brain areas examined. Immunofluorescence for AON and glial fibrillary acidic protein or tyrosine hydroxylase confirmed that the AON immunoreactivity in the ARH was limited to astrocytes. Confocal microscopy revealed close appositions of AON-positive astrocytes to dopaminergic neurons in the ARH. The decrease in AON mRNA in the midcaudal ARH during lactation coupled with decreased ARH ANG II receptor binding suggests that lactating rats are less subject to ANG II-mediated inhibition of prolactin secretion.