Rationale Neurogenic hypertension is characterized by an increase in sympathetic activity and often resistance to drug treatments. We previously reported that it is also associated with a reduction of Angiotensin Converting Enzyme 2 (ACE2) and an increase in A Disintegrin And Metalloprotease 17 (ADAM17) activity in experimental hypertension. In addition, while multiple cells within the central nervous system have been involved in the development of neurogenic hypertension, the contribution of ADAM17 has not been investigated. Objective To assess the clinical relevance of this ADAM17-mediated ACE2 shedding in hypertensive patients and further identify the cell types and signaling pathways involved in this process. Methods and Results Using a mass spectrometry-based assay, we identified ACE2 as the main enzyme converting Ang II into Ang-(1–7) in human cerebrospinal fluid (CSF). We also observed an increase in ACE2 activity in the CSF of hypertensive patients, which was correlated with systolic blood pressure. Moreover, the increased level of tumor necrosis factor (TNF)-α in those CSF samples confirmed that ADAM17 was up-regulated in the hypertensive patients’ brain. To further assess the interaction between brain renin-angiotensin system and ADAM17, we generated mice lacking Angiotensin II type 1 receptors (AT1R) specifically on neurons. Our data reveal that despite expression on astrocytes and other cells types in the brain, ADAM17 up-regulation during DOCA-salt hypertension occurs selectively on neurons and neuronal AT1R are indispensable to this process. Mechanistically, reactive oxygen species (ROS) and extracellular signal-regulated kinase (ERK) were found to mediate ADAM17 activation. Conclusions Our data demonstrate that AT1R promote ADAM17-mediated ACE2 shedding in the brain of hypertensive patients, leading to a loss in compensatory activity during neurogenic hypertension.
The recent outbreak of 2019 coronavirus disease , caused by a novel coronavirus, has now spread quickly worldwide. Like the severe acute respiratory syndrome coronavirus (SARS-CoV), this novel type of coronavirus, SARS-CoV-2, has been demonstrated to utilize angiotensin-converting enzyme 2 (ACE2) as an entry point to the cells. There is a growing body of reports indicating that COVID-19 patients, especially those in severe condition, exhibit neurological symptoms, thus supporting the possibility that SARS-CoV-2 could infect and damage neurons within the central nervous system in humans. Using human pluripotent stem cells-derived neurons, here we show the expression of ACE2 in human neurons via immunocytochemistry. From this perspective, we elaborate on the idea that the neuro-invasive potential of SARS-CoV-2 should be considered as a possible contributory factor, as well as a therapeutic target, for the severe respiratory symptoms in critical COVID-19 cases.
Brain renin angiotensin system within the paraventricular nucleus plays a critical role in balancing excitatory and inhibitory inputs to modulate sympathetic output and blood pressure regulation. We previously identified ACE2 and ADAM17 as a compensatory enzyme and a sheddase, respectively, involved in brain renin angiotensin system regulation. Here, we investigated the opposing contribution of ACE2 and ADAM17 to hypothalamic presympathetic activity and ultimately neurogenic hypertension. New mouse models were generated where ACE2 and ADAM17 were selectively knocked down from all neurons (AC-N) or Sim1 neurons (SAT), respectively. Neuronal ACE2 deletion revealed a reduction of inhibitory inputs to AC-N presympathetic neurons relevant to blood pressure regulation. Primary neuron cultures confirmed ACE2 expression on GABAergic neurons synapsing onto excitatory neurons within the hypothalamus but not on glutamatergic neurons. ADAM17 expression was shown to colocalize with angiotensin-II type 1 receptors on Sim1 neurons, and the pressor relevance of this neuronal population was demonstrated by photoactivation. Selective knockdown of ADAM17 was associated with a reduction of FosB gene expression, increased vagal tone, and prevented the acute pressor response to centrally administered angiotensin-II. Chronically, SAT mice exhibited a blunted blood pressure elevation and preserved ACE2 activity during development of salt-sensitive hypertension. Bicuculline injection in those models confirmed the supporting role of ACE2 on GABAergic tone to the paraventricular nucleus. Together, our study demonstrates the contrasting impact of ACE2 and ADAM17 on neuronal excitability of presympathetic neurons within the paraventricular nucleus and the consequences of this mutual regulation in the context of neurogenic hypertension.
The Ca(2+) activated Cl(-) channels (CaCCs) play a multitude of important physiological functions. A number of candidate proteins have been proposed to form CaCC, but only two families, the bestrophins and the TMEM16 proteins, recapitulate the properties of native CaCC in expression systems. Studies of endogenous CaCCs are hindered by the lack of specific pharmacology as most Cl(-) channel modulators lack selectivity and a systematic comparison of the effects of these modulators on TMEM16A and bestrophin is missing. In the present study, we studied seven Cl(-) channel inhibitors: niflumic acid (NFA), NPPB, flufenamic acid (FFA), DIDS, tannic acid, CaCCinh-A01 and T16Ainh-A01 for their effects on TMEM16A and bestrophin-1 (Best1) stably expressed in CHO (Chinese hamster ovary) cells using patch clamp technique. Among seven inhibitors studied, NFA showed highest selectivity for TMEM16A (IC50 of 7.40 ± 0.95 μM) over Best1 (IC50 of 102.19 ± 15.05 μM). In contrast, DIDS displayed a reverse selectivity inhibiting Best1 with IC50 of 3.93 ± 0.73 μM and TMEM16A with IC50 of 548.86 ± 25.57 μM. CaCCinh-A01 was the most efficacious blocker for both TMEM16A and Best1 channels. T16Ainh-A01 partially inhibited TMEM16A currents but had no effect on Best1 currents. Tannic acid, NPPB and FFA had variable intermediate effects. Potentiation of channel activity by some of these modulators and the effects on TMEM16A deactivation kinetics were also described. Characterization of Cl(-) channel modulators for their effects on TMEM16A and Best1 will facilitate future studies of native CaCCs.
ADAM17 is a metalloprotease and disintegrin that lodges in the plasmatic membrane of several cell types and is able to cleave a wide variety of cell surface proteins. It is somatically expressed in mammalian organisms and its proteolytic action influences several physiological and pathological processes. This review focuses on the structure of ADAM17, its signaling in the cardiovascular system and its participation in certain disorders involving the heart, blood vessels, and neural regulation of autonomic and cardiovascular modulation.
Flow cytometry has the potential to facilitate understanding of the heterogeneous responses of diverse brain cell populations to a variety of stimuli. However, existing methods of applying flow cytometry to brain tissues are each limited in certain ways. They either require genetically labeled cells to achieve separation of specific populations, are not applicable to previously fixed tissue, or are not compatible with downstream mRNA analysis. Here, we describe a group of related methods that overcome many previous limitations and allow robust sorting and downstream molecular analysis of highly enriched populations of specific neuronal and non-neuronal cells from any mammalian brain. We illustrate these techniques, which are compatible with antibodies for both nuclear and non-nuclear epitopes and do not require transgenic animals, with three examples. First, we describe the separation and downstream mRNA analysis of four types of cortical interneurons (somatostatin, parvalbumin, calretinin, and calbindin) from paraformaldehyde-fixed rat brain sections. Second, we demonstrate separation of neurons and non-neurons from zinc-fixed mouse brain cortical sections followed by analysis of enzymatic activity (ACE2 activity) and mRNA expression. Third, we show that routinely fixed post-mortem human brain can be analyzed by isolating parvalbumin-containing neurons from cortical samples that were fixed for periods of up to 8 weeks in formalin. In each case, sorted cell identity was confirmed with mRNA analysis. The neurocytometry methodology described here has the potential to significantly expand studies to analyze the effects of drugs, environmental manipulations, and disease states on the nucleic acid and protein content of specific brain cell populations.
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