Introduction
Mechanical stimulation of the medulla oblongata produces sympathoexcitation and increase in arterial pressure (AP) 1 triggering Cushing reflex targeted for the maintenance of adequate brain perfusion pressure and cerebral blood flow (CBF). While mechanisms of autoregulation of cerebral blood flow (CBF) remain elusive, data indicate participation of medullary structures 2 . These findings suggest that medullary neurons may possess mechanosensitive properties. Since epithelial sodium channels (ENaC) have been proposed to participate in barosensing of arterial pressure (AP) 3 , we hypothesized that mechanosensitive properties of the medulla are due to the presence of ENaCs, which also may contribute to CBF autoregulation.
Methods
Deeply anesthetized Sprague Dawley rats (230–270 g) were transcardially perfused, brains were removed and sectioned coronally. Medullary sections from the rostral pole of the facial nerve nucleus to 1 mm caudal to the obex were processed for ENaC beta subunit (affinity purified polyclonal antibodies, n=5) and tyrosine hydroxylase immunoreactivity (IR). To establish involvement of ENaCs in CBF autoregulation rats were anesthetized, artificially ventilated, while body temperature and blood gases were monitored and maintained at normal levels. Parietal cortex CBF was recorded using laser Doppler flowmeter. Autoregulatory curves were reconstructed by observing CBF changes while AP was varied from ∼60 to ∼140 mmHg by continuous i/v infusion of nitroprusside or phenylephrine, resp. Blockers of ENaCs (amiloride or benzamil) were microinjected into medulla using micropipettes with subsequent histological identification of the injection sites.
Results
ENaC IR, mostly associated with large neuronal cells, was observed in nucleus of the solitary tract, gigantocellular and hypoglossal nuclei, and rostral ventrolateral medulla (RVLM). To establish whether ENaC IR-positive cells are catecholaminergic neurons we analyzed colocalization of ENaC IR and TH-IR. Catecholaminergic neurons of the caudal RVLM demonstrated ENaC IR. To establish possible involvement of ENaCs in CBF autoregulation we microinjected amiloride (1.5 μM/100 nl, n=3) or benzamil (1 μM/100 nl, n=5), bilaterally into caudal RVLM. Microinjections of ENaC blockers did not affect baseline levels of CBF or AP. However as a result of the microinjections the slope of CBF autoregulatory curve increased significantly (p<0.05) indicating attenuation of the autoregulatory response.
Conclusions
Our experiments demonstrated the presence of ENaCs in the medullary nuclei, including nucleus of solitary tract, which is know to be involved in autoregulation 2 and RVLM, which is involved in CBF regulation 4 . In RVLM ENaC positive neurons were also TH positive indicating that RVLM catecholaminergic neurons, which participate in AP regulation, may possess mechanosensitive properties. Attenuation of the autoregulatory responses by specific ENaC blockers suggests involvement of mechanosensitive channels in CBF autoregulation. We conclude that ENaC proteins might be expressed in medullary neurons and participate in the autoregulatory control of CBF and medullary mechanosensitivity.