Cerebral blood flow is dynamically regulated by neurovascular coupling to meet the dynamic metabolic demands of the brain. We hypothesized that TRPA1 channels in capillary endothelial cells are stimulated by neuronal activity and instigate a propagating retrograde signal that dilates upstream parenchymal arterioles to initiate functional hyperemia. We find that activation of TRPA1 in capillary beds and post-arteriole transitional segments with mural cell coverage initiates retrograde signals that dilate upstream arterioles. These signals exhibit a unique mode of biphasic propagation. Slow, short-range intercellular Ca2+ signals in the capillary network are converted to rapid electrical signals in transitional segments that propagate to and dilate upstream arterioles. We further demonstrate that TRPA1 is necessary for functional hyperemia and neurovascular coupling within the somatosensory cortex of mice in vivo. These data establish endothelial cell TRPA1 channels as neuronal activity sensors that initiate microvascular vasodilatory responses to redirect blood to regions of metabolic demand.

The angiotensin II receptor type 1b (AT R ) is the primary sensor of intraluminal pressure in cerebral arteries. Pressure or membrane-stretch induced stimulation of AT R activates the TRPM4 channel and results in inward transient cation currents that depolarize smooth muscle cells, leading to vasoconstriction. Activation of either AT R or AT R with angiotensin II stimulates TRPM4 currents in cerebral artery myocytes and vasoconstriction of cerebral arteries. The expression of AT R mRNA is ∼30-fold higher than AT R in whole cerebral arteries and ∼45-fold higher in isolated cerebral artery smooth muscle cells. Higher levels of expression are likely to account for the obligatory role of AT R for pressure-induced vasoconstriction ABSTRACT: Myogenic vasoconstriction, which reflects the intrinsic ability of smooth muscle cells to contract in response to increases in intraluminal pressure, is critically important for the autoregulation of blood flow. In smooth muscle cells from cerebral arteries, increasing intraluminal pressure engages a signalling cascade that stimulates cation influx through transient receptor potential (TRP) melastatin 4 (TRPM4) channels to cause membrane depolarization and vasoconstriction. Substantial evidence indicates that the angiotensin II receptor type 1 (AT R) is inherently mechanosensitive and initiates this signalling pathway. Rodents express two types of AT R - AT R and AT R - and conflicting studies provide support for either isoform as the primary sensor of intraluminal pressure in peripheral arteries. We hypothesized that mechanical activation of AT R increases TRPM4 currents to induce myogenic constriction of cerebral arteries. However, we found that development of myogenic tone was greater in arteries from AT R knockout animals compared with controls. In patch-clamp experiments using native cerebral arterial myocytes, membrane stretch-induced cation currents were blocked by the TRPM4 inhibitor 9-phenanthrol in both groups. Further, the AT R blocker losartan (1 μm) diminished myogenic tone and blocked stretch-induced cation currents in cerebral arteries from both groups. Activation of AT R with angiotensin II (30 nm) also increased TRPM4 currents in smooth muscle cells and constricted cerebral arteries from both groups. Expression of AT R mRNA was ∼30-fold greater than AT R in cerebral arteries, and knockdown of AT R selectively diminished myogenic constriction. We conclude that AT R , acting upstream of TRPM4 channels, is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.

Junctophilin proteins maintain close contacts between the endoplasmic/sarcoplasmic reticulum (ER/SR) and the plasma membrane in many types of cells, as typified by junctophilin-2 (JPH2), which is necessary for the formation of the cardiac dyad. Here, we report that JPH2 is the most abundant junctophilin isotype in native smooth muscle cells (SMCs) isolated from cerebral arteries and that acute knockdown diminishes the area of sites of interaction between the SR and plasma membrane. Superresolution microscopy revealed nanometer-scale colocalization of JPH2 clusters with type 2 ryanodine receptor (RyR2) clusters near the cell surface. Knockdown of JPH2 had no effect on the frequency, amplitude, or kinetics of spontaneous Ca2+ sparks generated by transient release of Ca2+ from the SR through RyR2s, but it did nearly abolish Ca2+ spark-activated, large-conductance, Ca2+-activated K+ (BK) channel currents. We also found that JPH2 knockdown was associated with hypercontractility of intact cerebral arteries. We conclude that JPH2 maintains functional coupling between RyR2s and BK channels and is critically important for cerebral arterial function.

Activation of the brain renin-angiotensin system (RAS) is a pivotal step in the pathogenesis of hypertension. The paraventricular nucleus (PVN) of the hypothalamus is a critical part of the angiotensinergic sympatho-excitatory neuronal network involved in neural control of blood pressure and hypertension. However, the importance of the PVN (pro)renin receptor (PVN-PRR)—a key component of the brain RAS—in hypertension development has not been examined. In this study, we investigated the involvement and mechanisms of the PVN-PRR in DOCA-salt-induced hypertension, a mouse model of hypertension. Using nanoinjection of adeno-associated virus-mediated Cre recombinase expression to knock down the PRR specifically in the PVN, we report here that PVN-PRR knockdown attenuated the enhanced blood pressure and sympathetic tone associated with hypertension. Mechanistically, we found that PVN-PRR knockdown was associated with reduced activation of ERK (extracellular signal-regulated kinase)-1/2 in the PVN and rostral ventrolateral medulla during hypertension. In addition, using the genetically encoded Ca2+ biosensor GCaMP6 to monitor Ca2+-signaling events in the neurons of PVN brain slices, we identified a reduction in angiotensin II type 1 receptor-mediated Ca2+ activity as part of the mechanism by which PVN-PRR knockdown attenuates hypertension. Our study demonstrates an essential role of the PRR in PVN neurons in hypertension through regulation of ERK1/2 activation and angiotensin II type 1 receptor-mediated Ca2+ activity. NEW & NOTEWORTHY PRR knockdown in PVN neurons attenuates the development of DOCA-salt hypertension and autonomic dysfunction through a decrease in ERK1/2 activation in the PVN and RVLM during hypertension. In addition, PRR knockdown reduced AT1aR expression and AT1R-mediated calcium activity during hypertension. Furthermore, we characterized the neuronal targeting specificity of AAV serotype 2 in the mouse PVN and validated the advantages of the genetically encoded calcium biosensor GCaMP6 in visualizing neuronal calcium activity in the PVN.

SignificanceDuchenne muscular dystrophy (DMD) is a hereditary neuromuscular disease that results from mutations in the gene encoding dystrophin. The effects of the disease on cardiac and skeletal muscle have been intensely investigated, but much less is known about how DMD impacts vascular smooth muscle cells (SMCs). Using superresolution nanoscopy, we demonstrate that clusters of ryanodine receptors (RyR2s) on the sarcoplasmic reticulum (SR) of cerebral artery SMCs from the mdx mouse model of DMD are larger compared with controls. Increased RyR2 cluster size is associated with augmented SR Ca2+ release and Ca2+-activated K+ channel activity, resulting in impaired vasoconstriction of cerebral microvessels. Our findings demonstrate that remodeling of RyR2 clusters at the molecular level results in cerebral microvascular dysfunction during DMD.