Major features of the transcellular signaling mechanism responsible for endothelium-dependent regulation of vascular smooth muscle tone are unresolved. We identified local calcium (Ca2+) signals (“sparklets”) in the vascular endothelium of resistance arteries that represent Ca2+ influx through single TRPV4 cation channels. Gating of individual TRPV4 channels within a four-channel cluster was cooperative, with activation of as few as three channels per cell causing maximal dilation through activation of endothelial cell intermediate (IK)- and small (SK)-conductance, Ca2+-sensitive potassium (K+) channels. Endothelial-dependent muscarinic receptor signaling also acted largely through TRPV4 sparklet-mediated stimulation of IK and SK channels to promote vasodilation. These results support the concept that Ca2+ influx through single TRPV4 channels is leveraged by the amplifier effect of cooperative channel gating and the high Ca2+ sensitivity of IK and SK channels to cause vasodilation.

Blood flow into the brain is dynamically regulated to satisfy the changing metabolic requirements of neurons, but how this is accomplished has remained unclear. Here, we demonstrate a central role for capillary endothelial cells in sensing neural activity and communicating it to upstream arterioles in the form of an electrical vasodilatory signal. We further demonstrate that this signal is initiated by extracellular potassium (K+)—a byproduct of neural activity—which activates capillary endothelial cell inward-rectifier K+ (KIR2.1) channels to produce a rapidly propagating retrograde hyperpolarization that causes upstream arteriolar dilation, increasing blood flow into the capillary bed. Our results establish brain capillaries as an active sensory web that converts changes in external K+ into rapid, ‘inside-out’ electrical signaling to direct blood flow to active brain regions.

Endothelial cell dysfunction, characterized by a diminished response to endothelial cell–dependent vasodilators, is a hallmark of hypertension. TRPV4 channels play a major role in endothelial-dependent vaso-dilation, a function mediated by local Ca2+ influx through clusters of functionally coupled TRPV4 channels rather than by a global increase in endothelial cell Ca2+. We showed that stimulation of muscarinic acetylcholine receptors on endothelial cells of mouse arteries exclusively activated TRPV4 channels that were localized at myoendothelial projections (MEPs), specialized regions of endothelial cells that contact smooth muscle cells. Muscarinic receptor–mediated activation of TRPV4 depended on protein kinase C (PKC) and the PKC-anchoring protein AKAP150, which was concentrated at MEPs. Cooperative opening of clustered TRPV4 channels specifically amplified Ca2+ influx at MEPs. Cooperativity of TRPV4 channels at non-MEP sites was much lower, and cooperativity at MEPs was greatly reduced by chelation of intracellular Ca2+ or AKAP150 knockout, suggesting that Ca2+ entering through adjacent channels underlies the AKAP150-dependent potentiation of TRPV4 activity. In a mouse model of angiotensin II–induced hypertension, MEP localization of AKAP150 was disrupted, muscarinic receptor stimulation did not activate TRPV4 channels, cooperativity among TRPV4 channels at MEPs was weaker, and vasodilation in response to muscarinic receptor stimulation was reduced. Thus, endothelial-dependent dilation of resistance arteries is enabled by MEP-localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and the coupled channel gating necessary for efficient communication from endothelial to smooth muscle cells in arteries. Disruption of this molecular assembly may contribute to altered blood flow in hypertension.

The essential function of the circulatory system is to continuously and efficiently supply the O2 and nutrients necessary to meet the metabolic demands of every cell in the body, a function in which vast capillary networks play a key role. Capillary networks serve an additional important function in the central nervous system: acting as a sensory network, they detect neuronal activity in the form of elevated extracellular K+ and initiate a retrograde, propagating, hyperpolarizing signal that dilates upstream arterioles to rapidly increase local blood flow. Yet, little is known about how blood entering this network is distributed on a branch-to-branch basis to reach specific neurons in need. Here, we demonstrate that capillary-enwrapping projections of junctional, contractile pericytes within a postarteriole transitional region differentially constrict to structurally and dynamically determine the morphology of capillary junctions and thereby regulate branch-specific blood flow. We further found that these contractile pericytes are capable of receiving propagating K+-induced hyperpolarizing signals propagating through the capillary network and dynamically channeling red blood cells toward the initiating signal. By controlling blood flow at junctions, contractile pericytes within a functionally distinct postarteriole transitional region maintain the efficiency and effectiveness of the capillary network, enabling optimal perfusion of the brain.

In the CNS, astrocytes are sensory and regulatory hubs that play important roles in cerebral homeostatic processes, including matching local cerebral blood flow to neuronal metabolism (neurovascular coupling). These cells possess a highly branched network of processes that project from the soma to neuronal synapses as well as to arterioles and capillaries, where they terminate in "endfeet" that encase the blood vessels. Ca 2+ signaling within the endfoot mediates neurovascular coupling; thus, these functional microdomains control vascular tone and local perfusion in the brain. Transient receptor potential vanilloid 4 (TRPV4) channels-nonselective cation channels with considerable Ca 2+ conductance-have been identified in astrocytes, but their function is largely unknown. We sought to characterize the influence of TRPV4 channels on Ca 2+ dynamics in the astrocytic endfoot microdomain and assess their role in neurovascular coupling. We identified local TRPV4-mediated Ca 2+ oscillations in endfeet and further found that TRPV4 Ca 2+ signals are amplified and propagated by Ca 2+ -induced Ca 2+ release from inositol trisphosphate receptors (IP 3 Rs). Moreover, TRPV4-mediated Ca 2+ influx contributes to the endfoot Ca 2+ response to neuronal activation, enhancing the accompanying vasodilation. Our results identify a dynamic synergy between TRPV4 channels and IP 3 Rs in astrocyte endfeet and demonstrate that TRPV4 channels are engaged in and contribute to neurovascular coupling.calcium | parenchymal arteriole A strocytes are glial cells in the brain that are essential for the structural and functional integrity of the central nervous system. Astrocytes maintain cerebral homeostasis by acting as "switchboards," receiving and integrating communication from the surrounding microenvironment and translating that information into physiological and homeostatic responses. Numerous astrocytic projections make contact with neighboring synapses, while other projections terminate in "endfeet" that spread out and wrap around parenchymal arterioles and capillaries within the brain (1, 2). This structural orientation allows astrocytes to monitor synaptic activity in neuronal networks and mediate communication between neurons and the cerebral microcirculation.Calcium (Ca 2+ ) signaling is critical for astrocyte function. Transient increases in intracellular Ca 2+ concentration ([Ca 2+ ] i ) mediated by inositol 1,4,5-trisphosphate (IP 3 ) receptor Ca 2+ release channels (IP 3 Rs) in endoplasmic reticulum (ER) membranes drive the release of chemical transmitters like glutamate, adenosine triphosphate (ATP), and D-Serine that modulate synaptic transmission and neuronal excitability (3, 4). IP 3 Rdependent Ca 2+ signaling in astrocytes is also critical for neurovascular coupling (NVC), the process by which local cerebral blood flow (CBF) is matched to neuronal metabolism (5, 6). As neuronal activity increases, synaptically released glutamate binds to metabotropic glutamate receptors (mGluRs) on perisynaptic astrocytic projections, stimula...