David C. Hill-Eubanks

Elementary Ca2+ Signals Through Endothelial TRPV4 Channels Regulate Vascular Function

Blood Pressure Gauge Endothelial cells line blood vessels and, by interacting with smooth muscle, can help to control blood flow. Sonkusare et al. (p. 597; see the Perspective by Lederer et al.) describe how signaling in endothelial cells controls contraction of surrounding smooth muscle cells, which provides an important mechanism for control of blood pressure. A calcium-sensitive fluorescent protein was expressed in endothelial cells of mouse arteries to image small changes in calcium concentration that appear to represent opening of single TRPV4 ion channels and consequent influx of calcium into the cell. Clustering of the channels allowed cooperative activation of a handful of channels, which appeared to produce a sufficient calcium signal to open another set of calcium-sensitive potassium channels. The resulting depolarization of the endothelial cells then passes an electrical connection to smooth muscle cells through gap junctions. Imaging reveals single-channel openings of cation channels at the heart of endothelial cell–mediated blood pressure control. 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.

Calcium Signaling in Smooth Muscle

Changes in intracellular Ca(2+) are central to the function of smooth muscle, which lines the walls of all hollow organs. These changes take a variety of forms, from sustained, cell-wide increases to temporally varying, localized changes. The nature of the Ca(2+) signal is a reflection of the source of Ca(2+) (extracellular or intracellular) and the molecular entity responsible for generating it. Depending on the specific channel involved and the detection technology employed, extracellular Ca(2+) entry may be detected optically as graded elevations in intracellular Ca(2+), junctional Ca(2+) transients, Ca(2+) flashes, or Ca(2+) sparklets, whereas release of Ca(2+) from intracellular stores may manifest as Ca(2+) sparks, Ca(2+) puffs, or Ca(2+) waves. These diverse Ca(2+) signals collectively regulate a variety of functions. Some functions, such as contractility, are unique to smooth muscle; others are common to other excitable cells (e.g., modulation of membrane potential) and nonexcitable cells (e.g., regulation of gene expression).

Opposing actions of inositol 1,4,5-trisphosphate and ryanodine receptors on nuclear factor of activated T-cells regulation in smooth muscle.

The nuclear factor of activated T-cells (NFAT), originally identified in T-cells, has since been shown to play a role in mediating Ca2+-dependent gene transcription in diverse cell types outside of the immune system. We have previously shown that nuclear accumulation of NFATc3 is induced in ileal smooth muscle by platelet-derived growth factor in a manner that depends on Ca2+ influx through L-type, voltage-dependent Ca2+ channels. Here we show that NFATc3 is also the predominant NFAT isoform expressed in cerebral artery smooth muscle and is induced to accumulate in the nucleus by UTP and other Gq/11-coupled receptor agonists. This induction is mediated by calcineurin and is dependent on sarcoplasmic reticulum Ca2+ release through inositol 1,4,5-trisphosphate receptors and extracellular Ca2+ influx through L-type, voltage-dependent Ca2+ channels. Consistent with results obtained in ileal smooth muscle, depolarization-induced Ca2+ influx fails to induce NFAT nuclear accumulation in cerebral arteries. We also provide evidence that Ca2+release by ryanodine receptors in the form of Ca2+ sparks may exert an inhibitory influence on UTP-induced NFATc3 nuclear accumulation and further suggest that UTP may act, in part, by inhibiting Ca2+ sparks. These results are consistent with a multifactorial regulation of NFAT nuclear accumulation in smooth muscle that is likely to involve several intracellular signaling pathways, including local effects of sarcoplasmic reticulum Ca2+release and effects attributable to global elevations in intracellular Ca2+.

TRPV4 channels stimulate Ca2+-induced Ca2+ release in astrocytic endfeet and amplify neurovascular coupling responses

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. Ca2+ 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 Ca2+ conductance—have been identified in astrocytes, but their function is largely unknown. We sought to characterize the influence of TRPV4 channels on Ca2+ dynamics in the astrocytic endfoot microdomain and assess their role in neurovascular coupling. We identified local TRPV4-mediated Ca2+ oscillations in endfeet and further found that TRPV4 Ca2+ signals are amplified and propagated by Ca2+-induced Ca2+ release from inositol trisphosphate receptors (IP3Rs). Moreover, TRPV4-mediated Ca2+ influx contributes to the endfoot Ca2+ response to neuronal activation, enhancing the accompanying vasodilation. Our results identify a dynamic synergy between TRPV4 channels and IP3Rs in astrocyte endfeet and demonstrate that TRPV4 channels are engaged in and contribute to neurovascular coupling.

Chapter 12: Muscarinic acetylcholine receptor subtypes: localization and structure/function

Based on the sequence of the five cloned muscarinic receptor subtypes (m1-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues. The distributions of these subtypes and their potential physiological roles are discussed. By use of molecular genetic manipulation of cloned muscarinic receptor cDNAs, the regions of muscarinic receptors that specify G-protein coupling and ligand binding have been defined in several recent studies. Overall, these studies have shown that amino acids within the third cytoplasmic loop of the receptors define their selectivities for different G-proteins and that multiple discontinuous epitopes contribute to their selectivities for different ligands. The residues that contribute to ligand binding and G-protein coupling are described, as well as the implied structures of these functional domains.

NFAT regulation in smooth muscle.

First identified in activated T cells, the calcium (Ca2+)-dependent transcription factor, nuclear factor of activated T cells (NFAT), has since been shown to play a role in nonimmune cells, including cells of the cardiovascular system. In arterial smooth muscle, the diverse array of calcium-signaling modalities, the functional interplay between smooth muscle and endothelial cells, and the influence of intravascular pressure on calcium and other signaling pathways creates a calcium-regulatory environment that is arguably unique. This review focuses on mechanisms that control the initial Ca2+/calcineurin-dependent events in NFAT activation, with a particular emphasis on NFAT regulation in native vascular smooth muscle. Also addressed is the role of additional mechanisms that act to modulate calcineurin-dependent NFAT nuclear import/export, mechanisms that may have particular relevance in this tissue.

AKAP150-dependent cooperative TRPV4 channel gating is central to endothelium-dependent vasodilation and is disrupted in hypertension

Local Ca2+ signaling at specialized regions of endothelial cell–smooth muscle contact is impaired in hypertension. Concentrating the Signal to Relax Regulation of blood flow involves both the endothelial cells lining the blood vessels and the surrounding smooth muscle cells, with impaired communication between these cell types leading to vascular dysfunction. Sonkusare et al. monitored endothelial cell calcium signals through single TRPV4 channels in arterial preparations from mice and found that these channels were more active at sites where the endothelial cells make intimate contact with the smooth muscle. Endothelial-dependent vasodilators, such as acetylcholine, activated TRPV4 channels only at these sites and required the colocalized scaffolding protein AKAP150. In a mouse model of hypertension, AKAP150 localization to these contact sites was lost, leading to lower TRPV4 channel activity, loss of agonist-induced activation of TRPV4 channels, and diminished vasodilation. 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 vasodilation, 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.

Structure of a G-protein-coupling Domain of a Muscarinic Receptor Predicted by Random Saturation Mutagenesis

The third intracellular loop (i3) plays a critical role in the coupling of many receptors to G-proteins. In muscarinic receptor subtypes, the N- and C-terminal regions (Ni3 and Ci3) of this loop are sufficient to direct appropriate G-protein coupling. The relative functional contributions of all amino acids within Ni3 was evaluated by constructing libraries of m5 muscarinic receptors containing random mutations in Ni3 and screening them using high throughput assays based on ligand-dependent transformation of NIH 3T3 cells. In receptors that retained a wild type phenotype, the pattern of functionally tolerated substitutions is consistent with the presence of three turns of an α helix extending from the transmembrane domain. All of the amino acid positions that tolerate radical substitutions face away from a conserved hydrophobic face that ends with an arginine, and helix-disrupting proline substitutions were not observed. All of the mutant receptors with significantly compromised phenotypes had amino acid substitutions in residues predicted to form the hydrophobic face. Similar data from the Ci3 region (Burstein, E. S., Spalding, T. A., Hill-Eubanks, D., and Brann, M. R.(1995) J. Biol. Chem. 270, 3141-3146) are consistent with the presence of a single helical turn extending from the transmembrane domain, with an alanine that defines G-protein affinity. Functionally critical residues of Ni3 and Ci3 are predicted to be in close proximity where they form the G-protein-coupling domain.

A PLCγ1-Dependent, Force-Sensitive Signaling Network in the Myogenic Constriction of Cerebral Arteries

The signaling pathway that links the sensing of increased blood pressure to constriction in cerebral arteries is delineated. Maintaining Blood Flow to the Brain Cerebral arteries continually adjust to changes in blood pressure to ensure constant blood flow to the brain. In response to increased blood pressure, the smooth muscle cells in cerebral arteries contract, resulting in blood vessel constriction. This response requires two cell surface ion channels—TRPC6, a channel that is activated by the stretch caused by increased blood pressure, and TRPM4, a channel that triggers the electrical impulses necessary for blood vessel constriction. Gonzales et al. found that activation of TRPC6 stimulated TRPM4 through calcium-dependent pathways. TRPC6, TRPM4, and the enzyme PLCγ1 were located in close proximity to each other in smooth muscle cells, indicating that a pressure-sensitive signaling network keeps blood flowing in the brain. Maintaining constant blood flow in the face of fluctuations in blood pressure is a critical autoregulatory feature of cerebral arteries. An increase in pressure within the artery lumen causes the vessel to constrict through depolarization and contraction of the encircling smooth muscle cells. This pressure-sensing mechanism involves activation of two types of transient receptor potential (TRP) channels: TRPC6 and TRPM4. We provide evidence that the activation of the γ1 isoform of phospholipase C (PLCγ1) is critical for pressure sensing in cerebral arteries. Inositol 1,4,5-trisphosphate (IP3), generated by PLCγ1 in response to pressure, sensitized IP3 receptors (IP3Rs) to Ca2+ influx mediated by the mechanosensitive TRPC6 channel, synergistically increasing IP3R-mediated Ca2+ release to activate TRPM4 currents, leading to smooth muscle depolarization and constriction of isolated cerebral arteries. Proximity ligation assays demonstrated colocalization of PLCγ1 and TRPC6 with TRPM4, suggesting the presence of a force-sensitive, local signaling network comprising PLCγ1, TRPC6, TRPM4, and IP3Rs. Src tyrosine kinase activity was necessary for stretch-induced TRPM4 activation and myogenic constriction, consistent with the ability of Src to activate PLCγ isoforms. We conclude that contraction of cerebral artery smooth muscle cells requires the integration of pressure-sensing signaling pathways and their convergence on IP3Rs, which mediate localized Ca2+-dependent depolarization through the activation of TRPM4.

Ion channel networks in the control of cerebral blood flow.

One hundred and twenty five years ago, Roy and Sherrington made the seminal observation that neuronal stimulation evokes an increase in cerebral blood flow.1 Since this discovery, researchers have attempted to uncover how the cells of the neurovascular unit—neurons, astrocytes, vascular smooth muscle cells, vascular endothelial cells and pericytes—coordinate their activity to control this phenomenon. Recent work has revealed that ionic fluxes through a diverse array of ion channel species allow the cells of the neurovascular unit to engage in multicellular signaling processes that dictate local hemodynamics. In this review we center our discussion on two major themes: (1) the roles of ion channels in the dynamic modulation of parenchymal arteriole smooth muscle membrane potential, which is central to the control of arteriolar diameter and therefore must be harnessed to permit changes in downstream cerebral blood flow, and (2) the striking similarities in the ion channel complements employed in astrocytic endfeet and endothelial cells, enabling dual control of smooth muscle from either side of the blood–brain barrier. We conclude with a discussion of the emerging roles of pericyte and capillary endothelial cell ion channels in neurovascular coupling, which will provide fertile ground for future breakthroughs in the field.