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Significance Phosphatidylinositol 4,5-bisphosphate (PIP 2 ), a plasma membrane lipid, is hydrolyzed by G q -protein–coupled receptor (G q PCR) signaling into inositol 1,4,5-trisphosphate and diacylglycerol—extensively studied second messengers with profound regulatory effects in the vasculature. However, there is extensive evidence that PIP 2 directly regulates ion channels, a finding with significant implications for vascular function. Beyond providing a previously unexplored perspective on how vascular G q PCR signaling influences vascular function, the concept of PIP 2 -mediated ion channel regulation helps to explain how vascular cell excitability is coordinated to support cerebral blood flow control mechanisms. Importantly, the link between the metabolic state of vascular cells and PIP 2 content may provide insight into how metabolism affects vascular ion channel activity and, ultimately, vascular function in health and disease.

Brain capillary calcium signaling controls blood flow., Healthy brain function depends on the finely tuned spatial and temporal delivery of blood-borne nutrients to active neurons via the vast, dense capillary network. Here, using in vivo imaging in anesthetized mice, we reveal that brain capillary endothelial cells control blood flow through a hierarchy of IP3 receptor–mediated Ca2+ events, ranging from small, subsecond protoevents, reflecting Ca2+ release through a small number of channels, to high-amplitude, sustained (up to ~1 min) compound events mediated by large clusters of channels. These frequent (~5000 events/s per microliter of cortex) Ca2+ signals are driven by neuronal activity, which engages Gq protein–coupled receptor signaling, and are enhanced by Ca2+ entry through TRPV4 channels. The resulting Ca2+-dependent synthesis of nitric oxide increases local blood flow selectively through affected capillary branches, providing a mechanism for high-resolution control of blood flow to small clusters of neurons.

SUMMARYThe dense network of capillaries composed of capillary endothelial cells (cECs) and pericytes lies in close proximity to all neurons, ideally positioning it to sense neuro/glial-derived compounds that regulate regional and global cerebral perfusion. The membrane potential (VM) of vascular cells serves as the essential output in this scenario, linking brain activity to vascular function. The ATP-sensitive K+ channel (KATP) is a key regulator of vascular VM in other beds, but whether brain capillaries possess functional KATP channels remains unknown. Here, we demonstrate that brain capillary ECs and pericytes express KATP channels that robustly control VM. We further show that the endogenous mediator adenosine acts through A2A receptors and the Gs/cAMP/PKA pathway to activate capillary KATP channels. Moreover, KATP channel stimulation in vivo causes vasodilation and increases cerebral blood flow (CBF). These findings establish the presence of KATP channels in cECs and pericytes and suggest their significant influence on CBF.HIGHLIGHTSCapillary network cellular components—endothelial cells and pericytes—possess functional KATP channels.Activation of KATP channels causes profound hyperpolarization of capillary cell membranes.Capillary KATP channels are activated by exogenous adenosine via A2A receptors and cAMP-dependent protein kinase.KATP channel activation by adenosine or synthetic openers increases cerebral blood flow.

Dementia resulting from small vessel diseases of the brain (SVDs) is an emerging epidemic for which there is no treatment. Hypertension is the major risk factor for SVDs, but how hypertension damages the brain microcirculation is unclear. Here, we show that chronic hypertension in a mouse model progressively disrupts on-demand delivery of blood to metabolically active areas of the brain (functional hyperemia) through diminished activity of the capillary endothelial cell inward-rectifier potassium channel, Kir2.1. Despite similar efficacy in reducing blood pressure, amlodipine, a voltage-dependent calcium-channel blocker, prevented hypertension-related damage to functional hyperemia whereas losartan, an angiotensin II type-1 receptor blocker, did not. We attribute this drug class effect to losartan-induced 'aldosterone breakthrough', a phenomenon triggered by pharmacological interruption of the renin-angiotensin pathway leading to elevated plasma aldosterone levels. This hypothesis is supported by the finding that combining losartan with the aldosterone receptor antagonist eplerenone prevented the hypertension-related decline in functional hyperemia. Collectively, these data suggest Kir2.1 as a possible therapeutic target in vascular dementia and indicate that concurrent mineralocorticoid aldosterone receptor blockade may aid in protecting against late-life cognitive decline in hypertensive patients treated with angiotensin II type-1 receptor blockers.

Alzheimer’s disease (AD) is a leading cause of dementia and a substantial healthcare burden. Despite this, few treatment options are available for controlling AD symptoms. Notably, neuronal activity-dependent increases in cortical cerebral blood flow (CBF; functional hyperemia) are attenuated in AD patients, but the associated pathological mechanisms are not fully understood at the molecular level. A fundamental mechanism underlying functional hyperemia is activation of capillary endothelial inward-rectifying K+ (Kir2.1) channels by neuronally derived potassium (K+), which evokes a retrograde capillary-to-arteriole electrical signal that dilates upstream arterioles, increasing blood delivery to downstream active regions. Here, using a mouse model of familial AD (5xFAD), we tested whether this impairment in functional hyperemia is attributable to reduced activity of capillary Kir2.1 channels. In vivo CBF measurements revealed significant reductions in whisker stimulation (WS)-induced and K+-induced hyperemic responses in 5xFAD mice compared with age-matched controls. Notably, measurements of whole-cell currents in freshly isolated 5xFAD capillary endothelial cells showed that Kir2.1 current density was profoundly reduced, suggesting a defect in Kir2.1 function. Because Kir2.1 activity absolutely depends on binding of phosphatidylinositol 4,5-bisphosphate (PIP2) to the channel, we hypothesized that capillary Kir2.1 channel impairment could be corrected by exogenously supplying PIP2. As predicted, a PIP2 analog restored Kir2.1 current density to control levels. More importantly, systemic administration of PIP2 restored K+-induced CBF increases and WS-induced functional hyperemic responses in 5xFAD mice. Collectively, these data provide evidence that PIP2-mediated restoration of capillary endothelial Kir2.1 function improves neurovascular coupling and CBF in the setting of AD.

The essential function of the circulatory system is to continuously and efficiently supply the O(2) 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.

As the urinary bladder fills, the rate of rise (but not the frequency) of spontaneous contractions of smooth muscle in the bladder wall increases, thereby eliciting increased afferent nerve activity that signals the fullness of the bladder., Activation of afferent nerves during urinary bladder (UB) filling conveys the sensation of UB fullness to the central nervous system (CNS). Although this sensory outflow is presumed to reflect graded increases in pressure associated with filling, UBs also exhibit nonvoiding, transient contractions (TCs) that cause small, rapid increases in intravesical pressure. Here, using an ex vivo mouse bladder preparation, we explored the relative contributions of filling pressure and TC-induced pressure transients to sensory nerve stimulation. Continuous UB filling caused an increase in afferent nerve activity composed of a graded increase in baseline activity and activity associated with increases in intravesical pressure produced by TCs. For each ∼4-mmHg pressure increase, filling pressure increased baseline afferent activity by ∼60 action potentials per second. In contrast, a similar pressure elevation induced by a TC evoked an ∼10-fold greater increase in afferent activity. Filling pressure did not affect TC frequency but did increase the TC rate of rise, reflecting a change in the length-tension relationship of detrusor smooth muscle. The frequency of afferent bursts depended on the TC rate of rise and peaked before maximum pressure. Inhibition of small- and large-conductance Ca2+-activated K+ (SK and BK) channels increased TC amplitude and afferent nerve activity. After inhibiting detrusor muscle contractility, simulating the waveform of a TC by gently compressing the bladder evoked similar increases in afferent activity. Notably, afferent activity elicited by simulated TCs was augmented by SK channel inhibition. Our results show that afferent nerve activity evoked by TCs represents the majority of afferent outflow conveyed to the CNS during UB filling and suggest that the maximum TC rate of rise corresponds to an optimal length-tension relationship for efficient UB contraction. Furthermore, our findings implicate SK channels in controlling the gain of sensory outflow independent of UB contractility.

Brain capillaries play a critical role in sensing neural activity and translating it into dynamic changes in cerebral blood flow to serve the metabolic needs of the brain. The molecular cornerstone of this mechanism is the capillary endothelial cell inward rectifier K+ (Kir2.1) channel, which is activated by neuronal activity–dependent increases in external K+ concentration, producing a propagating hyperpolarizing electrical signal that dilates upstream arterioles. Here, we identify a key regulator of this process, demonstrating that phosphatidylinositol 4,5-bisphosphate (PIP2) is an intrinsic modulator of capillary Kir2.1-mediated signaling. We further show that PIP2 depletion through activation of Gq protein-coupled receptors (GqPCRs) cripples capillary-to-arteriole signal transduction in vitro and in vivo, highlighting the potential regulatory linkage between GqPCR-dependent and electrical neurovascular-coupling mechanisms. These results collectively show that PIP2 sets the gain of capillary-initiated electrical signaling by modulating Kir2.1 channels. Endothelial PIP2 levels would therefore shape the extent of retrograde signaling and modulate cerebral blood flow.

Studies of stress effects on the brain have traditionally focused on neurons, without considering the cerebral microcirculation. Here we report that stress impairs neurovascular coupling (NVC), the process that matches neuronal activity with increased local blood flow. A stressed phenotype was induced in male rats by administering a 7-d heterotypical stress paradigm. NVC was modeled by measuring parenchymal arteriole (PA) vasodilation in response to neuronal stimulation in amygdala brain slices. After stress, vasodilation of PAs to neuronal stimulation was greatly reduced, and dilation of isolated PAs to external K(+) was diminished, suggesting a defect in smooth muscle inwardly rectifying K(+) (KIR) channel function. Consistent with these observations, stress caused a reduction in PA KIR2.1 mRNA and smooth muscle KIR current density, and blocking KIR channels significantly inhibited NVC in control, but not in stressed, slices. Delivery of corticosterone for 7 d (without stressors) or RU486 (before stressors) mimicked and abrogated NVC impairment by stress, respectively. We conclude that stress causes a glucocorticoid-mediated decrease in functional KIR channels in amygdala PA myocytes. This renders arterioles less responsive to K(+) released from astrocytic endfeet during NVC, leading to impairment of this process. Because the fidelity of NVC is essential for neuronal health, the impairment characterized here may contribute to the pathophysiology of brain disorders with a stress component.

Activation of Ca(2+)-sensitive, large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) by local, ryanodine receptor-mediated Ca(2+) signals (Ca(2+) sparks) acts as a brake on pressure-induced (myogenic) vasoconstriction-a fundamental mechanism that regulates blood flow in small resistance arteries. We report that physiological intraluminal pressure within resistance arteries activated cGMP-dependent protein kinase (PKG) in VSMCs through oxidant-induced formation of an intermolecular disulfide bond between cysteine residues. Oxidant-activated PKG was required to trigger Ca(2+) sparks, BK channel activity, and vasodilation in response to pressure. VSMCs from arteries from mice expressing a form of PKG that could not be activated by oxidants showed reduced Ca(2+) spark frequency, and arterial preparations from these mice had decreased pressure-induced activation of BK channels. Thus, the absence of oxidative activation of PKG disabled the BK channel-mediated negative feedback regulation of vasoconstriction. Our results support the concept of a negative feedback control mechanism that regulates arterial diameter through mechanosensitive production of oxidants to activate PKG and enhance Ca(2+) sparks.

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.

The nuclear factor of activated T-cells (NFAT) is a Ca2+-dependent transcription factor that has been reported to regulate the expression of smooth muscle contractile proteins and ion channels. Here we report that large conductance Ca2+-sensitive potassium (BK) channels and voltage-gated K+(KV) channels may be regulatory targets of NFATc3 in urinary bladder smooth muscle (UBSM). UBSM myocytes from NFATc3-null mice displayed a reduction in iberiotoxin (IBTX)-sensitive BK currents, a decrease in mRNA for the pore-forming α-subunit of the BK channel, and a reduction in BK channel density compared with myocytes from wild-type mice. Tetraethylammonium chloride-sensitive KVcurrents were elevated in UBSM myocytes from NFATc3-null mice, as was mRNA for the Shab family member KV2.1. Despite KVcurrent upregulation, bladder strips from NFATc3-null mice displayed an elevated contractile response to electrical field stimulation relative to strips from wild-type mice, but this difference was abrogated in the presence of the BK channel blocker IBTX. These results support a role for the transcription factor NFATc3 in regulating UBSM contractility, primarily through an NFATc3-dependent increase in BK channel activity.

Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn2+-sensitive proton conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K+ current. We identify KIR2.1 as the acid-sensitive K+ channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of KIR2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K+ current in amplifying the sensory response, entry of protons through the Zn2+-sensitive conductance produces a transient block of the KIR2.1 current. The identification in sour taste cells of an acid-sensitive K+ channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.

Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn(2+)-sensitive proton conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K(+) current. We identify KIR2.1 as the acid-sensitive K(+) channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of Kir2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K(+) current in amplifying the sensory response, entry of protons through the Zn(2+)-sensitive conductance produces a transient block of the KIR2.1 current. The identification in sour taste cells of an acid-sensitive K(+) channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.

The transcription factor NFAT (nuclear factor of activated T-cells) is implicated in cardiac hypertrophy and vasculogenesis. NFAT activation, reflecting dephosphorylation by the calcium-dependent phosphatase, calcineurin, and subsequent nuclear localization, is generally thought to require a sustained increase in intracellular calcium. However, in smooth muscle we have found that elevation of calcium by membrane depolarization fails to induce an increase in nuclear localization of the NFATc3 isoform. Here, we demonstrate that physiological intravascular pressure (100 mm Hg) induces an increase in NFATc3 nuclear localization in mouse cerebral arteries. Pressure-induced NFATc3 nuclear accumulation is abrogated by endothelial denudation and by nitric-oxide synthase, cGMP-dependent kinase (PKG), and voltage-dependent calcium channels inhibition. We further show that exogenous nitric oxide, in combination with an elevation in calcium, is an effective stimulus for NFATc3 nuclear accumulation. c-Jun terminal kinase 2 (JNK) activity, which has been shown to regulate NFATc3 nuclear export, is also reduced by pressure, an effect that is prevented by pretreatment with a PKG inhibitor. Consistent with this, pressure-induced NFATc3 nuclear accumulation is independent of PKG in arteries from JNK2(-/-) mice. Collectively, our results indicate that both activation of the NO/PKG pathway and elevation of smooth muscle calcium are required for NFATc3 nuclear accumulation and that PKG inhibits JNK2 to decrease NFAT nuclear export. Our findings suggest that at physiological intravascular pressures NFATc3 is localized to the nucleus in smooth muscle cells of intact arteries and indicate a novel and unexpected role for nitric oxide/PKG in NFAT activation.

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.

Endothelial cells and smooth muscle cells of resistance arteries mediate opposing responses to mechanical forces acting on the vasculature, promoting dilation in response to flow and constriction in response to pressure, respectively. In this review, we explore the role of TRP channels, particularly endothelial TRPV4 and smooth muscle TRPC6 and TRPM4 channels, in vascular mechanosensing circuits, placing their putative mechanosensitivity in context with other proposed upstream and downstream signaling pathways.

Endothelial cell (EC) dysfunction is a hallmark of hypertension. We recently discovered that local Ca2+ influx through clusters of functionally coupled TRPV4 channels (detected optically as “TRPV4 sparklets”) drives physiological vasodilation. Here, we show that stimulation of EC muscarinic receptors activated TRPV4 sparklets exclusively at discrete sites at myoendothelial projections (MEPs)—specialized regions of ECs that contact adjacent smooth muscle cells. This activation was dependent on protein kinase C (PKC) and was absent in mice lacking the PKC-anchoring protein AKAP150 (A-kinase anchoring protein 150), which was localized predominantly to MEPs. Cooperative gating of TRPV4 channels within a cluster amplified Ca2+ influx at MEPs by more than 2-fold. This cooperativity was largely absent at non-MEP sites and was virtually eliminated by chelation of intracellular Ca2+ or AKAP150 knockout, suggesting AKAP150-dependent potentiation of TRPV4 activity by Ca2+ influx via adjacent channels. Notably, MEP-l...

The transcription factor NFAT (nuclear factor of activated T-cells) plays a central role in mediating Ca(2+)-dependent gene transcription in a variety of cell types. Sustained increases in intracellular calcium concentration ([Ca(2+)]i) are presumed to be required for NFAT dephosphorylation by the Ca(2+)/calmodulin-dependent protein calcineurin and its subsequent nuclear translocation. Here, we provide the first identification and characterization of NFAT in native smooth muscle, showing that NFAT4 is the predominant isoform detected by reverse transcriptase-polymerase chain reaction and Western blot analysis. PDGF induces NFAT4 translocation in smooth muscle, leading to an increase in NFAT transcriptional activity. NFAT4 activation by PDGF depends on Ca(2+) entry through voltage-dependent Ca(2+) channels, because its nuclear accumulation is prevented by the Ca(2+) channel blocker nisoldipine and the K(+) channel opener pinacidil. Interestingly, elevation of [Ca(2+)]i by membrane depolarization or ionomycin treatment are not effective stimuli for NFAT4 nuclear accumulation, indicating that Ca(2+) influx is necessary but not sufficient for NFAT4 activation. In contrast, membrane depolarization readily activates the Ca(2+)-dependent transcription factor CREB (cAMP-responsive element-binding protein). The calcineurin blockers CsA and FK506 also prevented the PDGF-induced NFAT4 nuclear localization. These results indicate that both the nature of the calcium signal and PDGF-induced modulation of nuclear import-export of NFAT are critical for NFAT4 activation in this tissue.

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 alpha 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.

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.

It has been proposed that prostaglandin E2 (PGE2) is released from astrocytic endfeet to dilate parenchymal arterioles through activation of prostanoid (EP4) receptors during neurovascular coupling. However, the direct effects of PGE2 on isolated parenchymal arterioles have not been tested. Here, we examined the effects of PGE2 on the diameter of isolated pressurized parenchymal arterioles from rat and mouse brain. Contrary to the prevailing assumption, we found that PGE2 (0.1, 1, and 5 μmol/L) constricted rather than dilated parenchymal arterioles. Vasoconstriction to PGE2 was prevented by inhibitors of EP1 receptors. These results strongly argue against a direct role of PGE2 on arterioles during neurovascular coupling.

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).

The nuclear factor of activated T-cells (NFAT), originally identified in T-cells, has since been shown to play a role in mediating Ca(2+)-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 Ca(2+) influx through L-type, voltage-dependent Ca(2+) 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 G(q/11)-coupled receptor agonists. This induction is mediated by calcineurin and is dependent on sarcoplasmic reticulum Ca(2+) release through inositol 1,4,5-trisphosphate receptors and extracellular Ca(2+) influx through L-type, voltage-dependent Ca(2+) channels. Consistent with results obtained in ileal smooth muscle, depolarization-induced Ca(2+) influx fails to induce NFAT nuclear accumulation in cerebral arteries. We also provide evidence that Ca(2+) release by ryanodine receptors in the form of Ca(2+) sparks may exert an inhibitory influence on UTP-induced NFATc3 nuclear accumulation and further suggest that UTP may act, in part, by inhibiting Ca(2+) 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 Ca(2+) release and effects attributable to global elevations in intracellular Ca(2+).

Publisher Summary Muscarinic receptors are purified to apparent homogeneity from the both porcine brain and heart. Sufficient peptide sequence is obtained from these preparations to allow the cloning of the ml and m2 muscarinic receptor subtypes from these tissues. Using homology cloning, the human and rat forms of these receptors as well as three additional subtypes (m3-m5) are identified. Based on the sequence of the five cloned muscarinic receptor subtypes (ml-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 the 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 selectivity for different ligands. In this chapter, the residues that contribute to ligand binding and G-protein coupling are described, as well as the implied structures of these functional domains.

Coagulation factor VIII (fVIII) is isolated from porcine blood as a set of three heterodimers because of proteolytic cleavages in the middle, or B region, of the parent single-chain molecule. A single 80-kDa COOH-terminal fragment, the light chain (fVIIILC), is associated with one of three forms of heavy chain (fVIIIHCs) by a calcium-dependent linkage. The purified heterodimers were dissociated using EDTA and fVIIILC, and fVIIIHCs were isolated by high pressure liquid chromatography under nondenaturing conditions. The association of fVIII, fVIIILC, and fVIIIHCs with multimeric human von Willebrand factor (vWF) was studied using analytical velocity sedimentation. A previous study using this method with an intact, single heterodimeric species of fVIII has shown that one molecule of fVIII can bind to each subunit of vWF (Lollar, P., and Parker, C.G. (1987) J. Biol. Chem. 262, 17572-17576). fVIIILC bound vWF as judged by the increase in the plateau height and sedimentation coefficient of the fVIIILC.vWF complex compared to vWF at 42,000 x g and by the decrease in the plateau height of the 4.8 S fVIIILC boundary sedimenting at 240,000 x g. Titration of a fixed concentration of fVIIILC with vWF yielded a stoichiometry of one fVIIILC molecule per subunit of vWF. Proteolytic cleavage by thrombin to remove an acidic 41-residue NH2-terminal peptide from fVIIILC completely abolished its binding to vWF. In contrast, no binding of fVIIIHCs to vWF was observed. Additionally, intact fVIII bound to vWF was completely dissociated after proteolysis by thrombin. These data are consistent with the hypothesis that a critical step in blood coagulation is the release of all regions of fVIII from vWF following a single proteolytic cleavage of fVIIILC.

Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. We isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO4/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated fVIII to function as a cofactor in the activation of factor X by factor IXa, we found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.

We recently reported that the inward-rectifier Kir2.1 channel in brain capillary endothelial cells (cECs) plays a major role in neurovascular coupling (NVC) by mediating a neuronal activity-dependent, propagating vasodilatory (hyperpolarizing) signal. We further demonstrated that Kir2.1 activity is suppressed by depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2). Whether cECs express depolarizing channels that intersect with Kir2.1-mediated signaling remains unknown. Here, we report that Ca2+/Na+-permeable TRPV4 (transient receptor potential vanilloid 4) channels are expressed in cECs and are tonically inhibited by PIP2. We further demonstrate that depletion of PIP2 by agonists, including putative NVC mediators, that promote PIP2 hydrolysis by signaling through Gq-protein-coupled receptors (GqPCRs) caused simultaneous disinhibition of TRPV4 channels and suppression of Kir2.1 channels. These findings collectively support the concept that GqPCR activation functions as a molecular switch to favor capillary TRPV4 activity over Kir2.1 signaling, an observation with potentially profound significance for the control of cerebral blood flow., eLife digest Capillaries form branching networks that surround all cells of the body. They allow oxygen and nutrient exchange between blood and tissue, but this is not their only role. Capillaries in the brain form a tight barrier that prevents components carried in the blood from easily reaching the brain compartment. They also detect the activity of neurons and trigger on-demand increases in blood flow to active regions of the brain. This role, revealed only recently, depends upon ion channels on the surface of the capillary cells. Active neurons release potassium ions, which open a type of ion channel called Kir2.1 that allows potassium inside the cell to flow out. This process is repeated in neighboring capillary cells until it reaches an upstream vessel, where it causes the vessel to relax and increase the blood flow. Kir2.1 channels sit astride the membranes of capillary cells, where they can interact with other membrane molecules. One such molecule, called PIP2, plays several roles in relaying signals from the outside to the inside of cells. It also physically interacts with channels in the membrane, including Kir2.1 channels. If PIP2 levels are low, Kir2.1 channel activity decreases. Here, Harraz et al. discovered that capillary cells contain another type of ion channel, called TRPV4, which is also regulated by PIP2. But unlike Kir2.1, its activity increases when PIP2 levels drop. Moreover, TRPV4 channels allow sodium and calcium ions to flow into the cell, which has an effect opposite to that of potassium flowing out of the cell. Capillary cells also have receptor proteins called GqPCRs that are activated by chemical signals released by active neurons in the brain. GqPCRs break down PIP2, so their activity turns Kir2.1 channels off and TRPV4 channels on. This resets the system so that it is ready to respond to new signals from active neurons. GqPCRs work as molecular switches to control the balance between Kir2.1 and TRPV4 channels and turn brain blood flow up and down. GqPCRs and ion channels that depend on PIP2 can also be found in other types of cells. These findings could reveal clues about how signals are switched on and off in different cells. Understanding the role of PIP2 in signaling could also unveil what happens when signaling go wrong.