Impaired intracellular Ca2+ signaling contributes to age-related cerebral small vessel disease in Col4a1 mutant mice.

Humans and mice with mutations in COL4A1 and COL4A2 manifest hallmarks of cerebral small vessel disease (cSVD). Mice with a missense mutation in Col4a1 at amino acid 1344 (Col4a1+/G1344D) exhibit age-dependent intracerebral hemorrhages (ICHs) and brain lesions. Here, we report that this pathology was associated with the loss of myogenic vasoconstriction, an intrinsic vascular response essential for the autoregulation of cerebral blood flow. Electrophysiological analyses showed that the loss of myogenic constriction resulted from blunted pressure-induced smooth muscle cell (SMC) membrane depolarization. Furthermore, we found that dysregulation of membrane potential was associated with impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ (BK) and transient receptor potential melastatin 4 (TRPM4) cation channels linked to disruptions in sarcoplasmic reticulum (SR) Ca2+ signaling. Col4a1 mutations impair protein folding, which can cause SR stress. Treating Col4a1+/G1344D mice with 4-phenylbutyrate, a compound that promotes the trafficking of misfolded proteins and alleviates SR stress, restored SR Ca2+ signaling, maintained BK and TRPM4 channel activity, prevented loss of myogenic tone, and reduced ICHs. We conclude that alterations in SR Ca2+ handling that impair ion channel activity result in dysregulation of SMC membrane potential and loss of myogenic tone and contribute to age-related cSVD in Col4a1+/G1344D mice.

PI3K block restores age-dependent neurovascular coupling defects associated with cerebral small vessel disease.

Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report a Col4a1 mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) in brain capillary endothelial cells, leading to the loss of inwardly rectifying K+ (Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP2 by converting it to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of aged Col4a1 mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD.

PI3K block restores age-dependent neurovascular coupling defects associated with cerebral small vessel disease.

Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report a Col4a1 mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP 2 ) in brain capillary endothelial cells, leading to the loss of inwardly rectifier K + (Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP 2 by converting it to phosphatidylinositol (3,4,5)-trisphosphate (PIP 3 ), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of aged Col4a1 mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD. One-sentence summary: PI3K inhibition rescues neurovascular coupling defects in cerebral small vessel disease.

Faulty TRPM4 channels underlie age-dependent cerebral vascular dysfunction in Gould syndrome.

Gould syndrome is a rare multisystem disorder resulting from autosomal dominant mutations in the collagen-encoding genes COL4A1 and COL4A2. Human patients and Col4a1 mutant mice display brain pathology that typifies cerebral small vessel diseases (cSVDs), including white matter hyperintensities, dilated perivascular spaces, lacunar infarcts, microbleeds, and spontaneous intracerebral hemorrhage. The underlying pathogenic mechanisms are unknown. Using the Col4a1+/G394V mouse model, we found that vasoconstriction in response to internal pressure-the vascular myogenic response-is blunted in cerebral arteries from middle-aged (12 mo old) but not young adult (3 mo old) animals, revealing age-dependent cerebral vascular dysfunction. The defect in the myogenic response was associated with a significant decrease in depolarizing cation currents conducted by TRPM4 (transient receptor potential melastatin 4) channels in native cerebral artery smooth muscle cells (SMCs) isolated from mutant mice. The minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) is necessary for TRPM4 activity. Dialyzing SMCs with PIP2 and selective blockade of phosphoinositide 3-kinase (PI3K), an enzyme that converts PIP2 to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), restored TRPM4 currents. Acute inhibition of PI3K activity and blockade of transforming growth factor-beta (TGF-ß) receptors also rescued the myogenic response, suggesting that hyperactivity of TGF-ß signaling pathways stimulates PI3K to deplete PIP2 and impair TRPM4 channels. We conclude that age-related cerebral vascular dysfunction in Col4a1+/G394V mice is caused by the loss of depolarizing TRPM4 currents due to PIP2 depletion, revealing an age-dependent mechanism of cSVD.

STIM1-dependent peripheral coupling governs the contractility of vascular smooth muscle cells.

Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs). The mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal interaction molecule 1), a single-transmembrane-domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER-PM junctions in response to depletion of ER Ca2+ stores and initiates store-operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR-PM interacting sites were decreased, and SR-dependent Ca2+-signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen-inducible, SMC-specific STIM1-knockout (Stim1-smKO) mice. VSMCs from Stim1-smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+-release sites on the SR and Ca2+-activated ion channels on the PM, accompanied by diminished channel activity. Stim1-smKO mice were hypotensive, and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 - independent of SR Ca2+ store depletion - is critically important for stable peripheral coupling in contractile VSMCs.

Nitric Oxide Signals Through IRAG to Inhibit TRPM4 Channels and Dilate Cerebral Arteries.

Nitric oxide (NO) relaxes vascular smooth muscle cells (SMCs) and dilates blood vessels by increasing intracellular levels of cyclic guanosine monophosphate (cGMP), which stimulates the activity of cGMP-dependent protein kinase (PKG). However, the vasodilator mechanisms downstream of PKG remain incompletely understood. Here, we found that transient receptor potential melastatin 4 (TRPM4) cation channels, which are activated by Ca2+ released from the sarcoplasmic reticulum (SR) through inositol triphosphate receptors (IP3Rs) under native conditions, are essential for SMC membrane depolarization and vasoconstriction. We hypothesized that signaling via the NO/cGMP/PKG pathway causes vasodilation by inhibiting TRPM4. We found that TRPM4 currents activated by stretching the plasma membrane or directly activating IP3Rs were suppressed by exogenous NO or a membrane-permeable cGMP analog, the latter of which also impaired IP3R-mediated release of Ca2+ from the SR. The effects of NO on TRPM4 activity were blocked by inhibition of soluble guanylyl cyclase or PKG. Notably, upon phosphorylation by PKG, IRAG (IP3R-associated PKG substrate) inhibited IP3R-mediated Ca2+ release, and knockdown of IRAG expression diminished NO-mediated inhibition of TRPM4 activity and vasodilation. Using superresolution microscopy, we found that IRAG, PKG, and IP3Rs form a nanoscale signaling complex on the SR of SMCs. We conclude that NO/cGMP/PKG signaling through IRAG inhibits IP3R-dependent activation of TRPM4 channels in SMCs to dilate arteries. SIGNIFICANCE STATEMENT: Nitric oxide is a gaseous vasodilator produced by endothelial cells that is essential for cardiovascular function. Although NO-mediated signaling pathways have been intensively studied, the mechanisms by which they relax SMCs to dilate blood vessels remain incompletely understood. In this study, we show that NO causes vasodilation by inhibiting the activity of Ca2+-dependent TRPM4 cation channels. Probing further, we found that NO does not act directly on TRPM4 but instead initiates a signaling cascade that inhibits its activation by blocking the release of Ca2+ from the SR. Thus, our findings reveal the essential molecular pathways of NO-induced vasodilation-a fundamental unresolved concept in cardiovascular physiology.

Brain endothelial cell TRPA1 channels initiate neurovascular coupling.

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 intracellular Ca2+ release channel TRPML1 regulates lower urinary tract smooth muscle contractility.

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel that is predominantly localized to the membranes of late endosomes and lysosomes (LELs). Intracellular release of Ca2+ through TRPML1 is thought to be pivotal for maintenance of intravesicular acidic pH as well as the maturation, fusion, and trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice (Mcoln1-/- ) induces a hyperdistended/hypertrophic bladder phenotype. Here, we investigated this phenomenon further by exploring an unconventional role for TRPML1 channels in the regulation of Ca2+-signaling activity and contractility in bladder and urethral smooth muscle cells (SMCs). Four-dimensional (4D) lattice light-sheet live-cell imaging showed that the majority of LELs in freshly isolated bladder SMCs were essentially immobile. Superresolution microscopy revealed distinct nanoscale colocalization of LEL-expressing TRPML1 channels with ryanodine type 2 receptors (RyR2) in bladder SMCs. Spontaneous intracellular release of Ca2+ from the sarcoplasmic reticulum (SR) through RyR2 generates localized elevations of Ca2+ ("Ca2+ sparks") that activate plasmalemmal large-conductance Ca2+-activated K+ (BK) channels, a critical negative feedback mechanism that regulates smooth muscle contractility. This mechanism was impaired in Mcoln1-/- mice, which showed diminished spontaneous Ca2+ sparks and BK channel activity in bladder and urethra SMCs. Additionally, ex vivo contractility experiments showed that loss of Ca2+ spark-BK channel signaling in Mcoln1-/- mice rendered both bladder and urethra smooth muscle hypercontractile. Voiding activity analyses revealed bladder overactivity in Mcoln1-/- mice. We conclude that TRPML1 is critically important for Ca2+ spark signaling, and thus regulation of contractility and function, in lower urinary tract SMCs.

TRPML1 channels initiate Ca2+ sparks in vascular smooth muscle cells.

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel localized to the membranes of endosomes and lysosomes and is not present or functional on the plasma membrane. Ca2+ released from endosomes and lysosomes into the cytosol through TRPML1 channels is vital for trafficking, acidification, and other basic functions of these organelles. Here, we investigated the function of TRPML1 channels in fully differentiated contractile vascular smooth muscle cells (SMCs). In live-cell confocal imaging studies, we found that most endosomes and lysosomes in freshly isolated SMCs from cerebral arteries were essentially immobile. Using nanoscale super-resolution microscopy, we found that TRPML1 channels present in late endosomes and lysosomes formed stable complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Spontaneous Ca2+ signals resulting from the release of SR Ca2+ through RyR2s ("Ca2+ sparks") and corresponding Ca2+-activated K+ channel activity are critically important for balancing vasoconstriction. We found that these signals were essentially absent in SMCs from TRPML1-knockout (Mcoln1-/- ) mice. Using ex vivo pressure myography, we found that loss of this critical signaling cascade exaggerated the vasoconstrictor responses of cerebral and mesenteric resistance arteries. In vivo radiotelemetry studies showed that Mcoln1-/- mice were spontaneously hypertensive. We conclude that TRPML1 is crucial for the initiation of Ca2+ sparks in SMCs and the regulation of vascular contractility and blood pressure.

Differential expression of angiotensin II type 1 receptor subtypes within the cerebral microvasculature.

Arteries and arterioles constrict in response to intraluminal pressure to generate myogenic tone, but the molecular nature of the vascular force-sensing mechanism is not fully characterized. Here, we investigated the role of angiotensin II type 1 receptors (AT1Rs) on vascular smooth muscle cells in the development of myogenic tone in cerebral parenchymal arterioles from mice. We found that pretreatment with the AT1R blocker losartan inhibited the development of myogenic tone in these vessels but did not alter the luminal diameter of arterioles with preestablished tone. Rodents express two AT1R isotypes: AT1Ra and AT1Rb. We previously demonstrated that AT1Rb is expressed at much higher levels compared with AT1Ra in cerebral pial arteries and is required for myogenic contractility in these vessels, whereas AT1Ra is unnecessary for this function. Here, we found that AT1Ra and AT1Rb are expressed at similar levels in parenchymal arterioles and that genetic knockout of AT1Ra blunted the ability of these vessels to generate myogenic tone. We also found that AT1Rb and total AT1R expression levels are much lower in parenchymal arterioles compared with pial arteries and that parenchymal arterioles are less sensitive to the vasoconstrictive effects of the endogenous AT1R ligand angiotensin II (ANG II). We conclude that 1) AT1Rs are critical for the initiation, but not the maintenance, of myogenic tone in parenchymal arterioles, and 2) lower levels of AT1Rb and total AT1R in parenchymal arterioles compared with pial arteries result in differences in myogenic and ANG II-induced vasoconstriction between these vascular segments.NEW & NOTEWORTHY Myogenic tone is critical for appropriate regulation of cerebral blood flow, but the mechanisms used by vascular smooth muscle cells to detect changes in intraluminal pressure are not fully characterized. Here, we demonstrate angiotensin II receptor type 1 (AT1R) is indispensable to initiation, but not maintenance, of myogenic tone in cerebral parenchymal arterioles. Furthermore, we demonstrate differences in AT1R expression levels lead to critical differences in contractile regulation between parenchymal arterioles and cerebral pial arteries.

Nanoscale coupling of junctophilin-2 and ryanodine receptors regulates vascular smooth muscle cell contractility.

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.

(Pro)renin receptor knockdown in the paraventricular nucleus of the hypothalamus attenuates hypertension development and AT1 receptor-mediated calcium events.

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.

Nanoscale remodeling of ryanodine receptor cluster size underlies cerebral microvascular dysfunction in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) results from mutations in the gene encoding dystrophin which lead to impaired function of skeletal and cardiac muscle, but little is known about the effects of the disease on vascular smooth muscle cells (SMCs). Here we used the mdx mouse model to study the effects of mutant dystrophin on the regulation of cerebral artery and arteriole SMC contractility, focusing on an important Ca2+-signaling pathway composed of type 2 ryanodine receptors (RyR2s) on the sarcoplasmic reticulum (SR) and large-conductance Ca2+-activated K+ (BK) channels on the plasma membrane. Nanoscale superresolution image analysis revealed that RyR2 and BKα were organized into discrete clusters, and that the mean size of RyR2 clusters that colocalized with BKα was larger in SMCs from mdx mice (∼62 RyR2 monomers) than in controls (∼40 RyR2 monomers). We further found that the frequency and signal mass of spontaneous, transient Ca2+-release events through SR RyR2s ("Ca2+ sparks") were greater in SMCs from mdx mice. Patch-clamp electrophysiological recordings indicated a corresponding increase in Ca2+-dependent BK channel activity. Using pressure myography, we found that cerebral pial arteries and parenchymal arterioles from mdx mice failed to develop appreciable spontaneous myogenic tone. Inhibition of RyRs with tetracaine and blocking of BK channels with paxilline restored myogenic tone to control levels, demonstrating that enhanced RyR and BK channel activity is responsible for the diminished pressure-induced constriction of arteries and arterioles from mdx mice. We conclude that increased size of RyR2 protein clusters in SMCs from mdx mice increases Ca2+ spark and BK channel activity, resulting in cerebral microvascular dysfunction.

The angiotensin II receptor type 1b is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.

KEY POINTS: The angiotensin II receptor type 1b (AT1 Rb ) is the primary sensor of intraluminal pressure in cerebral arteries. Pressure or membrane-stretch induced stimulation of AT1 Rb activates the TRPM4 channel and results in inward transient cation currents that depolarize smooth muscle cells, leading to vasoconstriction. Activation of either AT1 Ra or AT1 Rb with angiotensin II stimulates TRPM4 currents in cerebral artery myocytes and vasoconstriction of cerebral arteries. The expression of AT1 Rb mRNA is ∼30-fold higher than AT1 Ra 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 AT1 Rb 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 (AT1 R) is inherently mechanosensitive and initiates this signalling pathway. Rodents express two types of AT1 R - AT1 Ra and AT1 Rb - and conflicting studies provide support for either isoform as the primary sensor of intraluminal pressure in peripheral arteries. We hypothesized that mechanical activation of AT1 Ra increases TRPM4 currents to induce myogenic constriction of cerebral arteries. However, we found that development of myogenic tone was greater in arteries from AT1 Ra 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 AT1 R blocker losartan (1 µm) diminished myogenic tone and blocked stretch-induced cation currents in cerebral arteries from both groups. Activation of AT1 R with angiotensin II (30 nm) also increased TRPM4 currents in smooth muscle cells and constricted cerebral arteries from both groups. Expression of AT1 Rb mRNA was ∼30-fold greater than AT1 Ra in cerebral arteries, and knockdown of AT1 Rb selectively diminished myogenic constriction. We conclude that AT1 Rb , acting upstream of TRPM4 channels, is the primary sensor of intraluminal pressure in cerebral artery smooth muscle cells.

UTP activates small-conductance Ca2+-activated K+ channels in murine detrusor PDGFRα+ cells.

Purines induce transient contraction and prolonged relaxation of detrusor muscles. Transient contraction is likely due to activation of inward currents in smooth muscle cells, and prolonged relaxation may be due to activation of small-conductance Ca(2+)-activated K(+) (SK) channels via P2Y1 receptors expressed by detrusor PDGF receptor (PDGFR)α(+) cells. We investigated whether other subtypes of P2Y receptors are involved in the activation of SK channels in PDGFRα(+) cells of detrusor muscles. Quantitative analysis of transcripts revealed that P2ry2, P2ry4, and P2ry14 are expressed in PDGFRα(+) cells of P2ry1-deficient/enhanced green fluorescent protein (P2ry1(-/-)/eGFP) mice at similar levels as in wild-type mice. UTP, a P2Y2/P2Y4 agonist, activated large outward currents in detrusor PDGFRα(+) cells. SK channel blockers and an inhibitor of phospholipase C completely abolished currents activated by UTP. In contrast, UTP activated nonselective cation currents in smooth muscle cells. Under current-clamp (current = 0), UTP induced significant hyperpolarization of PDGFRα(+) cells. MRS2500, a selective P2Y1 antagonist, did not affect UTP-activated outward currents in PDGFRα(+) cells from wild-type mice, and activation of outward currents by UTP was retained in P2ry1(-/-)/eGFP mice. As a negative control, we tested the effect of MRS2693, a selective P2Y6 agonist. This compound did not activate outward currents in PDGFRα(+) cells, and currents activated by UTP were unaffected by MRS2578, a selective P2Y6 antagonist. The nonselective P2Y receptor blocker suramin inhibited UTP-activated outward currents in PDGFRα(+) cells. Our data demonstrate that P2Y2 and/or P2Y4 receptors function, in addition to P2Y1 receptors, in activating SK currents in PDGFRα(+) cells and possibly in mediating purinergic relaxation responses in detrusor muscles.