Objective— Intermediate and small conductance K_Ca channels IK1 (K_Ca3.1) and SK3 (K_Ca2.3) are primary targets of endothelial Ca²⁺ signals in the arterial vasculature, and their ablation results in increased arterial tone and hypertension. Activation of IK1 channels by local Ca²⁺ transients from internal stores or plasma membrane channels promotes arterial hyperpolarization and vasodilation. Here, we assess arteries from genetically altered IK1 knockout mice (IK1^−/−) to determine whether IK1 channels exert a positive feedback influence on endothelial Ca²⁺ dynamics. Approach and Results— Using confocal imaging and custom data analysis software, we found that although the occurrence of basal endothelial Ca²⁺ dynamics was not different between IK1^−/− and wild-type mice (P>0.05), the frequency of acetylcholine-stimulated (2 μmol/L) Ca²⁺ dynamics was greatly decreased in IK1^−/− endothelium (515±153 versus 1860±319 events; P<0.01). In IK1^−/−/SK3^T/T mice, ancillary suppression (+Dox) or overexpression (−Dox) of SK3 channels had little additional effect on the occurrence of events under basal or acetylcholine-stimulated conditions. However, SK3 overexpression did restore the decreased event amplitudes. Removal of extracellular Ca²⁺ reduced acetylcholine-induced Ca²⁺ dynamics to the same level in wild-type and IK1^−/− arteries. Blockade of IK1 and SK3 with the combination of charybdotoxin (0.1 μmol/L) and apamin (0.5 μmol/L) or transient receptor potential vanilloid 4 channels with HC-067047 (1 μmol/L) reduced acetylcholine Ca²⁺ dynamics in wild-type arteries to the level of IK1^−/−/SK3^T/T+Dox arteries. These drug effects were not additive. Conclusions— IK1, and to some extent SK3, channels exert a substantial positive feedback influence on endothelial Ca²⁺ dynamics.

Rationale:Recent data from mesenteric and cerebral beds have revealed spatially restricted Ca2+ transients occurring along the vascular intima that control effector recruitment and vasodilation. Although Ca2+ is pivotal for coronary artery endothelial function, spatial and temporal regulation of functional Ca2+ signals in the coronary endothelium is poorly understood. Objective:We aimed to determine whether a discrete spatial and temporal profile of Ca2+ dynamics underlies endothelium-dependent relaxation of swine coronary arteries. Methods and Results:Using confocal imaging, custom automated image analysis, and myography, we show that the swine coronary artery endothelium generates discrete basal Ca2+ dynamics, including isolated transients and whole-cell propagating waves. These events are suppressed by depletion of internal stores or inhibition of inositol 1,4,5-trisphosphate receptors but not by inhibition of ryanodine receptors or removal of extracellular Ca2+. In vessel rings, inhibition of specific...

Although acute and chronic vasoregulation is inherently driven by endothelial Ca2+, control and targeting of Ca2+-dependent signals are poorly understood. Recent studies have revealed localized and dynamic endothelial Ca2+ events comprising an intricate signaling network along the vascular intima. Discrete Ca2+ transients emerging from both internal stores and plasmalemmal cation channels couple to specific membrane K+ channels, promoting endothelial hyperpolarization and vasodilation. The spatiotemporal tuning of these signals, rather than global Ca2+ elevation, appear to direct endothelial functions under physiologic conditions. In fact, altered patterns of dynamic Ca2+ signaling may underlie essential endothelial dysfunction in a variety of cardiovascular diseases. Advances in imaging approaches and analyses in recent years have allowed for detailed detection, quantification, and evaluation of Ca2+ dynamics in intact endothelium. Here, we discuss recent insights into these signals, including their sources of origination and their functional encoding. We also address key aspects of data acquisition and interpretation, including broad applications of automated high-content analysis.

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within...

Transient receptor potential vanilloid 4 channels (TRPV4) are pivotal regulators of vascular homeostasis. Altered TRPV4 signaling has recently been implicated in various cardiovascular diseases, including hypertension and atherosclerosis. These versatile nonselective cation channels increase endothelial Ca²⁺ influx in response to various stimuli including shear stress and G protein-coupled receptor (GPCR) activation. Recent findings suggest TRPV4 channels produce localized Ca²⁺ transients at the endothelial cell plasma membrane that may allow targeted effector recruitment and promote large-scale Ca²⁺ events via release from internal stores (endoplasmic reticulum). However, the specific impact of TRPV4 channels on Ca²⁺ signaling in the intact arterial intima remains unknown. In the current study, we employ an endothelium-specific TRPV4 knockout mouse model (ecTRPV4^−/−) to identify and characterize TRPV4-dependent endothelial Ca²⁺ dynamics. We find that carotid arteries from both ecTRPV4^−/− and WT mice exhibit a range of basal and acetylcholine (ACh)-induced Ca²⁺ dynamics, similar in net frequency. Analysis of discrete Ca²⁺ event parameters (amplitude, duration, and spread) and event composite values reveals that while ecTRPV4^−/− artery endothelium predominantly produces large Ca²⁺ events comparable to and in excess of those produced by WT endothelium, they are deficient in a particular population of small events, under both basal and ACh-stimulated conditions. These findings support the concept that TRPV4 channels are responsible for generating a distinct population of focal Ca²⁺ transients in the intact arterial endothelium, likely underlying their essential role in vascular homeostasis.

Organized uterine contractions, including those necessary for parturition, are dependent on calcium entry through voltage-gated calcium channels in myometrial smooth muscle cells. Recent evidence suggests that small-conductance Ca²⁺-activated potassium channels (K_Ca2), specifically isoforms K_Ca2.2 and 2.3, may control these contractions through negative feedback regulation of Ca²⁺ entry. We tested whether selective pharmacologic activation of K_Ca2.2/2.3 channels might depress uterine contractions, providing a new strategy for preterm labor intervention. Western blot analysis and immunofluorescence microscopy revealed expression of both K_Ca2.2 and K_Ca2.3 in the myometrium of nonpregnant (NP) and pregnant (gestation day 10 and 16; D10 and D16, respectively) mice. Spontaneous phasic contractions of isolated NP, D10, and D16 uterine strips were all suppressed by the K_Ca2.2/2.3-selective activator CyPPA in a concentration-dependent manner. This effect was antagonized by the selective K_Ca2 inhibitor apamin. Whereas CyPPA sensitivity was reduced in D10 and D16 versus NP strips (pIC₅₀ 5.33 ± 0.09, 4.64 ± 0.03, 4.72 ± 0.10, respectively), all contractions were abolished between 30 and 60 μM. Blunted contractions were associated with CyPPA depression of spontaneous Ca²⁺ events in myometrial smooth muscle bundles. Augmentation of uterine contractions with oxytocin or prostaglandin F_2α did not reduce CyPPA sensitivity or efficacy. Finally, in an RU486-induced preterm labor model, CyPPA significantly delayed time to delivery by 3.4 h and caused a 2.5-fold increase in pup retention. These data indicate that pharmacologic stimulation of myometrial K_Ca2.2/2.3 channels effectively suppresses Ca²⁺-mediated uterine contractions and delays preterm birth in mice, supporting the potential utility of this approach in tocolytic therapies.