Polarized localization of phosphatidylserine in the endothelium regulates Kir2.1

Lipid regulation of ion channels is largely explored using in silico modeling with minimal experimentation in intact tissue; thus, the functional consequences of these predicted lipid-channel interactions within native cellular environments remain elusive. The goal of this study is to investigate how lipid regulation of endothelial Kir2.1 - an inwardly rectifying potassium channel that regulates membrane hyperpolarization - contributes to vasodilation in resistance arteries. First, we show that phosphatidylserine (PS) localizes to a specific subpopulation of myoendothelial junctions (MEJs), crucial signaling microdomains that regulate vasodilation in resistance arteries, and in silico data have implied that PS may compete with phosphatidylinositol 4,5-bisphosphate (PIP2) binding on Kir2.1. We found that Kir2.1-MEJs also contained PS, possibly indicating an interaction where PS regulates Kir2.1. Electrophysiology experiments on HEK cells demonstrate that PS blocks PIP2 activation of Kir2.1 and that addition of exogenous PS blocks PIP2-mediated Kir2.1 vasodilation in resistance arteries. Using a mouse model lacking canonical MEJs in resistance arteries (Elnfl/fl/Cdh5-Cre), PS localization in endothelium was disrupted and PIP2 activation of Kir2.1 was significantly increased. Taken together, our data suggest that PS enrichment to MEJs inhibits PIP2-mediated activation of Kir2.1 to tightly regulate changes in arterial diameter, and they demonstrate that the intracellular lipid localization within the endothelium is an important determinant of vascular function

Pharmacological inhibitors of TRPV4 channels reduce cytokine production, restore endothelial function and increase survival in septic mice

Sepsis is characterized by systemic inflammation, edema formation and hypo-perfusion leading to organ dysfunction and ultimately death. Activation of the transient receptor potential vanilloid type 4 (TRPV4) channel is associated with edema formation and circulatory collapse. Here, we show that TRPV4 channels are involved in the hyper-inflammatory response and mortality associated with sepsis. Pharmacological inhibition of TRPV4 channels in mice reduced mortality in lipopolysaccharide and cecal-ligation-and-puncture models of sepsis, but not in a tumor necrosis factor-α (TNFα)-induced sepsis model. These protective effects of TRPV4 channel inhibition were attributable to prevention of the sepsis-induced surge of a broad spectrum of pro-inflammatory cytokines, including TNFα, interleukin (IL)-1 and IL-6, and subsequent preservation of endothelial cell function, including Ca(2+) signaling, integrity and endothelium-dependent vasodilation. These results suggest that TRPV4 antagonists may be of therapeutic utility in the management of sepsis

Opposing roles of smooth muscle BK channels and ryanodine receptors in the regulation of nerve-evoked constriction of mesenteric resistance arteries

In depolarized smooth muscle cells of pressurized cerebral arteries, ryanodine receptors (RyRs) generate “Ca(2+) sparks” that activate large-conductance, Ca(2+)-, and voltage-sensitive potassium (BK) channels to oppose pressure-induced (myogenic) constriction. Here, we show that BK channels and RyRs have opposing roles in the regulation of arterial tone in response to sympathetic nerve activation by electrical field stimulation. Inhibition of BK channels with paxilline increased both myogenic and nerve-induced constrictions of pressurized, resistance-sized mesenteric arteries from mice. Inhibition of RyRs with ryanodine increased myogenic constriction, but it decreased nerve-evoked constriction along with a reduction in the amplitude of nerve-evoked increases in global intracellular Ca(2+). In the presence of L-type voltage-dependent Ca(2+) channel (VDCC) antagonists, nerve stimulation failed to evoke a change in arterial diameter, and BK channel and RyR inhibitors were without effect, suggesting that nerve- induced constriction is dependent on activation of VDCCs. Collectively, these results indicate that BK channels and RyRs have different roles in the regulation of myogenic versus neurogenic tone: whereas BK channels and RyRs act in concert to oppose myogenic vasoconstriction, BK channels oppose neurogenic vasoconstriction and RyRs augment it. A scheme for neurogenic vasoregulation is proposed in which RyRs act in conjunction with VDCCs to regulate nerve-evoked constriction in mesenteric resistance arteries

Vascular TRP channels: Performing under pressure and going with the flow

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

Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization

Intracerebral (parenchymal) arterioles are morphologically and physiologically unique compared with pial arteries and arterioles. The ability of subarachnoid hemorrhage (SAH) to induce vasospasm in large-diameter pial arteries has been extensively studied, although the contribution of this phenomenon to patient outcome is controversial. Currently, little is known regarding the impact of SAH on parenchymal arterioles, which are critical for regulation of local and global cerebral blood flow. Here diameter, smooth muscle intracellular Ca2+ concentration ([Ca2+]i), and membrane potential measurements were used to assess the function of intact brain parenchymal arterioles isolated from unoperated (control), sham-operated, and SAH model rats. At low intravascular pressure (5 mmHg), membrane potential and [Ca2+]i were not different in arterioles from control, sham-operated, and SAH animals. However, raising intravascular pressure caused significantly greater membrane potential depolarization, elevation in [Ca2+]i, and constriction in SAH arterioles. This SAH-induced increase in [Ca2+]i and tone occurred in the absence of the vascular endothelium and was abolished by the L-type voltage-dependent calcium channel (VDCC) inhibitor nimodipine. Arteriolar [Ca2+]i and tone were not different between groups when smooth muscle membrane potential was adjusted to the same value. Protein and mRNA levels of the L-type VDCC CaV1.2 were similar in parenchymal arterioles isolated from control and SAH animals, suggesting that SAH did not cause VDCC upregulation. We conclude that enhanced parenchymal arteriolar tone after SAH is driven by smooth muscle membrane potential depolarization, leading to increased L-type VDCC-mediated Ca2+ influx