Thomas A. Longden

Vascular Inward Rectifier K+ Channels as External K+ Sensors in the Control of Cerebral Blood Flow

For decades it has been known that external K+ ions are rapid and potent vasodilators that increase CBF. Recent studies have implicated the local release of K+ from astrocytic endfeet—which encase the entirety of the parenchymal vasculature—in the dynamic regulation of local CBF during NVC. It has been proposed that the activation of KIR channels in the vascular wall by external K+ is a central component of these hyperemic responses; however, a number of significant gaps in our knowledge remain. Here, we explore the concept that vascular KIR channels are the major extracellular K+ sensors in the control of CBF. We propose that K+ is an ideal mediator of NVC, and discuss KIR channels as effectors that produce rapid hyperpolarization and robust vasodilation of cerebral arterioles. We provide evidence that KIR channels, of the KIR2 subtype in particular, are present in both the endothelial and SM cells of parenchymal arterioles and propose that this dual positioning of KIR2 channels increases the robustness of the vasodilation to external K+, enables the endothelium to be actively engaged in NVC, and permits electrical signaling through the endothelial syncytium to promote upstream vasodilation to modulate CBF.

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.

Stress-induced glucocorticoid signaling remodels neurovascular coupling through impairment of cerebrovascular inwardly rectifying K+ channel function

Significance When neurons become active, they signal to local arterioles via intermediate glial cells, called astrocytes, to evoke dilation. This increases local blood flow and provides the oxygen and glucose necessary to support ongoing neuronal function. This process is termed neurovascular coupling. We demonstrate that chronic stress—which is a contributing factor for many diseases—impairs neurovascular coupling in the amygdala, a region involved in stressor processing. Our results further indicate that this dysfunction is due to the loss of arteriolar inwardly rectifying potassium (K+) channel function, which makes vessels less able to respond to vasodilatory K+ ions released by astrocytes during periods of increased neuronal activity. This neurovascular coupling impairment may contribute to the pathology of a range of brain disorders. 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.

Dysfunction of mouse cerebral arteries during early aging

Aging leads to a gradual decline in the fidelity of cerebral blood flow (CBF) responses to neuronal activation, resulting in an increased risk for stroke and dementia. However, it is currently unknown when age-related cerebrovascular dysfunction starts or which vascular components and functions are first affected. The aim of this study was to examine the function of microcirculation throughout aging in mice. Microcirculation was challenged by inhalation of 5% and 10% CO2 or by forepaw stimulation in 6-week, 8-month, and 12-month-old FVB/N mice. The resulting dilation of pial vessels and increase in CBF was measured by intravital fluorescence microscopy and laser Doppler fluxmetry, respectively. Neurovascular coupling and astrocytic endfoot Ca2+ were measured in acute brain slices from 18-month-old mice. We did not reveal any changes in CBF after CO2 reactivity up to an age of 12 months. However, direct visualization of pial vessels by in vivo microscopy showed a significant, age-dependent loss of CO2 reactivity starting at 8 months of age. At the same age neurovascular coupling was also significantly affected. These results suggest that aging does not affect cerebral vessel function simultaneously, but starts in pial microvessels months before global changes in CBF are detectable.

Intermediate-conductance calcium-activated potassium channels participate in neurovascular coupling

BACKGROUND AND PURPOSE Controlling vascular tone involves K+ efflux through endothelial cell small‐ and intermediate‐conductance calcium‐activated potassium channels (KCa2.3 and KCa3.1, respectively). We investigated the expression of these channels in astrocytes and the possibility that, by a similar mechanism, they might contribute to neurovascular coupling.

Traumatic Brain Injury Disrupts Cerebrovascular Tone Through Endothelial Inducible Nitric Oxide Synthase Expression and Nitric Oxide Gain of Function

Background Traumatic brain injury (TBI) has been reported to increase the concentration of nitric oxide (NO) in the brain and can lead to loss of cerebrovascular tone; however, the sources, amounts, and consequences of excess NO on the cerebral vasculature are unknown. Our objective was to elucidate the mechanism of decreased cerebral artery tone after TBI. Methods and Results Cerebral arteries were isolated from rats 24 hours after moderate fluid‐percussion TBI. Pressure‐induced increases in vasoconstriction (myogenic tone) and smooth muscle Ca2+ were severely blunted in cerebral arteries after TBI. However, myogenic tone and smooth muscle Ca2+ were restored by inhibition of NO synthesis or endothelium removal, suggesting that TBI increased endothelial NO levels. Live native cell NO, indexed by 4,5‐diaminofluorescein (DAF‐2 DA) fluorescence, was increased in endothelium and smooth muscle of cerebral arteries after TBI. Clamped concentrations of 20 to 30 nmol/L NO were required to simulate the loss of myogenic tone and increased (DAF‐2T) fluorescence observed following TBI. In comparison, basal NO in control arteries was estimated as 0.4 nmol/L. Consistent with TBI causing enhanced NO‐mediated vasodilation, inhibitors of guanylyl cyclase, protein kinase G, and large‐conductance Ca2+‐activated potassium (BK) channel restored function of arteries from animals with TBI. Expression of the inducible isoform of NO synthase was upregulated in cerebral arteries isolated from animals with TBI, and the inducible isoform of NO synthase inhibitor 1400W restored myogenic responses following TBI. Conclusions The mechanism of profound cerebral artery vasodilation after TBI is a gain of function in vascular NO production by 60‐fold over controls, resulting from upregulation of the inducible isoform of NO synthase in the endothelium.

Uncoupling of neurovascular communication after transient global cerebral ischemia is caused by impaired parenchymal smooth muscle Kir channel function.

Transient global cerebral ischemia is often followed by delayed disturbances of cerebral blood flow, contributing to neuronal injury. The pathophysiological processes underlying such disturbances are incompletely understood. Here, using an established model of transient global cerebral ischemia, we identify dramatically impaired neurovascular coupling following ischemia. This impairment results from the loss of functional inward rectifier potassium (KIR) channels in the smooth muscle of parenchymal arterioles. Therapeutic strategies aimed at protecting or restoring cerebrovascular KIR channel function may therefore improve outcomes following ischemia.

Endothelial GqPCR activity controls capillary electrical signaling and brain blood flow through PIP2 depletion

Significance Capillaries, the smallest blood vessels, mediate the on-demand delivery of oxygen and nutrients required to support the function of active cells throughout the brain. But how blood flow is directed to cells in active brain regions to satisfy their energy needs is poorly understood. We demonstrate that the plasma membrane phospholipid, PIP2, is fundamental to sustaining the activity of inwardly rectifying potassium channels—the molecular feature that allows capillary endothelial cells to sense ongoing neuronal activity and trigger an increase in local blood flow. We further show that chemical factors released in the brain, including those associated with neuronal activity, cause changes in the levels of PIP2, thereby altering endothelial potassium channel signaling and controlling cerebral blood flow. 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.

Channeling stress: inwardly-rectifying K+ channels in stress and disease.

From the pressure of an impending deadline to the frustration of sitting in traffic, stress is a normal feature of daily life. In most cases, the stressors are mild and we have both the mental and physical resources with which to cope. However, chronic exposure to high-stress situations is associated with many disease states and psychopathologies, and unfortunately, we don’t have to look far for real-life examples of this. Indeed, posttraumatic stress disorder affects up to 8% of Americans, and cardiovascular disease is rife in occupations that are characterized by high workloads with low controllability. For example, female healthcare professionals who rate their jobs as ‘highly stressful’ are 38% more likely to experience heart problems compared with those with lowstress roles. The clear interaction between stress and disease makes furthering our understanding of stress physiology crucial for the future prevention and treatment of disorders with a stress component. Recently, we identified a novel effect of stress on inwardly-rectifying potassium (KIR) channels located on the smooth muscle of small brain arterioles. These ‘parenchymal’ arterioles penetrate deep into the brain tissue, and dilate in response to nearby neuronal activity to deliver a surge of blood to the active region. This increase in blood flow provides the oxygen and glucose needed to offset increased local metabolic demand and importantly, failure of this process can lead to neuronal dysfunction. To achieve dilation, signaling molecules are released from the specialized projections of a type of glial cell (known as astrocytes, due to their highly branching processes which give them a star-shaped morphology) that contact the vascular smooth muscle. These projections are termed ‘astrocytic endfeet’ and the molecules they release act through various parallel signaling pathways to hyperpolarize the adjacent smooth muscle membrane, which ultimately leads to dilation of the arteriole and increased blood flow. This process is termed ‘neurovascular coupling’. KIR channels appear to have an important part to play in this phenomenon: Potassium (K+) ions are released from the astrocytic endfeet in response to neuronal activity, and this causes opening of smooth muscle KIR2 channels (which are activated by a small increases in extracellular K+ and membrane potential hyperpolarization.) Activation of smooth muscle KIR channels rapidly hyperpolarizes the membrane from about -35 mV to the new K+ equilibrium potential, and this consequently decreases intracellular calcium (Ca2+) through closure of tonically active voltage-gated Ca2+ channels. This fall in intracellular Ca2+ causes smooth muscle relaxation, thereby leading to vasodilation and increased blood flow. Intriguingly, after we exposed rats to a one-week chronic stress paradigm—in which rats were exposed to one stressor each day for 7 days and developed an anxiety-like phenotype—we observed a decrease in K IR channel expression and a marked impairment of smooth muscle K IR channel function in parenchymal arterioles from the amygdala, a stress-related brain region. This loss of K IR channel Channeling stress Inwardly-rectifying K channels in stress and disease

PIP2 depletion promotes TRPV4 channel activity in mouse brain capillary endothelial cells

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.