Electro-metabolic signaling.

Precise matching of energy substrate delivery to local metabolic needs is essential for the health and function of all tissues. Here, we outline a mechanistic framework for understanding this critical process, which we refer to as electro-metabolic signaling (EMS). All tissues exhibit changes in metabolism over varying spatiotemporal scales and have widely varying energetic needs and reserves. We propose that across tissues, common signatures of elevated metabolism or increases in energy substrate usage that exceed key local thresholds rapidly engage mechanisms that generate hyperpolarizing electrical signals in capillaries that then relax contractile elements throughout the vasculature to quickly adjust blood flow to meet changing needs. The attendant increase in energy substrate delivery serves to meet local metabolic requirements and thus avoids a mismatch in supply and demand and prevents metabolic stress. We discuss in detail key examples of EMS that our laboratories have discovered in the brain and the heart, and we outline potential further EMS mechanisms operating in tissues such as skeletal muscle, pancreas, and kidney. We suggest that the energy imbalance evoked by EMS uncoupling may be central to cellular dysfunction from which the hallmarks of aging and metabolic diseases emerge and may lead to generalized organ failure states-such as diverse flavors of heart failure and dementia. Understanding and manipulating EMS may be key to preventing or reversing these dysfunctions.

Pericytes and the Control of Blood Flow in Brain and Heart.

Pericytes, attached to the surface of capillaries, play an important role in regulating local blood flow. Using optogenetic tools and genetically encoded reporters in conjunction with confocal and multiphoton imaging techniques, the 3D structure, anatomical organization, and physiology of pericytes have recently been the subject of detailed examination. This work has revealed novel functions of pericytes and morphological features such as tunneling nanotubes in brain and tunneling microtubes in heart. Here, we discuss the state of our current understanding of the roles of pericytes in blood flow control in brain and heart, where functions may differ due to the distinct spatiotemporal metabolic requirements of these tissues. We also outline the novel concept of electro-metabolic signaling, a universal mechanistic framework that links tissue metabolic state with blood flow regulation by pericytes and vascular smooth muscle cells, with capillary KATP and Kir2.1 channels as primary sensors. Finally, we present major unresolved questions and outline how they can be addressed.

Brain capillary pericytes are metabolic sentinels that control blood flow through a KATP channel-dependent energy switch.

Despite the abundance of capillary thin-strand pericytes and their proximity to neurons and glia, little is known of the contributions of these cells to the control of brain hemodynamics. We demonstrate that the pharmacological activation of thin-strand pericyte KATP channels profoundly hyperpolarizes these cells, dilates upstream penetrating arterioles and arteriole-proximate capillaries, and increases capillary blood flow. Focal stimulation of pericytes with a KATP channel agonist is sufficient to evoke this response, mediated via KIR2.1 channel-dependent retrograde propagation of hyperpolarizing signals, whereas genetic inactivation of pericyte KATP channels eliminates these effects. Critically, we show that decreasing extracellular glucose to less than 1 mM or inhibiting glucose uptake by blocking GLUT1 transporters in vivo flips a mechanistic energy switch driving rapid KATP-mediated pericyte hyperpolarization to increase local blood flow. Together, our findings recast capillary pericytes as metabolic sentinels that respond to local energy deficits by increasing blood flow to neurons to prevent energetic shortfalls.

Prostaglandin E2 Dilates Intracerebral Arterioles When Applied to Capillaries: Implications for Small Vessel Diseases.

Prostaglandin E2 (PGE2) has been widely proposed to mediate neurovascular coupling by dilating brain parenchymal arterioles through activation of prostanoid EP4 receptors. However, our previous report that direct application of PGE2 induces an EP1-mediated constriction strongly argues against its direct action on arterioles during neurovascular coupling, the mechanisms sustaining functional hyperemia. Recent advances have highlighted the role of capillaries in sensing neuronal activity and propagating vasodilatory signals to the upstream penetrating parenchymal arteriole. Here, we examined the effect of capillary stimulation with PGE2 on upstream arteriolar diameter using an ex vivo capillary-parenchymal arteriole preparation and in vivo cerebral blood flow measurements with two-photon laser-scanning microscopy. We found that PGE2 caused upstream arteriolar dilation when applied onto capillaries with an EC50 of 70 nM. The response was inhibited by EP1 receptor antagonist and was greatly reduced, but not abolished, by blocking the strong inward-rectifier K+ channel. We further observed a blunted dilatory response to capillary stimulation with PGE2 in a genetic mouse model of cerebral small vessel disease with impaired functional hyperemia. This evidence casts previous findings in a different light, indicating that capillaries are the locus of PGE2 action to induce upstream arteriolar dilation in the control of brain blood flow, thereby providing a paradigm-shifting view that nonetheless remains coherent with the broad contours of a substantial body of existing literature.

Differential restoration of functional hyperemia by antihypertensive drug classes in hypertension-related cerebral small vessel disease.

Dementia resulting from small vessel diseases (SVDs) of the brain 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.

Local IP3 receptor-mediated Ca2+ signals compound to direct blood flow in brain capillaries.

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.

PIP2 corrects cerebral blood flow deficits in small vessel disease by rescuing capillary Kir2.1 activity.

Cerebral small vessel diseases (SVDs) are a central link between stroke and dementia-two comorbidities without specific treatments. Despite the emerging consensus that SVDs are initiated in the endothelium, the early mechanisms remain largely unknown. Deficits in on-demand delivery of blood to active brain regions (functional hyperemia) are early manifestations of the underlying pathogenesis. The capillary endothelial cell strong inward-rectifier K+ channel Kir2.1, which senses neuronal activity and initiates a propagating electrical signal that dilates upstream arterioles, is a cornerstone of functional hyperemia. Here, using a genetic SVD mouse model, we show that impaired functional hyperemia is caused by diminished Kir2.1 channel activity. We link Kir2.1 deactivation to depletion of phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid essential for Kir2.1 activity. Systemic injection of soluble PIP2 rapidly restored functional hyperemia in SVD mice, suggesting a possible strategy for rescuing functional hyperemia in brain disorders in which blood flow is disturbed.

Impaired capillary-to-arteriolar electrical signaling after traumatic brain injury.

Traumatic brain injury (TBI) acutely impairs dynamic regulation of local cerebral blood flow, but long-term (>72 h) effects on functional hyperemia are unknown. Functional hyperemia depends on capillary endothelial cell inward rectifier potassium channels (Kir2.1) responding to potassium (K+) released during neuronal activity to produce a regenerative, hyperpolarizing electrical signal that propagates from capillaries to dilate upstream penetrating arterioles. We hypothesized that TBI causes widespread disruption of electrical signaling from capillaries-to-arterioles through impairment of Kir2.1 channel function. We randomized mice to TBI or control groups and allowed them to recover for 4 to 7 days post-injury. We measured in vivo cerebral hemodynamics and arteriolar responses to local stimulation of capillaries with 10 mM K+ using multiphoton laser scanning microscopy through a cranial window under urethane and α-chloralose anesthesia. Capillary angio-architecture was not significantly affected following injury. However, K+-induced hyperemia was significantly impaired. Electrophysiology recordings in freshly isolated capillary endothelial cells revealed diminished Ba2+-sensitive Kir2.1 currents, consistent with a reduction in channel function. In pressurized cerebral arteries isolated from TBI mice, K+ failed to elicit the vasodilation seen in controls. We conclude that disruption of endothelial Kir2.1 channel function impairs capillary-to-arteriole electrical signaling, contributing to altered cerebral hemodynamics after TBI.

Vascular control of the CO2/H+-dependent drive to breathe.

Respiratory chemoreceptors regulate breathing in response to changes in tissue CO2/H+. Blood flow is a fundamental determinant of tissue CO2/H+, yet little is known regarding how regulation of vascular tone in chemoreceptor regions contributes to respiratory behavior. Previously, we showed in rat that CO2/H+-vasoconstriction in the retrotrapezoid nucleus (RTN) supports chemoreception by a purinergic-dependent mechanism (Hawkins et al., 2017). Here, we show in mice that CO2/H+ dilates arterioles in other chemoreceptor regions, thus demonstrating CO2/H+ vascular reactivity in the RTN is unique. We also identify P2Y2 receptors in RTN smooth muscle cells as the substrate responsible for this response. Specifically, pharmacological blockade or genetic deletion of P2Y2 from smooth muscle cells blunted the ventilatory response to CO2, and re-expression of P2Y2 receptors only in RTN smooth muscle cells fully rescued the CO2/H+ chemoreflex. These results identify P2Y2 receptors in RTN smooth muscle cells as requisite determinants of respiratory chemoreception.

The capillary Kir channel as sensor and amplifier of neuronal signals: Modeling insights on K+-mediated neurovascular communication.

Neuronal activity leads to an increase in local cerebral blood flow (CBF) to allow adequate supply of oxygen and nutrients to active neurons, a process termed neurovascular coupling (NVC). We have previously shown that capillary endothelial cell (cEC) inwardly rectifying K+ (Kir) channels can sense neuronally evoked increases in interstitial K+ and induce rapid and robust dilations of upstream parenchymal arterioles, suggesting a key role of cECs in NVC. The requirements of this signal conduction remain elusive. Here, we utilize mathematical modeling to investigate how small outward currents in stimulated cECs can elicit physiologically relevant spread of vasodilatory signals within the highly interconnected brain microvascular network to increase local CBF. Our model shows that the Kir channel can act as an "on-off" switch in cECs to hyperpolarize the cell membrane as extracellular K+ increases. A local hyperpolarization can be amplified by the voltage-dependent activation of Kir in neighboring cECs. Sufficient Kir density enables robust amplification of the hyperpolarizing stimulus and produces responses that resemble action potentials in excitable cells. This Kir-mediated excitability can remain localized in the stimulated region or regeneratively propagate over significant distances in the microvascular network, thus dramatically increasing the efficacy of K+ for eliciting local hyperemia. Modeling results show how changes in cEC transmembrane current densities and gap junctional resistances can affect K+-mediated NVC and suggest a key role for Kir as a sensor of neuronal activity and an amplifier of retrograde electrical signaling in the cerebral vasculature.

Ion channels in capillary endothelium.

Vascular beds are anatomically and functionally compartmentalized into arteries, capillaries, and veins. The bulk of the vasculature consists of the dense, anastomosing capillary network, composed of capillary endothelial cells (cECs) that are intimately associated with the parenchyma. Despite their abundance, the ion channel expression and function and Ca2+ signaling behaviors of capillaries have only recently begun to be explored in detail. Here, we discuss the established and emerging roles of ion channels and Ca2+ signaling in cECs. By mining a publicly available RNA-seq dataset, we outline the wide variety of ion channel genes that are expressed in these cells, which potentially imbue capillaries with a broad range of sensing and signal transduction capabilities. We also underscore subtle but critical differences between cEC and arteriolar EC ion channel expression that likely underlie key functional differences in ECs at these different levels of the vascular tree. We focus our discussion on the cerebral vasculature, but the findings and principles being elucidated in this area likely generalize to other vascular beds.

The Ion Channel and GPCR Toolkit of Brain Capillary Pericytes.

Brain pericytes reside on the abluminal surface of capillaries, and their processes cover ~90% of the length of the capillary bed. These cells were first described almost 150 years ago (Eberth, 1871; Rouget, 1873) and have been the subject of intense experimental scrutiny in recent years, but their physiological roles remain uncertain and little is known of the complement of signaling elements that they employ to carry out their functions. In this review, we synthesize functional data with single-cell RNAseq screens to explore the ion channel and G protein-coupled receptor (GPCR) toolkit of mesh and thin-strand pericytes of the brain, with the aim of providing a framework for deeper explorations of the molecular mechanisms that govern pericyte physiology. We argue that their complement of channels and receptors ideally positions capillary pericytes to play a central role in adapting blood flow to meet the challenge of satisfying neuronal energy requirements from deep within the capillary bed, by enabling dynamic regulation of their membrane potential to influence the electrical output of the cell. In particular, we outline how genetic and functional evidence suggest an important role for Gs-coupled GPCRs and ATP-sensitive potassium (KATP) channels in this context. We put forth a predictive model for long-range hyperpolarizing electrical signaling from pericytes to upstream arterioles, and detail the TRP and Ca2+ channels and Gq, Gi/o, and G12/13 signaling processes that counterbalance this. We underscore critical questions that need to be addressed to further advance our understanding of the signaling topology of capillary pericytes, and how this contributes to their physiological roles and their dysfunction in 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.

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

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.

Endothelial signaling and the dynamic regulation of arterial tone: A surreptitious relationship.

The endothelium is an integrated layer of cells whose dynamic regulation governs arterial tone development and the matching of blood flow delivery to tissue energetic demand. Investigating the structural and electrical properties of native endothelial cells has been a challenging prospect, with efforts often restricted to traditional myographic techniques. Concerted experimental attention, along with recent technical advances, has broadened the investigative tool kit, deepening mechanistic insights. This overview in part of a STI centered on the endothelium and how key structural/electrical properties guide arterial tone development and integrated network behavior. Articles are written in a provocative, opinionated manner to provoke deeper thought and to highlight areas of investigative deficiency.

Capillary K+-sensing initiates retrograde hyperpolarization to increase local cerebral blood flow.

Blood flow into the brain is dynamically regulated to satisfy the changing metabolic requirements of neurons, but how this is accomplished has remained unclear. Here we demonstrate a central role for capillary endothelial cells in sensing neural activity and communicating it to upstream arterioles in the form of an electrical vasodilatory signal. We further demonstrate that this signal is initiated by extracellular K+ -a byproduct of neural activity-which activates capillary endothelial cell inward-rectifier K+ (KIR2.1) channels to produce a rapidly propagating retrograde hyperpolarization that causes upstream arteriolar dilation, increasing blood flow into the capillary bed. Our results establish brain capillaries as an active sensory web that converts changes in external K+ into rapid, 'inside-out' electrical signaling to direct blood flow to active brain regions.

Inhibition of vascular smooth muscle inward-rectifier K+ channels restores myogenic tone in mouse urinary bladder arterioles.

Prolonged decreases in urinary bladder blood flow are linked to overactive and underactive bladder pathologies. However, the mechanisms regulating bladder vascular reactivity are largely unknown. To investigate these mechanisms, we examined myogenic and vasoactive properties of mouse bladder feed arterioles (BFAs). Unlike similar-sized arterioles from other vascular beds, BFAs failed to constrict in response to increases in intraluminal pressure (5-80 mmHg). Consistent with this lack of myogenic tone, arteriolar smooth muscle cell membrane potential was hyperpolarized (-72.8 ± 1.4 mV) at 20 mmHg and unaffected by increasing pressure to 80 mmHg (-74.3 ± 2.2 mV). In contrast, BFAs constricted to the thromboxane analog U-46619 (100 nM), the adrenergic agonist phenylephrine (10 µM), and KCl (60 mM). Inhibition of nitric oxide synthase or intermediate- and small-conductance Ca2+-activated K+ channels did not alter arteriolar diameter, indicating that the dilated state of BFAs is not attributable to overactive endothelium-dependent dilatory influences. Myocytes isolated from BFAs exhibited BaCl2 (100 µM)-sensitive K+ currents consistent with strong inward-rectifier K+ (KIR) channels. Notably, block of these KIR channels "restored" pressure-induced constriction and membrane depolarization. This suggests that these channels, in part, account for hyperpolarization and associated absence of tone in BFAs. Furthermore, smooth muscle-specific knockout of KIR2.1 caused significant myogenic tone to develop at physiological pressures. This suggests that 1) the regulation of vascular tone in the bladder is independent of pressure, insofar as pressure-induced depolarizing conductances cannot overcome KIR2.1-mediated hyperpolarization; and 2) maintenance of bladder blood flow during bladder filling is likely controlled by neurohumoral influences.

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.

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.