Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Cerebral blood flow is conveyed by vascular resistance arteries and downstream parenchymal arterioles. Steady-state vascular resistance to blood flow ...

Loma Linda University

Electrical dynamics of freshly isolated cerebral endothelium have not been determined independently of perivascular nerves and smooth muscle. We tested the hypothesis that endothelium of cerebral and skeletal muscle arteries differentially utilizes purinergic and muscarinic signaling pathways to activate endothelium-derived hyperpolarization. Changes in membrane potential () were recorded in intact endothelial tubes freshly isolated from posterior cerebral and superior epigastric arteries of male and female C57BL/6 mice (age: 3-8 months).  was measured in response to activation of purinergic (P2Y) and muscarinic (M) receptors in addition to small- and intermediate-conductance Ca-activated K (SK/IK) and inward rectifying K (K) channels using ATP (100 μmol·L), acetylcholine (ACh; 10 μmol·L), NS309 (0.01-10 μmol·L), and 15 mmol·L KCl, respectively. Intercellular coupling was demonstrated via transfer of propidium iodide dye and electrical current (±0.5-3 nA) through gap junctions. With similarities observed across gender, peak hyperpolarization to ATP and ACh in skeletal muscle endothelial tubes was ~twofold and ~sevenfold higher, respectively, vs cerebral endothelial tubes, whereas responses to NS309 were similar (from resting  ~-30 mV to maximum ~-80 mV). Hyperpolarization (~8 mV) occurred during 15 mmol·L KCl treatment in cerebral but not skeletal muscle endothelial tubes. Despite weaker hyperpolarization during endothelial GPCR stimulation in cerebral vs skeletal muscle endothelium, the capability for robust SK/IK activity is preserved across brain and skeletal muscle. As vascular reactivity decreases with aging and cardiovascular disease, endothelial K channel activity may be calibrated to restore blood flow to respective organs regardless of gender.

Electrical dynamics of isolated cerebral and skeletal muscle endothelial tubes: Differential roles of G-protein-coupled receptors and K channels.

Effective delivery of oxygen and essential nutrients to vital organs and tissues throughout the body requires adequate blood flow supplied through resistance vessels. The intimate relationship between intracellular calcium ([Ca]) and regulation of membrane potential (V) is indispensable for maintaining blood flow regulation. In particular, Ca-activated K⁺ (K) channels were ascertained as transducers of elevated [Ca] signals into hyperpolarization of V as a pathway for decreasing vascular resistance, thereby enhancing blood flow. Recent evidence also supports the reverse role for K channels, in which they facilitate Ca influx into the cell interior through open non-selective cation (e.g., transient receptor potential; TRP) channels in accord with robust electrical (hyperpolarization) and concentration (~20,000-fold) transmembrane gradients for Ca. Such an arrangement supports a feed-forward activation of V hyperpolarization while potentially boosting production of nitric oxide. Furthermore, in vascular types expressing TRP channels but deficient in functional K channels (e.g., collecting lymphatic endothelium), there are profound alterations such as downstream depolarizing ionic fluxes and the absence of dynamic hyperpolarizing events. Altogether, this review is a refined set of evidence-based perspectives focused on the role of the endothelial K and TRP channels throughout multiple experimental animal models and vascular types. We discuss the diverse interactions among K and TRP channels to integrate Ca, oxidative, and electrical signaling in the context of cardiovascular physiology and pathology. Building from a foundation of cellular biophysical data throughout a wide and diverse compilation of significant discoveries, a translational narrative is provided for readers toward the treatment and prevention of chronic, age-related cardiovascular disease.

Functional Interaction among K and TRP Channels for Cardiovascular Physiology: Modern Perspectives on Aging and Chronic Disease.

Age-related dementia entails impaired blood flow to and throughout the brain due, in part, to reduced endothelial nitric oxide signaling. However, it is unknown whether sex affects cerebrovascular Gq-protein-coupled receptors (GPCRs) and K+ channels underlying endothelium-derived hyperpolarization (EDH) during progressive aging. Thus, we simultaneously evaluated intracellular Ca2+ ([Ca2+]i) and membrane potential (Vm) of intact endothelial tubes freshly isolated from posterior cerebral arteries of young (4-6 mo), middle-aged (12-16 mo), and old (24-28 mo) male and female C57BL/6 mice. Purinergic receptor function (vs. muscarinic) was dominant and enhanced for [Ca2+]i increases in old females versus old males. However, Ca2+-sensitive K+ channel function as defined by NS309-evoked Vm hyperpolarization was mildly impaired in females versus males during old age. This sex-based contrast in declined function of GPCRs and K+ channels to produce EDH may support a greater ability for physiological endothelial GPCR function to maintain optimal cerebral blood flow in females versus males during old age. As reflective of the pattern of cerebral blood flow decline in human subjects, inward-rectifying K+ (KIR) channel function decreased with progressive age regardless of sex. Combined age-related analyses masked male versus female aging and, contrary to expectation, hydrogen peroxide played a minimal role. Altogether, we conclude a sex-based divergence in cerebrovascular endothelial GPCR and K+ channel function while highlighting a previously unidentified form of age-related endothelial dysfunction as reduced KIR channel function.

Aging Alters Cerebrovascular Endothelial GPCR and K+ Channel Function: Divergent Role of Biological Sex.

Development of Alzheimer's disease (AD) pathology is associated with impaired blood flow delivery of oxygen and nutrients throughout the brain. Cerebrovascular endothelium regulates vasoreactivity of blood vessel networks for optimal cerebral blood flow.

Development of Alzheimer's Disease Progressively Alters Sex-Dependent KCa and Sex-Independent KIR Channel Function in Cerebrovascular Endothelium.

As the sixth-leading cause of death in the United States, Alzheimer's disease (AD) entails deteriorating endothelial control of blood flow throughout the brain. In particular, reduced inward-rectifying K (K) channel function in animal models of aging and AD compromises endothelial function and optimal perfusion of brain parenchyma. Deficient endothelial K channels may result from aberrant interaction with plasma membrane cholesterol as a primary regulator of membrane fluidity and ion channels.

Methyl-Beta-Cyclodextrin Restores K Channel Function in Brain Endothelium of Female Alzheimer's Disease Mice.

Emerging evidence demonstrates association of Alzheimer's disease (AD) with impaired delivery of blood oxygen and nutrients to and throughout the brain. The cerebral circulation plays multiple roles underscoring optimal brain perfusion and cognition entailing moment-to-moment blood flow control, vascular permeability, and angiogenesis. With currently no effective treatment to prevent or delay the progression of AD, cerebrovascular microRNA (miRNA) markers corresponding to post-transcriptional regulation may distinguish phases of AD.

Md A. Hakim

Department of Basic Sciences

Loma Linda University

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Md A. Hakim

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Basic Sciences

Loma Linda University

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

Department of Basic Sciences