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

Abstract

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 (V m) 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). V m was measured in response to activation of purinergic (P2Y) and muscarinic (M3) receptors in addition to small‐ and intermediate‐conductance Ca2+‐activated K+ (SKC a/IKC a) and inward rectifying K+ (KIR) channels using ATP (100 μmol·L−1), acetylcholine (ACh; 10 μmol·L−1), NS309 (0.01‐10 μmol·L−1), and 15 mmol·L−1 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 V m ~−30 mV to maximum ~−80 mV). Hyperpolarization (~8 mV) occurred during 15 mmol·L−1 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 SKC a/IKC a 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.

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

“…The V m must be as high as −90 mV at physiological temperature (37 °C) in order to bring transmembrane K + flux to equilibrium (E K ). For reference, vascular endothelial cells typically have a V m of ~−30 to −40 mV during rest, and activation of SK Ca /IK Ca channels alone using NS309 can increase V m to E K [37,41]. As a dominant form of K + channel expressed in the endothelial membrane, the SK Ca /IK Ca channels are tetrameric (four subunits), voltage-independent (from V m ~−80 to +10 mV [37]), and are constitutively bound to calmodulin binding sites for [Ca 2+ ] i [42].…”

G Protein-Coupled Receptor Systems as Crucial Regulators of DNA Damage Response Processes

“…In addition to cellular/tissue damage caused by accumulated protein/DNA damage, molecular aging ‘programs’ (i.e., coherent and repeated pathological patterns of protein expression leading to stress-related damage) can also generate age-related increases in cellular senescence. Cell growth arrest and hyporesponsiveness to extrinsic stimuli via cell surface receptors, such as GPCRs, are hallmarks of senescent cells [ 122 , 123 , 124 , 125 ]. Cell senescence describes the process in which cells cease dividing, but do not enter an apoptotic state.…”

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

“…In particular, during endothelium-derived hyperpolarization (commonly known as EDH), intracellular Ca 2+ ([Ca 2+ ] i ) and electrical signaling in endothelial cells coordinate vasodilation among endothelial cells and their surrounding smooth muscle cells through gap junctions for arterial relaxation 4 . Physiological initiation of EDH sequentially entails stimulation of G q -coupled receptors (GPCRs), an increase in [Ca 2+ ] i , and activation of endothelial small- and intermediate-Ca 2+ -activated K + (SK Ca /IK Ca ) channels to hyperpolarize cerebral endothelial membrane potential (V m ) 5,6,7 . Thus, the intimate relationship of endothelial [Ca 2+ ] i and V m is integral to blood flow regulation and indispensable to cardio- and cerebrovascular function 6,8 .…”

“…Physiological initiation of EDH sequentially entails stimulation of G q -coupled receptors (GPCRs), an increase in [Ca 2+ ] i , and activation of endothelial small- and intermediate-Ca 2+ -activated K + (SK Ca /IK Ca ) channels to hyperpolarize cerebral endothelial membrane potential (V m ) 5,6,7 . Thus, the intimate relationship of endothelial [Ca 2+ ] i and V m is integral to blood flow regulation and indispensable to cardio- and cerebrovascular function 6,8 . Throughout the broader literature, numerous studies have reported the association of vascular endothelial dysfunction with the development of chronic diseases (e.g., hypertension, diabetes, heart failure, coronary artery disease, chronic renal failure, peripheral artery disease) 9,10 , indicating the significance of studying endothelial function in physiological as well as pathological conditions.…”

“…Vascular endothelium is integral to the production of hyperpolarization, vasodilation, and tissue perfusion and thus, examination of its native cellular properties is crucial. As a general study model, preparation of the mouse arterial endothelial tube model has been published before for skeletal muscle 11,12 , gut 13 , lung 14 , and recently for the brain 6 . Studies of simultaneous [Ca 2+ ] i and V m measurements in particular have been published for skeletal muscle arterial endothelium 15,16 as well as lymphatic vessel endothelium 17 .…”