Erik J. Behringer

Aging Impairs Electrical Conduction Along Endothelium of Resistance Arteries Through Enhanced Ca2+-Activated K+ Channel Activation

Objective—Intercellular conduction of electrical signals underlies spreading vasodilation of resistance arteries. Small- and intermediate-conductance Ca2+-activated K+ channels of endothelial cells serve a dual function by initiating hyperpolarization and modulating electrical conduction. We tested the hypothesis that regulation of electrical signaling by small- and intermediate-conductance Ca2+-activated K+ channels is altered with advancing age. Approach and Results—Intact endothelial tubes (60 µm wide; 1–3 mm long) were freshly isolated from male C57BL/6 mouse (Young: 4–6 months; Intermediate: 12–14 months; Old: 24–26 months) superior epigastric arteries. Using dual intracellular microelectrodes, current was injected (±0.1–3 nA) at site 1 while recording membrane potential (Vm) at site 2 (separation distance: 50–2000 µm). Across age groups, greatest differences were observed between Young and Old. Resting Vm in Old (−38±1 mV) was more negative (P<0.05) than Young (−30±1 mV). Maximal hyperpolarization to both direct (NS309) and indirect (acetylcholine) activation of small- and intermediate-conductance Ca2+-activated K+ channels was sustained (&Dgr;Vm ≈−40 mV) with age. The length constant (&lgr;) for electrical conduction was reduced (P<0.05) from 1630±80 µm (Young) to 1320±80 µm (Old). Inhibiting small- and intermediate-conductance Ca2+-activated K+ channels with apamin+charybdotoxin or scavenging hydrogen peroxide (H2O2) with catalase improved electrical conduction (P<0.05) in Old. Exogenous H2O2 (200 µmol/L) in Young evoked hyperpolarization and impaired electrical conduction; these effects were blocked by apamin+charybdotoxin. Conclusions—Enhanced current loss through Ca2+-activated K+ channel activation impairs electrical conduction along the endothelium of resistance arteries with aging. Attenuating the spatial domain of electrical signaling will restrict the spread of vasodilation and thereby contribute to blood flow limitations associated with advanced age.

Tuning Electrical Conduction Along Endothelial Tubes of Resistance Arteries Through Ca2+-Activated K+ Channels

Rationale: Electrical conduction through gap junction channels between endothelial cells of resistance vessels is integral to blood flow control. Small and intermediate-conductance Ca2+-activated K+ channels (SKCa/IKCa) initiate electrical signals in endothelial cells, but it is unknown whether SKCa/IKCa activation alters signal transmission along the endothelium. Objective: We tested the hypothesis that SKCa/IKCa activity regulates electrical conduction along the endothelium of resistance vessels. Methods and Results: Freshly isolated endothelial cell tubes (60 &mgr;m wide; 1–3 mm long; cell length, ≈35 &mgr;m) from mouse skeletal muscle feed (superior epigastric) arteries were studied using dual intracellular microelectrodes. Current was injected (±0.1–3 nA) at site 1 while recording membrane potential (Vm) at site 2 (separation distance=50–2000 &mgr;m). SKCa/IKCa activation (NS309, 1 &mgr;mol/L) reduced the change in Vm along endothelial cell tubes by ≥50% and shortened the electrical length constant (&lgr;) from 1380 to 850 &mgr;m (P<0.05) while intercellular dye transfer (propidium iodide) was maintained. Activating SKCa/IKCa with acetylcholine or SKA-31 also reduced electrical conduction. These effects of SKCa/IKCa activation persisted when hyperpolarization (>30 mV) was prevented with 60 mmol/L [K+]o. Conversely, blocking SKCa/IKCa (apamin+charybdotoxin) depolarized cells by ≈10 mV and enhanced electrical conduction (ie, changes in Vm) by ≈30% (P<0.05). Conclusions: These findings illustrate a novel role for SKCa/IKCa activity in tuning electrical conduction along the endothelium of resistance vessels by governing signal dissipation through changes in membrane resistance. Voltage-insensitive ion channels can thereby tune intercellular electrical signaling independent from gap junction channels.

Electrical conduction along endothelial cell tubes from mouse feed arteries: confounding actions of glycyrrhetinic acid derivatives

BACKGROUND AND PURPOSE Electrical conduction along endothelium of resistance vessels has not been determined independently of the influence of smooth muscle, surrounding tissue or blood. Two interrelated hypotheses were tested: (i) Intercellular conduction of electrical signals is manifest in endothelial cell (EC) tubes; and (ii) Inhibitors of gap junction channels (GJCs) have confounding actions on EC electrical and Ca2+ signalling.

Age-dependent changes in Ca2+ homeostasis in peripheral neurones: implications for changes in function.

Calcium ions represent universal second messengers within neuronal cells integrating multiple cellular functions, such as release of neurotransmitters, gene expression, proliferation, excitability, and regulation of cell death or apoptotic pathways. The magnitude, duration and shape of stimulation‐evoked intracellular calcium ([Ca2+]i) transients are determined by a complex interplay of mechanisms that modulate stimulation‐evoked rises in [Ca2+]i that occur with normal neuronal function. Disruption of any of these mechanisms may have implications for the function and health of peripheral neurones during the aging process. This review focuses on the impact of advancing age on the overall function of peripheral adrenergic neurones and how these changes in function may be linked to age‐related changes in modulation of [Ca2+]i regulation. The data in this review suggest that normal aging in peripheral autonomic neurones is a subtle process and does not always result in dramatic deterioration in their function. We present studies that support the idea that in order to maintain cell viability peripheral neurones are able to compensate for an age‐related decline in the function of at least one of the neuronal calcium‐buffering systems, smooth endoplasmic reticulum calcium ATPases, by increased function of other calcium‐buffering systems, namely, the mitochondria and plasmalemma calcium extrusion. Increased mitochondrial calcium uptake may represent a ‘weak point’ in cellular compensation as this over time may contribute to cell death. In addition, we present more recent studies on [Ca2+]i regulation in the form of the modulation of release of calcium from smooth endoplasmic reticulum calcium stores. These studies suggest that the contribution of the release of calcium from smooth endoplasmic reticulum calcium stores is altered with age through a combination of altered ryanodine receptor levels and modulation of these receptors by neuronal nitric oxide containing neurones.

Spreading the signal for vasodilatation: implications for skeletal muscle blood flow control and the effects of ageing

Abstract  Blood flow control requires coordinated contraction and relaxation of smooth muscle cells (SMCs) along and among the arterioles and feed arteries that comprise vascular resistance networks. Whereas smooth muscle contraction of resistance vessels is enhanced by noradrenaline release along perivascular sympathetic nerves, the endothelium is integral to coordinating smooth muscle relaxation. Beyond producing nitric oxide in response to agonists and shear stress, endothelial cells (ECs) provide an effective conduit for conducting hyperpolarization along vessel branches and into surrounding SMCs through myoendothelial coupling. In turn, bidirectional signalling from SMCs into ECs enables the endothelium to moderate adrenergic vasoconstriction in response to sympathetic nerve activity. This review focuses on the endothelium as the cellular pathway that coordinates spreading vasodilatation. We discuss the nature and regulation of cell‐to‐cell coupling through gap junctions, bidirectional signalling between ECs and SMCs, and how oxidative stress during ageing may influence respective signalling pathways. Our recent findings illustrate the role of small (SKCa) and intermediate (IKCa) Ca2+ activated K+ channels as modulators of electrical conduction along the endothelium. Gaps in current understanding indicate the need to determine mechanisms that regulate intracellular Ca2+ homeostasis and ion channel activation in the resistance vasculature with advancing age.

Coordination of Intercellular Ca2+ Signaling in Endothelial Cell Tubes of Mouse Resistance Arteries

To test the hypothesis that Ca2+ responses to GPCR activation are coordinated between neighboring ECs of resistance arteries.

Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation

Endothelial function in resistance vessels entails Ca2+ and electrical signalling to promote vasodilatation and increase tissue blood flow. Whether membrane potential (Vm) governs intracellular calcium concentration ([Ca2+]i) of the endothelium remains controversial. [Ca2+]i and Vm were evaluated simultaneously during intracellular current injection using intact endothelial tubes freshly isolated from mouse skeletal muscle resistance arteries. [Ca2+]i did not change during hyperpolarization or depolarization under resting conditions. However in the presence of 100 nM ACh (∼EC50), [Ca2+]i increased during hyperpolarization and decreased during depolarization. These responses required extracellular Ca2+ and were attenuated by half with genetic ablation of TRPV4 channels. In native microvascular endothelium, half‐maximal stimulation of muscarinic receptors enables Vm to govern [Ca2+]i by activating Ca2+‐permeable channels in the plasma membrane. This effect of Vm is absent at rest and can be masked during maximal receptor stimulation.

Advanced age protects microvascular endothelium from aberrant Ca2+ influx and cell death induced by hydrogen peroxide

Calcium signalling in endothelial cells of resistance arteries is integral to blood flow regulation. Oxidative stress and endothelial dysfunction can prevail during advanced age and we questioned how calcium signalling may be affected. Intact endothelium was freshly isolated from superior epigastric arteries of Young (∼4 months) and Old (∼24 months) male C57BL/6 mice. Under resting conditions, with no difference in intracellular calcium levels, hydrogen peroxide (H2O2) availability was ∼1/3 greater in endothelium of Old mice while vascular catalase activity was reduced by nearly half. Compared to Old, imposing oxidative stress (200 μm H2O2) for 20 min increased intracellular calcium to 4‐fold greater levels in endothelium of Young in conjunction with twice the calcium influx. Prolonged (60 min) exposure to H2O2 induced 7‐fold greater cell death in endothelium of Young. Microvascular adaptation to advanced age may protect endothelial cells during elevated oxidative stress to preserve functional viability of the intima.

Maturation and long-term hypoxia alters Ca2+-induced Ca2+ release in sheep cerebrovascular sympathetic neurons

The contribution of sympathetic nerves arising from the superior cervical ganglia (SCG) toward the growth and function of cerebral blood vessels is pertinent throughout maturation as well as in response to cardiovascular stress imposed by high-altitude long-term hypoxia (LTH). The function of SCG sympathetic neurons is dependent on intracellular Ca2+ concentration ([Ca2+]i) signaling, which is strongly influenced by a process known as Ca(2+)-induced Ca2+ release (CICR) from the smooth endoplasmic reticulum (SER). In this study, we used the sheep SCG neuronal model to test the hypotheses that maturation decreases CICR and high-altitude LTH depresses CICR in fetal SCG neurons but not in those of the adult. We found that the contribution of CICR to electric field stimulation (EFS)-evoked [Ca2+]i transients was greatest in SCG cells from normoxic fetuses and was abolished by LTH. The decline in CICR was associated with a reduction in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) function in fetal SCG cells during LTH, reducing SER Ca2+ levels below the threshold needed for the coupling of Ca2+ influx and CICR. With respect to the maturation from the fetus to adult, the decrease in CICR may reflect both a reduction in the levels of ryanodine receptor isoforms 2 and 3 and SERCA function. In response to LTH and in contrast to the fetus, CICR function in adult SCG cells is maintained and may reflect alterations in other mechanisms that modulate the CICR process. As CICR is instrumental in the function of sympathetic neurons within the cerebrovasculature, the loss of this signaling mechanism in the fetus may have consequences for the adaptation to LTH in terms of fetal susceptibility to vascular insults.

Calcium and electrical signaling in arterial endothelial tubes: new insights into cellular physiology and cardiovascular function

The integral role of the endothelium during the coordination of blood flow throughout vascular resistance networks has been recognized for several decades now. Early examination of the distinct anatomy and physiology of the endothelium as a signaling conduit along the vascular wall has prompted development and application of an intact endothelial “tube” study model isolated from rodent skeletal muscle resistance arteries. Vasodilatory signals such as increased endothelial cell (EC) Ca2+ ([Ca2+]i) and hyperpolarization take place in single ECs while shared between electrically coupled ECs through gap junctions up to distances of millimeters (≥2 mm). The small‐ and intermediate‐conductance Ca2+ activated K+ (SKCa/IKCa or KCa2.3/KCa3.1) channels function at the interface of Ca2+ signaling and hyperpolarization; a bidirectional relationship whereby increases in [Ca2+]i activate SKCa/IKCa channels to produce hyperpolarization and vice versa. Further, the spatial domain of hyperpolarization among electrically coupled ECs can be finely tuned via incremental modulation of SKCa/IKCa channels to balance the strength of local and conducted electrical signals underlying vasomotor activity. Multifunctional properties of the voltage‐insensitive SKCa/IKCa channels of resistance artery endothelium may be employed for therapy during the aging process and development of vascular disease.