Mordecai P. Blaustein

Calcium transport and buffering in neurons

Abstract Calcium ions serve as a signal for numerous neuronal functions. This requires a very low, modulated, resting cytosolic free Ca 2+ concentration, and diverse mechanisms to regulate the time course and spatial distribution of transient Ca 2+ increases, so that Ca 2+ can separately activate multiple processes within the same neuron. The interplay of several systems that release Ca 2+ into the cytosol, and multiple mechanisms that buffer Ca 2+ in the cytosol, sequester Ca 2+ in intracellular organelles, and extrude Ca 2+ across the plasmalemma, provide the requisite control. These mechanisms are utilized differently in different neurons to meet specific demands such as variations in excitation patterns.

Salt-sensitive hypertension is triggered by Ca2+ entry via Na+/Ca2+ exchanger type-1 in vascular smooth muscle.

Excessive salt intake is a major risk factor for hypertension. Here we identify the role of Na+/Ca2+ exchanger type 1 (NCX1) in salt-sensitive hypertension using SEA0400, a specific inhibitor of Ca2+ entry through NCX1, and genetically engineered mice. SEA0400 lowers arterial blood pressure in salt-dependent hypertensive rat models, but not in other types of hypertensive rats or in normotensive rats. Infusion of SEA0400 into the femoral artery in salt-dependent hypertensive rats increases arterial blood flow, indicating peripheral vasodilation. SEA0400 reverses ouabain-induced cytosolic Ca2+ elevation and vasoconstriction in arteries. Furthermore, heterozygous NCX1-deficient mice have low salt sensitivity, whereas transgenic mice that specifically express NCX1.3 in smooth muscle are hypersensitive to salt. SEA0400 lowers the blood pressure in salt-dependent hypertensive mice expressing NCX1.3, but not in SEA0400-insensitive NCX1.3 mutants. These findings indicate that salt-sensitive hypertension is triggered by Ca2+ entry through NCX1 in arterial smooth muscle and suggest that NCX1 inhibitors might be useful therapeutically.

How NaCl raises blood pressure: a new paradigm for the pathogenesis of salt-dependent hypertension

Excess dietary salt is a major cause of hypertension. Nevertheless, the specific mechanisms by which salt increases arterial constriction and peripheral vascular resistance, and thereby raises blood pressure (BP), are poorly understood. Here we summarize recent evidence that defines specific molecular links between Na(+) and the elevated vascular resistance that directly produces high BP. In this new paradigm, high dietary salt raises cerebrospinal fluid [Na(+)]. This leads, via the Na(+)-sensing circumventricular organs of the brain, to increased sympathetic nerve activity (SNA), a major trigger of vasoconstriction. Plasma levels of endogenous ouabain (EO), the Na(+) pump ligand, also become elevated. Remarkably, high cerebrospinal fluid [Na(+)]-evoked, locally secreted (hypothalamic) EO participates in a pathway that mediates the sustained increase in SNA. This hypothalamic signaling chain includes aldosterone, epithelial Na(+) channels, EO, ouabain-sensitive α(2) Na(+) pumps, and angiotensin II (ANG II). The EO increases (e.g.) hypothalamic ANG-II type-1 receptor and NADPH oxidase and decreases neuronal nitric oxide synthase protein expression. The aldosterone-epithelial Na(+) channel-EO-α(2) Na(+) pump-ANG-II pathway modulates the activity of brain cardiovascular control centers that regulate the BP set point and induce sustained changes in SNA. In the periphery, the EO secreted by the adrenal cortex directly enhances vasoconstriction via an EO-α(2) Na(+) pump-Na(+)/Ca(2+) exchanger-Ca(2+) signaling pathway. Circulating EO also activates an EO-α(2) Na(+) pump-Src kinase signaling cascade. This increases the expression of the Na(+)/Ca(2+) exchanger-transient receptor potential cation channel Ca(2+) signaling pathway in arterial smooth muscle but decreases the expression of endothelial vasodilator mechanisms. Additionally, EO is a growth factor and may directly participate in the arterial structural remodeling and lumen narrowing that is frequently observed in established hypertension. These several central and peripheral mechanisms are coordinated, in part by EO, to effect and maintain the salt-induced elevation of BP.

Sodium pump α2 subunits control myogenic tone and blood pressure in mice

A key question in hypertension is: How is long‐term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain‐like compound (EOLC) and induces salt‐dependent hypertension mediated by Na+/Ca2+ exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na+ pump α1 (α1+/−) or α2 (α2+/−) catalytic subunits. Both low‐dose ouabain (1–100 nm; inhibits only α2) and high‐dose ouabain (≥1 μm; inhibits α1) elevate myocyte Ca2+ and constrict arteries from α1+/−, as well as α2+/− and wild‐type mice. Nevertheless, only mice with reduced α2 Na+ pump activity (α2+/−), and not α1 (α1+/−), have elevated blood pressure. Also, isolated, pressurized arteries from α2+/−, but not α1+/−, have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB‐R7943) normalize myogenic tone in ouabain‐treated arteries. Only the NCX blockers normalize the elevated myogenic tone in α2+/− arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt‐dependent and ouabain‐induced hypertension. Thus, chronically reduced α2 activity (α2+/− or chronic ouabain) apparently regulates myogenic tone and long‐term blood pressure whereas reduced α1 activity (α1+/−) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt‐dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing α2 Na+ pump activity and promoting Ca2+ entry via NCX in myocytes.

Sodium Transport Inhibition, Cell Calcium, and Hypertension: The Natriuretic Hormone/Na+-Ca2+ Exchange/Hypertension Hypothesis

Sodium plays a critical role in the etiology of essential hypertension, but the mechanism by which excess dietary sodium actually leads to the elevation of blood pressure is not understood. The hypothesis described shows how an excessive sodium load can lead to the development of hypertension. The underlying factor must be a genetic or acquired deficiency or limitation in renal sodium excretion that may be undetectable by standard renal function tests. The resultant tendency towards sodium, water, and extracellular fluid volume expansion is compensated by the secretion of a natriuretic hormone that promotes sodium excretion by inhibiting sodium pumps in the kidney tubule cells. The hormone also inhibits sodium pumps in other cells, including vascular smooth muscle cells, causing intracellular sodium to increase. Then, because the vascular smooth muscle cells contain a Na+-Ca2+ exchange transport system in their plasma membranes, more calcium than normal is delivered to these cells. This causes the increased contractility and reactivity that underlies the increased vascular tone and peripheral vascular resistance that elevates the blood pressure.

Regulation of cell calcium and contractility in mammalian arterial smooth muscle: the role of sodium‐calcium exchange.

1. The contraction and relaxation of rings of rat thoracic aorta and bovine tail artery were examined as a function of changes in the Na+ electrochemical gradient in order to determine the role of Na‐Ca exchange in the control of contractility. 2. Inhibition of the Na+ pump in rat aorta by K+‐free media or a low concentration (5 x 10(‐5) M) of strophanthidin reversibly increased the contractile responses to caffeine and noradrenaline. These effects were dependent upon external Ca2+ and were observed even in the presence of a Ca2+ channel blocker (10 microM‐verapamil or 10 microM‐diltiazem) and an alpha‐receptor blocker (10 microM‐phentolamine). 3. Reduction of external Na+ concentration, [Na+]o (replaced by N‐methylglucamine, tetramethylammonium or Tris), also caused an external Ca2+‐dependent increase in tonic tension and, in rat aorta, an increase in the response to caffeine. These effects were also observed in the presence of verapamil and phentolamine. 4. Caffeine relaxed the bovine tail artery, but increased the sensitivity of the rat aorta to reduced [Na+]o. The latter effect was presumably due to block of Ca2+ sequestration in the sarcoplasmic reticulum, so that entering Ca2+ was more effective in raising the intracellular free Ca2+ level, [Ca2+]i. 5. Relaxation from K+‐free or low‐Na+ contractions, in Ca2+‐free media, depended upon [Na+]o. Reduction of [Na+]o to 1.2 or 7.5 mM slowed the relaxation of rat aorta (5 mM‐caffeine present) 3‐ to 5‐fold, and the relaxation of bovine tail artery (without caffeine) 5‐ to 10‐fold. These effects were seen in the presence of verapamil and phentolamine. 6. These observations are all consistent with an Na‐Ca exchange transport system that can move Ca2+ either into or out of the arterial smooth muscle cells. Ca2+ entry is enhanced by raising [Na+]i (by Na+ pump inhibition) and/or lowering [Na+]o. Ca2+ extrusion from the contracted muscles is largely dependent upon external Na+. The latter observation implies that, when [Ca2+] exceeds the contraction threshold, Ca2+ efflux is mediated primarily by the Na‐Ca exchanger, rather than by the sarcolemmal ATP‐driven Ca2+ pump. 7. When bovine tail artery was treated with verapamil and phentolamine, and [Na+]o was reduced from 139.2 to 43.9 mM, substitution of K+ for Na+ induced a larger external Ca2+‐dependent contraction than did substitution of Tris for Na+. The amplitudes of these contractions were greatly increased when the Na+ pump was inhibited by 5 x 10(‐5) M‐strophanthidin, presumably because of the rise in [Na+]i.(ABSTRACT TRUNCATED AT 400 WORDS)

The Pump, the Exchanger and Endogenous Ouabain: Signaling Mechanisms that Link Salt Retention to Hypertension

The central roles of salt (NaCl) and the kidneys in the pathogenesis of most forms of hypertension are well established.1,2 The linkage between NaCl retention and blood pressure (BP) elevation is often referred to as “whole body autoregulation.”3,4 Surprisingly, however, the precise mechanisms that underlie this linkage (ie, the signaling pathway) have escaped elucidation. Here, we examined the evidence that endogenous ouabain (EO), Na+ pumps (Na,K-ATPase), and the Na/Ca exchanger (NCX) are critical molecular mechanisms in this pathway. At constant cardiac output (CO), mean arterial BP=CO×TPR (where TPR is total peripheral vascular resistance).5 In most (chronic) hypertension, in humans and animals, the CO is relatively normal, and the high BP is maintained by an elevated TPR.1,4 TPR is controlled dynamically by vasoconstriction/dilation in small “resistance” arteries, which exhibit tonic constriction (“tone”). This can be studied in isolated, cannulated small arteries that develop spontaneous (myogenic) tone (MT),6 under constant or increasing intraluminal pressure. Indeed, the level of tone in isolated arteries “is often comparable to that observed in the same vessels in vivo,”6 and may even be used to predict BP changes7 (see below). MT is triggered by Ca2+ entry, primarily through voltage-gated Ca2+ channels in arterial smooth muscle (SM; ASM) cells,6 and contraction is activated by the rise in cytosolic Ca2+ concentration ([Ca2+]CYT).8 In NaCl-dependent hypertension, the enhanced vasoconstriction and increased tone and TPR are, at least in part, functional and reversible phenomena.9 Numerous mechanisms contribute to the regulation of myocyte [Ca2+]CYT and vasoconstriction, but the plasma membrane (PM) NCX provides an unique, direct link between Na+ and [Ca2+]CYT and, thus, Ca2+ signaling and contraction in ASM cells.10 NCX-mediated Ca2+ transport is …

Plasma Membrane-Cytoskeleton-Endoplasmic Reticulum Complexes in Neurons and Astrocytes

The possibility that certain integral plasma membrane (PM) proteins involved in Ca2+ homeostasis form junctional units with adjacent endoplasmic reticulum (ER) in neurons and glia was explored using immunoprecipitation and immunocytochemistry. Rat brain membranes were solubilized with the mild, non-ionic detergent, IGEPAL CA-630. Na+/Ca2+ exchanger type 1 (NCX1), a key PM Ca2+ transporter, was immunoprecipitated from the detergent-soluble fraction. Several abundant PM proteins co-immunoprecipitated with NCX1, including the α2 and α3 isoforms of the Na+ pump catalytic (α) subunit, and the α2 subunit of the dihydropyridine receptor. The adaptor protein, ankyrin 2 (Ank 2), and the cytoskeletal proteins, α-fodrin and β-spectrin, also selectively co-immunoprecipitated with NCX1, as did the ER proteins, Ca2+ pump type 2 (SERCA 2), and inositol-trisphosphate receptor type 1 (IP3R-1). In contrast, a number of other abundant PMs, adaptors, and cytoskeletal proteins did not co-immunoprecipitate with NCX1, including the Na+ pump α1 isoform, PM Ca2+ pump type 1 (PMCA1), β-fodrin, and Ank 3. In reciprocal experiments, immunoprecipitation with antibodies to the Na+ pump α2 and α3 isoforms, but not α1, co-immunoprecipitated NCX1; the antibodies to α1 did, however, co-immunoprecipitate PMCA1. Antibodies to Ank 2, α-fodrin, β-spectrin and IP3R-1 all co-immunoprecipitated NCX1. Immunocytochemistry revealed partial co-localization of β-spectrin with NCX1, Na+ pump α3, and IP3R-1 in neurons and of α-fodrin with NCX1 and SERCA2 in astrocytes. The data support the idea that in neurons and glia PM microdomains containing NCX1 and Na+ pumps with α2 or α3 subunits form Ca2+ signaling complexes with underlying ER containing SERCA2 and IP3R-1. These PM and ER components appear to be linked through the cytoskeletal spectrin network, to which they are probably tethered by Ank 2.

Physiological Roles of the Sodium‐Calcium Exchanger in Nerve and Musclea

Sodium-calcium exchange has been well studied in cardiac muscle and in photoreceptors; the physiological roles of the exchanger in these tissues are widely recognized and appreciated (see other articles in this volume). A prominent Na-Ca exchanger has also been identified in both vertebrate and invertebrate neurons, but the physiological role(s) of the exchanger in neurons is (are) poorly understood. In other types of cells, such as vascular smooth muscle (VSM) cells, the physiological significance of an Na-Ca exchanger, and even its presence, have been questioned. This uncertainty has arisen because even large changes in the trans-plasmalemmal Na+ electrochemical gradient, ApNa, often do not markedly alter the resting cytosolic free-Ca2+ concentration, [Ca2+]c(rest), or, in VSM, “resting” tension. Here we review the evidence that there is a prominent, physiologically important Na-Ca exchanger in the plasmalemma of mammalian neurons as well as in astrocytes and VSM cells, and in barnacle “skeletal” muscle fibers. The exchangers in these cells clearly modulate [Ca2f]c(mt), even though this parameter is mainly under the control of the ATP-driven Ca2+ pumps in the plasmalemma and in the endoplasmic reticulum (ER) or, in muscle, the sarcoplasmic reticulum (SR). A very different situation prevails during cell activation, however, because even small effects of the exchanger on [Ca2+Idr,,) are reflected by relatively large changes in the amount of Caz+ stored in the ER or SR. Thus, a key role of the exchanger in many types of cells appears to be

Location of calcium transporters at presynaptic terminals

The plasma membrane ATP‐driven Ca2+ pump (PMCA) and the Na+/Ca2+ exchanger (NCX) are the major means of Ca2+ extrusion at presynaptic nerve terminals, but little is know about the location of these transporters relative to the major sites of Ca2+ influx, the transmitter release sites. We used immunocytochemistry to identify these transport proteins in a calyx‐type presynaptic nerve terminal from the ciliary ganglion of the chick. The PMCA clusters were localized to the transmitter release sites, as identified by staining for the secretory vesicle‐specific protein synaptotagmin I. This colocalization was not due to the presence of the pump on the secretory vesicle itself because membrane fractionation of chick brain synaptosomes demonstrated comigration of the pump with surface membrane and not vesicle markers. In contrast, the NCX did not colocalize with synaptotagmin but tended to be located at nonsynaptic regions of the terminal. The PMCA location, near the transmitter release sites, suggests that it plays a role in priming the release site by maintaining a low free Ca2+ level, facilitating the dissociation of the ion from its binding sites. The PMCA may also replenish external Ca2+ in the synaptic cleft following periods of synaptic activity. In contrast, the NCX location suggests a role in the rapid emptying of cytoplasmic Ca2+ uptake organelles which serve as the main line of defence against high free Ca2+.