Hema Raina

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 …

Sympathetic neurogenic Ca2+ signalling in rat arteries: ATP, noradrenaline and neuropeptide Y

The sympathetic nervous system (SNS) plays an essential role in the control of total peripheral vascular resistance by controlling the contraction of small arteries. The SNS also exerts long‐term trophic influences in health and disease; SNS hyperactivity accompanies most forms of human essential hypertension, obesity and heart failure. At their junctions with smooth muscle cells, the peri‐arterial sympathetic nerves release ATP, noradrenaline (NA) and neuropeptide Y (NPY) onto smooth muscle cells. Confocal Ca2+ imaging studies reveal that ATP and NA each produce unique types of postjunctional Ca2+ signals and consequent smooth muscle cell contractions. Neurally released ATP activates postjunctional P2X1 receptors to produce local, non‐propagating Ca2+ transients, termed ‘junctional Ca2+ transients’, or ‘jCaTs’. Neurally released NA binds to α1‐adrenoceptors and can activate Ca2+ waves or more uniform global changes in [Ca2+]. Neurally released NPY does not appear to produce Ca2+ transients directly, but significantly modulates NA‐induced Ca2+ signalling. The neural release of ATP and NA, as judged by postjunctional Ca2+ signals, electrical recording of excitatory junction potentials and carbon fibre amperometry to measure NA, varies markedly with the pattern of nerve activity. This probably reflects both pre‐ and postjunctional mechanisms, which are not yet fully understood. These phenomena, together with different temporal patterns of sympathetic nerve activity in different regional circulations, are probably an important mechanistic basis of the important selective regulation of regional vascular resistance and blood flow by the sympathetic nervous system.

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats.

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na(+) reabsorption. Recently we demonstrated that Ca(2+) signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na(+)/Ca(2+) exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca(2+) signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca(2+) signals in response to 5 μM PE or ATP in the absence and presence of extracellular Ca(2+). These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca(2+) release and increased Ca(2+) entry, respectively. The increased SR Ca(2+) release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca(2+) signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca(2+) signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension.

Low-dose ouabain constricts small arteries from ouabain-hypertensive rats: implications for sustained elevation of vascular resistance.

Prolonged ouabain administration to normal rats causes sustained blood pressure (BP) elevation. This ouabain-induced hypertension (OH) has been attributed, in part, to the narrowing of third-order resistance arteries (approximately 320 microm internal diameter) as a result of collagen deposition in the artery media. Here we describe the structural and functional properties of fourth-order mesenteric small arteries from control and OH rats, including the effect of low-dose ouabain on myogenic tone in these arteries. Systolic BP in OH rats was 138 +/- 3 versus 124 +/- 4 mmHg in controls (P < 0.01). Pressurized (70 mmHg) control and OH arteries, with only a single layer of myocytes, both had approximately 165-microm internal diameters and approximately 20-microm wall thicknesses. Even after fixation, despite vasoconstriction, the diameters and wall thicknesses did not differ between control and OH fourth-order arteries, whereas in third-order arteries, both parameters were significantly smaller in OH than in controls. Myogenic reactivity was significantly augmented in OH fourth-order arteries. Nevertheless, phenylephrine- (1 microM) and high K(+)-induced vasoconstrictions and acetylcholine-induced vasodilation were comparable in control and OH arteries. Vasoconstrictions induced by 5 microM phenylephrine and by 10 mM caffeine in Ca(2+)-free media indicated that releasable sarcoplasmic reticulum Ca(2+) stores were normal in OH arteries. Importantly, 100 nM ouabain constricted both control and OH arteries by approximately 26 microm, indicating that this response was not downregulated in OH rats. This maximal ouabain-induced constriction corresponds to a approximately 90% increase in resistance to flow in these small arteries; thus ouabain at EC(50) of approximately 0.66 nM should raise resistance by approximately 35%. We conclude that dynamic constriction in response to circulating nanomolar ouabain in small arteries likely makes a major contribution to the increased vascular tone and BP in OH rats.

Activation by Ca2+/calmodulin of an exogenous myosin light chain kinase in mouse arteries

Activation of myosin light chain kinase (MLCK) and other kinases was studied in the arteries of transgenic mice that express an optical fluorescence resonance energy transfer (FRET) MLCK activity biosensor. Binding of Ca2+/calmodulin (Ca2+/CaM) induces an increase in MLCK activity and a change in FRET. After exposure to high external [K+], intracellular [Ca2+] (fura‐2 ratio or fluo‐4 fluorescence) and MLCK activity both increased rapidly to an initial peak and then declined, rapidly at first and then very slowly. After an initial peak (‘phasic’) force was constant or increased slowly (termed ‘tonic’ force). Inhibition of rho‐kinase (Y‐27632) decreased tonic force more than phasic, but had little effect on [Ca2+] and MLCK activation. Inhibition of PKCα and PKCβ with Gö6976 had no effect. KN‐93, an inhibitor of CaMK II, markedly reduced force, MLCK FRET and [Ca2+]. Applied during tonic force, forskolin caused a rapid decrease in MLCK FRET ratio and force, but no change in Ca2+, suggesting a cAMP mediated decrease in affinity of MLCK for Ca2+/CaM. However, receptor (β‐adrenergic) activated increases in cAMP during KCl were ineffective in causing relaxation, changes in [Ca2+], or MLCK FRET. At the same tonic force, MLCK FRET ratio activated by α1‐adrenoceptors was ∼60% of that activated by KCl. In conclusion, MLCK activity of arterial smooth muscle during KCl‐induced contraction is determined primarily by Ca2+/CaM. Rho‐kinase is activated, by unknown mechanisms, and increases ‘Ca2+ sensitivity’ significantly. Forskolin mediated increases in cAMP, but not receptor mediated increases in cAMP cause a rapid decrease in the affinity of MLCK for Ca2+/CaM.

A technique for simultaneous measurement of Ca2+, FRET fluorescence and force in intact mouse small arteries

FRET (Forster resonance energy transfer)‐based biosensor molecules are powerful tools to reveal specific molecular interactions in cells. Typically however, they are used in cultured cells that (inevitably) express different genes than their counterparts in intact organisms. In such cells it may be impossible to administer physiological stimuli and measure physiological outputs. Here, through the use of transgenic mice that express a FRET‐based myosin light chain kinase (MLCK) biosensor molecule, we report a technique for dynamically observing activation and regulation of MLCK within the smooth muscle cells of intact, functioning small arteries, together with measurement of arterial force production and intracellular [Ca2+].

High Vascular Tone of Mouse Femoral Arteries In Vivo Is Determined by Sympathetic Nerve Activity Via α1A- and α1D-Adrenoceptor Subtypes

Background and purpose Determining the role of vascular receptors in vivo is difficult and not readily accomplished by systemic application of antagonists or genetic manipulations. Here we used intravital microscopy to measure the contributions of sympathetic receptors, particularly α1-adrenoceptor subtypes, to contractile activation of femoral artery in vivo. Experimental approach Diameter and intracellular calcium ([Ca2+]i) in femoral arteries were determined by intravital fluorescence microscopy in mice expressing a Myosin Light Chain Kinase (MLCK) based calcium-calmodulin biosensor. Pharmacological agents were applied locally to the femoral artery to determine the contributions of vascular receptors to tonic contraction and [Ca2+]i,. Key results In the anesthetized animal, femoral arteries were constricted to a diameter equal to 54% of their passive diameter (i.e. tone = 46%). Of this total basal tone, 16% was blocked by RS79948 (0.1 µM) and thus attributable to α2-adrenoceptors. A further 46% was blocked by prazosin (0.1 µM) and thus attributable to α1-adrenoceptors. Blockade of P2X and NPY1 receptors with suramin (0.5 mM) and BIBP3226 (1.0 µM) respectively, reduced tone by a further 22%, leaving 16% of basal tone unaffected at these concentrations of antagonists. Application of RS100329 (α1A-selective antagonist) and BMY7378 (α1D-selective) decreased tone by 29% and 26%, respectively, and reduced [Ca2+]i. Chloroethylclonidine (1 µM preferential for α1B-) had no effect. Abolition of sympathetic nerve activity (hexamethonium, i.p.) reduced basal tone by 90%. Conclusion and Implications Tone of mouse femoral arteries in vivo is almost entirely sympathetic in origin. Activation of α1A- and α1D-adrenoceptors elevates [Ca2+]i and accounts for at least 55% of the tone.

Effects of deoxycholylglycine, a conjugated secondary bile acid, on myogenic tone and agonist-induced contraction in rat resistance arteries.

Background Bile acids (BAs) regulate cardiovascular function via diverse mechanisms. Although in both health and disease serum glycine-conjugated BAs are more abundant than taurine-conjugated BAs, their effects on myogenic tone (MT), a key determinant of systemic vascular resistance (SVR), have not been examined. Methodology/Principal Findings Fourth-order mesenteric arteries (170–250 µm) isolated from Sprague-Dawley rats were pressurized at 70 mmHg and allowed to develop spontaneous constriction, i.e., MT. Deoxycholylglycine (DCG; 0.1–100 µM), a glycine-conjugated major secondary BA, induced reversible, concentration-dependent reduction of MT that was similar in endothelium-intact and -denuded arteries. DCG reduced the myogenic response to stepwise increase in pressure (20 to 100 mmHg). Neither atropine nor the combination of L-NAME (a NOS inhibitor) plus indomethacin altered DCG-mediated reduction of MT. K+ channel blockade with glibenclamide (KATP), 4-aminopyradine (KV), BaCl2 (KIR) or tetraethylammonium (TEA, KCa) were also ineffective. In Fluo-2-loaded arteries, DCG markedly reduced vascular smooth muscle cell (VSM) Ca2+ fluorescence (∼50%). In arteries incubated with DCG, physiological salt solution (PSS) with high Ca2+ (4 mM) restored myogenic response. DCG reduced vascular tone and VSM cytoplasmic Ca2+ responses (∼50%) of phenylephrine (PE)- and Ang II-treated arteries, but did not affect KCl-induced vasoconstriction. Conclusion In rat mesenteric resistance arteries DCG reduces pressure- and agonist-induced vasoconstriction and VSM cytoplasmic Ca2+ responses, independent of muscarinic receptor, NO or K+ channel activation. We conclude that BAs alter vasomotor responses, an effect favoring reduced SVR. These findings are likely pertinent to vascular dysfunction in cirrhosis and other conditions associated with elevated serum BAs.

Sympathetic neurogenic Ca2+signalling in rat arteries: ATP, noradrenaline and neuropeptide Y: Sympathetic neurogenic Ca2+signalling in arteries

The sympathetic nervous system (SNS) plays an essential role in the control of total peripheral vascular resistance by controlling the contraction of small arteries. The SNS also exerts long‐term trophic influences in health and disease; SNS hyperactivity accompanies most forms of human essential hypertension, obesity and heart failure. At their junctions with smooth muscle cells, the peri‐arterial sympathetic nerves release ATP, noradrenaline (NA) and neuropeptide Y (NPY) onto smooth muscle cells. Confocal Ca2+ imaging studies reveal that ATP and NA each produce unique types of postjunctional Ca2+ signals and consequent smooth muscle cell contractions. Neurally released ATP activates postjunctional P2X1 receptors to produce local, non‐propagating Ca2+ transients, termed ‘junctional Ca2+ transients’, or ‘jCaTs’. Neurally released NA binds to α1‐adrenoceptors and can activate Ca2+ waves or more uniform global changes in [Ca2+]. Neurally released NPY does not appear to produce Ca2+ transients directly, but significantly modulates NA‐induced Ca2+ signalling. The neural release of ATP and NA, as judged by postjunctional Ca2+ signals, electrical recording of excitatory junction potentials and carbon fibre amperometry to measure NA, varies markedly with the pattern of nerve activity. This probably reflects both pre‐ and postjunctional mechanisms, which are not yet fully understood. These phenomena, together with different temporal patterns of sympathetic nerve activity in different regional circulations, are probably an important mechanistic basis of the important selective regulation of regional vascular resistance and blood flow by the sympathetic nervous system.

Sympathetic Neurogenic Ca2+ Signaling in Arteries: ATP, Noradrenaline and NPY

The sympathetic nervous system (SNS) plays an essential role in the control of total peripheral vascular resistance by controlling the contraction of small arteries. The SNS also exerts long‐term trophic influences in health and disease; SNS hyperactivity accompanies most forms of human essential hypertension, obesity and heart failure. At their junctions with smooth muscle cells, the peri‐arterial sympathetic nerves release ATP, noradrenaline (NA) and neuropeptide Y (NPY) onto smooth muscle cells. Confocal Ca2+ imaging studies reveal that ATP and NA each produce unique types of postjunctional Ca2+ signals and consequent smooth muscle cell contractions. Neurally released ATP activates postjunctional P2X1 receptors to produce local, non‐propagating Ca2+ transients, termed ‘junctional Ca2+ transients’, or ‘jCaTs’. Neurally released NA binds to α1‐adrenoceptors and can activate Ca2+ waves or more uniform global changes in [Ca2+]. Neurally released NPY does not appear to produce Ca2+ transients directly, but significantly modulates NA‐induced Ca2+ signalling. The neural release of ATP and NA, as judged by postjunctional Ca2+ signals, electrical recording of excitatory junction potentials and carbon fibre amperometry to measure NA, varies markedly with the pattern of nerve activity. This probably reflects both pre‐ and postjunctional mechanisms, which are not yet fully understood. These phenomena, together with different temporal patterns of sympathetic nerve activity in different regional circulations, are probably an important mechanistic basis of the important selective regulation of regional vascular resistance and blood flow by the sympathetic nervous system.