Activation of Na+,H+ exchanger produces vasoconstriction of renal resistance vessels

To evaluate the influence of the sodium/proton exchanger (Na⁺,H⁺ exchanger) on the constriction of rat resistance vessels and on the iliac artery, the isometric vasoconstrictions of renal resistance vessels and strips from iliac artery derived from Wistar-Kyoto rats were measured using a vessel myograph. The Na⁺,H⁺ exchanger was activated by intracellular acidification using propionic acid. Cytosolic pH (pH_i) and cytosolic free sodium concentration ([Na⁺]_i) in vascular smooth muscle cells were measured using the fluorescent dye technique. The activation of the Na⁺,H⁺ exchanger increased the [Na⁺]_i by 12.4 ± 1.3 mmol/L (n = 8). The activation of the Na⁺,H⁺ exchanger caused a contractile response of the renal resistance vessels (increase of tension, 1.5 ± 0.1 × 10⁻³ N; n = 13) and of the rat iliac artery (increase of tension, 7.5 ± 0.8 × 10⁻³ N; n = 5). The contractile response after activation of the Na⁺,H⁺ exchanger was significantly inhibited in the absence of external sodium or in the presence of amiloride, confirming the involvement of the Na⁺,H⁺ exchanger. The contractile response after activation of the Na⁺,H⁺ exchanger was significantly reduced in the absence of external calcium, after inhibition of calcium channels by nifedipine, and in the presence of an intracellular calcium antagonist 8-(diethylamino-)-octyl-3,4,5-trimethoxybenzoate (TMB-8), indicating that the activation of the Na⁺,H⁺ exchanger consecutively caused transplasma membrane calcium influx. On the other hand, the inhibition of the Na⁺,Ca²⁺ exchanger by NiCl₂ significantly increased the vasoconstriction of renal resistance vessels after activation of the Na⁺,H⁺ exchanger. The activation of the Na⁺,H⁺ exchanger produces vasoconstriction by an increased cytosolic sodium concentration, inhibition of the Na⁺,Ca²⁺ exchanger, and activation of transplasma membrane calcium influx through potential dependent calcium channels.     

Role of sodium in antigen-induced contraction of tracheal smooth muscle in dogs

We examined the role of Na⁺ influx in the airway response to antigen (ragweed pollen extract) in sensitized dogs, using amiloride analogs to block Na⁺-dependent processes. In in vivo studies, respiratory resistance was measured in amiloride treated and untreated groups. The resistance increased by 9.3 cmH₂O·L⁻¹·sec in response to ragweed aerosol in the untreated group, but increased only by 5.2 cmH₂O·L⁻¹·sec in the treated group. In in vitro studies, isometric tension was measured in ragweed pollen sensitized tracheal strips. Tissues were treated with amiloride or its derivatives (50 μM) for specifically blocking Na⁺ channels (phenamil), Na⁺-H⁺ exchanger [5-(N-methylN-guanidinocarbonyl methyl)-amiloride] r Na⁺-Ca²⁺ exchanger [5(4-chlorobenzyl)-2′,4′-dimethylbenzamil]. In untreated strips, tension increased in response to ragweed by 1.9 ± 0.5 mN/mg. The increase was reduced by phenamil (95.2 ± 2.5%; P<0.01 and amiloride (41.7 ± 13.1%; P<0.01), but not by the other two agents. Furthermore, phenamil also inhibited histamine-induced tension response and histamine-induced ²²Na⁺ uptake of the muscle. We conclude that antigen-induced airway response is attenuated blocking Na⁺ influx in smooth muscle.     

Nongenomic Actions of Thyroid Hormone and Intracellular Calcium Metabolism

Nongenomic actions of thyroid hormone include several that involve or require calcium. Actions of thyroid hormone at the plasma or intracellular membranes include stimulation of membrane glucose transport and of the Na⁺/H⁺ antiporter (exchanger) by mechanisms that require liberation of intracellular calcium and stimulation of the cell membrane and sarcoplasmic reticulum calcium pumps (Ca²⁺-ATPases). These pumps not only transport Ca²⁺, but also are regulated by the intracellular calmodulin-Ca²⁺ complex (plasma membrane/sarcolemma) or calmodulin-dependent protein kinase II phosphorylation of phospholamban (sarcoplasmic reticulum). Intracellular calcium ion concentration may also be subject to regulation by other nongenomic effects of iodothyronines, such as those on the Na⁺/H⁺ antiporter or sodium current, that secondarily affect the Na⁺/Ca²⁺ exchanger. Certain of these nongenomic actions of thyroid hormone, e.g., Na⁺/H⁺ exchanger, Ca²⁺-ATPase, are now recognized to begin at a recently described hormone receptor on a heterodimeric structural membrane protein, integrin αvβ3. The thyroid hormone signal at this receptor is further transduced by the mitogen-activated protein kinase (MAPK; extracellular regulated kinase1/2, ERK1/2) pathway.     

Hyperinsulinaemia and Na+, K+ -ATPase activity in thyrotoxic periodic paralysis

OBJECTIVE Thyrotoxic periodic paralysis (TPP) usually follows a heavy carbohydrate meal and this may be explained by hyperinsulinaemia stimulating Na⁺, K⁺ -ATPase activity. To clarify this the effect of glucose load on serum insulin concentration and platelet Na⁺, K⁺ -ATPase activity In thyrotoxic periodic paralysis (TPP) was examined. DESIGN In all subjects a standard 75-g glucose tolerance test was done and blood samples were taken at 0, 1 and 2 hours. SUBJECTS Twenty-five healthy controls (8 M and 17 F), 17 uncomplicated thyrotoxic patients (7M and 10 F), 15 TPP patients who presented with paralysis and 4 TPP patients after treatment with antithyrold drugs. MEASUREMENTS Plasma glucose was measured by the glucose oxidase method, serum insulin by radioimmunoassay and platelet Na⁺, K⁺ -ATPase by the release of phosphate from ATP. RESULTS TPP patients showed glucose intolerance (area under the curve (AUC) 16·5 ± 4·4 (mean ± SD) In TPP compared to 12·9 ± 4·5 In controls (P < 0·01) and hyperinsulinaemia (AUC 189·6 ±100·6 vs 98·5 ±53·4, P < 0·001). In uncomplicated thyrotoxicosis the results were similar to that in healthy controls. Platelet Na⁺, K⁺ -ATPase were significantly higher in thyrotoxic patients compared to controls and In TPP patients were even higher. Ingestion of glucose increased platelet Na⁺, K⁺ -ATPase in all groups. AUC for platelet Na⁺, K⁺ -ATPase in TPP patients were significantly higher than in uncomplicated thyrotoxicosis (601 ±99·3 vs 482 ± 109·4, P < 0·01) or healthy controls (320 ± 107·3). In the 4 TPP patients studied after antithyroid treatment the results were similar to healthy controls. CONCLUSION Patients with thyrotoxic periodic paralysis have hyperinsulinaemia and this is accompanied by higher Na⁺, K⁺-ATPase activity.     

Na+/H+ antiporter properties in peripheral blood lymphocytes from normotensive obese and type 2 diabetic patients do not differ significantly from controls

It has been hypothesized that some genetic factors link different conditions characterized by the presence of insulin resistance: among them, obesity, type 2 (non-insulin-dependent) diabetes mellitus and arterial hypertension. A good candidate could be the Na⁺/H⁺ exchanger, the increased activity of which is considered a genetic marker of essential hypertension. In this study we looked at whether the Na⁺ dependence of the Na⁺/H⁺ antiporter is modified in obese and type 2 diabetic patients, in the absence of arterial hypertension. The activity of this ion exchanger was measured in peripheral blood lymphocytes by acidifying them in Na⁺-free buffer and then monitoring the recovery of intracellular pH after Na⁺ addition. Quiescent lymphocytes were used because they do not have insulin receptors, thus ruling out the effects of the elevated insulin concentrations on the Na⁺/H⁺ exchanger activity. Antiport activity, measured as the ability to extrude H⁺ in the presence of external Na⁺, showed no differences in normotensive obese and type 2 diabetic patients when compared with healthy subjects. Our data therefore suggest that an altered Na⁺/H⁺ exchange activity cannot be considered a common feature of insulin-resistant states.     

Functional and Cellular Regulation of the Myocardial Na+/H+ Exchanger

The Na⁺/H⁺ exchanger is a pH-regulatory protein present in the plasma membrane of cardiomyocytes and other cell types. In response to intracellular acidosis, the protein removes one intracellular proton in exchange for an extracellular sodium. The protein consists of a membrane transporting domain and a regulatory cytosolic domain. The regulatory cytosolic domain mediates the stimulation of the membrane domain. Hormonal stimulation of myocardial cells results in activation of the antiporter, possibly through protein kinases and other regulatory proteins. Several hormones and growth factors have been shown to stimulate the antiporter in the myocardium, including endothelin, thrombin, angiotensin II, and ₁-adrenergic stimulation. The exact mechanisms involved in this stimulation are as yet unclear, and may be important in regulation of the Na⁺/H⁺ exchanger during ischemia and reperfusion.     

Enhanced red cell sodium-hydrogen exchange in microvascular angina

OBJECTIVES: Enhanced calcium content in arterial smooth muscle cells andaltered reactivity of coronary vessels to alkalinization havebeen reported in angina pectoris due to impaired motility ofcoronary arteries. An altered function of sodium-hydrogen exchange,a ubiquitous membrane transport system that links proton effluxto calcium drifts, may mediate these phenomena. DESIGN AND SUBJECTS: Twenty patients with microvascular angina (stable effort angina,reversible perfusion defects during effort thallium 201 heartscintigraphy, and angio-graphically normal coronary arteries)were compared to 20 patients with stable effort angina due tocoronary atherosclerosis and 20 healthy subjects. The sodium-hydrogenexchange was defined as the initial fraction of the amiloride-sensitiveproton efflux from red cells with inhibited anion exchanger(pHi 6·00–6·05) into an Na⁺-containing medium(pHo 8·00–8·05). 12-0-tetradecanoylphorbol-13-acetate(TPA, 600 nmol. 1^–1) and staurosporine (100 nmol. 1^–1)were used as phosphorylation modulators in vitro. RESULTS: The mean red blood cell Na⁺/H⁺ exchange was increased in patientswith microvascular angina (451±37 vs 142±17 and124±21 µmol H⁺. 1 cells^–1. min^–1, P<0·01).TPA and staurosporine abolished differences between the groups. CONCLUSION: Microvascular angina is associated with enhanced Na⁺/H⁺ exchangein erythrocytes, probably due to more extensive phosphorylationof the membrane antiporter sites.     

The role of Na+/H+ exchange in ischemia-reperfusion

In ischemia the cytosol of cardiomyocytes acidifies; this is reversed upon reperfusion. One of the major pH_i-regulating transport systems involved is the Na⁺/H⁺ exchanger. Inhibitors of the Na⁺/H⁺ exchanger have been found to more effectively protect ischemic-reperfused myocardium when administered before and during ischemia than during reperfusion alone. It has been hypothesized that the protection provided by pre-ischemic administration is due to a reduction in Na⁺ and secondary Ca²⁺ influx. Under reperfusion conditions Na⁺/H⁺ exchange inhibition also seems protective since it prolongs intracellular acidosis which can prevent hypercontracture. In detail, however, the mechanisms by which Na⁺/H⁺ exchange inhibition provides protection in ischemic-reperfused myocardium are still not fully identified.     

Altered expression of Na+ transporters at the mRNA level in rat normal and hypertrophic myocardium

Intracellular Na⁺ ([Na⁺]_i) regulation plays a crucial role in the structural, mechanical, and electrical properties of myocardium. It is assumed that the [Na⁺]_i handling system may differ not only between normal and diseased hearts but also regionally within a heart. To gain new insight concerning disease- and region-dependent differences in the [Na⁺]_i-regulatory system, we investigated mRNA expression of Na+ transporters, the principal determinants of [Na⁺]_i. Nonischemic pressure-overloaded hypertrophy was created by suprarenal abdominal aortic constriction of 50% for 7 weeks. mRNA abundances of Na⁺-Ca²⁺ exchanger (NCX1), Na⁺-H⁺ exchanger (NHE1), Na⁺-K⁺-2Cl⁻ exchanger (NKCC1) and Na⁺, K⁺-ATPase multigene family(α₁, α₂, α₃, and β₁ isoforms) were measured by the real-time quantitative polymerase chain reaction method. mRNA abundance of all transporters mediating Na⁺ influx (NCX1, NHE1, and NKCC1) was significantly upregulated as compared to normal. In contrast, Na⁺-efflux-mediating transporter (Na⁺, K⁺-ATPase) mRNA expression was unaltered between normal and hypertrophic hearts. Losartan, an angiotensin II AT1 receptor antagonist, significantly attenuated upregulation of Na⁺-influx-mediating transporters induced by aortic constriction. The onset of Na⁺-influx-mediating transporter upregulation occurred within 5 days following constriction. In normal and hypertrophied hearts, mRNA of all Na⁺-influx-mediating transporters was expressed in order of abundance as: apex > septum ∼ free wall of left ventricles. A transmural gradient in expression was also evident in normal hearts (midcardium > endo- and epicardium), which was attenuated under hypertrophic development. Myocardial hypertrophy is associated with significant changes in the spatial distribution and expression levels of Na⁺ transporters. The upregulation of Na influx transporters during hypertrophy may contribute to the remodeling process, modulate contractility and promote arrhythmias.     

Insulin stimulation of K+ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta

Summary Despite the important physiological role of insulin in the regulation of ionic homeostasis, primarily mediated by the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter, the intracellular signalling molecules mediating this effect of insulin have not been elucidated. Treatment of 3T3-L1 fibroblasts with insulin increased total ⁸⁶Rb⁺ (K⁺) uptake from 0.8 ± 0.04 to 1.02 ± 0.05 nmol · mg^–1· protein^–1· min^–1 (p < 0.005). These changes in K⁺ flux, though small, can alter the membrane potential. Uptake occurred through both the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter and both were stimulated by insulin. Interestingly, when bumetanide was used to inhibit the Na⁺/K⁺/2Cl^– cotransporter prior to insulin action, no increase in ⁸⁶Rb⁺ uptake via the Na⁺/K⁺-ATPase was observed. The structurally distinct phosphatidylinositol 3-kinase inhibitors wortmannin (50–200 nmol/l) and LY294 002 (50 μmol/l) attenuated both total insulin-stimulated ⁸⁶Rb⁺ uptake as well as uptake via the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter. Neither the inhibitor of p70 S6 kinase activation, rapamycin (30 ng/ml) nor the mitogen activated protein kinase kinase inhibitor, PD098 059 (50 μmol/l), had any effect on insulin's stimulation of K⁺ influx. A 10 μmol/l concentration of the protein kinase C (PKC) inhibitor bisindolylmaleimide attenuated insulin action but at 1 μmol/l it was ineffective, suggesting involvement of the atypical PKC-ζ isoform. We conclude that insulin-stimulated K⁺ uptake in 3T3-L1 fibroblasts appears to involve concerted regulation of both the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter and we show for the first time that this process is signalled via a pathway involving phosphatidylinositol 3-kinase and PKC-ζ. [Diabetologia (1998) 41: 1199–1204] Received: 20 March 1998 and in revised form: 2 June 1998     

Aldosterone increases Na+ -K+ -ATPase activity in skeletal muscle of patients with Conn's syndrome

Objective In Conn’s syndrome, hypokalaemia normally results from renal potassium loss because of the effect of excess aldosterone on Na⁺‐K⁺‐ATPase in principal cells. Little is known about the effect of aldosterone on cellular potassium redistribution in skeletal muscle. Our study determined the effect of aldosterone on muscle Na⁺‐K⁺‐ATPase. Design Muscle biopsies were taken from six patients immediately before and 1 month after adrenalectomy. Ten age‐matched subjects with normal levels of circulating aldosterone served as controls. Results Average plasma aldosterone was significantly higher in presurgery (235·0 ± 51·1 pg/ml) than postsurgery (64·5 ± 25·1 pg/ml) patients. Similarly, Na⁺‐K⁺‐ATPase activity, relative mRNA expression of α₂ (not α₁ or α₃) and β₁ (not β₂ or β₃), and protein abundance of α₂ and β₁ subunits were greater in pre‐ than postsurgery samples (128·7 ± 12·3 vs 79·4 ± 13·3 nmol·mg/protein/h, 2·45 ± 0·31 vs 1·04 ± 0·17, 1·92 ± 0·22 vs1·02 ± 0·14, 2·17 ± 0·33 vs 0·98 ± 0·09 and 1·70 ± 0·17 vs 0·90 ± 0·17, respectively, all P < 0·05). The activity and mRNA expression of the α₂ and β₁ subunits correlated well with plasma aldosterone levels (r = 0·71, r = 0·75 and r = 0·78, respectively, all P < 0·01). Conclusions Our study provides the first evidence in human skeletal muscle that increased plasma aldosterone leads to increased Na⁺‐K⁺‐ATPase activity via increases in α₂ and β₁ subunit mRNAs and their protein expressions. The increased activity may contribute in part to the induction of hypokalaemia in patients with Conn’s syndrome.     

Acute effects of glucose and insulin on vascular endothelium

Aims/hypothesis Chronic exposure to high concentrations of glucose has consistently been demonstrated to impair endothelium-dependent, nitric oxide (NO)-mediated vasodilation. In contrast, several clinical investigations have reported that acute exposure to high glucose, alone or in combination with insulin, triggers vasodilation. The aim of this study was to examine whether elevated glucose itself stimulates endothelial NO formation or enhances insulin-mediated endothelial NO release.Methods We measured NO release and vessel tone ex vivo in porcine coronary conduit arteries (PCAs). Intracellular Ca²⁺ was monitored in porcine aortic endothelial cells (PAECs) by fura-2 fluorescence. Expression of the Na⁺/glucose cotransporter-1 (SGLT-1) was assayed in PAECs and PCA endothelium by RT-PCR.Results Stimulation of PCAs with d-glucose, but not the osmotic control l-glucose, induced a transient increase in NO release (EC₅₀10 mmol/l), mediated by a rise in intracellular Ca²⁺ levels due to an influx from the extracellular space. This effect was abolished by inhibitors of the plasmalemmal Na⁺/Ca²⁺ exchanger (dichlorobenzamil) and the SGLT-1 (phlorizin), which was found to be expressed in aortic and coronary endothelium. Alone, d-glucose did not relax PCA, but did augment the effect of insulin on NO release and vasodilation.Conclusions/interpretation An increased supply of extracellular d-glucose appears to enhance the activity of the endothelial isoform of nitric oxide synthase by increasing intracellular Na⁺ concentrations via SGLT-1, which in turn stimulates an extracellular Ca²⁺ influx through the Na⁺/Ca²⁺ exchanger. This mechanism may be responsible for glucose-enhanced, insulin-dependent increases in tissue perfusion (including coronary blood-flow), thus accelerating glucose extraction from the blood circulation to limit the adverse vascular effects of prolonged hyperglycaemia.     

Insulin resistant subjects lack islet adaptation to short-term dexamethasone-induced reduction in insulin sensitivity

Aims/hypothesis. To establish whether islet compensation to deterioration of insulin action depends on inherent insulin sensitivity. Methods. We examined insulin and glucagon secretion after iv arginine (5 g) at fasting, 14 and greater than 25 mmol/l glucose concentrations before and after lowering of insulin sensitivity by oral dexamethasone (3 mg twice daily for 2 1/2 days) in 10 women with normal glucose tolerance, aged 58 or 59 years. Five women had high insulin sensitivity as shown by euglycaemic, hyperinsulinaemic clamp (99 ± 12 nmol glucose · kg body weight^–1· min^–1/pmol insulin · l^–1; means ± SD) whereas five women had low insulin sensitivity (34 ± 15 nmol glucose · kg body weight^–1· min^–1/pmol insulin · l^–1). Results. Dexamethasone reduced insulin sensitivity in both groups. Fasting insulin concentration increased by dexamethasone in high insulin sensitivity (72 ± 10 vs 49 ± 9 pmol/l, p = 0.043) but not in low insulin sensitivity (148 ± 63 vs 145 ± 78 pmol/l) whereas the fasting glucose concentration increased in low insulin sensitivity (6.5 ± 0.8 vs 5.8 ± 0.6 mmol/l, p = 0.043) but not in high insulin sensitivity (5.3 ± 0.8 vs 5.3 ± 0.6 mmol/l). Fasting glucagon concentration was not changed. Plasma insulin concentrations after raising glucose to 14 and more than 25 mmol/l and the insulin response to arginine at more than 25 mmol/l glucose were increased by dexamethasone in high insulin sensitivity (p < 0.05) but not changed by dexamethasone in low insulin sensitivity. Furthermore, in high but not in low insulin sensitivity, dexamethasone reduced the glucagon response to arginine (p = 0.043). Conclusion/interpretation. The results show that adaptation in islets function to dexamethasone-induced short-term reduction in insulin sensitivity is lacking in subjects with low inherent insulin sensitivity. [Diabetologia (1999) 42: 936–943] Received: 26 January 1999 and in revised form: 1 March 1999     

Hyperlipidaemia is associated with increased insulin-mediated glucose metabolism, reduced fatty acid metabolism and normal blood pressure in transgenic mice overexpressing human apolipoprotein C1

Aims/hypothesis. Insulin resistance for glucose metabolism is associated with hyperlipidaemia and high blood pressure. In this study we investigated the effect of primary hyperlipidaemia on basal and insulin-mediated glucose and on non-esterified fatty acid (NEFA) metabolism and mean arterial pressure in hyperlipidaemic transgenic mice overexpressing apolipoprotein C1 (APOC1). Previous studies have shown that APOC1 transgenic mice develop hyperlipidaemia primarily because of an impaired hepatic uptake of very low density lipoprotein (VLDL). Methods. Basal and hyperinsulinaemic (6 mU · kg^–1· min^–1), euglycaemic (7 mmol/l) clamps with 3-³H-glucose or 9,10-³H-palmitic acid infusions and in situ freeze clamped tissue collection were carried out. Results. The APOC1 mice showed increased basal plasma cholesterol, triglyceride, NEFA and decreased glucose concentrations compared with wild-type mice (7.0 ± 1.2 vs 1.6 ± 0.1, 9.1 ± 2.3 vs 0.6 ± 0.1, 1.9 ± 0.2 vs 0.9 ± 0.1 and 7.0 ± 1.0 vs 10.0 ± 1.1 mmol/l, respectively, p < 0.05). Basal whole body glucose clearance was increased twofold in APOC1 mice compared with wild-type mice (18 ± 2 vs 10 ± 1 ml · kg^–1· min^–1, p < 0.05). Insulin-mediated whole body glucose uptake, glycolysis (generation of ³H₂O) and glucose storage increased in APOC1 mice compared with wild-type mice (339 ± 28 vs 200 ± 11; 183 ± 39 vs 128 ± 17 and 156 ± 44 vs 72 ± 17 μmol · kg^–1· min^–1, p < 0.05, respectively), corresponding with a twofold to threefold increase in skeletal muscle glycogenesis and de novo lipogenesis from 3-³H-glucose in skeletal muscle and adipose tissue (p < 0.05). Basal whole body NEFA clearance was decreased threefold in APOC1 mice compared with wild-type mice (98 ± 21 vs 314 ± 88 ml · kg^–1· min^–1, p < 0.05). Insulin-mediated whole body NEFA uptake, NEFA oxidation (generation of ³H₂O) and NEFA storage were lower in APOC1 mice than in wild-type mice (15 ± 3 vs 33 ± 6; 3 ± 2 vs 11 ± 4 and 12 ± 2 vs 22 ± 4 μmol · kg^–1· min^–1, p < 0.05) in the face of higher plasma NEFA concentrations (1.3 ± 0.3 vs 0.5 ± 0.1 mmol/l, p < 0.05), respectively. Mean arterial pressure and heart rate were similar in APOC1 vs wild-type mice (82 ± 4 vs 85 ± 3 mm Hg and 459 ± 14 vs 484 ± 11 beats · min^–1). Conclusions/interpretation. 1) Hyperlipidaemic APOC1 mice show reduced NEFA and increased glucose metabolism under both basal and insulin-mediated conditions, suggesting an intrinsic defect in NEFA metabolism. Primary hyperlipidaemia alone in APOC1 mice does not lead to insulin resistance for glucose metabolism and high blood pressure. [Diabetologia (2001) 44: 437–443] Received: 14 September 2000 and in revised form: 23 November 2000     

Transport proteins NHA1 and NHA2 are essential for survival,but have distinct transport modalities

The cation/proton antiporter (CPA) family includes the well-known sodium/proton exchanger (NHE; SLC9A) family of Na⁺/H⁺ exchangers, and the more recently discovered and less well understood CPA2s (SLC9B), found widely in living organisms. In Drosophila, as in humans, they are represented by two genes, Nha1 (Slc9b1) and Nha2 (Slc9b2), which are enriched and functionally significant in renal tubules. The importance of their role in organismal survival has not been investigated in animals, however. Here we show that single RNAi knockdowns of either Nha1 or Nha2 reduce survival and in combination are lethal. Knockdown of either gene alone results in up-regulation of the other, suggesting functional complementation of the two genes. Under salt stress, knockdown of either gene decreases survival, demonstrating a key role for the CPA2 family in ion homeostasis. This is specific to Na⁺ stress; survival on K⁺ intoxication is not affected by sodium/hydrogen antiporter (NHA) knockdown. A direct functional assay in Xenopus oocytes shows that Nha2 acts as a Na⁺/H⁺ exchanger. In contrast, Nha1 expressed in Xenopus oocytes shows strong Cl⁻ conductance and acts as a H⁺-Cl⁻ cotransporter. The activity of Nha1 is inhibited by chloride-binding competitors 4,4′-diiso-thiocyano-2,2′-disulfonic acid stilbene and 4,4′-dibenzamido-2,2′-stilbenedisulphonate. Salt stress induces a massive up-regulation of NHA gene expression not in the major osmoregulatory tissues of the alimentary canal, but in the crop, cuticle, and associated tissues. Thus, it is necessary to revise the classical view of the coordination of different tissues in the coordination of the response to osmoregulatory stress.     

Differential Effects of Amiloride on the Basal Rate and the Pressure Overload-induced Increase in Protein Synthesis in Perfused Rat Heart

The purposes of the present study were to determine the contribution of Na⁺/H⁺ exchange to pressure overload-induced cardiac hypertrophy and to examine its potential interaction with cAMP-dependent signaling pathway.

Isolated rat hearts were perfused as Langendorff preparations with aortic pressure of 60 mmHg. In pressure overload group, aortic pressure was increased to 120 mmHg. cAMP contents in the heart perfused at 2 min were examined by RIA. Rates of protein synthesis were examined by ¹⁴C-phenylalanine incorporation into myocardial protein during the second hour of perfusion. Expression of c-fos mRNA in the heart perfused at 1 hour was analyzed by Northern blotting.

Elevation of aortic pressure from 60 mmHg to 120 mmHg in perfused rat hearts increased cAMP contents from 4.89±0.09 to 6.30±0.28 pmol/mg protein and accelerated rates of protein synthesis from 644±13 to 860±49 nmol Phe/g dry heart/hr. Expression of c-fos mRNA was induced by elevated aortic pressure. Amiloride, an inhibitor of Na⁺/H⁺ exchange, decreased rates of protein synthesis in a concentration-dependent manner (12.5, 25, 50, 100 μM) but did not change cAMP content (5.25±0.11 pmol/mg protein) or expression of c-fos mRNA. Furthermore, amiloride did not prevent the increases in cAMP (6.99±0.34 pmol/mg protein), protein synthesis rates (476±18 to 689±31 nmolPhe/g dry heart/hr) and expressions of c-fos mRNA that were induced by elevation of aortic pressure.     

Alteration of Na+ homeostasis as a critical step in the development of irreversible hepatocyte injury after adenosine triphosphate depletion

The exposure of isolated hepatocytes to the redox-cycling quinone menadione caused an early loss of mitochondrial membrane potential, adenosine triphosphate (ATP) depletion, and decreased intracellular pH. These alterations were followed by an increase in intracellular Na⁺ and, ultimately, cell death. If HCO₃⁻ was omitted from the incubation buffer, or the hepatocytes were incubated in an acidic medium (pH 6.5) the accumulation of Na⁺ was markedly reduced. Inhibition of the Na⁺/H⁺ exchanger and of the Na⁺/HC0₃⁻ cotransporter by, respectively, amiloride and 4,4′-di-isothiocyano-2,2′-disulfonic acid stilbene (DIDS) suppressed the initial Na⁺ influx but did not prevent subsequent Na⁺ accumulation, because amiloride and DIDS inhibited the Na⁺/K⁺ pump. The omission of HCO₃⁻ from the extracellular medium or the incubation in acidic conditions also prevented menadione toxicity, without interfering with the loss of mitochondrial membrane potential and with ATP depletion. A similar protection was evident when hepatocytes were incubated with menadione in a medium without Na⁺. The preservation of adequate levels of ATP by supplementing hepatocytes with fructose allowed the initial Na⁺ load to be recovered and provided partial protection against menadione toxicity. These effects were suppressed if Na⁺/K⁺-ATPase was inhibited with ouabain. Taken together, these results indicated that the activation of the Na⁺/HCO₃⁻ cotransporter and of the Na⁺/K⁺ exchanger in response to the decrease of intracellular pH stimulated an enhanced influx of Na⁺. When the activity of the Na⁺/K⁺ pump was not able to control Na⁺ levels because of ATP depletion, such an uncontrolled Na⁺ influx precipitated irreversible injury and caused hepatocyte death.     

23Na NMR demonstrates prolonged increase of intracellular sodium following transient regional ischemia in the in situ pig heart

This study tests the hypothesis that Na⁺ _i increases during regional ischemia in the in situ pig heart. An extracorporeal shunt was created between the carotid artery and the left anterior descending artery of 14 open chest pigs. ²³Na and ³¹P NMR spectroscopy measured myocardial Na⁺ _i and high energy phosphates (HEPs). The protocol consisted of three 40 min periods: pre-ischemia (shunt pressure, 76±23 mmHg (S.D.)), ischemia (shunt pressure, 25±7 mmHg), and post-ischemia (shunt pressure, 53±11 mmHg). The pre-ischemia Na⁺ _i concentration was 6.7±4.2 mM. Phosphocreatine (PCr) was 15.3±0.5 mM, ATP 9.4±0.4 mM, inorganic phosphate (Pi) 1.5±0.2 mM, and pH_i 7.16±0.09. At the end of ischemia Na⁺ _i had increased to 10.5±2.8 mM (p<0.0002); PCr decreased to 5.9±2.1 mM (p<0.0002); ATP was 6.5±0.5 mM (p<0.003); Pi had increased to 6.3±1.0 mM (p<0.0002), and pH_i was 6.41±0.06 (p<0.0002). During the first 10 min of the reperfusion, Na⁺ _i increased further to 12.4±2.8 mM (p<0.025), whereas HEPs all returned to pre-ischemic values. Na⁺ _i increases during regional ischemia in the in situ pig heart, suggesting reduced Na⁺/K⁺ ATPase activity. While ATP probably does not limit Na⁺/K⁺ ATPase activity, increases in Pi and decreases in pH_i may reduce Na⁺/K⁺ ATPase activity. Additional Na⁺ _i increases during reperfusion suggest either augmented Na⁺ influx or decreased Na⁺ efflux. Received: 25 May 1998, Returned for revision: 22 June 1998, Revision received: 20 August 1998, Accepted: 15 September 1998     

Regulation of myocardial Na+/H+ exchanger activity

The Na⁺/H⁺ exchanger is a plasma membrane protein, present in the myocardium, which removes intracellular protons and exchanges them with extracellular Na⁺. The protein comprises an N-terminal, hydrophobic, integral membrane domain that transports the ions and a C-terminal, hydrophilic region that regulates the N-terminal domain. The C-terminal domain has several sub-domains, including one region that binds calmodulin and another that is phosphorylated by protein kinases. The Na⁺/H⁺ exchanger is activated by angiotensin, endothelin and α1-adrenergic stimulation. These effectors increase phosphorylation of the C-terminal domain by protein kinases, and G proteins have been implicated in this, but their role remains to be defined. It has recently been shown that ischemia and other stimuli lead to an increased expression of the Na⁺/H⁺ exchanger in the myocardium. The role of this increased expression in the pathology of ischemia and reperfusion-mediated myocardial damage has yet to be determined. Recent evidence suggests that the Na⁺/H⁺ exchanger may play a key role in hypertrophy of the myocardium, and that its activation through G protein-coupled receptors may be important in mediating its effects. Received: 23 April 2001 / Accepted: 14 May 2001