Effects of Na(+)-HCO3- symport on ischemia/reperfusion injury; Na+/H+ exchanger inhibitor and buffer composition

We studied the impact of perfusate buffer composition on the relative degree of protection afforded by Na+/H+ exchanger (NHE) inhibition during ischemia as opposed to during reperfusion. Isolated rat hearts were perfused with bicarbonate- or HEPES-buffered medium. There was infusion of HOE 694 immediately before ischemia, during initial reperfusion, or during both of these periods. With bicarbonate-buffered medium, HOE 694 improved the post-ischemic recovery of left ventricular developed pressure (LVDP) when given before ischemia and before ischemia plus during reperfusion. In the presence of HEPES-buffered medium, however, HOE 694 significantly improved recovery of LVDP in all protocols. HOE 694 also provided an almost complete recovery of LVDP (88 +/- 9% vs 30 +/- 7% in controls) when given before ischemia plus during reperfusion. In conclusion, our results suggest that the influence of NHE activity during reperfusion on the extent of functional recovery is modulated significantly by perfusate buffer composition.     

Ischemic preconditioning and Na+/H+ exchange inhibition improve reperfusion ion homeostasis

BACKGROUND: Intramyocyte sodium (Na+) increases during ischemia and reperfusion, which causes myocardial calcium (Ca2+) uptake and leads to myocyte injury or death. This study determines if ischemic preconditioning and myocyte sodium-hydrogen ion (Na+-H+) exchange (NHE) inhibition decreases Na+ gain that otherwise occurs with cardioplegic arrest and reperfusion. METHODS: Pigs had 1 hour of cardioplegic arrest followed by reperfusion. Group 1 had no intervention (controls). Group 2 received dimethyl amiloride (DMA, an NHE inhibitor), and group 3 had ischemic preconditioning before cardioplegic arrest. Precardioplegia to postreperfusion change in intramyocyte ion content was measured with atomic absorption spectrometry. The time to initial electrical activity and number of defibrillations needed to establish an organized rhythm postreperfusion were used as electrophysiologic variables to measure ischemia-reperfusion injury. RESULTS: Intramyocyte Na+ content for group 1 increased from 45.9+/-6.7 to 61.9+/-22.5 micromol/g (p = 0.02). Group 2 had an insignificant decrease in intramyocyte Na+ of 27.7+/-19.58 micromol/g (p = 0.06), and group 3 had an insignificant decrease of 10.8+/-46.33 micromol/g (p = 0.48). Interstitial water increased significantly in all groups, but there were no significant increases in intramyocyte water content. Electrophysiologic recovery was similar for all three groups. CONCLUSIONS: The NHE inhibition and ischemic preconditioning each eliminated the increase in intramyocyte Na+ content that otherwise occurred with cardioplegic arrest and reperfusion in this porcine model. Because their mechanisms are distinct, it is possible that an additive beneficial effect against ischemia-reperfusion injury can be achieved by using NHE inhibition together with a preconditioning stimulus as prereperfusion therapy.     

Long-term myocardial preservation: effects of hyperkalemia, sodium channel, and Na/K/2Cl cotransport inhibition on extracellular potassium accumulation during hypothermic storage.

OBJECTIVES: We previously demonstrated improved myocardial preservation with polarized (tetrodotoxin-induced), compared with depolarized (hyperkalemia-induced), arrest and hypothermic storage. This study was undertaken to determine whether polarized arrest reduced ionic imbalance during ischemic storage and whether this was influenced by Na+/K +/2Cl- cotransport inhibition. METHODS: We used the isolated crystalloid perfused working rat heart preparation (1) to measure extracellular K+ accumulation (using a K+-sensitive intramyocardial electrode) during ischemic (control), depolarized (K+ 16 mmol/L), and polarized (tetrodotoxin, 22 micromol/L) arrest and hypothermic (7.5 degrees C) storage (5 hours), (2) to determine dose-dependent (0.1, 1.0, 10 and 100 micromol/L) effects of the Na +/K+/2Cl- cotransport inhibitor, furosemide, on extracellular K+ accumulation during polarized arrest and 7.5 degrees C storage, and (3) to correlate extracellular K+ accumulation to postischemic recovery of cardiac function. RESULTS: Characteristic triphasic profiles of extracellular K+ accumulation were observed in control and depolarized arrested hearts; a significantly attenuated profile with polarized arrested hearts demonstrated reduced extracellular K+ accumulation, correlating with higher postischemic function (recovery of aortic flow was 54% +/-4% [P =.01] compared with 39% +/-3% and 32% +/-3% in depolarized and control hearts, respectively). Furosemide (0.1, 1.0, 10, and 100 micromol/L) modified extracellular K+ accumulation by -18%, -38%, -0.2%, and +9%, respectively, after 30 minutes and by -4%, -27%, +31%, and +42%, respectively, after 5 hours of polarized storage. Recovery of aortic flow was 53% +/-4% (polarized arrest alone), 56% +/-8%, 70% +/-2% (P =.04 vs control), 69% +/-4% (P =.04 vs control), and 65% +/-3% ( P =. 04 vs control), respectively. CONCLUSIONS: Polarized arrest was associated with a reduced ionic imbalance (demonstrated by reduced extracellular K+ accumulation) and improved recovery of cardiac function. Further attenuation of extracellular K + accumulation (by furosemide) resulted in additional recovery.     

Inhibition of Na(+)/H(+) isoform-1 exchange protects hearts perfused after 6-hour cardioplegic cold storage.

OBJECTIVES: Cardiac ischemia-reperfusion activates Na(+)/H(+) exchange; excess Na(+) and the resulting Ca(2+) overload, through reverse Na(+)/Ca(2+) exchange, cause cellular injury and cardiac dysfunction. We postulated that inhibiting the Na(+)/H(+) isoform-1 exchanger would add to the protection of hearts after long-term cold storage in acidic cardioplegic solution. METHODS: Guinea pig hearts were isolated and perfused at 37 degrees C with Krebs-Ringer's solution (KRS) and then switched to an acidic St. Thomas solution (STS) at 25 degrees C. Perfusion was stopped at 10 degrees C, and hearts were stored for 6 hours in STS at 3.4 degrees C. On reperfusion to 25 degrees C, hearts were perfused with KRS for 60 minutes. Hearts were divided into 4 groups: sham control (SHAM); eniporide (EPR, EMD96785) IV, 1 mg/kg given IV over 15 minutes before heart isolation; EPR intracoronary, 1 micromol/liter in STS given intracoronary after heart isolation; and EPR IV and intracoronary. RESULTS: Values at 60 minutes reperfusion (the percentage of control [100%] before cold storage) are given, respectively, for EPR IV, EPR intracoronary, and EPR IV and intracoronary vs drug-free SHAM (SEM, *p < 0.05 vs SHAM): 72% +/- 3%*, 65% +/- 3%*, and 81% +/- 2%* vs 55% +/- 3% for left ventricular pressure; 94% +/- 3%*, 96% +/- 5%*, and 102% +/- 2%* vs 81% +/- 3% for coronary flow; 60% +/- 2%, 58% +/- 3%, and 74%* +/- 3% vs 58% +/- 4% for cardiac efficiency; 106% +/- 2%*, 108% +/- 3%*, and 107% +/- 2%* vs 116% +/- 4% for percentage of O(2) extraction. Infarct size as percentage of ventricular weight was 20% +/- 3%*, 31% +/- 3%, and 6% +/- 2%* vs 35% +/- 3% (SHAM) after 60 minutes of reperfusion. CONCLUSIONS: Na(+)/H(+) isoform-1 exchanger inhibition, particularly if given IV before storage and intracoronary during cooling and rewarming, adds to the protection of cardioplegic solutions.     

Hypertension, Na+/Ca2+ exchanger, and Na+, K+-ATPase

Hypertension is the most prevalent risk factor for stroke, myocardial infarction, or end-stage renal failure. The critical importance of excess salt intake in the pathogenesis of hypertension is widely recognized, but the mechanisms whereby salt intake elevates blood pressure have puzzled researchers. Recent studies using Na+/Ca2+ exchange inhibitors and genetically engineered mice provide evidence that vascular Na+/Ca2+ exchanger type 1 (NCX1) is involved in the development of salt-dependent hypertension. Endogenous cardiac glycosides, which may contribute to salt-dependent hypertension, seem to be necessary for NCX1-mediated hypertension. Intriguingly, studies using knock-in mice with modified cardiac glycoside binding affinity of Na+,K+-ATPases provide a clear demonstration that this cardiac glycoside-binding site plays an important role in blood pressure regulation. Taken all together: (1) endogenous cardiac glycosides are secreted after high salt intake; (2) these cardiac glycosides inhibit Na+,K+-ATPase in vascular smooth muscle cells; (3) this inhibition results in the elevation of local Na+ on the submembrane area; and (4) this elevation of local Na+ facilitates Ca2+ entry through NCX1, resulting in vasoconstriction. This proposed pathway may have enabled us to explain how to link dietary salt to hypertension.     

Effects of ifenprodil on voltage-gated tetrodotoxin-resistant Na+ channels in rat sensory neurons

BACKGROUND AND OBJECTIVE: To examine a possible mechanism for the antinociceptive action of the N-methyl-D-aspartate receptor antagonist ifenprodil, we compared its effects with those of ketamine on tetrodotoxin-resistant Na+ channels in rat dorsal root ganglion neurons, which play an important role in the nociceptive pain pathway. METHODS: Experiments were performed on dorsal root ganglion neurons from Sprague-Dawley rats, recordings of whole-cell membrane currents being made using patch-clamp technique. RESULTS: Both drugs blocked tetrodotoxin-resistant Na+ currents dose dependently, their half-maximal inhibitory concentrations being 145+/-12.1 micromol (ketamine) and 2.6+/-0.95 micromol (ifenprodil). Ifenprodil shifted the inactivation curve for tetrodotoxin-resistant Na+ channels in the hyperpolarizing direction and shifted the activation curve in the depolarizing direction. Use-dependent blockade of tetrodotoxin-resistant Na+ channels was more marked with ifenprodil than with ketamine. When paired with lidocaine, these drugs produced similar additive inhibitions of tetrodotoxin-resistant Na+ channel activity. CONCLUSIONS: The observed suppressive effects on tetrodotoxin-resistant Na+ channel activity may, at least in part, underlie the antinociceptive effects of these N-methyl-D-aspartate receptor antagonists.     

Choroid plexus Na+/K+-activated adenosine triphosphatase and cerebrospinal fluid formation

The quantitative relationship between intraventricular fluid formation and choroid plexus Na+/K+-activated (transport) adenosine triphosphatase (ATPase) was studied in rabbit and dog by perfusing the ventricular system with a solution containing ouabain (10(-8) to 10(-3) M). The effect of ouabain in the same range of concentrations on ATPase activity was also measured by the release of inorganic orthophosphate from in vitro choroid plexus tissue. Normally, about 20 to 25% of ATPase activity in lateral ventricle plexus is Na+/K+-activated, and this component is almost completely inhibited by ouabain in a concentration of 10(-4) M. At this concentration, the rate of intraventricular cerebrospinal fluid (CSF) formation is decreased some 70 to 80% in dog and rabbit. The results of this study suggest that a portion of the intraventricular fluid formation is insensitive to cardiac glycoside inhibition and that either there are two different mechanisms responsible for choroid plexus fluid formation or there is a significant non-ATPase dependent extrachoroidal source of CSF.     

Na+/H+ exchange inhibition improves post-transplant myocardial compliance in 4-hour stored donor hearts

Na⁺/H⁺ exchange inhibitors have cardioprotective properties. The effects of the new Na⁺/H⁺ exchange inhibitor, HOE642 on myocardial function were assessed after transplantation of canine brain-dead and non-brain-dead donor hearts preserved for 4 h. Four groups were studied: brain-dead donors; non-brain-dead donors; brain-dead donors and recipients treated with HOE642 (2 mg/kg); and treated non brain-dead donors and recipients. Donor hearts were stored in NIH2. At the end of 60 min reperfusion after transplantation, pressure–volume curves were constructed. Biopsies were analysed histologically and ultrastructurally. Afterwards, weaning from cardiopulmonary bypass was accomplished. HOE642 improved compliance in hearts from both brain-dead and non-brain-dead donors. No differences in myocardial water content nor in myocardial performance were detected. No irreversible damage was seen ultrastructurally. It is concluded that myocardial compliance after transplantation was improved by administration of HOE642. The use of this inhibitor might improve the current myocardial preservation technique for transplantation.     

Regulation of the Na+/H+ antiporter in patients with mild chronic renal failure: effect of glucose.

BACKGROUND: The aim of this study was to determine the glucose-dependent regulation of the sodium-proton-antiporter (Na+/H+ antiporter) in patients with mild chronic renal failure (CRF). METHODS: We measured plasma glucose concentrations, plasma insulin concentrations, plasma C peptide concentrations, arterial blood pressure, cytosolic pH (pHi), cellular Na+/H+ antiporter activity, and cytosolic sodium concentration ([Na+]i) in 19 patients with CRF and 41 age-matched healthy control subjects (control) during a standardized oral glucose tolerance test. Intracellular pHi, [Na+]i, and Na+/H+ antiporter activity was measured in lymphocytes using fluorescent dye techniques. RESULTS: Under resting conditions, the pHi was significantly lower, whereas the Na+/H+ antiporter activity was significantly higher in CRF patients compared with controls (each P < 0.0001). The oral administration of 100 g glucose significantly increased the Na+/H+ antiporter activity in CRF patients from 13.35 +/- 1.26 x 10-3 pHi/second to 16.44 +/- 1.37 x 10-3 pHi/second after one hour and to 14.06 +/- 1.36 x 10-3 pHi/second after two hours (mean +/- SEM, P = 0.008 by Friedmans's two-way analysis of variance). In controls, the administration of 100 g glucose significantly increased the Na+/H+ antiporter activity from 4.23 +/- 0.20 x 10-3 pHi/second to 6.00 +/- 0.56 x 10-3 pHi/second after one hour and to 6.65 +/- 0.64 x 10-3 pHi/second after two hours (P = 0.0003). The glucose-induced enhancement of the Na+/H+ antiporter activity was more pronounced in CRF patients compared with controls (P = 0.011). Resting [Na+]i was not significantly different between the two groups. CONCLUSIONS: CRF patients show an intracellular acidosis leading to an increased Na+/H+ antiporter activity. In addition, high glucose levels exaggerate the differences in Na+/H+ antiporter activity already present between cells from patients with mild CRF and those from control subjects.     

Differential regulation of collecting duct Na+, K+-ATPase and K+ excretion by furosemide and piretanide: role of bradykinin

In response to chronic treatment with furosemide, collecting ducts adapt their function to the initial loss of Na+ to prevent further Na+ loss and extracellular volume decrease. This adaptation, which includes the overexpression of Na+, K+-ATPase, is thought to account for most of the kaliuretic effect of furosemide. Because piretanide is reported to be less kaliuretic than equidiuretic doses of furosemide, the authors compared the effects of 1-wk treatment with the two loop diuretics on urinary potassium excretion and on Na+, K+-ATPase activity in the collecting duct. At equidiuretic and equinatriuretic doses, furosemide increased urinary potassium excretion as well as collecting duct Na+, K+-ATPase activity, whereas piretanide had no effect on either parameter. These effects of furosemide were curtailed by concomitant administration of the angiotensin-converting enzyme inhibitor enalapril, but they were not altered either by clamping changes in plasma aldosterone or by blocking type I angiotensin receptors. Treatment with the antagonist of bradykinin B2 receptors Hoe140 mimicked the two effects of furosemide. In addition, the effects of Hoe140 and furosemide were not additive. Finally, piretanide increased urinary bradykinin excretion, whereas furosemide did not. These results suggest that induction of collecting duct Na+, K+-ATPase (a) accounts for the kaliuretic effect of furosemide, (b) is independent of the renin/angiotensin/aldosterone system, (c) results from increased Na+ delivery to the collecting duct and enhanced intracellular Na+ concentration, and (d) is prevented in piretanide treated rats by increased bradykinin production that may limit apical Na+ entry in collecting duct principal cells.     

Effects of alpha2-adrenoceptor agonists on tetrodotoxin-resistant Na+ channels in rat dorsal root ganglion neurons

BACKGROUND AND OBJECTIVE: When intrathecally or epidurally administered, alpha2-adrenoceptor agonists produce potent antinociception by affecting the activity of primary afferent fibres and spinal cord neurons. Recent reports have indicated that in dorsal root ganglion neurons, tetrodotoxin-resistant Na+ channels play important roles in the conduction of nociceptive sensation. We therefore investigated the effects of alpha2-adrenoceptor agonists on tetrodotoxin-resistant Na+ currents. METHODS: Using the whole-cell patch-clamp technique, we recorded tetrodotoxin-resistant Na+ currents from rat dorsal root ganglion neurons. RESULTS: Both clonidine and dexmedetomidine reduced the peak amplitude of the tetrodotoxin-resistant Na+ current concentration- and use-dependently. The concentration required for a half-maximal effect was significantly lower for dexmedetomidine (58.0 +/- 10.2 micromol) than for clonidine (257.2 +/- 30.9 micromol) at holding potential -70 mV. The current inhibitions induced by these agonists were not prevented by 1 micromol yohimbine, an alpha2-adrenoceptor antagonist. Both clonidine and dexmedetomidine shifted the inactivation curve for the tetrodotoxin-resistant Na+ current in the hyperpolarizing direction. The combinations clonidine with lidocaine and dexmedetomidine with lidocaine produced an additive blockade-type interaction on the tetrodotoxin-resistant Na+ current. CONCLUSIONS: The results suggest that a direct inhibition of tetrodotoxin-resistant Na+ channels may contribute to the antinociceptive effects of clonidine and dexmedetomidine when used as additives to regional anaesthesia.     

pH dependence of Na+/myo-inositol cotransporters in rat thick limb cells

BACKGROUND: To balance medullary interstitium hypertonicity generated by transepithelial NaCl absorption, medullary thick ascending limb (MTAL) cells accumulate myo-inositol (MI). Expression of Na+-MI cotransporter (SMIT) mRNA in TAL is correlated with the NaCl absorption rate. Our present study aimed to determine the plasma membrane location and functional properties of the Na+-MI cotransporter in MTAL cells. METHODS: Preparation of basolateral (BLMV) and luminal (LMV) membrane vesicles were simultaneously isolated from purified rat MTAL suspension, and uptake of [3H]myo-inositol ([3H]MI) was used to assess Na+-MI cotransport activity. RESULTS: In the presence of an inside-negative membrane potential, imposing an inwardly-directed Na+-gradient versus tetramethylammonium (TMA) stimulated the initial [3H]MI uptake in BLMV and LMV. Phlorizin inhibited Na+ gradient-dependent initial [3H]MI uptake in both preparations, with IC50 values of 565 and 29 micromol/L in BLMV and LMV, respectively. 2-0,C-methylene myo-inositol (MMI), a competitive inhibitor of MI transport, only inhibited the BLMV Na+-MI cotransporter. Phlorizin-sensitive Na+ gradient-dependent initial [3H]MI uptake showed Michaelis-Menten kinetics in both preparations, with similar Vmax but different Km values of 51 and 107 micromol/L in BLMV and LMV, respectively. Finally, BLMV but not LMV Na+-MI cotransporter exhibited a marked pH dependence with sigmoidal patterns of activation, as intravesicular pH (pHi) was decreased from 8.0 to 6.0 at extravesicular pH (pHe) 8.0, and as pHe was increased from 6.0 to 8.0 at pHi 6.0. Maximal activation was observed at pHi 6.5 and pHe 7.5. CONCLUSIONS: In rat MTAL cells, Na+-MI cotransporter activity is present in both BLM and LM, and has markedly different functional properties, indicating the presence of distinct transporters. Basolateral Na+-MI cotransporter activity is maximal at physiological pH values of MTAL cells and interstitium, and a powerful modulation of the transporter activity may be exerted by pHe and pHi.     

Red cell Na+/Li+ countertransport and Na+/H+ exchanger isoforms in human proximal tubules

BACKGROUND: Increased activity of the Na+/Li+ countertransporter (SLC) is a well-recognized intermediate phenotype of hypertension and diabetic nephropathy and may indicate a predisposition to hypertension. Previous work has attempted to link this membrane transport marker to altered Na+ reabsorption in the proximal tubule. Since the Na+/H+ exchanger (NHE) isoforms 1 and 3 are expressed in the basolateral and apical membranes of the proximal tubule, respectively, we investigated the relationship between these transport proteins and red cell SLC to examine whether the peripheral blood transport phenotype is associated with altered levels of transport proteins in the proximal tubule. METHODS: Proximal tubules were prepared from human nephrectomy specimens. NHE-1 and NHE-3 were detected on Western blots by specific antibodies. Red cell SLC was also measured. RESULTS: Both NHE-1 and NHE-3 proteins were demonstrated, with molecular weights of 97 and 85 kD, respectively. SLC was very strongly correlated with the level of NHE-3 protein (r = 0.78, P < 0.001) and was negatively related to NHE-1 protein (r = -0.32). In multiple regression analysis, only NHE-3 and NHE-1 protein levels were significant predictors of red cell SLC, accounting for up to about 70% of the variance of this parameter. CONCLUSIONS: We conclude that red cell SLC may be a marker of increased NHE-3 protein expression in the proximal tubule, which may account for the blunted pressure natriuresis and predisposition to hypertension.     

Optimal dose and mode of delivery of Na+/H+ exchange-1 inhibitor are critical for reducing postsurgical ischemia-reperfusion injury

BACKGROUND: In clinical trials, perioperative intravenous Na(+)/H(+) exchange isoform-1 (NHE1) inhibitors were only moderately effective in high-risk patients undergoing surgical reperfusion (GUARDIAN trial). However, effective myocardial concentrations of NHE1 inhibitor may not have been achieved by parenteral administration alone. We tested the hypothesis that increasing doses of NHE1 inhibitor EMD 87580 ((2-methyl-4,5-di-(methylsulfonyl)-benzoyl)-guanidine) delivered in blood cardioplegia (BCP) and by parenteral route at reperfusion reduce myocardial injury after surgical reperfusion of evolving infarction. METHODS: Twenty-six anesthetized dogs underwent 75 minutes of left anterior descending coronary artery occlusion, followed by cardiopulmonary bypass and 60 minutes of arrest with multidose 10 degrees C BCP. In the control group (n = 8), BCP was not supplemented. In the three EMD-BCP groups, BCP was supplemented with 10 micromol/L EMD 87580 (EMD-10, n = 5), 20 micromol/L EMD 87580 (EMD-20, n = 5), or 20 micromol/L EMD 87580 combined with an immediate reperfusion bolus (5 mg/kg intravenously) (EMD-20R, n = 8). The left anterior descending coronary artery occlusion was released just before the second infusion of BCP. Reperfusion continued for 120 minutes after discontinuation of cardiopulmonary bypass. RESULTS: Postischemic systolic and diastolic function in the area at risk was dyskinetic in all groups. Infarct size (percentage of area at risk) was not significantly reduced in the EMD-10 (26.2% +/- 3.6%) and EMD-20 (22.5% +/- 2.4%) groups versus control (30.7% +/- 2.4%); however, infarct size was significantly reduced in the EMD-20R group (16.1% +/- 2.8%, p = 0.003). Edema in the area at risk in the EMD-10 (81.1% +/- 0.5% water content), EMD-20 (81.7% +/- 0.3%), and EMD-20R (81.9% +/- 0.3%) groups was less than in controls (83.2% +/- 0.2%), (p < 0.056). Neutrophil accumulation (myeloperoxidase activity) in postischemic area-at-risk myocardium was less in the EMD-20R group versus the control group (5.3 +/- 0.7 versus 8.7 +/- 1.4 absorbance units x min(-1) x g(-1); p = 0.05), which suggests an attenuated postischemic inflammatory response. CONCLUSIONS: Optimal delivery of NHE1 inhibitor to the heart through combined cardioplegia and parenteral routes significantly attenuates myocardial injury after surgical reperfusion of regional ischemia. Timing, dose, and mode of delivery of NHE1 inhibitors are important to their efficacy.     

Red cell Na+/Li+ countertransport in non-insulin-dependent diabetics with diabetic nephropathy

Genetic predisposition to essential hypertension, as indicated by increased maximal velocity of Na+/Li+ countertransport in red cells, has been suggested as a marker for the risk of developing diabetic nephropathy. To evaluate the validity of this concept in non-insulin-dependent diabetics, we measured the maximal velocity of Na+/Li+ countertransport in red cells in 18 male diabetics suffering from proteinuria due to biopsy proven diabetic glomerulosclerosis (GFR: 51 [range 27 to 146] ml/min/1.73 m2), 17 male diabetics with normoalbuminuria, and in 18 sex-, age-, and body mass index-matched healthy control subjects. Na+/Li+ countertransport was identical in patients with and without diabetic nephropathy, 0.43 (0.24 to 0.92) versus 0.44 (0.20 to 0.83) mmol/(liter cells x hr), but was elevated compared to control subjects, 0.32 (0.09 to 0.73; P less than 0.05). Arterial blood pressure was elevated in patients with nephropathy (162/92 +/- 21/9 mm Hg) compared to normoalbuminuric patients (132/82 +/- 15/7) and control subjects (133/83 +/- 14/7 mm Hg; P less than 0.001). Our study does not support the hypothesis that the risk of diabetic nephropathy in non-insulin-dependent diabetes is associated with a genetic predisposition to hypertension. Diabetes per se seems to enhance Na+/Li+ countertransport activity.     

The effects of Na movement on surgical myocardial protection: the role of the Na+-H+ exchange system and Na-channel in the development of ischemia and reperfusion injury.

OBJECTIVES: We investigated whether the Na+-H+ exchange inhibitor, HOE642 (Hoe), and/or the Na channel blocker, mexiletine (Mex), enhance a cardioprotective effect on St. Thomas' Hospital cardioplegic solution (STS) to clarify the mechanism by which intracellular Na+ is accumulated after cardioplegic arrest. MATERIALS AND METHODS: Isolated working rat hearts were perfused with Krebs-Henseleit bicarbonate buffer (KHBB). The hearts were then arrested with STS and subjected to normothermic global ischemia (30 min). This was followed by Langendorff reperfusion (15 min) and then a working reperfusion (20 min). In study A, we added Hoe (5, 10, and 20 microM), Mex (70 microM), or a combination of Hoe (20 microM) and Mex (70 microM), to STS. In study B, we added Hoe (20 microM), Mex (70 microM), or a combination of Hoe (20 microM) and Mex (70 microM) to KHBB during the first 3 min of Langendorff reperfusion. RESULTS: In study A, the addition of Hoe (10 and 20 microM) to STS showed a significantly greater postischemic recovery of cardiac output compared to the control group [63.1+/-5.7% (10 microM), 62.7+/-4.7% (20 microM), and 55.5+/-4.6% (control), respectively]. The postischemic recovery of cardiac output was significantly greater in the group of the combined addition (Hoe and Mex) to STS than that in the control, 20 microM Hoe, 70 microM Mex groups [70.3+/-3.7 (Hoe and Mex), 55.5+/-4.6% (control), 62.7+/-4.7% (Hoe 20 microM), and 60.2+/-4.7% (Mex 70 microM), respectively]. The myocardial water content in the postischemic period was 565.1+/-29.1, 525.8+/-2.9, 509.4+/-19.6, and 532.2+/-20.1; it was 497.3+/-9.1 mL/100 g dry weight in the control; and 10 microM Hoe, 20 microM Hoe, and 70 microM Mex in the combined use groups. In study B, there was no significant difference in the postischemic recovery of cardiac output in all experimental groups. CONCLUSION: The combined use of the Na+-H+ exchange inhibitor and Na+ channel blocker during cardioplegia may achieve a superior cardioprotective effect on myocardial damage because of ischemia and reperfusion.     

Neural blockade in basal and postreceptor-stimulated intestinal transport

Postreceptor protein stimulation significantly alters the transport state of the ex vivo small intestine. This study investigated the effects of neural blockade on basal and stimulated ionic transport. Rabbit ileal segments (n = 46) were arterially perfused with an oxygenated sanguinous buffered electrolyte solution. The lumen was perfused with an isotonic solution containing [14C]polyethylene glycol as a nonabsorbable marker. Net fluxes of H2O, Na+, and Cl- were calculated. Tetrodotoxin (TTX) was used to block enteric neural transmission. Forskolin (FOR) was used to activate adenylate cyclase, and phorbol 12,13-dibutyrate (PDB) served to activate protein kinase C. Two groups were studied. Group A preparations had no TTX pretreatment, while group B preparations were pretreated with TTX. In the Group A preparations, TTX at 10(-6) M and PDB at 10(-5) M caused significant proabsorptive effects with a delta FH2O of +20 +/- 7 and +15 +/- 2 microliters/min, respectively (P less than 0.05), while FOR stimulated significant secretion with a delta FH2O of -14 +/- 3 microliter/min (P less than 0.05). In the Group B TTX-pretreated preparations, FOR did not cause secretion and PDB maintained an absorptive state. These results indicate that neural blockade with TTX reverses basal secretion in the ex vivo intestine, suggesting that an intact enteric nervous system maintains the secretory status of the intestine. FOR-induced adenylate cyclase-activated secretion does not occur in the presence of TTX, implying that intact neural transmission is required for the FOR effect. PDB-induced protein kinase C-activated absorption occurs despite neural blockade, suggesting that the PDB-induced proabsorptive effect is mediated without neural intermediaries.     

Modulation of pulmonary NA+ pump gene expression during cold storage and reperfusion

BACKGROUND: Reperfusion injury with pulmonary edema continues to be a major complication after lung transplantation. Alveolar fluid homeostasis is regulated by Na+/K+-ATPase activity on the basolateral surface of alveolar epithelial cells. Intact Na+/K+-ATPase is essential to the resolution of pulmonary edema. We characterized the effects of cold ischemia and reperfusion on expression of Na+/K+-ATPase mRNA and protein. METHODS: Baseline values for Na+/K+-ATPase mRNA and protein were determined from freshly harvested lungs with no cold storage time or reperfusion (group I). Group II lungs were analyzed after cold storage times of 12 or 24 hr without subsequent reperfusion. Group III lungs were analyzed after cold storage times of 12 or 24 hr with subsequent reperfusion. Lungs were flushed with either Euro-Collins (EC) or University of Wisconsin (UW) solution in each group. All samples were quantified for Na+/K+-ATPase mRNA and Na+/K+-ATPase protein. Physiological parameters including oxygenation and compliance were also measured. RESULTS: There were no significant differences in the level of mRNA and protein for samples that were cold stored without reperfusion (group II). With reperfusion (group III) there was a significant increase in the level of the Na+/K+-ATPase mRNA after 12 hr of storage for both EC and UW. After 24 hr of storage and subsequent reperfusion, lungs flushed with EC had significantly decreased Na+/K+-ATPase protein and mRNA, although lungs preserved with UW maintained their increased levels of Na+/K+-ATPase protein and mRNA. CONCLUSIONS: Our data suggest that ischemia-reperfusion injury results in an initial up-regulation of Na+/K+-ATPase mRNA. With prolonged injury in lungs preserved with EC, the level of the mRNA decreased with a corresponding decrease in the Na+/K+-ATPase protein. The different response seen in EC versus UW may be explained by better preservation of pump function with UW than EC and correlates with improved physiological function in lungs preserved with UW solution.     

Endothelin-1 stimulates the Na+/H+ and Na+/HCO3- transporters in rabbit renal cortex.

Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor identified to date, raising the strong possibility of its involvement in the pathogenesis of systemic hypertension. Whether ET-1 exerts a direct stimulating effect on sodium reabsorption in the renal proximal convoluted tubule, the dominant locus of sodium reabsorption in the nephron, is currently unknown. Such an effect would suggest yet another mechanism by which ET-1 might mediate systemic hypertension. In studies on membrane vesicles prepared from rabbit renal cortex, we show that ET-1 (10(-8) to 10(-11) M) exerts dose-dependent stimulation of the apical Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter; preincubation of vesicles with 10(-10) M ET-1 for five minutes enhanced the activity of each transporter by approximately 25%. This stimulation reflected an increase in the Vmax of each transporter but no change in the Km for sodium. The stimulatory effect of ET-1 was blocked in the presence of an ET-1 antiserum. Moreover, the stimulation of the apical Na+/H+ exchanger and the basolateral Na+/HCO3- cotransporter by ET-1 displayed specificity as indicated by the lack of effects on the activities of the apical Na(+)-glucose transporter and the basolateral Na(+)-succinate transporter. The data implicate ET-1 as a novel, direct and specific modulator of sodium reabsorption in the proximal tubule. As such, ET-1 might be a direct determinant of extracellular fluid volume under normal and pathophysiologic circumstances, including hypertensive disorders.     

Na+/H+ exchange inhibition in hypertrophied myocardium subjected to cardioplegic arrest: an effective cardioprotective approach.

OBJECTIVE: This study was designed to assess whether the protective effects of Na+/H+ exchange (NHE) inhibition, which have been largely demonstrated in normal hearts, are also manifest in a more surgically relevant model of hypertrophied myocardium subjected to cardioplegic arrest. METHODS: Left ventricular hypertrophy was created in 3-week-old rats by coarctation of the ascending thoracic aorta with a hemoclip. Eight weeks later, hearts were excised, isovolumetrically perfused and subjected to 1 h of potassium cardioplegic arrest followed by 2 h of reperfusion. Hearts were allocated to one of the following four groups: sham-operated and aortic banding hearts without any treatment or treated with the NHE inhibitor cariporide (1 micromol/L) given as an additive to cardioplegia and over the first 15 min of reperfusion. RESULTS: The major effect of cariporide was to reduce ischemic peak contacture and to improve post-ischemic diastolic function in both sham-operated and hypertrophied hearts. Total creatine kinase release over the first 45 min of reperfusion was significantly reduced in hypertrophied hearts treated with cariporide. The endothelium-dependent coronary vasodilation to 5-hydroxytryptamine was observed in all sham-operated hearts before cardiac arrest, however, it was significantly impaired following cardioplegic ischemia and reperfusion. Hypertrophied hearts demonstrated markedly impaired endothelium-dependent and -independent coronary vasodilations during both pre- and post-ischemic period that were not affected by the treatment with cariporide. CONCLUSIONS: The cardioprotective effects of the NHE inhibitor cariporide are also manifest in hypertrophied myocardium, which supports the potential usefulness of NHE inhibition in the setting of cardiac surgery.