Cardiovascular remodelling and red blood cell Li+/Na+ countertransport in patients with type 1 diabetes and essential hypertension

Patients with type 1 (insulin-dependent) diabetes may develop a specific cardiac disease characterized by functional and structural abnormalities. The pathogenesis of the cardiac disease is poorly understood but cardiac and renal complications may coexist. Patients with overt diabetic nephropathy have increased red cell Li⁺/Na⁺ countertransport (CT), which reflects abnormal kinetic properties of the red cell membrane Na⁺/H⁺ exchange. Since the activation of Na⁺/H⁺ exchange has a key role in cell proliferation and cell growth, as well as in the regulation of cell sodium and cell pH and in the renal reabsorption of Na⁺ and bicarbonate, we have looked for relationships between red cell Li⁺/Na⁺ CT, Na⁺/H⁺ exchange and cardiovascular remodeling in patients with type 1 diabetes, essential hypertension and idiopathic familiar cardiomyopathy. In type 1 diabetes the maximal velocity of Li⁺/Na⁺ CT is positively correlated with interventricular septum thickness and the left ventricular wall to lumen ratio. Similar results were obtained in patients with essential hypertension. In these patients an increased Li⁺/Na⁺ CT is also associated with severe and drug-resistant hypertension and with significant vascular remodelling, estimated by the minimal post-ischaemic vascular resistance in the calf. Finally, Li⁺/Na⁺ CT is negatively correlated with diastolic relaxation of the left ventricle in familiar hypertrophic cardiomyopathy. From these data it appears that widespread abnormal kinetic properties of Na⁺/H⁺ exchange, estimated by increased red cell Li⁺/Na⁺ CT, may have epistatic effects on the pathogenesis of cardiac complications of type 1 diabetes and essential hypertension.     

Endothelin-1 induced hypertrophic effect in neonatal rat cardiomyocytes: involvement of Na+/H+ and Na+/Ca2+ exchangers

Endothelin-1 (ET-1) is a potent agonist of cell growth that also stimulates Na(+)/H(+) exchanger isoform 1 (NHE-1) activity. It was hypothesized that the increase in intracellular Na(+) ([Na(+)](i)) mediated by NHE-1 activity may induce the reverse mode of Na(+)/Ca(2+) exchanger (NCX(rev)) increasing intracellular Ca(2+) ([Ca(2+)](i)) which in turn will induce hypertrophy. The objective of this work was to test whether the inhibition of NHE-1 or NCX(rev) prevents ET-1 induced hypertrophy in neonatal rat cardiomyocytes (NRVMs). NRVMs were cultured (24 h) in the absence (control) and presence of 5 nmol/L ET-1 alone, or combined with 1 mumol/L HOE 642 or 5 mumol/L KB-R7943. Cell surface area, (3)H-phenylalanine incorporation and atrial natriuretic factor (ANF) mRNA expression were increased to 131 +/- 3, 220 +/- 12 and 190 +/- 25% of control, respectively (P < 0.05) by ET-1. [Na(+)](i) and total [Ca(2+)](i) were higher (8.1 +/- 1.2 mmol/L and 636 +/- 117 nmol/L, respectively) in ET-1-treated than in control NRVMs (4.2 +/- 1.3 and 346 +/- 85, respectively, P < 0.05), effects that were cancelled by NHE-1 inhibition with HOE 642. The rise in [Ca(2+)](i) induced by extracellular Na(+) removal (NCX(rev)) was higher in ET-1-treated than in control NRVMs and the effect was prevented by co-treatment with HOE 642 or KB-R7943 (NCX(rev) inhibitor). The ET-1-induced increase in cell area, ANF mRNA expression and (3)H-phenylalanine incorporation in ET-1-treated NRVM were decreased by NHE-1 or NCX(rev) inhibition. Our results provide the first evidence that NCX(rev) is, secondarily to NHE-1 activation, involved in ET-1-induced hypertrophy in NRVMs.     

Evidence for apical Na+/H+ exchanger in bovine main pancreatic duct

The finding of a high Pco ₂ in basally secreted pancreatic juice of man and dog raises the hypothesis of proton secretion from ductal epithelial cells presumably through a Na⁺/H⁺ exchanger. To test this possibility, H⁺ luminal secretion and Na⁺ movements were measuredin vitro on samples of bovine pancreatic ducts mounted in Ussing-type chambers. The rate of luminal acidification measured by the pH stat method, using bicarbonate-free media gassed with 100% O₂, reached 2.75 Eq/cm²/hr. Proton secretion was blocked in the presence of 1 mM amiloride or in the absence of Na⁺ (replaced by choline) in the mucosal solution. Study of transepithelial²²Na fluxes in short-circuited tissue, bathed on both sides by control Ringer solution, gassed by 95% O₂-5% CO₂ demonstrated a net sodium transport from the mucosal to the interstitial side of the duct (net²²Na flux=3.23±0.8 Eq/cm²/hr). This net sodium transport was electroneutral and blocked by mucosal amiloride (0.5–1 mM/liter) or by interstitial ouabain (1 mM/liter). These results are consistent with the existence of a Na⁺/H⁺ exchanger on the luminal side of the bovine main pancreatic duct.     

Na+/H+ exchange mediates postprandial ileal water and electrolyte transport

Feeding stimulates fluid and electrolyte absorption in the small intestine. Previous studies have suggested that Na⁺/glucose cotransport is important in initiating this response in the jejunum. The purpose of this study was to determine whether Na⁺/H⁺ exchange plays a role in meal-induced absorption. Exteriorized, neurovascularly intact jejunal and ileal loops (25 cm) were constructed in dogs. Following a two-week period of postoperative recovery, the loops of awake dogs were perfused with standard buffer alone or with increasing concentrations of amiloride, a Na⁺/H⁺ exchange inhibitor. Water, sodium, and chloride fluxes were calculated following a meal using [¹⁴C]PEG as a volume marker. The meal significantly increased absorption in both the jejunum (P<0.001) and ileum (P<0.01) in those animals perfused with buffer alone. More significantly, amiloride suppressed the increased absorption seen following a meal in the ileum (P<0.001) but not the jejunum. The response in the ileum was dose dependent. These findings suggest that a major mediator of postprandial sodium and water absorption in the ileum is the Na⁺/H⁺ exchanger.Supported by NIH R29-DK-47326 (S.W.A.), R01-DK-39879 (M.J.Z.), and a VA Merit Review (D.W.M.).Portions of this work were presented at the Annual Meeting of the American Gastroenterological Association, May 1993, Boston, Massachusetts.     

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.     

Na+/H+ exchange and its inhibition in cardiac ischemia and reperfusion

Summary The characterization of various ion transport systems has led to a better understanding of the effects, which seem to take part in the impairment of ischemic and reperfused cardiac tissue. This review discusses the role of the Na⁺/H⁺ exchange system in the pathophysiology of ischemia and reperfusion and the beneficial effects of its inhibition.At the onset of ischemia intracellular pH (pH_i) decreases due to anaerobic metabolism and ATP hydrolysis, leading to an activation of Na⁺/H⁺ exchange. This in turn increases intracellular Na⁺ (Na⁺ _i) and activates Na⁺/K⁺ ATPase, with a consecutive increase of energy consumption. Since cellular Na⁺ and Ca⁺⁺ transport are coupled by the Na⁺/Ca⁺⁺ exchange system, which depends on the Na⁺ gradient, the high Na⁺ _i leads to increased intracellular Ca⁺⁺ (Ca⁺⁺ _i). After a certain period, Na⁺/H⁺ exchange is inactivated by a decrease of extracellular pH.In case of reperfusion the acid extracellular fluid is washed out, which reactivates Na⁺/H⁺ exchange, leading to an unfavourably fast restoration of pH_i and a second time to Na⁺ and Ca⁺⁺ _i overflow.High Ca⁺⁺ _i is assumed to be one of the main reasons for ischemic and reperfusion injury, like arrhythmias, myocardial contracture, stunning and necrosis.It seems that the inhibition of Na⁺/H⁺ exchange can interrupt this process at an early phase and prevent or delay the consequences of ischemia and reperfusion as demonstrated by numerous investigators.     

Cell shrinkage activates Na+/H+ exchange in dog red cells by relieving inhibition of exchange by Na+ in isotonic medium

Na⁺/H⁺ exchangers (NHE) are widely distributed transporters responsible for regulation of cell volume and pH. In isotonic medium, NHE is often low or negligible, and is strongly activated by osmotic cell shrinkage. It is reported here that extracellular Na⁺ inhibits NHE in isotonic medium, and cell shrinkage stimulates NHE by relieving this inhibition. There is more than one inhibitory Na⁺ site on each transporter. Shrinkage activates NHE by decreasing the apparent affinity for Na⁺ at the inhibitory sites. Shrinkage has no effect on the apparent affinity for Na⁺ at the substrate sites for activation of NHE.     

Crystal structure of Ca2+/H+ antiporter protein YfkE reveals the mechanisms of Ca2+ efflux and its pH regulation

Ca²⁺ efflux by Ca²⁺ cation antiporter (CaCA) proteins is important for maintenance of Ca²⁺ homeostasis across the cell membrane. Recently, the monomeric structure of the prokaryotic Na⁺/Ca²⁺ exchanger (NCX) antiporter NCX_Mj protein from Methanococcus jannaschii shows an outward-facing conformation suggesting a hypothesis of alternating substrate access for Ca²⁺ efflux. To demonstrate conformational changes essential for the CaCA mechanism, we present the crystal structure of the Ca²⁺/H⁺ antiporter protein YfkE from Bacillus subtilis at 3.1-Å resolution. YfkE forms a homotrimer, confirmed by disulfide crosslinking. The protonated state of YfkE exhibits an inward-facing conformation with a large hydrophilic cavity opening to the cytoplasm in each protomer and ending in the middle of the membrane at the Ca²⁺-binding site. A hydrophobic “seal” closes its periplasmic exit. Four conserved α-repeat helices assemble in an X-like conformation to form a Ca²⁺/H⁺ exchange pathway. In the Ca²⁺-binding site, two essential glutamate residues exhibit different conformations compared with their counterparts in NCX_Mj, whereas several amino acid substitutions occlude the Na⁺-binding sites. The structural differences between the inward-facing YfkE and the outward-facing NCX_Mj suggest that the conformational transition is triggered by the rotation of the kink angles of transmembrane helices 2 and 7 and is mediated by large conformational changes in their adjacent transmembrane helices 1 and 6. Our structural and mutational analyses not only establish structural bases for mechanisms of Ca²⁺/H⁺ exchange and its pH regulation but also shed light on the evolutionary adaptation to different energy modes in the CaCA protein family.     

Reduced infarct size in the rabbit heartin vivo by ethylisopropyl-amiloride. A role for Na+/H+ exchange

Inhibition of Na⁺/H⁺ exchange with amiloride analogues has been shown to offer functional protection during ischemia and reperfusion and reduce infarct size in isolated rat hearts and intact pigs. The aim of the present study was to examine if pre- or postischemic treatment with ethylisopropylamiloride (EIPA), a selective Na⁺/H⁺ exchange inhibitor, could reduce infarct size in anin situ rabbit model of regional ischemia and reperfusion. Anesthetized, open-chest rabbits were subjected to 30 min of regional ischemia and 180 min of reperfusion. The risk zone was determined by fluorescent particles, and infarct size was determined by TTC staining. Preischemic treatment with EIPA (0.65 mg/kg) significantly reduced infarct size from 45.8±3.5% of the risk zone in the control group to 10.6±3.1% (p<0.01). EIPA-treatment during the first part of the reperfusion period did not reduced infarct size compared to controls (41.9±3.5%). We conclude that EIPA, when administered prior to ischemia, reduces infarct size in the rabbit heartin situ, a protection most likely due to inhibition of Na⁺/H⁺ exchange.     

Inhibition of Na+/H+ exchange preserves viability,restores mechanical function,and prevents the pH paradox in reperfusion injury to rat neonatal myocytes

Summary Rat neonatal myocytes exposed to 2.5 mM CaCN and 20 mM 2-deoxyglucose at pH 6.2 (chemical hypoxia) quickly lose viability when pH is increased to 7.4, with or without washout of inhibitors — a pH paradox. In this study, we evaluated the effect of two Na⁺/H⁺ exchange inhibitors (dimethylamiloride and HOE694) and a Na⁺/Ca²⁺ exchange inhibitor (dichlorobenzamil) on pH-dependent reperfusion injury. Intracellular free Ca²⁺ and electrical potential were monitored by laser scanning confocal microscopy of rat neonatal cardiac myocytes grown on coverslips and co-loaded with Fluo-3 and tetramethylrhodamine methylester. After 30–60 min of chemical hypoxia at pH 6.2, mitochondria depolarized and Ca²⁺ began to increase uniformly throughout the cell. Free Ca²⁺ reached levels estimated to exceed 2 M by 4h. Washout of inhibitors at pH 7.4 (reperfusion), with or without dichlorobenzamil, killed most cells within 60 min, despite a marked reduction of Ca²⁺ in dichloroben zamil-treated cells. Reperfusion at pH 7.4 in the presence of 75 M dimethylamiloride or 20 M HOE694, or at pH 6.2, prevented cell death. HOE694-treated cells placed into culture medium recovered mitochondrial membrane potential. In most cells, this occurred before normal Ca²⁺ was restored. Contracted myocytes re-extended over a 24-h-period. By 48 hours, most cells contracted spontaneously and showed normal Ca²⁺ transients. Our results indicate that Na⁺/H⁺ exchange inhibition protects against pH-dependent reperfusion injury and facilitates full recovery of cell function.Abbreviations DCB dichlorobenzamil - DMA dimethylamiloride - 2-DOG 2-deoxy-D-glucose - HOE694 3-methylsulfonyl-4-piperidinobenzoyl guanidine hydrochloride - KRH Krebs-Ringer-HEPES buffer containing 115 mM NaCl, 5 mM KCl, 1 mM KH₂PO₄, 1,2 mM MgSO₄, 2 mM CaCl₂, and 25 mM Na-HEPES buffer - PI propidium iodide - TMRM tetramethylrhodamine methylester - electrical potential difference - electric potential This work was supported, in part, by Grant N00014-89-J-1433 from the Office of Naval Research and Grants AG07218 an DK37034 from the National Institutes of Health. I.S.H. was the recipient of a Post-Doctoral Scholarship from the Medical Research Council of South Africa. A preliminary report of portions of this work was presented at Experimental Biology '93, New Orleans, March 28–April 1, 1993 (15)     

A study on the action of vitamin E supplementation on plasminogen activator inhibitor type 1 and platelet nitric oxide production in type 2 diabetic patients

Background and aimType 2 diabetic (T2DM) patients show decreased fibrinolysis, mainly linked to high plasminogen activator inhibitor type 1 (PAI-1) production, together with a reduced bioavailability of nitric oxide and an impairment in Na⁺/K⁺-ATPase activity possibly involved in increased cardiovascular risk. Vitamin E is the major natural lipid-soluble antioxidant in human plasma. The present work was conducted in order to measure PAI-1, ICAM and VCAM-1 plasma levels, platelet nitric oxide production and membrane Na⁺/K⁺-ATPase activity in type 2 diabetic subjects treated with vitamin E (500 IU/day) for 10 weeks and then followed for other 20 weeks.Methods and resultsThirty-seven T2DM patients (24 males and 13 females) were studied. None of them were affected by any other disease or diabetic complications. Significant differences were detected for PAI-1 antigen (p < 0.001), PAI-1 activity (p < 0.001), nitric oxide (NO) production (p < 0.001), and Na⁺/K⁺-ATPase activity (p < 0.001) among the 4 phases of the study. A significant decrease both in ICAM and VCAM-1 plasma levels was also found at the 10th week compared with baseline (respectively p < 0.001 and p < 0.05).ConclusionOur data suggest that vitamin E counteracts endothelial activation in T2DM patients possibly representing a new tool for endothelial protection.     

Myocardial recovery during post-ischemic reperfusion: Optimal concentrations of Na+ and Ca2+ in the reperfusate and protective effects of amiloride added to cardioplegic solution

The effects of Na⁺ and Ca²⁺ concentrations in the reperfusate on post-ischemic myocardial recovery were examined. Also, the myocardial protective effects of amiloride, an inhibitor of the Na⁺/Ca²⁺ and Na⁺/H⁺ exchange systems, added to cardioplegic solutions were assessed, using an isolated working rat heart perfusion system. Global myocardial ischemia was induced by 30-min normothermic cardioplegic arrest, using St. Thomas’ solution. The concentration of Na⁺ in the reperfusate varied, stepwise, from 75 to 145 mM/l, and that of Ca²⁺, from 0.1 to 2.5 mM/l. In this study post-ischemic functional recovery was best at 110 mM/l Na⁺ and 1.2–1.8 mM/l Ca²⁺ in the reperfusate. A significantly greater postischemic functional recovery and a lower creatine kinase release were observed when amiloride was added to the cardioplegic solution. Ca²⁺ overload via Na⁺/Ca²⁺ and Na⁺/H⁺ exchange systems would, thus, appear to be due, at least in part, to post-ischemic reperfusion injury.     

ATP-dependent Ca2+ Transport and Na+/Ca2+ Exchange System in Renal Basolateral Plasmamembranes of Spontaneously Hypertensive Rats

A common abnormality of cellular Ca²⁺ handling in most tissues of spontaneously hypertensive rats (SHR) has been suggested. Therefore we investigated the ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange system in basolateral membrane vesicles (BLMV) of renal cortices from SHR and normotensive Wistar-Kyoto rats (WKY) at 12 and 20 weeks of age. In WKY the maximal transport rate of the ATP-dependent Ca²⁺ transport was 5.7 nmol/min/mg prot with an affinity for Ca²⁺ of 0.14 µM. These values were not significantly different in SHR at both ages studied. High concentrations of Na⁺ inhibited ATP-dependent Ca²⁺ uptake by 40% in BLMV of SHR and WKY. Low concentrations of Na⁺ stimulated ATP-dependent Ca²⁺ transport 20% in both rats. These findings suggest equal Na⁺/Ca²⁺ exchange activity in WKY and SHR. The present study failed to show a significant change in ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange activity in renal BLMV in SHR, suggesting that the Ca²⁺ homeostasis of the cortical cells is normal in SHR as far as the plasmamembrane is concerned.     

Localization and function of the Na+/Ca2+-exchanger in normal and detubulated rat cardiomyocytes

It is controversial whether the Na+/Ca2+-exchanger (NCX) can induce cardiomyocyte contraction through reverse-mode exchange and Ca2+-induced Ca2+ release (CICR). Information about the spatial distribution and functional activity within different sarcolemmal (SL) regions could shed light on this potential role. We raised a new antibody to the NCX and showed by confocal laser scanning microscopy (CLSM) that immunoreactivity is strongly expressed throughout the surface SL and intercalated disk regions with punctate labeling of the vertical transverse (T)-tubules but not the longitudinal T-tubules. Immuno-electron microscopy confirmed CLSM observations. Gold particles associated with the exchanger were within nanometer range of particles signaling ryanodine receptors. A similar close association was found between the L-type Ca2+ channel (known to be concentrated in the dyad) and ryanodine receptors. In whole-cell patch-clamped cardiomyocytes, peak I(NCX) (measured at 90 mV) decreased by approximately 40% (497 +/- 32 vs. 304 +/- 12 pA, P < 0.001) after detubulation, while membrane capacitance decreased by 27% (204 +/- 11 vs. 150 +/- 7 pF, P < 0.01) thus giving a small but significant 16% reduction in current density. Thus, the density and/or functional activity of the NCX is greater in the vertical T-tubules than in the longitudinal T-tubules, surface SL or disk regions, pointing to important functional differences between these plasma membrane domains. Our combined co-immunolocalization and physiological data suggest that the NCX has multiple functions depending upon membrane location. We suggest the possibility that NCX modulates CICR, sarcoplasmic reticulum Ca2+ load, and that it also serves to regulate Ca2+ handling in neighboring cells.     

Reduction of erythrocyte (Na+-K+)ATPase activity in Type 1 (insulin-dependent) diabetic subjects and its activation by homologous plasma

Summary The (Na⁺-K⁺)ATPase and (Mg²⁺)ATPase activities of erythrocyte membranes of Type 1 (insulin-dependent) diabetic patients were found to be significantly reduced compared to matched controls (p < 0.005). On the contrary, erythrocyte Na⁺ and K⁺ contents were similar in diabetic patients and in normal subjects. When erythrocyte membranes from diabetic patients were incubated with their own plasma, a significant increase was observed in sodium-potassium ATPase activity (p < 0.005), whereas (Mg²⁺)ATPase activity was not affected. The plasma stimulatory effect showed saturation kinetics. Maximum average stimulation was 96% (±21.3). A similar stimulation pattern, although more limited in extent (maximum 48.3 % ± 12.2), was found when erythrocyte membranes from normal subjects were incubated with diabetic plasma. Normal plasma exhibited a modest stimulatory effect on erythrocyte (Na⁺-K⁺ATPase activity. Similar stimulatory effects by diabetic plasma were observed on a (Na⁺-K⁺) ATPase preparation from beef heart. It is proposed that diabetic plasma contains a specific (Na⁺-K⁺)ATPase activator in a higher concentration than normal plasma. This may explain why a normal cellular electrolyte content was found in diabetic erythrocytes in spite of a reduced Na⁺-K⁺ pump activity. Purification experiments indicate that the plasma activator is a protein with a molecular weight greater than 50,000. Both the (Na⁺-K⁺)ATPase activity and the stimulatory effect of diabetic plasma were not influenced by the metabolic control, since they did not correlate significantly with fasting blood glucose and daily insulin dosage. Moreover, no correlation was found with duration of diabetes or age at diagnosis of diabetes. It is suggested that the enzyme defect of erythrocyte membrane and the stimulation of (Na⁺-K⁺)ATPase activity by homologous plasma are early biochemical alterations in the course of diabetes mellitus.     

Serotonin inhibits Na+/H+ exchange activity via 5-HT4 receptors and activation of PKC alpha in human intestinal epithelial cells

BACKGROUND & AIMS: Increased serotonin levels have been implicated in the pathophysiology of diarrhea associated with celiac and inflammatory diseases. However, the effects of serotonin on Na+ /H+ exchange (NHE) activity in the human intestine have not been investigated fully. The present studies examined the acute effects of 5-hydroxytryptamine (5-HT) on NHE activity using Caco-2 cells as an in vitro model. METHODS: Caco-2 cells were treated with 5-HT (.1 micromol/L, 1 h) and NHE activity was measured as ethyl-isopropyl-amiloride (EIPA)-sensitive 22Na uptake. The effect of 5-HT receptor-specific agonists and antagonists was examined. The role of signaling intermediates in 5-HT-mediated effects on NHE activity was elucidated using pharmacologic inhibitors and immunoblotting. RESULTS: NHE activity was inhibited significantly (approximately 50%-75%, P < .05) by .1 micromol/L 5-HT via inhibition of maximal velocity (Vmax) without any changes in apparent affinity (Km) for the substrate Na+ . NHE inhibition involved a decrease of both NHE2 and NHE3 activities. Studies using specific inhibitors and agonists showed that the effects of 5-HT were mediated by 5-HT4 receptors. 5-HT-mediated inhibition of NHE activity was dependent on phosphorylation of phospholipase C gamma 1 (PLC gamma 1) via activation of src-kinases. Signaling pathways downstream of PLC gamma 1 involved increase of intracellular Ca 2+ levels and subsequent activation of protein kinase C alpha (PKC alpha). The effects of 5-HT on NHE activity were not cell-line specific because T84 cells also showed NHE inhibition. CONCLUSIONS: A better understanding of the regulation of Na+ absorption by 5-HT offers the potential for providing insights into molecular and cellular mechanisms involved in various diarrheal and inflammatory disorders.     

Quantification in crude homogenates of rat myocardial Na+, K+-and Ca2+-ATPase by K+ and Ca2+-dependent pNPPase. Age-dependent changes

Assays for complete quantification of Na⁺, K⁺-and Ca²⁺-ATPase in crude homogenates of rat ventricular myocardium by determination of K⁺-and Ca²⁺-dependentp-nitrophenyl phosphatase (pNPPase) activities were evaluated and optimized. Using these assays the total K⁺-and Ca²⁺-dependentpNPPase activities in ventricular myocardium of 11–12 week-old rats were found to be 2.98±0.10 and 0.29±0.02 mol×min^–1×g^–1 wet wt. (mean±SEM) (n=5), respectively. Coefficient of variance of interindividual determinations was 7 and 12%, respectively. The total Na⁺, K⁺-and Ca²⁺-ATPase concentrations were estimated to 2 and 10 nmol×g^–1 wet wt., respectively. Evaluation of a putative developmental variation revealed a biphasic age-related change in the rat myocardial Ca²⁺-dependentpNPPase activity with an increase from birth to around the third week of life followed by a decrease. By contrast, the K⁺-dependentpNPPase activity of the rat myocardium showed a decrease from birth to adulthood. It was excluded that the changes were simple out-come of variations in water and protein content of myocardium. Expressed per heart, the K⁺-and Ca²⁺-dependentpNPPase activity gradually increased to a plateau. The present assay for Na⁺, K⁺-ATPase quantification has the advantage over [³H] ouabain binding of being applicable on the ouabain-resistant rat myocardium, and is more simple and rapid than measurements of K⁺-dependent 3-0-methylfluorescein phosphatase (3-0-MFPase) in crude tissue homogenates. Furthermore, with few modifications thepNPPase assay allows quantification of Ca²⁺-ATPase on crude myocardial homogenates. Age-dependent changes in K⁺-and Ca²⁺-dependentpNPPase activities are of developmental interest and indicate the importance of close age match in studies of quantitative aspects of Na⁺, K⁺-and Ca²⁺-ATPase in excitable tissues.Abbreviations Na⁺, K⁺-ATPase sodium, potassium-dependent ATPase - Ca²⁺-ATPase caldium-dependent ATPase - pNP p-nitrophenyl - pNPP p-nitrophenyl phosphate - 3-0-MFP 3-0 methylfluorescein phosphate - DOC sodium deoxycholate     

Myocardial (Na+k+)-Atpase Activity in Daul Salt-Sensitive and Resistant Rats

Vascular (Na⁺K⁺)-punp activity (ouabain-sensitive ⁵⁶Rb⁺uptake) and myocardial (Na⁺K⁺)-ATPase activity are reduced in. animals with various forms of low renin, experimental hypertersion. On the other hand, vascular (Na⁺, K⁺)-puinp activity is increased in Dahl salt-sensitive relative to resistant rats (a genetic model of hypertension), regardless of salt intake or blood pressure and it is also increased in Dahl salt-sensitive rats on high salt (3% NaCl) relative to low salt (0.4% NaCl) diets. It has been suggested that this increase in vascular (Na⁺K⁺)-pump activity may be secondary to an increase in the vascular sarcolemmal permeability to Na⁺in these salt-sensitive rats. In the present study, (Na⁺K⁺)-ATPase activity of left ventricular microsomal fractions, was increased in Dahl salt-sensitive relative to resistant rats on low salt diets; however, this difference disappeared when these salt-sensitive and resistant rats were placed on high salt diets. In contrast, mvocardial (Na⁺K⁺)-ATPase activity was decrease in Dahl salt-sensitive rats on high relative to low salt diets. Evidence that this decrease in (Na⁺K⁺)-ATPase activity is not secondary to myocardial hypertrophy in the hypertensive salt-sensitive rats, and mechanisms by which decreased cardiovascular (Na⁺K⁺)-pump activity, increased sarcolemmal permeability or both, might contribute to elevated blood pressure, are discussed.     

肥胖2型糖尿病肾病患者微量白蛋白排泄率与胰岛素抵抗及血浆纤溶系统相关性研究

目的探讨肥胖2型糖尿病(T2DM)肾病患者胰岛素敏感性与血浆纤溶系统相关性。方法对108例肥胖T2DM患者及31例健康体检者进行体格检查,采用生物电阻抗法测定其体脂含量,测定其24h尿白蛋白排泄量、血脂、空腹血糖、糖基化血红蛋白(HbA1c)及胰岛素水平、血浆组织型纤溶酶原激活物(tPA)及其抑制物1(PAI1)活性和D二聚体(DD)水平。结果大量白蛋白尿组肥胖度、HbA1c和胰岛素敏感性指数(ISI)分别大于和小于微量白蛋白尿组和尿白蛋白正常组(均P<005)。与正常对照者比较,肥胖T2DM患者血浆PAI1活性明显升高(均P<005),大量白蛋白尿组更明显;糖尿病肾病患者血浆tPA活性及大量白蛋白尿组DD水平大于正常对照者(P<005,P<001)。多因素逐步回归分析显示,PAI1活性与BMI、腰围及血清TG水平呈正相关,与ISI呈负相关。结论肥胖T2DM肾病患者尤其是向心性者血浆PAI1活性及DD水平明显增加,而tPA活性减低,并随着肾病的加重而增加,与肥胖程度呈正相关、与ISI呈负相关。     

肥胖和非肥胖2型糖尿病患者的临床特点及影响其心功能的多因素探讨

目的 比较肥胖和非肥胖2型糖尿病患者的临床特点。探讨影响两组患者心功能的因素。方法 符合1999年糖尿病诊断标准的住院型糖尿病患者238例。根据有无肥胖将所有患者分为肥胖组和非肥胖组，胰岛素敏感指数采用QUICKI公式，用二维指导下的M型超声心动图检测患者在左心房大小，舒张末期左心室内径，左心室后壁厚度，室间隔厚度，射血分数，用超声多普勒检测E峰A峰比值，计算左心室质量指数。结果 （1）肥胖组胰岛素敏感指数较非肥胖组高，高密度脂蛋白胆固醇水平较非肥胖组低；（2）肥胖组左心房内径，舒张末期左心室内径，左心室后壁厚度，室间隔厚度，左心室质量指数均大于非肥胖组；（2）多元逐步回归分析显示；肥胖组射血分数与左心室质量指数，空腹C肽呈负相关；非肥胖组射血分数与左心室质量指数，收缩压，尿酸呈负相关，结论 肥胖糖尿病患者存在明显的左心肥大，并与低胰岛素敏感发性和低，高密度脂蛋白胆固醇并存；左心室肥大，无论在肥胖和非肥胖的糖尿病患者均影响左心室收缩功能；肥胖与非肥胖糖尿病患者还存在不同的收收缩功能有关的因素。