Relationship of red blood cell ion transport alterations and serum lipid abnormalities in Lyon genetically hypertensive rats

Alterations of Na+ and K+ transport in erythrocytes of hypertensive humans or animals are often associated with abnormal lipid metabolism. The aim of the present study was to investigate red blood cell ion transport in Lyon inbred strains selected from Sprague-Dawley rats for different blood pressure levels. Lyon strains are characterized by important metabolic changes, including plasma lipid abnormalities. Serum triglycerides, cholesterol, and uric acid as well as red blood cell Na+ and K+ (Rb+) transport mediated by Na(+)-K+ pump or Na(+)-K+ cotransport and cation leaks were studied in hypertensive (LH), normotensive (LN), and low blood pressure (LL) Lyon rats aged 12 weeks. Increased erythrocyte Na+ content (Nai+) and higher levels of serum triglycerides, cholesterol, and uric acid were demonstrated in LH rats compared with LN animals. Nevertheless, at this age serum triglycerides and erythrocyte Nai+ of LL rats were even higher than those of LH animals. There were no significant differences between Lyon strains in either Na(+)-K+ pump activity or bumetanide-resistant (BR) cation leaks. The activity of bumetanide-sensitive (BS) Na(+)-K+ cotransport mediating inward Na+ movement was highest in LL rats and lowest in LH animals. In Lyon rats, Nai+ was positively related to serum triglycerides, whereas blood pressure correlated positively with BR Na+ leak and negatively with BS net Na+ uptake. A similar association of erythrocyte Nai+ with serum triglycerides was also observed in Prague hereditary hypertriglyceridemic rats (HTG) that were selected from Wistar rats for high plasma triglycerides. The major difference of the two forms of genetic hypertension associated with abnormal lipid metabolism was in BS net Na+ uptake, which was enhanced in HTG but reduced in LH rats. This was probably due to differences in plasma cholesterol, which was elevated in LH but not in HTG animals. Our study in Lyon rats confirmed the positive association of blood pressure with Na+ leak as a characteristic feature of genetic hypertension.     

Dissimilar alterations of sodium-coupled uptake by platinum-coordination complexes in renal proximal tubular cells in primary culture

The potent anticancer drug cis-diamminedichloroplatinum (II) (CDDP) interferes early with electrolyte transport by the renal proximal tubule. To study the early effects of platinum coordination complexes on apical Na(+)-coupled transport systems, we examined the effect of increasing concentrations of CDDP, trans-diamminedichloroplatinum (II) (TDDP) and cis-diammine-1,1-cyclobutane-dicarboxylate platinum (II) (CBDCA) on Na(+)-coupled uptake of P(i), methyl-alpha-D-glucopyranoside (MGP) and L-alanine by rabbit proximal tubule cells in primary culture. At 17 microM CDDP and 540 microM CBDCA, 1) cell viability (lactate dehydrogenase release) and ATP content were unaffected, 2) Na(+)-K(+)-ATPase activity was reduced by 40%, 3) Na(+)-coupled uptake of MGP and P(i) was reduced, whereas 4) Na(+)-coupled uptake of alanine rose to twice the control value. Alterations of Na(+)-coupled uptake of P(i), MGP and alanine were due to changes in Km, with no significant change in Vmax. At 333 microM TDDP, Na(+)-dependent P(i) and MGP uptake decreased, whereas Na(+)-independent uptake increased markedly and was associated with a decline in cell viability and ATP content. We conclude that 1) the TDDP-induced decrease in Na+/P(i) and Na+/glucose cotransport was associated with reduced cell viability, 2) both CDDP and CBDCA had different effects on Na+/P(i), Na+/glucose and Na+/alanine cotransport, arguing against an alteration of the Na+ gradient due to reduced Na(+)-K(+)-ATPase activity and 3) CBDCA induced alterations of Na(+)-coupled uptake similar to those of CDDP at concentrations 20 to 30 times higher.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin 1 stimulates Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport through ETA receptors and protein kinase C-dependent pathway in cerebral capillary endothelium

The effect of endothelins (ET-1 and ET-3) on 86Rb+ uptake as a measure of K+ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K+ uptake (EC50 = 0.60 +/- 0.15 and 21.5 +/- 4.1 nM, respectively), which was inhibited by the selective ETA receptor antagonist BQ 123 (cyclo-D-Trp-D-Asp-Pro-D-Val-Leu). Neither the selective ETB agonists IRL 1620 [N-succinyl-(Glu9,-Ala11,15)-ET-1] and sarafotoxin S6c, nor the ETB receptor antagonist IRL 1038 [(Cys11,Cys15)-ET-1] had any effect on K+ uptake. Ouabain (inhibitor of Na+,K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K+ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport. ET-1- but not PMA-stimulated K+ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na+/H+ exchange system), suggesting a linkage of Na+/H+ exchange with ET-1-stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport activity that is not mediated by PKC.     

K+ fluxes mediated by Na(+)-K(+)-Cl- cotransport and Na(+)-K(+)-ATPase pumps in renal tubule cell lines transformed by wild-type and temperature-sensitive strains of Simian virus 40

The relative contributions of Na(+)-K(+)-ATPase pumps and Na(+)-K(+)-Cl- cotransport to total rubidium (Rb+) influx into primary cultures of renal tubule cells (PC.RC) and cells transformed either with the wild-type or a temperature-sensitive mutant of the simian virus 40 (SV40), were measured under various growth conditions. The Na(+)-K(+)-ATPase-mediated component represented 74% and 44-48% of total Rb+ influx into PC.RC and SV40-transformed cells, respectively. Proliferating transformed cells showed substantial ouabain-resistant bumetanide-sensitive (Or-Bs) Rb+ influx (41-45% of total) which indicated the presence of a Na(+)-K(+)-Cl- cotransport. The Or-Bs component of Rb+ influx was greatly reduced when temperature-sensitive transformed renal cells (RC.SVtsA58) grown in Petri dishes or on permeable filters were shifted from the permissive (33 degrees C) to the restrictive temperature (39.5 degrees C) to arrest cell growth. The ouabain-sensitive Rb+ influx mediated by the Na(+)-K(+)-ATPase, the total and amiloride-sensitive Na+ uptakes were not modified following inhibition of cell proliferation. A similar fall in the Or-Bs influx was obtained when renal tubule cells transformed by the wild-type SV40 (RC.SV) were incubated with the K+ channel blocker, tetraethylammonium (TEA) ion, which we had previously shown to arrest cell growth without affecting cell viability (Teulon et al.: J. Cell. Physiol., 151:113-125, 1992). Reinitiation of cell growth by removal of TEA or return to 33 degrees C of the temperature-sensitive cells restored the Or-Bs component of Rb influx. Taken together, these results indicate that the Na(+)-K(+)-Cl- cotransport activity is critically dependent on cell growth conditions.     

An effect of voltage on binding of Na+ at the cytoplasmic surface of the Na(+)-K+ pump

This work utilizes proteoliposomes reconstituted with renal Na(+)-K(+)-ATPase to study effects of electrical potential (40-80 mV) on activation of pump-mediated fluxes of Na+ or Rb+ (K+) ions and on inhibitory effects of Rb+ ions or organic cations. The latter include guanidinium derivatives that are competitive Na(+)-like antagonists (David, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1141-1149). Cytoplasmic side-positive diffusion potentials significantly decreased the K0.5 of Na+ at the cytoplasmic surface for activation of ATP-dependent Na(+)-K+ exchange but did not affect the inhibitory potency of Rb+ (K+) or any Na(+)-like antagonist. Diffusion potentials did not affect activation of Rb(+)-Rb+ exchange by Rb+ ions at the cytoplasmic surface and had only a minor effect on Rb+ activation at the extracellular surface. Previously, we proposed that the cation binding domain consists of two negatively charged sites, to which two K+ or two Na+ ions bind, and one neutral site for the third Na+ (Glynn, I. M., and Karlish, S.J.D. (1990) Annu. Rev. Biochem. 59, 171-205). The present experiments suggest that binding of a Na+ ion in the neutral site at the cytoplasmic surface is sensitive to voltage. By contrast, binding of Rb+ ions at the extracellular surface of renal pumps appears to be only weakly or insignificantly affected by voltage. Inferences on the identity of the charge-carrying steps, based on experiments using proteoliposomes, are discussed in relation to recent evidence that dissociation of Na+ or association of K+ ions, at the extracellular surface, represent the major charge-carrying steps.     

Reduced Na(+)-K(+)-ATPase activity and plasma lysophosphatidylcholine concentrations in diabetic patients

A fraction from normal human plasma inhibiting Na(+)-K(+)-ATPase has been recently identified as lysophosphatidylcholine (LPC). The aim of this study was to investigate the existence of a relationship between the activity of the cellular membrane Na(+)-K(+)-ATPase and plasma LPC in human diabetes. We studied 10 patients with insulin-dependent-diabetes mellitus (IDDM), 14 patients with non-insulin-dependent diabetes mellitus (NIDDM), and 10 sex- and age-matched control subjects. Plasma LPC concentrations were increased in both IDDM and NIDDM patients compared with control subjects. Na(+)-K(+)-ATPase activity was reduced in both groups of patients in erythrocyte and platelet membranes. There was a significant correlation between the concentrations of plasma LPC and Na(+)-K(+)-ATPase activity in both erythrocyte and platelet membranes (P < 0.01). To investigate the effect of LPC on the enzyme, Na(+)-K(+)-ATPase activity was determined in erythrocyte membranes obtained from six healthy subjects after in vitro incubation with increasing concentrations of LPC (1-10 microM). Enzymatic activity was significantly reduced by in vitro LPC at a concentration of 2.5 microM, with a further decrease at 5 microM. These data suggest that the decrease in Na(+)-K(+)-ATPase activity in diabetes might be due to increased LPC concentrations.     

Transjugular intrahepatic portosystemic shunt: a medical perspective

We studied the ability of cilostazol (CL), an antithrombotic and vasodilating agent, to prevent functional, structural and biochemical abnormalities including delayed motor nerve conduction velocity (MNCV), morphological changes in myelinated fibers, and decreased Na(+)-K(+) -ATPase activity in the peripheral nerves of rats with streptozotocin (STZ)-induced diabetes. Cilostazol treatment (30 mg/kg/day p.o.) for 10 weeks significantly prevented the delay in MNCV in the tail nerve, and morphometric analysis of the sural nerves revealed that this dose of cilostazol had a significant effect on reduction of average size of myelinated fibers. In untreated diabetic rats, cyclic AMP content and Na(+)-K(+)-ATPase activity of peripheral nerve were each significantly less than in normal control rats. Cilostazol (30 mg/kg/day) prevented reduction of Na(+)-K(+)-ATPase activity. Decrease in cyclic AMP content was completely prevented with both doses of cilostazol (30 and 10 mg/kg/day). These findings suggest that cilostazol may have beneficial effects in the treatment of diabetic neuropathy, possibly via improvement of nerve Na(+)-K(+) -ATPase activity and cyclic AMP content. Cilostazol may thus be a potent drug for the clinical treatment of diabetic neuropathy.     

Ethinylestradiol treatment induces multiple canalicular membrane transport alterations in rat liver

We investigated the effects of 17 alpha-ethinylestradiol treatment of rats on various transport functions in isolated basolateral and canalicular liver plasma membrane vesicles. Both membrane subfractions were purified to a similar degree from control and cholestatic livers. Although moderate membrane lipid alterations were predominantly observed in basolateral vesicles, no change in basolateral Na+/K(+)-ATPase activity was found. Furthermore, while Na(+)-dependent taurocholate uptake was decreased by approximately 40% in basolateral vesicles, the maximal velocity of ATP-dependent taurocholate transport was decreased by 63% in canalicular membranes. In contrast, only minimal changes or no changes at all were observed for electrogenic taurocholate transport in "cholestatic" canalicular membranes and total microsomes, respectively. However, canalicular vesicles from cholestatic livers also exhibited marked reductions in ATP-dependent transport of S-(2,4-dinitrophenyl)glutathione and in Na(+)-dependent uptake of adenosine, while in the same vesicles HCO3-/SO4- exchange and Na+/glycine cotransport activities were markedly stimulated. These data show that in addition to the previously demonstrated sinusoidal transport abnormalities ethinylestradiol-induced cholestasis is also associated with multiple canalicular membrane transport alterations in rat liver. Hence, functional transport alterations at both polar surface domains might ultimately be responsible for the inhibitory effects of estrogens on the organic anion excretory capacity and on bile formation in rat liver.     

Erythrocyte Na+/K(+)-ATPase and membrane and serum lipid profiles: as related to alcohol, body mass index and blood pressure

Erythrocyte Na+/K(+)-pump activities have been measured in hypertensives, alcohol consumers and obese persons, but the results have been variously reported as decreased, increased or unchanged. We analyzed the relationships between erythrocyte Na+/K(+)-ATPase activities and the membrane and serum lipid profiles in 83 middle-aged men, to clarify the reasons for these inconsistencies. Increases in erythrocyte Na+/K(+)-ATPase activity related closely to decreases in cholesterol to phospholipid (C/P) ratio of the erythrocyte membrane. Decreases in the C/P ratio in turn related closely to elevations of serum triglycerides (TG) with increasing body mass index, and weakly to the volume of alcohol consumed. Thus, erythrocyte Na+/K(+)-ATPase activities depend largely on the membrane and serum lipid profiles as related to body weight and alcohol consumption, and which may be a cause of the previous conflicting findings. Erythrocyte Na+/K(+)-ATPase showed a positive association with blood pressure, independently of age, body mass index and serum gamma-glutamyl transpeptidase levels. Although the biological link of elevated erythrocyte Na+/K(+)-ATPase with the rise in blood pressure remains unclear, it may be a reflection of hyperinsulinemia in the subjects with a higher blood pressure due to overweight or excessive alcohol consumption.     

Effect of human recombinant erythropoietin (Epo) on cellular Ca2+, Na+, and K+ regulation of vascular smooth muscle cells grown in vitro--a new insight into the pathogenesis of Epo induced hypertension

To gain an insight into the effect of erythropoietin (Epo) upon cation transporters and cytosolic free Ca2+ concentration ([Ca2+]i) of vascular smooth muscle cells (VSMC), we studied whether 1) Epo, per se, alters Ca2+ Na+, K+ fluxes and [Ca2+]i of VSMC, and 2) Epo may modify the effect of endothelin (ET-1). Using serially passaged quiescent cultured VSMC, the following results were obtained. 1) Epo had no direct effect on steady state Na(+)-K+ transporters (Na(+)-K+ pump, Na(+)-K+ cotransport and Na(+)-H+ antiport). 2) ET-1 alone substantially stimulated Na(+)-K+ pump, Na(+)-H+ antiport and 45Ca uptake, although these effects were not potentiated in the presence of Epo. 3) Epo alone substantially stimulated 45Ca uptake, leading to an increase in [Ca2+]i, which effect was not seen in Ca2+ deficient medium, and was partially inhibited with diltiazem but not with TMB-8. 4) Even in the presence of Epo, ET-1 and angiotensin II (A II) had substantial stimulatory effect on [Ca2+]i of cultured VSMC. The present data indicate that Epo, per se, elicits an increase in [Ca2+]i of VSMC through the stimulation of inward Ca2+ flux without affecting Na(+)-K+ transporters. In contrast, Epo did not potentiate ET-1's stimulatory effect on the transporters. Although the effect of Epo was subtle compared to ET-1 and A II, it may alter an overall characteristic of vascular smooth muscle cell contractility, possibly leading to blood pressure elevation in patients on maintenance dialysis.     

Stimulation of ouabain-sensitive 86Rb+ uptake and Na+,K+-ATPase alpha-subunit phosphorylation by a cAMP-dependent signalling pathway in intact cells from rat kidney cortex

We investigated in intact cortical kidney tubules the role of PKA-mediated phosphorylation in the short-term control of Na+,K+-ATPase activity. The phosphorylation level of Na+,K+-ATPase was evaluated after immunoprecipitation of the enzyme from 32P-labelled cortical tubules and the cation transport activity of Na+,K+-ATPase was measured by ouabain-sensitive 86Rb+ uptake. Incubation of cells with cAMP analogues (8-bromo-cAMP, dibutyryl-cAMP) or with forskolin plus 3-isobutyl-1-methylxanthine increased the phosphorylation level of the Na+,K+-ATPase alpha-subunit and stimulated ouabain-sensitive 86Rb+ uptake. Inhibition of PKA by H-89 blocked the effects of dibutyryl-cAMP on both phosphorylation and 86Rb+ uptake processes. The results suggest that phosphorylation by PKA stimulates the Na+,K+-ATPase activity.     

Pathways of Rb+ influx and their relation to intracellular [Na+] in the perfused rat heart. A 87Rb and 23Na NMR study

The aims of this study were to characterize the routes of influx of the K+ congener, Rb+, into cardiac cells in the perfused rat heart and to evaluate their links to the intracellular Na+ concentration ([Na+]i) using 87Rb and 23Na nuclear magnetic resonance (NMR) spectroscopy. The rate constant for Rb+ equilibration in the extracellular space was 8.5 times higher than that for the intracellular space. The sensitivity of the rate of Rb+ accumulation in the intracellular space of the perfused rat heart to the inhibitors of the K+ and Na+ transport systems has been analyzed. The Rb+ influx rates were measured in both beating and arrested hearts: both procaine (5 mmol/L) and lidocaine (1 mmol/L) halved the Rb+ influx rate. In procaine-arrested hearts, the Na+,K(+)-ATPase inhibitor ouabain (0.6 mmol/L) decreased Rb+ influx by 76 +/- 24% relative to that observed in untreated but arrested hearts. Rb+ uptake was insensitive to the K+ channel blocker 4-aminopyridine (1 mmol/L). The inhibitor of Na+/K+/2 Cl- cotransport bumetanide (30 mumol/L) decreased Rb+ uptake only slightly (by 9 +/- 8%). Rb+ uptake was dependent on [Na+]i: it increased by 58 +/- 34% when [Na+]i was increased with the Na+ ionophore monensin (1 mumol/L) and decreased by 48 +/- 9% when [Na+]i was decreased by the Na+ channel blockers procaine and lidocaine. Dimethylamiloride (15 to 20 mumol/L), an inhibitor of the Na+/H+ exchanger, slightly reduced [Na+]i and Rb+ entry into the cardiomyocytes (by 15 +/- 5%). 31P NMR spectroscopy was used to monitor the energetic state and intracellular pH (pHi) in a parallel series of hearts. Treatment of the hearts with lidocaine, 4-aminopyridine, dimethylamiloride, or bumetanide for 15 to 20 minutes at the same concentrations as used for the Rb+ and Na+ experiments did not markedly affect the levels of the phosphate metabolites or pHi. These data show that under normal physiological conditions, Rb+ influx occurs mainly through Na+,K(+)-ATPase; the contribution of the Na+/K+/2 Cl- cotransporter and K+ channels to Rb+ influx is small. The correlation between Rb+ influx and [Na+bdi during infusion of drugs that affect [Na+]i indicates that, in rat hearts at 37 degrees C, Rb+ influx can serve as a measure of Na+ influx. We estimate that, at normothermia, at least 50% of the Na+ entry into beating cardiac cells is provided by the Na+ channels, with only minor contributions (< 15%) from the Na+/K+/2 Cl- cotransporter and the Na+/H+ exchanger.     

Relationship between fluid transport and in situ inhibition of Na(+)-K+ adenosine triphosphatase in corneal endothelium

PURPOSE: To examine the relationship between the activity of the sodium pump of the corneal endothelium and corneal thickness. It was postulated that because inhibition pressure of the stroma decreases as thickness increases, a partially inhibited sodium pump would result in a new steady-state thickness of the cornea when reduced rates of fluid influx and efflux were equal. Measurements of physiologic behavior and biochemical activity were to be made in the same tissue and thus establish the relationship directly. METHODS: Rabbit corneas were superfused with a bicarbonate Ringer solution containing different concentrations of ouabain. Exposure to ouabain was either continuous for 4 hours or for an initial 10 minutes followed by ouabain-free superfusion. Thickness was measured, and, after superfusion, endothelium was removed from the corneas, sonicated, and assayed for Na(+)-K+ adenosine triphosphatase (ATPase) activity without further addition of ouabain to the assay medium. Thickness was also measured during superfusion with suboptimal concentrations of Na+ or HCO3- and with brefeldin A, an inhibitor of protein trafficking. RESULTS: Continuous exposure to ouabain caused corneas to swell, but no new steady-state thickness was reached. At low concentrations, swelling rates increased with time, as did the extent of inhibition of the Na(+)-K+ ATPase. With only a 10-minute exposure to ouabain, swelling rates with 10(-4) M to 10(-5) M decreased with the duration of ouabain-free superfusion. Similar swelling curves were obtained by reductions in Na+ or HCO3- concentrations in the superfusion medium, indicating that partial inhibition of the endothelial fluid transport processes, whether via the Na(+)-K+ ATPase or by suboptimal ionic conditions, led toward a new equilibrium thickness of the cornea. However, when superfusion was continued for more than 4 hours, the corneas exposed for 10 minutes to 3 x 10(-5) M or lower-concentration ouabain showed increasing Na(+)-K+ ATPase activity and began to thin, indicating a recovery of fluid transport capability. This recovery was blocked by addition of brefeldin A during the ouabain-free superfusion. CONCLUSIONS: Inhibition of Na(+)-K+ ATPase by low concentrations of ouabain increases with time. Temporary exposure to ouabain causes swelling at rates that decline with time as ouabain dissociates from enzyme sites. This dissociation, together with the turnover of Na(+)-K+ ATPase in the plasma membrane, can lead to recovery of normal thickness in ouabain-exposed corneas. Twenty percent of Na(+)-K+ ATPase in the endothelium is estimated to be intracellular, and about 20% of the activity can be inhibited without inducing swelling.     

Modulation of sodium-coupled uptake and membrane fluidity by cisplatin in renal proximal tubular cells in primary culture and brush-border membrane vesicles

The proximal tubule appears to be the main target for the adverse effects of cis-diamminedichloroplatinum (II) (cDDP). We evaluated the early effects of cDDP at concentrations (3 to 67 microM) lower that those which alter cell viability, on three apical transport systems and on the physical state of the brush border membrane (BBM) in rabbit proximal tubule (RPT) cells in primary culture. The maximal effect, corresponding to a 30% decrease in Na(+)-coupled uptake of phosphate (Pi) and alpha-methylglucopyranoside (MGP) and a twofold increase in Na(+)-coupled alanine uptake, was obtained at 17 microM (5 micrograms/ml) cDDP and occurred through a modification of their affinity. At this concentration, cDDP increased BBM fluidity and decreased the BBM cholesterol content by 28%, without increasing the permeability of tight junctions. To clarify the role of cDDP-induced increase in BBM fluidity on alterations of Na(+)-coupled uptake, these parameters were also investigated in BBM vesicles isolated from rabbit renal cortex directly exposed to cDDP. cDDP induced a concentration-dependent inhibition of Na(+)-coupled uptake of MGP, Pi and alanine in BBM vesicles from the renal cortex, associated with a decrease in protein sulfhydryl content, without modifying BBM fluidity. Our findings strongly suggest that the cDDP-induced increase in BBM fluidity in RPT cells results from an indirect mechanism, possibly an alteration of cholesterol metabolism, and did not play a major role in the cDDP-induced inhibition of Na+/Pi and Na+/glucose cotransport systems that may be mainly mediated through a direct chemical interaction with essential sulfhydryl groups of the transporters.     

Postprandial lipoprotein responses in hypertriglyceridemic subjects with and without cardiovascular disease

Three groups of age- and weight-matched men (aged 40 to 70 years) without diabetes were studied: controls (n = 10), plasma triglycerides (TG) less than 180 mg/dL and no cardiovascular disease (CVD); HTG-CVD (n = 11), hypertriglyceridemic (HTG) (TG > 240 mg/dL) without CVD; and HTG+CVD (n = 10), HTG (TG > 240 mg/dL) with documented CVD. HTG+CVD subjects had higher fasting and post-oral glucose tolerance test insulin levels than the other two groups, respectively. Very-low-density lipoprotein (VLDL)+chylomicrons (CMs), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and three high-density lipoprotein (HDL) subfractions (HDL-L, HDL-M, and HDL-D, from least to most dense) were isolated by gradient ultracentrifugation. Fasting lipoproteins were similar in HTG groups, except for higher VLDL lipid to apolipoprotein (apo) B ratios (P < .04) in the HTG+CVD group. Subjects were fed a high-fat mixed meal, and lipoprotein composition was determined at 3, 6, 9, and 12 hours postprandially. Postprandial responses of the core lipids (TG and cholesterol esters [CE]) in all of the lipoprotein subfractions were similar in the two HTG groups at each time point. However, both controls and HTG-CVD subjects had increases in HDL-M phospholipid (PL) at 9 and 12 hours with no change in HDL-D PL. The HTG+CVD group, on the other hand, had no increase in HDL-M PL and had a substantial reduction in HDL-D PL. These changes resulted in significant increases in HDL-M and HDL-D PL to apo A-I ratios in both controls and HTG-CVD subjects between 6 and 12 hours, whereas there was no increase seen in the HTG+CVD group. The HTG-CVD group also had a significantly greater increase in the VLDL+CM PL to apo B ratio (P = .038) at 3 hours than the HTG+CVD group. This diminished amount of surface lipid per VLDL particle may account for the late decrease in the HDL-D PL to apo A-I ratio seen in HTG+CVD patients. There were no other postprandial lipid or apolipoprotein differences between the two HTG groups. We conclude therefore that the major postprandial lipoprotein abnormality in these HTG+CVD patients was a failure to increase the PL content per particle in VLDL+CM, HDL-M, and HDL-D. This abnormality could prevent the usual increase in reverse cholesterol transport seen in postprandial plasma and therefore contribute to their increased incidence of CVD. The greater insulin resistance seen in these patients also appears to contribute significantly to their CVD.     

Epidermal growth factor increases lung liquid clearance in rat lungs

Epidermal growth factor (EGF) has been reported to stimulate the proliferation of epithelial cells and increase Na+ flux and Na+-K+-ATPase function in alveolar epithelial cell monolayers. Increases in Na+-K+-ATPase in alveolar type II cells (AT2) have been associated with increased active Na+ transport and lung edema clearance across the rat alveolar epithelium in a model of proliferative lung injury. Thus we tested whether administration of aerosolized EGF to rat lungs would increase active Na+ transport and lung liquid clearance. Sixteen adult Sprague-Dawley male rats were randomized to three groups. To a group of six rats, an aerosol generated from 20 microgram of EGF in saline was delivered to the lungs, to a second group of five rats only aerosolized saline was delivered, and a third group of five rats without treatment served as the control. Forty-eight hours postaerosolization of rat lungs with EGF there was an approximately 40% increase in active Na+ transport and lung liquid clearance compared with control rats, in the absence of changes in 22Na+, [3H]mannitol, and albumin permeabilities. The Na+-K+-ATPase activity in AT2 cells harvested from these lungs was increased in rats that received aerosolized EGF compared with AT2 cells from both control rats and rats receiving aerosolized saline. These results support the hypothesis that in vivo delivery of EGF aerosols upregulates alveolar epithelial Na+-K+-ATPase and increases lung liquid clearance in rats.     

Unique natriuretic properties of the ATP-sensitive K(+)-channel blocker glyburide in conscious rats

Small-conductance, ATP-sensitive K(+)-channels (KATP) localized in apical membranes of both thick ascending limb of the loop of Henle and cortical collecting duct cells may be involved in Na+ reabsorption and K+ secretion in the mammalian kidney. Possible pharmacologic tools to evaluate such an hypothesis may be the antidiabetic sulfonylureas which block K(+)-channels in pancreatic beta-cells. In saline-loaded conscious rats, glyburide (GLY) dose-dependently increased urinary Na+ excretion with little change in urinary K+ excretion after i.p. administration (10-100 mg/kg). In renal clearance studies, GLY at 25 mg/kg i.v. increased Na+ excretion 350% during the first hour post-treatment without affecting K+ excretion, glomerular filtration rate, mean arterial pressure or heart rate. GLY at 50 mg/kg was no more natriuretic than the 25 mg/kg dose, whereas 12.5 mg/kg of GLY increased Na+ excretion 200%. The change in Na+ excretion produced by 25 mg/kg of GLY in streptozotocin-induced diabetic rats was significantly greater than the change after drug vehicle in these animals. It is unlikely that the natriuresis produced by GLY is secondary to changes in plasma insulin and/or glucose because the doses used were far above GLY's insulin-releasing action (i.e., all natriuretic doses would have produced maximal insulin release) and GLY was natriuretic in streptozotocin-induced diabetic rats. It is possible that GLY interferes with reabsorption of Na+ by blocking KATP and thereby interrupting K+ recycling and Na(+)-2Cl(-)-K+ cotransport in the loop of Henle.     

Calbindin D-28k and monoamine oxidase A immunoreactive neurons in the nucleus basalis of Meynert in senile dementia of the Alzheimer type and Parkinson''s disease

The lungs must be kept "dry" for efficient gas exchange. The mechanisms that contribute to clear alveoli from fetal lung fluid at birth are still present during adult life and allow recovery from alveolar flooding. It has recently been shown with the use of different approaches in vitro, as well as in vivo, that alveolar epithelium performs solute-coupled fluid transport. Fluid absorption from alveoli occurs chiefly as a result of active transepithelial Na+ transport. The mechanisms of Na+ transport have been partly elucidated; Na+ enters alveolar cells through apical Na+ channels and Na(+)-coupled solute transporters and is pumped out at the basolateral membrane by a Na(+)-K(+)-adenosinetriphosphatase (ATPase). Transepithelial Na+ transport and fluid absorption are stimulated by beta-adrenergic agonists, with adenosine 3',5'-cyclic monophosphate being the likely intracellular second messenger. K+ is probably secreted into alveoli because its concentration in the epithelial lining fluid is larger than expected for passive distribution. K+ channels have been described that, in conjunction with Na(+)-K(+)-ATP-ase, might provide pathways for active transport. Active proton secretion or bicarbonate absorption have been reported, which may explain the low pH of the alveolar epithelial lining fluid. It is probable that active solute transports are the main determinants of epithelial lining fluid depth and composition. A challenge for the future is to understand how this homeostasis is achieved.     

Double disruptions of the superior shoulder suspensory complex

Previous studies indicate a particular sensitivity of red blood cell Na(+)-Li+ countertransport activity to small variations in the fatty acid composition of membrane phospholipids. To assess whether the interindividual variability of Na(+)-Li+ countertransport is related to differences in the species pattern of erythrocyte phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vivo, the molecular species composition of PC and PE as well as the kinetics of Na(+)-Li+ countertransport were analyzed in parallel in normo- and hyperlipidemic donors. Both in diacyl-PC and in diacyl-PE the species 16:0/20:4 and 16:0/18:2 were, respectively, positively and negatively related to the apparent maximal velocity of Na(+)-Li+ countertransport. The sum of all species with 20:4 at sn2 of diacyl-PE exhibited a strong positive (r = 0.82, 2p < 0.001), and those containing 18:2 a negative correlation (r = -0.63, 2p < 0.01) to the transport activity. Essentially similar connections were observed between these species and the apparent affinity of the transport system for intracellular Na+. To evaluate whether the associations between molecular species of membrane phospholipids and Na(+)-Li+ countertransport activity were indicative of a causal relationship, the species 16:0/20:4-PC and 16:0/18:2-PC were selectively introduced into the erythrocyte membrane by means of the PC-specific transfer protein. Replacement of 11% of native PC by 16:0/18:2-PC inhibited the transport rate by about 25%. Exchange of 6 and 9% of PC with 16:0/20:4-PC, in contrast, accelerated the transport rate by 30 and 60%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of the total concentration of functional Na+, K(+)-pumps in rumen epithelium

Using the technique of vanadate-facilitated [3H]ouabain binding we have developed a simple and reliable assay for measuring the concentration of [3H]ouabain binding sites in small fresh or frozen biopsies of rumen epithelium papillae. In bovine and ovine rumen epithelium obtained from the cranio-ventral rumen sac the concentration of [3H]ouabain binding sites was 1.6-4.9 nmol g dry wt-1 (n = 32) and 3.7-5.2 nmol g dry wt-1 (n = 6), respectively. When incubated in oxygenated Krebs-Ringer bicarbonate buffer fresh biopsies of rumen epithelium maintained a high K+ and low Na+ content for at least 6 h. Na+ loading of the biopsies induced about 20-fold increase of the Na+, K(+)-pump activity based on measurement of ouabain suppressible net [86Rb+] influx. The ouabain suppressible net influx of [86Rb+] measured in Na+ loaded biopsies showed a close correlation to the [3H]ouabain binding capacity (r = 0.80, P < 0.01) and corresponded to 47 +/- 2% (n = 9) of the theoretical maximum flux rate. The ouabain suppressible net influx of K+ and [86Rb+] were linearly related (r = 0.73; P < 0.001). The net Na+ efflux was 1.21 times the net K+ influx. It is concluded that rumen epithelium has a large capacity for active Na+/K+ transport and that there is agreement between the concentration of [3H]ouabain binding sites in the epithelium and the ouabain suppressible rate of net [86Rb+] influx in Na+ loaded biopsies in spite of some uncertainty about the maximum turnover number of the Na+, K(+)-pump in rumen epithelium.