Cardiovascular and renal characteristics, and responses to acute volume expansion of a rat model of diabetic pregnancy

The objective of this study was to characterize the cardiovascular and renal alterations that occur during diabetic pregnancy, and to evaluate the effect of insulin treatment in 12-14 days pregnant diabetic rats. Four groups of female Sprague Dawley rats were studied: virgin control group (NP), pregnant control group (CP), diabetic pregnant group (DP), and diabetic pregnant group with insulin treatment (DPI). Systolic arterial pressure (SAP) was increased on day 12, whereas heart rate (HR) decreased starting with day 3 in DP group of rats. DP rats exhibited marked renal hypertrophy with greater kidney weight (wt) and kidney wt/body wt ratio. Insulin treatment normalized blood glucose (BG) concentration, SAP and HR, and prevented the increase in kidney wt/body wt ratio in DPI rats. At the time of the terminal acute experiment, acute saline volume expansion (VE, 5% body wt/30 min) significantly increased renal interstitial hydrostatic pressure (RIHP), urinary sodium excretion (U(Na)V) and urine flow rate (V) in all groups, but the increases (Delta) were significantly attenuated in both CP (1.7 +/- 0.2mmHg, 12.0 +/- 1.5 microEq.min(-1).g kidney wt(-1) and 76.2 +/- 10.9 microl.min(-1).g kidney wt(-1) for DeltaRIHP, DeltaU(Na)V and DeltaV respectively) and DP (1.3 +/- 0.1 mmHg, 6.8 +/- 1.8 microEq.min(-1).g kidney wt(-1) and 32.3 +/- 9.3 microl.min(-1).g kidney wt(-1) for DeltaRIHP, DeltaU(Na)V and DeltaV respectively) group of rats as compared to NP (4.0 +/- 0.6 mmHg, 21.6 +/- 1.4 microEq.min(-1).g kidney wt(-1)and 136.8 +/- 10.5 microl.min(-1).g kidney wt(-1) for DeltaRIHP, DeltaU(Na)V and DeltaV respectively) group of rats. Although RIHP response to VE was similar in DP and CP group of rats, the natriuretic and diuretic responses to VE were significantly lower in DP as compared to CP group of rats. Insulin treatment had no effect on RIHP response (DeltaRIHP = 1.5 +/- 0.3 mmHg), but restored most of the natriuretic (DeltaU(Na)V = 15.7 +/- 2.9 microEq.min(-1).g kidney wt(-1)) and diuretic (DeltaV = 100.2 +/- 19.3 microl.min(-1).g kidney wt(-1)) responses to VE in DPI as compared with CP group of rats. These data suggest that with VE, the restoration of the increase in U(Na)V and V with insulin treatment in diabetic pregnant rats is not mediated by changes in RIHP.     

The Na+/Ca2+ exchanger is active and working in the reverse mode in human umbilical artery smooth muscle cells

The data presented in this work suggest that in human umbilical artery (HUA) smooth muscle cells, the Na(+)/Ca(2+) exchanger (NCX) is active and working in the reverse mode. This supposition is based on the following results: (i) microfluorimetry in HUA smooth muscle cells in situ showed that a Ca(2+)-free extracellular solution diminished intracellular Ca(2+) ([Ca(2+)](i)), and KB-R7943 (5microM), a specific inhibitor of the Ca(2+) entry mode of the exchanger, also decreased [Ca(2+)](i) (40.6+/-4.5% of Ca(2+)-free effect); (ii) KB-R7943 produced the relaxation of HUA rings (-24.7+/-7.3gF/gW, n=8, p<0.05); (iii) stimulation of the NCX by lowering extracellular Na(+) increases basal [Ca(2+)](i) proportionally to Na(+) reduction (Delta fluorescence ratio=0.593+/-0.141 for Na(+)-free solution, n=8) and HUA rings' contraction (peak force=181.5+/-39.7 for 130mM reduction, n=8), both inhibited by KB-R7943 and a Ca(2+)-free extracellular solution. In conclusion, the NCX represents an important Ca(2+) entry route in HUA smooth muscle cells.     

Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala

Na,K-ATPase containing the amino acid substitution glutamate to alanine at position 779 of the alpha subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+-Na+ exchange activity in the absence of K+. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 +/- 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K+. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K+. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K+-free solutions. Electrogenic Na+-Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+(o)) concentration. At all Na+(o) concentrations tested (3-148 mM), exchange current was maximal at negative membrane potentials (V(M)), but decreased as V(M) became more positive. Analyzing this current at each V(M) with a Hill equation showed that Na+-Na+ exchange had a high-affinity, low-capacity component with an apparent Na+(o) affinity at 0 mV (K0(0.5)) of 13.4 +/- 0.6 mM and a low-affinity, high-capacity component with a K0(0.5) of 120 +/- 13 mM (n = 17). Both high- and low-affinity exchange components were V(M) dependent, dissipating 30 +/- 3% and 82 +/- 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+-Na+ exchange could be explained by Na+(o) acting as a low-affinity K+ congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+(o) binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K-ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47-59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K-ATPase.     

Relaxin-induced changes in renal sodium excretion in the anesthetized male rat

Pregnancy is associated with profound changes in renal hemodynamics and electrolyte handling. Relaxin, a hormone secreted by the corpus luteum, has been shown to induce pregnancy-like increases in renal blood flow and glomerular filtration rate (GFR) and alter osmoregulation in nonpregnant female and male rats. However, its effects on renal electrolyte handling are unknown. Accordingly, the influence of short (2 h)- and long-term (7 day) infusion of relaxin on renal function was determined in the male rat. Short term infusion of recombinant human relaxin (rhRLX) at 4 microg.h(-1).100 g body wt(-1) induced a significant increase in effective renal blood flow (ERBF) within 45 min, which peaked at 2 h of infusion (vehicle, n = 6, 2.1 +/- 0.4 vs. rhRLX, n = 7, 8.1 +/- 1.1 ml.min(-1).100 g body wt(-1), P < 0.01). GFR and urinary excretion of electrolytes were unaffected. After a 7-day infusion of rhRLX at 4 microg/h, ERBF (1.4 +/- 0.2 vs. 2.5 +/- 0.4 ml.min(-1).100 g body wt(-1), P < 0.05), urine flow rate (3.1 +/- 0.3 vs. 4.3 +/- 0.4 microl.min(-1).100 g body wt(-1), P < 0.05) and urinary sodium excretion (0.8 +/- 0.1 vs. 1.2 +/- 0.1 micromol.min(-1).100 g body wt(-1), P < 0.05) were significantly higher; plasma osmolality and sodium concentrations were lower in rhRLX-treated rats. These data show that long-term relaxin infusion induces a natriuresis and diuresis in the male rat. The mechanisms involved are unclear, but they do not involve changes in plasma aldosterone or atrial natriuretic peptide concentrations.     

Uncoupled basal sodium absorption and chloride secretion in prairie dog (Cynomys ludovicianus) gallbladder.

1. Prairie dog gallbladders mounted in a Ussing-type chamber and bathed with symmetrical Ringer's solutions exhibited a transepithelial resistance (Rt) of 51 +/- 5 omega cm2, a lumen negative potential difference (Vms) of 11.5 +/- 0.7 mV and a short-circuit current (Isc) of 6.9 +/- 0.3 microEq/hr/cm2. 2. Radioisotopic ion flux experiments revealed that the basal Isc of 6.9 +/- 0.3 microEq/hr/cm2 was mostly accounted for by net Na+ absorption of 3.2 +/- 0.5 microEq/hr/cm2 and net Cl- secretion of 2.9 +/- 0.3 microEq/hr/cm2. 3. In HCO3- free Ringer's, net Na+ flux was virtually abolished, net Cl- flux decreased by 50% and Isc was reduced by 77%. 4. 10(-3) M mucosal amiloride and DIDS reduced Isc by 28 and 24%, respectively. 5. Mucosal NaCl diffusion potentials indicated that the paracellular pathway was cation selective. 6. Thin section electron micrographs showed a single cell population in this epithelium suggesting that net Na+ absorption and Cl- secretion may emerge from the same cells. 7. We conclude that prairie dog gallbladder epithelium is an electrogenic tissue and, in contrast to gallbladders of most other species, simultaneously but independently absorbs Na+ and secretes Cl-.     

Quercetin and NPPB-induced diminution of aldosterone action on Na+ absorption and ENaC expression in renal epithelium

In renal epithelial A6 cells, aldosterone applied for 24 h increased the transepithelial Cl- secretion over 30-fold due to activation of the Na+/K+/2Cl- cotransporter and stimulated the transepithelial Na+ absorption, activity of epithelial Na+ channel (ENaC), and alpha-ENaC mRNA expression. The stimulatory action of aldosterone on the transepithelial Na+ absorption, ENaC activity, and alpha-ENaC mRNA expression was diminished by 24h-pretreatment with quercetin (an activator of Na+/K+/2Cl- cotransporter participating in Cl- entry into the cytosolic space) or 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB) (a blocker of Cl- channel participating in Cl- release from the cytosolic space), while 24h-pretreatment with bumetanide (a blocker of Na+/K+/2Cl- cotransporter) enhanced the stimulatory action of aldosterone on transepithelial Na+ absorption. On the other hand, under the basal (aldosterone-unstimulated) condition, quercetin, NPPB or bumetanide had no effect on transepithelial Na+ absorption, activity of ENaC or alpha-ENaC mRNA expression. These observations suggest that although aldosterone shows overall its stimulatory action on ENaC (transepithelial Na+ transport), aldosterone has an inhibitory action on ENaC (transepithelial Na+ transport) via activation of the Na+/K+/2Cl- cotransporter, and that modification of activity of Cl- transporter/channel participating in the transepithelial Cl- secretion influences the aldosterone-stimulated ENaC (transepithelial Na+ transport).     

Stimulation of vascular Na-K pump with subpressor angiotensin II in rats.

The effect of subpressor doses of angiotensin II (ANG II) on vascular Na-K pump activity and Na-H exchange, two transmembrane signals of trophic stimulation of vascular muscle, was investigated. Male Sprague-Dawley rats (350-400 g) were given subpressor doses of ANG II by osmotic minipump intraperitoneally for 24 hr or 7-10 days. Control rats received sham procedure/vehicle infusion. Na-K pump activity (86Rb uptake), total and intracellular (Li exchange at 4 degrees C) Na content, and amiloride-sensitive and -insensitive Na uptake of aortas were measured ex vivo. Ouabain-sensitive 86Rb uptake of aortas of rats receiving 80-100, 160-180, and 240-260 ng/kg.min-1 of ANG II for 24 hr was 26.6 +/- 3.5, 28.8 +/- 3.4, and 29.1 +/- 2.6 nmol/mg dry wt.15 min-1 (mean +/- SD, n = 7-12), respectively, compared with 25.2 +/- 3.8 in controls (n = 23, P less than 0.01). These increases were maintained at 7-10 days. After 24 hr and 7-10 days of ANG II treatment, the total Na content of aortas was increased by 9.2% (P less than 0.01) and 7.6% (P less than 0.02), respectively, without a change in intracellular Na content, indicating accumulation of excess extracellular Na. Total and amiloride-sensitive Na uptake of the aorta was unchanged after 24 hr or 7-10 days of ANG II administration. The dry weight of anatomically defined segments of the aorta was 40 +/- 3.8 mg/kg body wt (n = 25) after 24 hr and 42 +/- 4.4 (n = 20) after 7-10 days of ANG II administration, compared with 37 +/- 4.8 (n = 15, P less than 0.05) and 37 +/- 4.9 (n = 17, P less than 0.01) in appropriate controls. Increased Na-K pump activity may signal the onset of trophic stimulation of vascular muscle by ANG II.     

Splenic denervation worsens lipopolysaccharide-induced hypotension, hemoconcentration, and hypovolemia

During lipopolysaccharide (LPS)-induced endotoxemia, increased intrasplenic fluid efflux contributes to a reduction in plasma volume. We hypothesized that splenic sympathetic nerve activity (SSNA), which increases during endotoxemia, limits intrasplenic fluid efflux. We reasoned that splenic denervation would exaggerate LPS-induced intrasplenic fluid efflux and worsen the hypotension, hemoconcentration, and hypovolemia. A nonlethal dose of LPS (150 microg x kg(-1) x h(-1) for 18 h) was infused into conscious male rats bearing transit time flow probes on the splenic artery and vein. Fluid efflux was estimated from the difference in splenic arterial inflow and venous outflow (A-V). LPS significantly increased the (A-V) flow differential (fluid efflux) in intact rats (saline -0.01 +/- 0.02 ml/min, n = 8 vs. LPS +0.21 +/- 0.06 ml/min, n = 8); this was exaggerated in splenic denervated rats (saline -0.03 +/- 0.01 ml/min, n = 7 vs. LPS +0.41 +/- 0.08 ml/min, n = 8). Splenic denervation also exacerbated the LPS-induced hypotension, hemoconcentration, and hypovolemia (peak fall in mean arterial pressure: denervated 19 +/- 3 mmHg, n = 10 vs. intact 12 +/- 1 mmHg, n = 8; peak rise in hematocrit: denervated 6.7 +/- 0.3%, n = 8 vs. intact 5.0 +/- 0.3%, n = 8; decrease in plasma volume at 90-min post-LPS infusion: denervated 1.08 +/- 0.15 ml/100 g body wt, n = 7 vs. intact 0.54 +/- 0.08 ml/100 g body wt, n = 8). The exaggerated LPS-induced hypovolemia associated with splenic denervation was mirrored in the rise in plasma renin activity (90 min post-LPS: denervated 11.5 +/- 0.8 ng x ml(-1) x h(-1), n = 9 vs. intact 6.6 +/- 0.7 ng x ml(-1) x h(-1), n = 8). These results are consistent with our proposal that SSNA normally limits LPS-induced intrasplenic fluid efflux.     

Regulation of endogenous and expressed Na+/K+ pumps in Xenopus oocytes by membrane potential and stimulation of protein kinases

Modulation of the current generated by the Na+/K+ pump by membrane potential and protein kinases was investigated in oocytes of Xenopus laevis. In addition to a positive slope region in the current-voltage (I-V) relationship of the Na+/K+ pump, a negative slope region has been described in these cells (Lafaire & Schwarz, 1986) and has been attributed to a voltage-dependent apparent Km value for pump stimulation by external [K+] (Rakowski et al., 1991). To study this feature in more detail, Xenopus oocytes were used for comparative analysis of the negative slope of the I-V relationship of the endogenous Na+/K+ pump and of the Na+/K+ pump of the electric organ of Torpedo californica expressed in the oocytes. The effects of stimulation of protein kinases A and C on the negative slope were also analyzed. To investigate the negative slope over a wide potential range, experiments were performed in Na(+)-free solution and in the presence of high concentrations of Ba2+ and tetraethylammonium, to block all nonpump related K(+)-sensitive currents. Pump currents and pump-mediated fluxes were determined as differences of currents or fluxes in solutions with and without extracellular K+. The voltage dependence of the Km value for stimulation of the Na+/K+ pump by external [K+] shows significant species differences. Over the entire voltage range from -140 to +20 mV, the Km value for the Na+/K+ pump of Torpedo electroplax is substantially higher than for the endogenous pump and exhibits more pronounced voltage dependence. For the Xenopus pump, the voltage dependence can be described by voltage-dependent stimulation by external [K+] and can be interpreted by voltage-dependent K+ binding, assuming that an effective charge between 0.37 and 0.56 of an elementary charge is moved in the electrical field. An analogous evaluation of the voltage dependence of the Torpedo pump requires the assumption of movement of two effective charges of 0.16 and 1.0 of an elementary charge. Application of 1,2-dioctanoyl-sn-glycerol (diC8, 10-50 microM), which is known to stimulate protein kinase C, reduces the maximum activity of the Xenopus pumps in the oocyte membrane by 40% and modulates the voltage dependence of K+ stimulation. For the endogenous Xenopus pump, the apparent effective charge increased from 0.37 to 0.51 of elementary charge and the apparent Km at 0 mV increased from 0.46 to 0.83 mM. For the Torpedo pump, one of the apparent effective charges increased from 1.0 to 2.5 of elementary charge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Antiinflammatory and chronic toxicity study of the leaves of Ageratum conyzoides L. in rats

The hydroalcoholic extract (HAE) of Ageratum conyzoides leaves was studied for its antiinflammatory effect on subacute (cotton pellet-induced granuloma) and chronic (formaldehyde-induced arthritis) models of inflammation in rats. The absence or presence of toxicity by prolonged use of HAE was also evaluated through biochemical and hematological analysis of rats blood samples using daily oral doses of 250 or 500 mg/kg body wt., during 90 days. The results showed that the group of rats treated with HAE (250 mg/kg body wt.; p.o.) had a 38.7% (p < 0.05) reduction in cotton-pellet granuloma. The development of chronically induced paw edema was also reduced significantly (p < 0.05) by the plant extract. The toxicity study did not show any treatment-related abnormalities in biochemical and hematological parameters. The biochemical analysis from blood samples drawn from group of rats treated orally with 500 mg/kg body wt. did, however, present 30.2% (p < 0.05) reduction of SGPT activity as compared to the corresponding control group. These results confirm the antiinflammatory properties of A. conyzoides, with no apparent hepatotoxicity.     

A critical analysis of transepithelial potential in intact killifish (Fundulus heteroclitus) subjected to acute and chronic changes in salinity

We investigated the in vivo salinity-dependent behavior of transepithelial potential (TEP) in Fundulus heteroclitus (3-9 g) using indwelling coelomic catheters, a technique which was validated against blood catheter measurements in a larger species (Opsanus beta; 35-70 g). In seawater (SW)-acclimated killifish, TEP was +23 mV (inside positive), but changed to -39 mV immediately after transfer to freshwater (FW). Acute transfer to dilute salinities produced a TEP profile, which rapidly attenuated as salinity increased (0, 2.5, 5 and 10% SW), with cross-over to positive values between 20 and 40% SW, and a linear increase thereafter (60, 80 and 100% SW). TEP response profiles were also recorded after acute transfer to comparable dilutions of 500 mmol L(-1) NaCl, NaNO3, Na gluconate, choline chloride, N-methyl-D-glutamate (NMDG) chloride, or 1,100 mosmol kg(-1) mannitol. These indicated high non-specific cation permeability and low non-specific anion permeability without influence of osmolality in SW-acclimated killifish. While there was a small electrogenic component in high salinity, a Na+ diffusion potential predominated at all salinities due to the low P Cl/P Na (0.23) of the gills. The very negative TEP in FW was attenuated in a linear fashion by log elevations in [Ca2+] such that P Cl/P Na increased to 0.73 at 10 mmol L(-1). SW levels of [K+] or [Mg2+] also increased the TEP, but none of these cations alone restored the positive TEP of SW-acclimated killifish. The very negative TEP in FW attenuated over the first 12 h of exposure and by 24-30 h reached +3 mV, representative of long-term FW-acclimated animals; this reflected a progressive increase in P Cl/P Na from 0.23 to 1.30, probably associated with closing of the paracellular shunt pathway. Thereafter, the TEP in FW-acclimated killifish was unresponsive to [Ca2+] (also to [K+], [Mg2+], or chloride salts of choline and NMDG), but became more positive at SW levels of [Na+]. Killifish live in a variable salinity environment and are incapable of gill Cl(-) uptake in FW. We conclude that the adaptive significance of the TEP patterns is that changeover to a very negative TEP in FW will immediately limit Na+ loss while not interfering with active Cl(-) uptake because there is none. Keeping the shunt permeability high for a few hours means that killifish can return to SW and instantaneously re-activate their NaCl excretion mechanism.     

Increased cathepsin D-like activity in cortex, tubules, and glomeruli isolated from rats with experimental nephrotic syndrome.

We have examined the activity and distribution of cathepsin D (EC 3.4.23.5), a major renal lysosomal endoproteinase, in the various anatomical and functional areas of normal rat kidney. Cathepsin D-like activities (delta A280/h per mg of protein) in normal rat tissues were: cortex, 0.78 +/- 0.05, n = 37; medulla, 0.62 +/- 0.03, n = 12; papilla, 0.63 +/- 0.04, n = 12; tubules, 0.74 +/- 0.04, n = 28; glomeruli, 0.59 +/- 0.03, n = 28; and liver, 0.41 +/- 0.02, n = 28. Enzyme activity was maximal at pH 3.0-3.5 and inhibited more than 90% by pepstatin (6.7 micrograms/ml), suggesting that the enzyme is cathepsin D. In subsequent experiments we measured cathepsin D-like activity in cortex, tubules and glomeruli isolated from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Treated animals (15 mg of PAN/100g body wt., intraperitoneally) developed proteinuria beginning 4 days after injection and exceeding 900 mg/24h on day 9. In two separate experiments involving 52 animals we observed a significant increase in cathepsin D-like activity in cortex (+82.7%), tubules (+109.6%) and glomeruli (+54.7%) isolated from PAN-treated rats killed during marked proteinuria (day 9, mean total urinary protein excretion: 937 +/- 94 mg/24h). This increase was observed whether the activity was expressed per mg of DNA or per mg of protein. Increased cathepsin D-like activity was first observed in cortex and tubules coincident with the onset of proteinurea (day 4, mean total urinary protein excretion: 112 +/- 23 mg/24h). In contrast with the significant elevation of renal cathepsin D-like activity, the activity (nmol/h per mg of protein) of alpha-L-fucosidase (EC 3.2.1.51), a non-proteolytic enzyme, was markedly decreased in the identical samples used for the measurement of cathepsin D-like activity: cortex (-46.4%); tubules (-46.1%); and glomeruli (-38.5%). In addition to changes in renal enzyme activities, PAN-treated rats excreted large amounts of cathepsin D-like activity in their urine (beginning on day 3) compared with nearly undetectable cathepsin D-like activity in the urine from control rats. The significant increases in glomerular and tubular cathepsin D activity may reflect an important role for this enzyme in the pathophysiology associated with PAN-induced nephrotic syndrome.     

High fat diet induced oxidative DNA damage estimated by 8-oxo-7,8-dihydro-2-deoxyguanosine excretion in rats

The role of dietary fats and energy in carcinogenesis has been partly related to oxidative damage to DNA. We have investigated the effect of dietary fat content and saturation on the urinary excretion of 8-oxo-7,8dihydro-2'-deoxyguanosine (8-oxodG) in male and female rats. Groups of Fischer F344 rats (n = 6-10) were fed control chow (3.4% fat) or diets containing 21.8% corn oil or 19.8% coconut oil + 2% corn oil for 12-15 weeks. At the end of the diet intervention period 24h urine was collected for determination of 8-oxodG by HPLC. In the male groups fed control, corn oil and coconut oil diet the excretion of 8-oxodG was 403+/-150, 932+/-198 and 954+/-367pmol/kg 24 h, respectively (p < 0.05). In the female groups fed control and corn oil diet the excretion of 8-oxodG was 752+/-80 and 2206+/-282 pmol/kg 24 h, respectively (p < 0.05). Calculated per whole animal the excretion was 137+/-51, 324+/-70 and 328+/-128 pmol/24 h in the control, corn and coconut oil male groups and 156+/-21 and 464+/-56 pmol/24 h in the control and corn oil female groups, respectively ( p < 0.05). Thus, per animal or per consumed energy there was much less difference in 8-oxodG excretion between the corresponding male and female groups and only significant difference between the high fat groups. There was a close correlation (r = 0.7; p < 0.05) between 8-oxodG excretion and the energy intake. The present study suggests that a high fat diet increases oxidative DNA modification substantially irrespective of the saturation level of the fat. Energy intake appears to be the major determinant of the rate of modification.     

Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation

The regulation of intracellular pH (pHi) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2',7'-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO3(-)-containing medium (25 mM NaHCO3/5% CO2; pH 7.4) have a steady-state pHi of 7.25 +/- 0.02. Acid loading of acinar cells using the NH4+/NH3 prepulse technique resulted in a Na(+)-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, Ki approximately 0.42 microM) pHi recovery, the kinetics of which were not influenced by the absence of extracellular Cl-. The rate and magnitude of the pHi recovery were dependent on the extracellular Na+ concentration, indicating that Na+/H+ exchange plays a critical role in maintaining pHi above the pH predicted for electrochemical equilibrium. When the NH4+/NH3 concentration was varied, the rate of pHi recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na+/H+ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl(-)-free medium did not significantly alter resting pHi, suggesting the absence of Cl-/HCO3- exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t 1/2 < 30 sec; 0.16 +/- 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37 +/- 0.05 pH unit) or in the absence of extracellular Na+ (0.34 +/- 0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO3(-)-free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl-. The Ca2+ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid) to chelate intracellular Ca2+ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na+/H+ exchange activity; (ii) is most likely mediated by HCO3- efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca2+].     

Acute and chronic cardiovascular effects of intermittent hypoxia in C57BL/6J mice.

We investigated the effects of 1) acute hypoxia and 2) 5 wk of chronic intermittent hypoxia (IH) on the systemic and pulmonary circulations of C57BL/6J mice. Mice were chronically instrumented with either femoral artery or right ventricular catheters. In response to acute hypoxia (4 min of 10% O2; n = 6), systemic arterial blood pressure fell (P < 0.005) from 107.7 +/- 2.5 to 84.7 +/- 6.5 mmHg, whereas right ventricular pressure increased (P < 0.005) from 11.7 +/- 0.8 to 14.9 +/- 1.3 mmHg. Another cohort of mice was then exposed to IH for 5 wk (O2 nadir = 5%, 60-s cycles, 12 h/day) and then implanted with catheters. In response to 5 wk of chronic IH, mice (n = 8) increased systemic blood pressure by 7.5 mmHg, left ventricle + septum weight by 32.2 +/- 7.5 x 10(-2) g/100 g body wt (P < 0.015), and right ventricle weight by 19.3 +/- 3.2 x 10(-2) g/100 g body wt (P < 0.001), resulting in a 14% increase in the right ventricle/left ventricle + septum weight (P < 0.005). We conclude that in C57BL/6J mice 1) acute hypoxia causes opposite effects on the pulmonary and systemic circulations, leading to preferential loading of the right heart; and 2) chronic IH in mice results in mild to moderate systemic and pulmonary hypertension, with resultant left- and right-sided ventricular hypertrophy.     

Influence of N-dodecyl-N,N-dimethylamine N-oxide on the activity of sarcoplasmic reticulum Ca(2+)-transporting ATPase reconstituted into diacylphosphatidylcholine vesicles: efects of bilayer physical parameters

Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 degrees C, the specific activity of ATPase displays a maximum (31.5+/-0.8 IU/mg) for dioleoylphosphatidylcholine (diC18:1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22:1PC), the zwitterionic surfactant N-dodecyl-N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2+/-0.6 to 32.5+/-0.8 IU/mg in the range of molar ratios C12NO:diC22:1PC=0/1.2. In dilauroylphosphatidylcholines (diC12:0PC) and diC18:1PC, the effect of C12NO is twofold-the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18:1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO:diC18:1PC< or =1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4+/-0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N(+)-P- accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO:diC18:1PC=1.3/2.5, where mixed tubular micelles are detected by SANS in C12NO+diC18:1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca(2+)-ATPase activity.     

H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two uncompetitive, activated, and transport sites of the Na+/H+ exchanger for pH regulation in perfused rat kidney

The purpose of this study is to assess the effect of an apparent alteration in intracellular pH and the effect of amiloride on the activity of the Na+/H+ antiporter in perfused rat kidney. Rat kidney-Na+ retention was determined using tracer 22Na in perfusate composed of HCl-glycine buffer (pH 3.80 to pH 5.92) or NH4OH-glycine buffer (pH 6.22-7.95) containing Na+ to match physiologic concentrations. Plotting renal Na+ retention for 10 min versus pH in absence of amiloride showed two classical uncompetitive activator curves for H+, one curve from pH 4.19 to 5.10 and another from pH 6.22 to 7.95. H+ acts as an uncompetitive reversible binding substrate with the receptor triggering activation of the exchanger already sequestered with Na+, thus yielding two Ka values for the exchanger suggesting non-first order kinetics. Using an equation derived for uncompetitive-activation binding of Nao+ and Hi+, plotting [mM Na+ mg protein-1 10 min-1]-1 versus [H+], two linear plots are observed on Cartesian coordinates with abscissa intersecting at 47 +/- 1 microM, pKa = 4.32 +/- 0.02 (pH 4.19-5.10) and 4.21 +/- 0.02 microM, pKa = 5.38 +/- 0.01 (pH 6.22-7.95), respectively. Perfusing buffer containing 2 mM amiloride, completely inactivated the antiporter showing stronger inhibition between pH 3.80 and 5.92. Results suggest the presence of two uncompetitive binding sites for H+ with the Na+/H+ exchanger. One is a high affinity binding site at physiological intracellular apparent pH, and another is a low affinity binding site at ischaemic apparent pH, implying the existence of two titration sites for intracellular pH regulation.     

Hyponatremia-induced brain edema in guinea pigs is reduced by treatment with the novel anion transport inhibitor L-644,711

Cerebral edema in various disease states may result from astroglial swelling due to increased NaCl uptake mediated by enhanced Cl-HC03 exchange. We evaluated this mechanism in the pathogenesis of cerebral edema in acute hyponatremia by administering L-644,711, a fluorenyloxyacetate derivative that functions as an anion exchange inhibitor, to guinea pigs with severe reductions in serum Na+ concentration. Acute hyponatremia was induced for 54 hr by daily injections of arginine vasopressin (10 U/day) and 5% dextrose in water (7.5% body wt/day). Experimental animals received L-644,711, 20 mg/kg/day, while controls were given an equal volume of the diluent. This regimen lowered the serum Na from normal levels to 108 +/- 3 and 109 +/- 4 mM in experimental and control animals, respectively. Drug treatment resulted in less cerebral edema characterized by a reduction in brain total tissue water 432 +/- 4 vs 466 +/- 8 ml/100 g dry wt experimental vs control, P less than 0.005. This difference was composed mainly of less expansion of the intracellular water space, 287 +/- 11 vs 323 +/- 9 ml/100 g dry wt experimental vs control, p less than 0.005. The cerebral cortical Na+ +Cl content was reduced from 55.5 +/- 1.3 (control) to 39.5 +/- 1.1 mEq/100 g dry wt (experimental), p less than 0.01. These results indicate that treatment of guinea pigs with L-644,711 decreases brain NaCl content and attenuates cerebral edema during severe acute hyponatremia without normalizing the serum Na+ concentration.