K+-Phosphatase activity of gill (Na+, K+)-ATPase from the blue crab, Callinectes danae: low-salinity acclimation and expression of the alpha-subunit

The kinetic properties of a microsomal gill (Na(+), K(+)) ATPase from the blue crab, Callinectes danae, acclimated to 15 per thousand salinity for 10 days, were analyzed using the substrate p-nitrophenylphosphate. The (Na(+), K(+))-ATPase hydrolyzed the substrate obeying Michaelian kinetics at a rate of V=102.9+/-4.3 U.mg(-1) with K(0.5)=1.7+/-0.1 mmol.L(-1), while stimulation by magnesium (V=93.7+/-2.3 U.mg(-1); K(0.5)=1.40+/-0.03 mmol.L(-1)) and potassium ions (V=94.9+/-3.5 U.mg(-1); K(0.5)=2.9+/-0.1 mmol.L(-1)) was cooperative. K(+)-phosphatase activity was also stimulated by ammonium ions to a rate of V=106.2+/-2.2 U. mg(-1) with K(0.5)=9.8+/-0.2 mmol.L(-1), following cooperative kinetics (n(H)=2.9). However, K(+)-phosphatase activity was not stimulated further by K(+) plus NH(4) (+) ions. Sodium ions (K(I)=22.7+/-1.7 mmol.L(-1)), and orthovanadate (K(I)=28.1+/-1.4 nmol.L(-1)) completely inhibited PNPPase activity while ouabain inhibition reached almost 75% (K(I)=142.0+/-7.1 micromol.L(-1)). Western blotting analysis revealed increased expression of the (Na(+), K(+))-ATPase alpha-subunit in crabs acclimated to 15 per thousand salinity compared to those acclimated to 33 per thousand salinity. The increase in (Na(+), K(+))-ATPase activity in C. danae gill tissue in response to low-salinity acclimation apparently derives from the increased expression of the (Na(+), K( (+) ))-ATPase alpha-subunit; phosphate-hydrolyzing enzymes other than (Na(+), K(+))-ATPase are also expressed. These findings allow a better understanding of the kinetic behavior of the enzymes that underlie the osmoregulatory mechanisms of euryhaline crustaceans.     

Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm

Electron probe analysis of dry cryosections was used to determine the composition of the cytoplasm and organelles of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle. All analytical values given are in mmol/kg wt +/- SEM. Cytoplasmic concentrations in normal, resting muscles were: K, 611 +/- 1.7; Na, 167 +/- 2.7; Cl, 278 +/- 1.0; Mg, 36 +/- 1.1; Ca, 1.9 +/- 0.5; and P, 247 +/- 1.1. Hence, the sum of intracellular Na + K exceeded cytoplasmic Cl by 500 mmol/kg dry wt, while the calculated total, nondiffusible solute was approximately 50 mmol/kg. Cytoplasmic K and Cl were increased in smooth muscles incubated in solutions containing an excess (80 mM) of KCl. Nuclear and cytoplasmic Na and Ca concentrations were not significantly different. The mitochondrial Ca content in normal fibers was low, 0.8 +/- 0.5, and there was no evidence of mitochondrial Ca sequestration in muscles frozen after a K contracture lasint 30 min. Transmitochondrial gradients of K, Na, and Cl were small (0.9--1.2). In damaged fibers, massive mitochondrial Ca accumulation of up to 2 mol/kg dry wt in granule form and associated with P could be demonstrated. Our findings suggest (a) that the nonDonnan distribution of Cl in smooth muscle is not caused by sequestration in organelles, and that considerations of osmotic equilibrium and electroneutrality suggest the existence of unidentified nondiffusible anions in smooth muscle, (b) that nuclei do not contain concentrations of Na or Ca in excess of cytoplasmic levels, (c) that mitochondria in PAMV smooth muscle do not play a major role in regulating cytoplasmic Ca during physiological levels of contraction but can be massively Ca loaded in damaged cells, and (d) that the in situ transmitochondrial gradients of K, Na, and Cl do not show these ions to be distributed according to a large electromotive Donnan force.     

Protection against cadmium toxicity and enzyme inhibition by dithiothreitol

In the present in vivo studies the alterations in cation transporting enzymes of the brain, kidney and liver tissues were assessed at intervals between 0 to 48 h after a single, acute (10 mg kg-1, i.p.) dose of cadmium (Cd). The inhibition of Na+-K+-ATPase during the first 24 h does not parallel the changes in K+-PNPPase suggesting differential effects on phosphorylation and dephosphorylation steps of the overall ATPase reaction. Between 30 min to 2 h the inhibition in enzyme activity was steep (27 per cent in brain, 54 per cent in liver) followed by a rapid reversal between 2-6 h. This critical period may correspond to the time of induction of metallothionein. This enzyme reversal was followed by a significant decrease in Na+-K+ ATPase (40-68 per cent) and K+-PNPPase (44-60 per cent) between 24 to 48 h. A similar pattern was observed in Ca2+-ATPase in all the three tissues. A 33 per cent mortality was observed in rats after 48 h of cadmium challenge. Administration of dithiothreitol (DTT, 20 mg kg-1, i.p.) to CdCl2 pretreated rats at 24 h resulted in mortality reduced from 33 per cent to 0 and reversal in the inhibition of Na+-K+-ATPase in brain and kidney and Ca2+-ATPase in brain. Since protection of brain and kidney enzymes by DTT paralleled its protection against Cd toxicity, their inhibition by Cd may, in part, constitute the biochemical basis of Cd toxicity.     

The osmotic concentration of parietal muscle of the hagfish Myxine glutinosa L., including estimates of free and bound constituents

The principal osmotic constituents of plasma and of muscle before and after ultracentrifugation have been determined. By analysing the muscle fluid and centrifuged muscle and determining their extracellular fluid (inulin space), ion-binding in the cells was estimated at Na 26%, K 0.3%, Ca 93%, Mg 24%, Cl 21% and P 10%. Muscle fluid was 4.9% (2.7-7.4%) hyperosmotic to plasma. This is discussed in relation to calculated osmolality of muscle and plasma.     

Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K(+)-ATPase through proteasomal and endo-/lysosomal proteolytic pathways.

The mechanisms of cadmium (Cd)-dependent nephrotoxicity were studied in a rat proximal tubule (PT) cell line. CdCl(2) (5 microM) increased the production of reactive oxygen species (ROS), as determined by oxidation of dihydrorhodamine 123 to fluorescent rhodamine 123. The levels of ubiquitin-conjugated cellular proteins were increased by Cd in a time-dependent fashion (maximum at 24-48 h). This was prevented by coincubation with the thiol antioxidant N-acetylcysteine (NAC, 15 mM). Cd also increased apoptosis (controls: 2.4+/-1.6%; Cd: 8.1+/-1.9%), but not necrosis (controls: 0.5 +/- 0.3%; Cd: 1.4+/- 2.5%). Exposure of PT cells with Cd decreased protein levels of the catalytic subunit (alpha1) of Na+/K(+)-ATPase, a long-lived membrane protein (t(1/2)>48 h) that drives reabsorption of ions and nutrients through Na(+)-dependent transporters in PT. Incubation of PT cells for 48 h with Cd decreased Na+/K(+)-ATPase alpha1-subunit, as determined by immunoblotting, by approximately 50%, and NAC largely prevented this effect. Inhibitors of the proteasome such as MG-132 (20 microM) or lactacystin (10 microM), as well as lysosomotropic weak bases such as chloroquine (0.2 mM) or NH(4)Cl (30 mM), significantly reduced the decrease of Na(+)/K(+)-ATPase alpha1-subunit induced by Cd, and in combination abolished the effect of Cd on Na+/K(+)-ATPase. Immunofluorescence labeling of Na+/K(+)-ATPase showed a reduced expression of the protein in the plasma membrane of Cd-exposed cells. After addition of lactacystin and chloroquine to Cd-exposed PT cells, immunoreactive material accumulated into intracellular vesicles. The data indicate that micromolar concentrations of Cd can increase ROS production and exert a toxic effect on PT cells. Oxidative damage increases the degradation of Na+/K(+)-ATPase through both the proteasomal and endo-/lysosomal proteolytic pathways. Degradation of oxidatively damaged Na+/K(+)-ATPase may contribute to the 'Fanconi syndrome'-like Na(+)-dependent transport defects associated with Cd-nephrotoxicity.     

Postexercise rehydration: effect of Na(+) and volume on restoration of fluid spaces and cardiovascular function.

Our purpose was to study the interaction between Na(+) content and fluid volume on rehydration (RH) and restoration of fluid spaces and cardiovascular (CV) function. Ten men completed four trials in which they exercised in a 35 degrees C environment until dehydrated by 2. 9% body mass, were rehydrated for 180 min, and exercised for an additional 20 min. Four RH regimens were tested: low volume (100% fluid replacement)-low (25 mM) Na(+) (LL), low volume-high (50 mM) Na(+) (LH), high volume (150% fluid replacement)-low Na(+) (HL), and high volume-high Na(+) (HH). Blood and urine samples were collected and body mass was measured before and after exercise and every hour during RH. Before and after the dehydration exercise and during the 20 min of exercise after RH, cardiac output was measured. Fluid compartment (intracellular and extracellular) restoration and percent change in plasma volume were calculated using the Cl(-) and hematocrit/Hb methods, respectively. RH was greater (P < 0.05) in HL and HH (102.0 +/- 15.2 and 103.7 +/- 14.7%, respectively) than in LL and LH (70.7 +/- 10.5 and 75.9 +/- 6.3%, respectively). Intracellular RH was greater in HL (1.12 +/- 0.4 liters) than in all other conditions (0.83 +/- 0.3, 0.69 +/- 0.2, and 0.73 +/- 0.3 liter for LL, LH, and HH, respectively), whereas extracellular RH (including plasma volume) was greater in HL and HH (1.35 +/- 0.8 and 1.63 +/- 0.4 liters, respectively) than in LL and LH (0.83 +/- 0.3 and 1.05 +/- 0.4 liters, respectively). CV function (based on stroke volume, heart rate, and cardiac output) was restored equally in all conditions. These data indicate that greater RH can be achieved through larger volumes of fluid and is not affected by Na(+) content within the range tested. Higher Na(+) content favors extracellular fluid filling, whereas intracellular fluid benefits from higher volumes of fluid with lower Na(+). Alterations in Na(+) and/or volume within the range tested do not affect the degree of restoration of CV function.     

The chemical composition and the osmotic pressure of the aqueous humor and plasma of the rabbit

Measurements were made of the osmotic pressure of plasma, and of aqueous humor taken from the anterior chamber of the right and left eyes and from the posterior chamber of unanesthetized rabbits. Aqueous humor from the anterior chamber was found to be hypertonic to the plasma by approximately 3 mM/liter equivalent of sodium chloride. The aqueous humor from the anterior and posterior chambers of the right and left eyes was isotonic. The concentration of chloride in the anterior and posterior chambers was the same. The concentration of all the major components of the aqueous humor and plasma has been determined by chemical analysis on fluid samples obtained from unanesthetized rabbits at approximately the same time. The calculated osmotic pressure of the total of these substances in terms of sodium chloride equivalent agrees to within better than 1 per cent of the total osmotic pressure as measured experimentally. The distribution of some individual anions and cations of the aqueous humor and plasma was determined. This distribution is widely different from that which would obtain at a state of equilibrium. The positive and negative charges carried by the ions in the aqueous humor were approximately equal. Sources of error in the experiments are discussed.     

Effect of calixarene-phosphonic acid on Na+, K+-ATPase activity in plasma membranes of the smooth-muscle cells

Effect of calix[4]arenes C-97, C-99, C-107, functionalized by fragments of alpha-hydroxy-phosphonic, alpha-aminophosphonic- and methylene-bisphosphonic acid on enzymatic activity of oubaine-sensitive Na+, K+-ATPase and oubaine-resistant basal Mg2+- ATPase (specific activity - 10.6 +/- 0.9 and 18.1 +/- 1.2 micromol Pi/h per 1 mg of protein, respectively; n = 7) was studied in experiments made on the suspension of myometrium cell plasma membranes treated by 0.1% solution of digitonin. It was found that calixarene-phosphonic acids in concentration of 100 microM inhibited enzymatic activity of Na+, K+-ATPase by 86-98% and did not practically affect activity of Mg2+-ATPase. These calixarenes were more efficient than oubaine in suppressing enzymatic activity of the sodium pump: in case of the effect of calixerenes the value of the appearence constant of inhibition I0.5 was < 0.1 microM. Calixarene-methylene-bisphosphonic acid (calixarene C-97; I0.5 =33 +/- 4 microM (n = 6) takes the most efficient inhibitory effect on Na+,K+-ATPase activity among the studied calixarenes. A phenomenon of negative cooperation: the Hill coefficient value etaH =0.1-0.5<1 is characteristic of both the inhibiting effect of calixarenes and oubaine. Reguliarities of calixarenes C-97 effect on enzymatic activity of Na+,K+-ATPase were studied. As it appeared its inhibiting effect cannot be caused by trivial factors - potentially possible binding of Mg ions by it and (or) this substance effect on Mg2+ interaction with ATP4- in the incubation medium. Calixerene C-97 does not also decrease the enzyme affinity for Mg ions or ATP. However this calixerenes decreases the affinity of Na+,K+-ATPase for Na ions (the value of activation constant K(Na+)) from 50 +/- 4 (control) to 76 +/- 6 microM in the control and under the effect of calixerene, respectively). A conclusion is made that calixerene C-97 is highly-efficient (with respect to oubaine) and selective (with respect to lack of its effect on basal Mg2+-ATPase) inhibitor of Na+,K+-ATPase of plasma membrane. In the practical aspect it may be used in concentration of 1-10 microM in biochemical membranology when testing and studying kinetic and catalytic properties of the sodium pump in case of such experimental model, as the plasma membrane fraction.     

Continuous collection and analysis of bovine oviduct fluid: preliminary results

Oviduct fluid was collected by cannulating the oviducts of nine cows. The fluid was analyzed for sodium (Na), potassium (K), chlorine (Cl), calcium (Ca), inorganic phosphorus (Pi), magnesium (Mg) concentration and osmolarity (Osm). The mean concentrations +/- the standard error of the means of the constituents were: Na (140.7 +/- 0.37 mEq/L), K (5.12 +/- 0.08 mEq/L), Cl (101.8 +/- 1.54 mEq/L), Ca (1.88 +/- 0.08 mEq/L), Pi (1.97 +/- 0.07 mEq/L), and Mg (1.00 +/- 0.03 mEq/L). Osmolarity was 281.0 +/- 2.56 m Osmols. Significantly lower concentrations of Na (117.4 +/- 2.58 mEq/L) were found in the fluid collected from the oviduct ipsilateral to the ovulating ovary on the day of ovulation. This decrease in Na concentration did not occur in fluid from the contralateral oviduct. The lowest concentrations of Ca were found during days 18 through 21, while the highest were found during days 2 through 6. No significant cyclic changes in the other constituents were observed although the concentrations tended to be highest during days 18 through 21 and lowest on day 1. The concentrations of many of the constituents analyzed were different than those previously reported for bovine oviduct fluid (1).     

Characterization of L-Glutamate Binding Sites in Rat Spinal Cord Synaptic Membranes: Evidence for Multiple Chloride Ion-Dependent Sites

The effects of various ions on L-glutamate (L-Glu) binding sites (Na+-dependent, Cl(-)-dependent, and Cl(-)-independent) in synaptic plasma membranes (SPM) isolated from rat spinal cord and forebrain were examined. Cl(-)-dependent binding sites were over twofold higher in spinal cord (Bmax = 152 +/- 34 pmol/mg protein) as compared to forebrain SPM (Bmax = 64 +/- 12 pmol/mg protein). Na+-dependent binding, on the other hand, was nearly sixfold less in spinal cord (Bmax = 74 +/- 10 pmol/mg protein) compared to forebrain SPM (408 +/- 26 pmol/mg protein). Uptake of L-Glu (Na+-dependent) was also eightfold less in the P2 fraction from spinal cord relative to forebrain (Vmax of 2.89 and 22.3 pmol/mg protein/min, respectively). The effects of Na+, K+, NH4+, and Ca2+ on L-Glu binding sites were similar in both regions of the CNS. In addition, in spinal cord membranes, Br-, I-, and NO3- were equivalent to Cl- in their capacity to stimulate L-Glu binding, whereas F- and CO3- were less effective. Cl(-)-dependent L-Glu binding in spinal cord membranes consisted of two distinct sites. The predominant site (74% of the total) had characteristics similar to the Cl(-)-dependent binding site in forebrain membranes [i.e., Ki values of 5.7 +/- 1.4 microM and 119 +/- 38 nM for 2-amino-4-phosphonobutyric acid (AP4) and quisqualic acid, (QUIS), respectively]. The other Cl(-)-dependent site was unaffected by AP4 but was blocked by QUIS (Ki = 14.2 +/- 4.8 microM).     

Diurnal rhythms of thyroid hormones in rooster chicks (Gallus domesticus). 2. Studies on the protein-bound radioiodine and the "free" hormone portion]

In four week old cockerels the plasma was investigated for cyclic changes in total radioactivity, PB131I, PI131I, and "free" thyroid hormones 24 hours after injection of Na131I. Maxima in total radioactivity were observed at 3.00 am and 9.00 pm. They were significant different from the minima at noon (2P less than 0.005). At the same points of the day maxima, resp. minima were found in the PB131I and the "free" hormones. The "free" hormones expressed in per cent of total hormones showed low values at 6.00 am, 3.00 pm, and 9.00 pm. The diurnal changes in haematocrit and albumin concentration were not responsible for the variation of PB131I. In order to eleminate effects of isotope dilution by ingested iodine the PB131I was expressed in per cent of total radioactivity of plasma (= conversion rate) or resp. in per cent of thyroidal radioiodine uptake. The obtained values showed maxima at 6.00 am and 9.00 pm. From that we conclude again on a stimulation of the thyroid at these times.     

Fluid reabsorption by the ductuli efferentes testis of the rat is dependent on both sodium and chlorine

The role of Na(+) and Cl(-) in fluid reabsorption by the efferent ducts was examined by perfusing individual ducts in vivo with preparations of 160 mM NaCl in which the ions were replaced, together or individually, with organic solutes while maintaining the osmolality at 300 mmol/kg. Progressively replacing NaCl with mannitol reduced net reabsorption of water and the ions in a concentration-dependent manner, and caused net movement into the lumen at concentrations of NaCl less than 80 mM. The net rates of flux were lower for Na(+) than for Cl(-). In collectates, [Na(+)] was greater than [Cl(-)], indicating that Cl(-) transport is probably linked with another anion. Replacing either Na(+) or Cl(-) in perfusates (with choline and isethionate, respectively) while maintaining the other inorganic ion at 160 mM also reduced net rates of reabsorption in a concentration-dependent manner to zero when either ion was completely replaced. There were no significant differences in the osmolality of perfusate and collectate, and collectates contained a mean of 3.4 mM K(+), indicating a backflux of K(+) into the lumen. It is concluded that fluid reabsorption from the efferent ducts is dependent on the transport of both Na(+) and Cl(-) from the lumen (from a luminal concentration of at least 70-80 mM), and that Cl(-) transport is dependent on another anion. The epithelium is permeable to K(+) and has a higher permeability to a range of organic solutes (mannitol, choline, and isethionate) than epithelium in the proximal kidney tubules.     

Thermal responses and body fluid balance of competitive male swimmers during a training session

Thermoregulatory and body fluid balance (BFB) responses of competitive swimmers were studied during a typical interval training session under natural field conditions. Subjects were 9 males (18.0 +/- 1.7 years; VO(2)max = 3.8 +/- 0.9 L x min(-1)) who covered 9,000 m in 180 minutes in an outdoor pool (mean water temperature = 26.8 +/- 0.3 degrees C; mean wet bulb globe temperature = 29.8 +/- 2.8 degrees C). Mean body weight (BWt) decreased by 1.8 +/- 0.5 kg (P < 0.05), and rectal temperature increased by 1.0 +/- 1.0 degrees C (P < 0.05). Volitional water intake (WI) (0.1 +/- 0.2 kg) did not maintain BFB (-0.5 kg per hour) and plasma volume decreased 10.7 +/- 5.4%. During a typical training session, swimmers experienced significant body fluid losses, and WI was not enough to prevent involuntary dehydration. The magnitude of the fluid losses (2.5% of BWt) was sufficient to compromise convective thermoregulation because of the decreased plasma volume. Hence, to prevent involuntary dehydration, swimmers should be encouraged to consume an amount of fluids that equals losses throughout the training sessions.     

Carrier-Mediated Transport of Chloride Across the Blood-Brain Barrier

36Cl concentrations in each of eight brain regions and in cisternal cerebrospinal fluid (CSF) were determined 30 min after the intravenous injection of 36Cl in dialyzed-nephrectomized rats with plasma Cl concentrations between 14 and 120 mumol X ml-1. CSF 36Cl exceeded 36Cl concentrations in brain extracellular fluid. The calculated blood-to-brain transfer constants for Cl, kCl, ranged from 1.8 X 10(-5) S-1 at the parietal cortex to 3.8 X 10(-5) S-1 at the thalamus-hypothalamus. kCl fell by 42-62% when mean plasma [Cl] was elevated from 16 to 114 mumol X ml-1. Brain uptake of [14C]mannitol or of 22Na was independent of plasma [Cl], but 22Na influx into CSF fell when plasma [Cl] was reduced. Cl flux into brain and CSF could be represented by Michaelis-Menten saturation kinetics, where, for the parietal cortex, Km = 43 mumol X ml-1 and Vmax = 2.5 X 10(-3) mumol X S-1 X g-1, and for CSF Km = 68 mumol X ml-1. At least 80% of 36Cl influx into the parietal cortex was calculated to occur at the cerebrovascular endothelium, whereas the remainder was derived from tracer that first entered CSF. The CSF contribution was greater at brain regions adjacent to cerebral ventricles. The results show that Cl transport at the cerebrovascular endothelium as well as at the choroid plexus epithelium is a saturable concentration-dependent process, and that the CSF is a significant intermediate pathway for Cl passage from blood to brain.     

Mechanisms behind Pb-induced disruption of Na+ and Cl- balance in rainbow trout (Oncorhynchus mykiss)

The mechanism of Pb-induced disruption of Na(+) and Cl(-) balance was investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Na(+) and Cl(-) influx rates were reduced immediately in the presence of 2.40 +/- 0.24 and 1.25 +/- 0.14 muM Pb, with a small increase in efflux rates occurring after 24-h exposure. Waterborne Pb caused a significant decrease in the maximal rate of Na(+) influx without a change in transporter affinity, suggesting a noncompetitive disruption of Na(+) uptake by Pb. Phenamil and bafilomycin markedly reduced Na(+) influx rate but did not affect Pb accumulation at the gill. Time-course analysis in rainbow trout exposed to 0, 0.48, 2.4, and 4.8 microM Pb revealed time- and concentration-dependent branchial Pb accumulation. Na(+)-K(+)-ATPase activity was significantly reduced, with 4.8 microM exposure resulting in immediate enzyme inhibition and 0.48 and 2.4 microM exposures inhibiting activity by 24 h. Reduced activity was weakly correlated with gill Pb accumulation after 3- and 8-h exposures; this relationship strengthened by 24 h. Reduced Na(+) uptake was correlated with gill Pb burden after exposures of 3, 8, and 24 h. Immediate inhibition of branchial carbonic anhydrase activity occurred after 3-h exposure to 0.82 +/- 0.05 or 4.30 +/- 0.05 microM Pb and continued for up to 24 h. We conclude that Pb-induced disruption of Na(+) and Cl(-) homeostasis is in part a result of rapid inhibition of carbonic anhydrase activity and of binding of Pb with Na(+)-K(+)-ATPase, causing noncompetitive inhibition of Na(+) and Cl(-) influx.     

Basolateral membrane Cl/HCO3 exchange in the rat proximal convoluted tubule. Na-dependent and -independent modes

To examine whether Cl-coupled HCO3 transport mechanisms were present on the basolateral membrane of the mammalian proximal tubule, cell pH was measured in the microperfused rat proximal convoluted tubule using the pH-sensitive, intracellularly trapped fluorescent dye (2',7')- bis(carboxyethyl)-(5,6)-carboxyfluorescein. Increasing the peritubular Cl concentration from 0 to 128.6 meq/liter caused cell pH to decrease from 7.34 +/- 0.04 to 7.21 +/- 0.04 (p less than 0.001). With more acid extracellular fluid (pH 6.62), a similar increase in the peritubular Cl concentration caused cell pH to decrease by a similar amount from 6.97 +/- 0.04 to 6.84 +/- 0.05 (p less than 0.001). This effect was blocked by 1 mM SITS. To examine the Na dependence of Cl/HCO3 exchange, the above studies were repeated in the absence of luminal and peritubular Na. In alkaline Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.57 +/- 0.06 to 7.53 +/- 0.06 (p less than 0.025); in acid Na-free solutions, peritubular Cl addition caused cell pH to decrease from 7.21 +/- 0.04 to 7.19 +/- 0.04 (p less than 0.05). The effect of Cl on cell pH was smaller in the absence of luminal and peritubular Na than in its presence. To examine whether the previously described Na/(HCO3)n greater than 1 cotransporter was coupled to or dependent on Cl, the effect of lowering the peritubular Na concentration from 147 to 25 meq/liter was examined in the absence of ambient Cl. Cell pH decreased from 7.28 +/- 0.03 to 7.08 +/- 0.03, a response similar to that observed previously in the presence of Cl. The results demonstrate that Cl/HCO3 (or Cl/OH) exchange is present on the basolateral membrane. Most of Cl/HCO3 exchange is dependent on the presence of Na and may be coupled to it. The previously described Na/(HCO3)n greater than 1 cotransporter is the major basolateral membrane pathway for the coupling of Na and HCO3 and is not coupled to Cl.     

Stimulation of thermoregulatory and respiratory functions with the help of EDTA and EGTA during deep hypothermia in rats

Influence of EDTA (C10H14N2Na2O8.2H2O) and EGTA (C14H24N2O10) on physiological functions homoiothermic organisms at deep hypothermia, was studied. White rats during cooling were in special sections without rigid fixing of head and limbs. In reply to intravenous introduction of EDTA and EGTA solutions, similar answers of the organisms were observed: raised breathing frequency and amplitude, intensity of electrical activity of muscles; these signs of activation of physiological functions lasted 8-10 minutes. Besides, of the 20th-30th minute after introduction of the second dose of preparations (at rectal temperature 17.1 +/- 0.5 degrees C), the secondary activation respiratory and thermoregulatory functions were registered. The termination of the cold shivering in experiments with introduction of EDTA and EGTA solutions occurred at lower temperatures in rectum and in a brain (16.7-17.3 degrees and 17.8-18.2 degrees C, resp.) than in control experiments (18.7 +/- 0.6 degrees C and 20.2 +/- 1.5 degrees C). The authors suppose that the activation of the thermoregulatory and respiratory functions is caused by a decrease in concentration of ions Ca2+ in the blood plasma.     

The effect of atrial natriuretic peptide on small intestinal contractility and transit.

Isometric tension in response to ANF (10(-10) to 10(-4) M) was recorded from longitudinally and circularly oriented rat jejunal smooth muscle strips. Conscious, fasted rats received an IV infusion of 1.25 nmol ANF/100 g body weight in 0.5 ml normal saline and controls received saline alone. Five minutes later 10 muCi Na2 51CrO4 in 0.5 ml saline was instilled through a jejunostomy. Fifteen minutes later animals were sacrificed, and the gut divided into 8 equal segments of small intestine, cecum and remaining colon. The radioactivity of each segment was measured and a geometric center of transit determined for each group. ANF induced relaxation of longitudinally oriented strips (Tm = -72.3 +/- 10.7 mN/g, ED50 7.3 +/- 3.6 x 10(-8) M), and contraction of circularly oriented strips (Tm = 35.0 +/- 5.0 mN/g, ED50 1.3 +/- 1.0 x 10(-8) M). This response was unaffected by 10(-6) M tetrodotoxin. The geometric mean center of transit was significantly (p less than 0.001) further aboral in ANF-treated compared to control animals (intestinal segment 4.2 +/- 0.2 vs. 3.2 +/- 0.2).     

Endotoxemia stimulates skeletal muscle Na+-K+-ATPase and raises blood lactate under aerobic conditions in humans

We assessed the hypothesis that the epinephrine surge present during sepsis accelerates aerobic glycolysis and lactate production by increasing activity of skeletal muscle Na(+)-K(+)-ATPase. Healthy volunteers received an intravenous bolus of endotoxin or placebo in a randomized order on two different days. Endotoxemia induced a response resembling sepsis. Endotoxemia increased plasma epinephrine to a maximum at t = 2 h of 0.7 +/- 0.1 vs. 0.3 +/- 0.1 nmol/l (P < 0.05, n = 6-7). Endotoxemia reduced plasma K(+) reaching a nadir at t = 5 h of 3.3 +/- 0.1 vs. 3.8 +/- 0.1 mmol/l (P < 0.01, n = 6-7), followed by an increase to placebo level at t = 7-8 h. During the declining plasma K(+), a relative accumulation of K(+) was seen reaching a maximum at t = 6 h of 8.7 +/- 3.8 mmol/leg (P < 0.05). Plasma lactate increased to a maximum at t = 1 h of 2.5 +/- 0.5 vs. 0.9 +/- 0.1 mmol/l (P < 0.05, n = 8) in association with increased release of lactate from the legs. These changes were not associated with hypoperfusion or hypoxia. During the first 24 h after endotoxin infusion, renal K(+) excretion was 27 +/- 7 mmol, i.e., 58% higher than after placebo. Combination of the well-known stimulatory effect of catecholamines on skeletal muscle Na(+)-K(+)-ATPase activity, with the present confirmation of an expected Na(+)-K(+)- ATPase-induced decline in plasma K(+), suggests that the increased lactate release was due to increased Na(+)-K(+)-ATPase activity, supporting our hypothesis. Thus increased lactate levels in acutely and severely ill patients should not be managed only from the point of view that it reflects hypoxia.