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In dilute seawater, Carcinus maenas hyperosmoregulates by actively absorbing Na, K, and Cl. Here we characterize K uptake using a novel technique. Rb was used as a tracer for K transport, and hemolymph Rb levels were measured using cation chromatography. Hemolymph Rb was detectable at 0.1 mmol L(-1), which enabled determination of initial rate of Rb uptake. Crabs maintained for 3 wk in dilute artificial seawater (35% ASW crabs) maintained Na and K above the level of the external media and had elevated Na-K-ATPase activity in the posterior gills. In assay conditions matched to 100% ASW, Rb uptake was the same in 35% ASW crabs (0.45+/-0.04 micromol g(-1) h(-1)) and in crabs acclimated to normal seawater (100% ASW crabs, 0.49+/-0.05 micromol g(-1) h(-1)). In assay conditions matched to 35% ASW, Rb uptake was greater in 35% ASW crabs (0.28+/-0.03 micromol g(-1) h(-1)) compared with 100% ASW crabs (0.10+/-0.04 micromol g(-1) h(-1)). Low external [Rb] or reduced salinity were found to contribute independently to the difference between 100% ASW and 35% ASW crabs. Thus, whole-body Rb uptake in crabs can be measured by cation chromatography, and Rb uptake is greater in 35% ASW crabs than in 100% ASW crabs.

We have studied the regulation of the K-Cl cotransporter KCC1 and its functional interaction with the Na-K-Cl cotransporter. K-Cl cotransporter activity was substantially activated in HEK-293 cells overexpressing KCC1 (KCC1-HEK) by hypotonic cell swelling, 50 mM external K, and pretreatment with N-ethylmaleimide (NEM). Bumetanide inhibited 86Rb efflux in KCC1-HEK cells after cell swelling [inhibition constant (Ki) approximately 190 microM] and pretreatment with NEM (Ki approximately 60 microM). Thus regulation of KCC1 is consistent with properties of the red cell K-Cl cotransporter. To investigate functional interactions between K-Cl and Na-K-Cl cotransporters, we studied the relationship between Na-K-Cl cotransporter activation and intracellular Cl concentration ([Cl]i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activity than controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells was activated <2-fold by low-Cl hypotonic prestimulation, compared with 10-fold activation in HEK-293 cells and >20-fold activation in cells overexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cells had lower resting [Cl]i than HEK-293 cells; cell volume was not different among cell lines. We found a steep relationship between [Cl]i and Na-K-Cl cotransport activity within the physiological range, supporting a primary role for [Cl]i in activation of Na-K-Cl cotransport and in apical-basolateral cross talk in ion-transporting epithelia.

We rapidly infused 234 +/- 3 mL of 5% human serum albumin in eight men while measuring haematocrit, haemoglobin concentration, plasma volume (PV), albumin concentration, total protein concentration, osmolality, sodium concentration, renin activity, aldosterone concentration, and atrial natriuretic peptide concentration to test the hypotheses that plasma volume expansion and plasma albumin content expansion will not persist for 24 h. Plasma volume and albumin content were expanded for the first 6 h after infusion (44.3 +/- 1.9-47.2 +/- 2.0 mL kg-1 and 1.9 +/- 0.1-2.1 +/- 0.1 g kg-1 at pre-infusion and 1 h, respectively, P < 0.05), but by 24 h plasma volume and albumin content decreased significantly from 1 h post-infusion and were not different from pre-infusion (44.8 +/- 1.9 mL kg-1 and 1.9 +/- 0.1 g kg-1, respectively). Plasma aldosterone concentration showed a significant effect of time over the 24 h after infusion (P < 0.05), and showed a trend to decrease at 2 h after infusion (167.6 +/- 32.5(-1) 06.2 +/- 13.4 pg mL-1, P = 0.07). These data demonstrate that a 6.8% expansion of plasma volume and 10.5% expansion of plasma albumin content by infusion does not remain in the vascular space for 24 h and suggest a redistribution occurs between the intravascular space and interstitial fluid space.

We report the cloning, sequence analysis, tissue distribution, and functional expression of the K-Cl cotransport protein, KCC1. KCC1 was identified by searching the human expressed sequence tag data base, based on the expectation that it would be distantly related to the Na-K-Cl cotransporter. Rabbit KCC1 (rbKCC1) and rat KCC1 (rtKCC1) were cloned by screening rabbit kidney and rat brain cDNA libraries using homologous cDNA probes. Human KCC1 (hKCC1) was obtained from I.M.A.G.E. clones and in part by reverse transcription-polymerase chain reaction; it exhibits 97% identity with rbKCC1. KCC1 encodes a 1085-residue polypeptide with substantial sequence homology (24-25% identity) to the bumetanide-sensitive Na-K-Cl cotransporter (NKCC or BSC) and the thiazide-sensitive Na-Cl cotransporter (NCC or TSC). Hydropathy analysis of KCC1 indicates structural homology to NKCC, including 12 transmembrane domains, a large extracellular loop with potential N-linked glycosylation sites, and cytoplasmic N- and C-terminal regions. Northern blot analysis revealed a ubiquitously expressed 3. 8-kilobase transcript. Much of the genomic sequence of hKCC1 is in the data base, and the gene has been previously localized to 16q22.1 (Larsen, F., Solhein, J., Kristensen, T., Kolsto, A. B., and Prydz, H.(1993) Hum. Mol. Genet. 2, 1589-1595). Epitope-tagged rbKCC1 was stably expressed in human embryonic kidney (HEK 293) cells, resulting in production of a approximately150-kDa glycoprotein. The initial rate of 86Rb efflux from cells expressing rbKCC1 was more than 7 times greater than efflux from control cells and was inhibited by 2 mM furosemide; 86Rb efflux was stimulated by cell swelling. Uptake of 86Rb into rbKCC1 cells after a 15-min pretreatment with 1 mM N-ethylmaleimide was dependent on external chloride but not on external sodium, and was inhibited by furosemide with a Ki of approximately 40 microM and by bumetanide with a Ki of approximately 60 microM. These data demonstrate that the KCC1 cDNAs encode a widely expressed K-Cl cotransporter with the characteristics of the K-Cl transporter that has been characterized in red cells.

To gain better insight into the interaction between thermoregulation and osmoregulation, we examined the thermal sweating response to drinking in cell-dehydrated humans. Cell dehydration (CDH) was induced by infusion of a 3% NaCl solution, at 1.2 ml/kg, for 2 h; infusion of a 0.9% NaCl solution in a separate experiment served as a control (euhydrated condition, EH). After infusion, subjects were heated by immersion of the lower legs in 42 degrees C water at an ambient temperature of 28 degrees C for 90 min. Subjects drank 4.3 ml/kg of H2O (approximately 38 degrees C) at 60 min of heating. The 3% NaCl infusion increased plasma osmolality by 13.6 +/- 0.8 mosmol/kgH2O and plasma arginine vasopressin concentration ([AVP]) by 3.3 +/- 0.7 pg/ml. Neither variable was altered with 0.9% NaCl infusion. Before drinking, esophageal temperature (Tes) had increased by 0.91 +/- 0.08 degrees C in CDH and by 0.40 +/- 0.11 degrees C in EH. Local chest sweating rate (SRch) had increased by 0.67 +/- 0.08 and 0.63 +/- 0.07 mg.min-1.cm-2 in CDH and EH, respectively. Thus the change in SRch per unit rise in Tes was much lower in CDH than in EH. Drinking immediately increased SRch and reduced Tes in CDH, with a reduction in plasma [AVP] and thirst rating. Drinking did not change thermoregulatory and osmoregulatory responses in EH. These results suggest that the act of drinking itself eliminates, at least partially, an osmotic inhibitory input to the thermoregulatory center, as well as osmotic AVP secretion and thirst.

To test the hypothesis that reduced baroreflex sensitivity is a direct result of exercise, we measured forearm vascular conductance (FVC) responses to graded lower body negative pressure (LBNP) 2, 20, and 44 h after intense exercise. Eight 4-min bouts of exercise at 85% of maximum oxygen uptake produced 3.5 +/- 0.7 and 3.9 +/- 1.0% blood volume (BV) expansions at 20 and 44 h of recovery, respectively. BV was unchanged from control values 2 h after exercise. The reduction in FVC was significantly less than control values during 30 and 40 mmHg of LBNP at 2 and 20 h of recovery, respectively, whereas heart rate and cardiac stroke volume responses were unchanged. Thus, a reduced FVC response to LBNP preceded BV expansion, demonstrating that exercise itself can elicit an attenuation of baroreflex function. To test the hypothesis that volume sensitivity of renal function is attenuated by intense exercise, we measured cardiovascular variables, plasma hormone concentrations, and renal output. At 20 h of recovery, resting mean arterial blood pressure and cardiac output were increased by 6 +/- 1 mmHg and 0.6 +/- 0.2 l/min, respectively, but resting plasma aldosterone and overnight Na+ excretion rate were unchanged. At 44 h of recovery, plasma aldosterone was decreased by 26 +/- 9% and overnight Na+ excretion rate was increased by 51 +/- 26%. Thus, appropriate endocrine and renal responses to increased BV were delayed until 44 h of recovery. Our findings suggest that a postexercise attenuation of baroreflex function participates in the induction of BV expansion by intense exercise.

After a 7-h H2O and Na+ depletion period (DP), produced by intermittent light exercise (8 bouts) at 35 degrees C, we examined thirst and taste palatability responses to 10 different NaCl solutions during 23 h of rehydration (RH) at 25 degrees C. During DP, net H2O and Na+ loss were 27.2 +/- 2.9 ml/kg and 3.29 +/- 0.45 meq/kg, respectively. Plasma osmolality (POsm) and plasma Na+ concentration ([Na+]p) increased significantly during DP by 3.4 +/- 1.2 mosmol/kgH2O and 3.0 +/- 1.0 meq/kgH2O, respectively. Plasma volume (PV) decreased by 6.5 +/- 1.9%. Thirst rating, renal fractional reabsorption of H2O, and plasma arginine vasopressin concentration (PAVP) increased as POsm increased. This increased thirst was accompanied by increased palatability ratings to H2O. During RH, subjects drank deionized H2O ad libitum and ate a Na(+)-free diet for 23 h. POsm and [Na+]p returned to control levels within 1 h RH and remained at or below the control thereafter. PV remained reduced by approximately 5% throughout RH. The increased thirst and PAVP returned to their respective control levels within 1 h of RH as POsm decreased, but thirst rating increased against between 17 and 23 h of RH without increase in POsm or PAVP. Palatability ratings to a 1 M NaCl solution at and after 3 h RH and palatability ratings to 0.3 M at 17 and 23 h RH were significantly higher than control. Plasma aldosterone concentration (PAldo) increased after DP, decreased with drinking, and increased again between 6 and 23 h of RH, accompanied by a marked decrease in fractional Na+ excretion to < 0.07%. Thus both Na+ preference and thirst in humans are influenced by body fluid and electrolyte status. The increased Na+ palatability (Na+ appetite) was preceded by osmotically induced thirst, and accompanied by nonosmotically driven thirst [extracellular fluid (ECF) thirst] and increased PAldo. The "Na+ appetite" and "ECF thirst" along with increased renal Na+ retention could contribute to ECF volume regulation after thermally induced H2O and Na+ depletion.

We examined osmotic control of thirst and free water clearance in healthy older (65+, n = 10) and younger (Y, n = 6) subjects during a 3-h rehydration period after an approximately 2.4% decrease in body weight. Plasma volume (PV), plasma osmolality (Posm), renal function, and thirst were measured before and after dehydration and during rehydration. In 65+, baseline PV was lower (43.1 +/- 1.6 vs. 48.1 +/- 2.5 ml/kg), Posm was higher (287 +/- 1 vs. 281 +/- 2 mosmol/kgH2O), and perceived thirst was lower than in Y. During dehydration, the osmotic threshold for increased thirst was shifted to a higher Posm in 65+. Total fluid intake was greater in Y than in 65+ (16.6 +/- 4.1 vs. 8.9 +/- 2.0 ml/kg); however, the relation between thirst and the rate of fluid intake was identical. Thus the blunted rehydration in 65+ is related to a lower overall sensation of thirst. The stimulus-response characteristics of osmotic control of free water clearance was similar in 65+ and Y; however, 65+ operated around a higher Posm and on a less-steep portion of the stimulus-response curve. These data support the hypothesis that the hyperosmotic hypovolemic state of healthy older individuals is not a result of a simple water deficit but represents a shift in the operating point for control of body fluid volume and composition.

We describe a method for measuring plasma volume (PV) in small animals that allows small sample sizes but does not require the use of radioisotopes and thus is a convenient approach for making repeated measurements. Texas Red covalently bound to albumin (TR-A) was used in a typical indicator-dilution technique to measure PV. The relative fluorescent intensity of TR-A is linear to its concentration (up to 0.15 mg/ml) at an excitation lambda of 590 nm and an emission lambda of 610 nm. Catheters were inserted through the right jugular vein of anesthetized rats and threaded into the vena cava. A 0.5-ml control blood sample was taken, a measured quantity of TR-A was injected, and the catheter was flushed with saline. A 0.5-ml postinjection sample was taken 5 min after TR-A injection. PV was calculated by comparing the difference between the relative fluorescent intensity of control and postinjection plasma samples to a standard. The PV of 22 rats [362 +/- 14 (SE) g] was 14.1 +/- 0.4 ml (39.6 +/- 0.9 ml/kg body wt) measured by the TR-A method and 12.8 +/- 0.4 ml (35.9 +/- 1.0 ml/kg body wt) measured by a standard radioiodinated albumin method. There was a strong correlation between PV measured by both methods in the same rat (r = 0.90, P < 0.01). Infusion experiments indicated that the TR-A method can detect acute changes in PV, and repeated measurements of PV made on a chronically instrumented rat demonstrated that the method can reliably measure PV on consecutive days.

The purpose of this study was to compare the cardiovascular control mechanisms that defend arterial blood pressure against blood pooling between rest and moderate dynamic exercise. We studied ten physically active men during rest and five 12-min graded supine cycle ergometer exercise bouts with and without application of LBNP in 25 degrees C and 35 degrees C. Exercise intensities were 10, 50, and 100 watts (W), each for 4 min, and LBNP was applied at 0 (control), -20, -40, and -60 mm Hg in 25 degrees C and -40 mm Hg in 35 degrees C. At rest, cardiac stroke volume (SV) decreased from 120 +/- 5 ml during control to 94 +/- 6, 67 +/- 5 and 49 +/- 3 ml during -20, -40, and -60 mm Hg LBNP, respectively, and to 55 +/- 3 ml during -40 mm Hg at 35 degrees C. Exercise elevated SV toward the control level during LBNP due to muscle pumping action. Heart rate (HR) did not increase significantly during application of LBNP until SV decreased by 20-25 ml during LBNP, both during rest and exercise. The magnitude of HR increase per decrease in SV, once an increase in HR occurred, was similar between rest and exercise, regardless of exercise intensity. The change in total peripheral resistance (TPR) with respect to SV was linear, confirming that peripheral vascular adjustments were proportional to changes in the heart's preload. The slopes of the TPR-SV relation were similar during rest and exercise, although shifted to the left with increasing exercise intensity.(ABSTRACT TRUNCATED AT 250 WORDS)

To examine the hypothesis that the skin blood flow response to body heating is not uniform over the entire body surface, we compared forearm (FBF) and calf (CBF) blood flow responses to an increase in core temperature (esophageal temperature, Tes) during dynamic exercise. We studied 13 physically active men during semi-recumbent one leg exercise and/or intermittent supine cycle exercise at 35 degrees C. During 30 min of one leg exercise, Tes, FBF, and CBF in the nonactive leg increased from 36.94 +/- 0.09 degrees C, 5.7 +/- 1.2, and 5.6 +/- 0.6 ml.(min.100 ml)-1 at rest to 37.97 +/- 0.10 degrees C, 27.0 +/- 2.4, and 11.1 +/- 0.8 ml.(min.100 ml)-1, respectively. The increase in blood flow per unit increase in Tes was much less in the calf than in the forearm. The ratio of the peak to resting blood flow averaged 6.5 in the forearm and 2.5 in the calf. During 60 min of intermittent supine two leg exercise, Tes, FBF, and CBF increased from 36.96 +/- 0.06 degrees C, 7.9 +/- 1.5, and 5.6 +/- 0.7 ml.(min.100 ml)-1 at rest to 37.91 +/- 0.07 degrees C, 23.6 +/- 3.0, and 11.4 +/- 1.9 ml.(min.100 ml)-1, respectively. Skin blood flow (SkBF) in the forearm and calf was estimated by using a simple cylindrical model, assuming skin thickness and resting muscle blood flow to be 0.2 cm and 2 ml.(min.100 ml)-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

We used intense intermittent exercise to produce a 10% expansion of plasma volume (PV) within 24 h and tested the hypothesis that PV expansion is associated with an increase in plasma albumin content. The protocol consisted of eight 4-min bouts of exercise at 85% maximal O2 uptake with 5-min recovery periods between bouts. PV, plasma concentrations of albumin and total protein (TP), and plasma osmolality were measured before and during exercise and at 1, 2, and 24 h of recovery from exercise. During exercise, PV decreased by 15%, while plasma TP and albumin content remained at control levels. At 1 h of recovery, plasma albumin content was elevated by 0.17 +/- 0.04 g/kg body wt, accounting for the entire increase in plasma TP content. PV returned to control level at 1 h of recovery without fluid intake by the subjects, despite a 820 +/- 120-g reduction in body weight. At 2 h of recovery, plasma TP content remained significantly elevated, and plasma TP and albumin concentration were significantly elevated. At 24 h of recovery, PV was expanded by 4.5 +/- 0.7 ml/kg body wt (10 +/- 1%), estimated from hematocrit and hemoglobin changes, and by 3.8 +/- 1.3 ml/kg body wt (8 +/- 3%), measured by Evans blue dye dilution. Plasma albumin content was increased by 0.19 +/- 0.05 g/kg body wt at 24 h of recovery. If 1 g of albumin holds 18 ml of water, this increase in plasma albumin content can account for a 3.4-ml/kg body wt expansion of the PV. No significant changes in plasma osmolality occurred during recovery, but total plasma osmotic content increased in proportion to PV.(ABSTRACT TRUNCATED AT 250 WORDS)