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To examine effects of free fatty acids (FFA) on insulin-stimulated glucose fluxes, euglycemic hyperinsulinemic (86 pmol . kg-1 . min-1) clamps were performed for 5 h in conscious rats with (n = 8) or without (n = 8) lipid-heparin infusion. Glucose infusion rate required to maintain euglycemia was not different between the two groups during the first 2 h of clamps but became significantly lower with lipid-heparin infusion in the 3rd h and thereafter. To investigate changes in intracellular glucose metabolism during lipid-heparin infusion, additional clamps (n = 8 each) were performed for 1, 2, 3, or 5 h with an infusion of [3-3H]glucose. Insulin-stimulated whole body glucose utilization (Rd), glycolysis, and glycogen synthesis were estimated on the basis of tracer concentrations in plasma during the final 40 min of each clamp. Similar to changes in glucose infusion rate, Rd was not different between the two groups in the 1st and 2nd h but was significantly lower with lipid-heparin infusion in the 3rd h and thereafter. Whole body glycolysis was significantly lower with lipid-heparin infusion in all time periods, i.e., 1st, 2nd, 3rd, and 5th h of clamps. In contrast, whole body glycogen synthesis was higher with lipid-heparin infusion in the 1st and 2nd h but lower in the 5th h. Similarly, accumulation of [3H]glycogen radioactivity in muscle glycogen was significantly higher with lipid-heparin during the 1st and 2nd h but lower during the 3rd and 5th h. Glucose 6-phosphate (G-6-P) concentrations in gastrocnemius muscles were significantly higher with lipid-heparin infusion throughout the clamps. Muscle glycogen synthase (GS) activity was not altered with lipid-heparin infusion at 1, 2, and 3 h but was significantly lower at 5 h. Thus increased availability of FFA significantly reduced whole body glycolysis, but compensatory increase in skeletal muscle glycogen synthesis in association with accumulation of G-6-P masked this effect, and Rd was not affected in the early phase (within 2 h) of lipid-heparin infusion. Rd was reduced in the later phase (>2 h) of lipid-heparin infusion, when glycogen synthesis was reduced in association with reduced skeletal muscle GS activity.

Renal responses to low doses of atrial natriuretic peptide (ANP) may be potentiated during water immersion-induced central hypervolemia. To test this hypothesis, ANP was infused in awake dogs in doses of 0, 5, and 25 ng.kg-1.min-1 either when the dogs were in air or during head-out water immersion (WI) under thermoneutral conditions (37 degrees C). In general, there were greater diuretic (V) and natriuretic responses (UNaV) at the same level of plasma ANP in WI, with the slopes (or sensitivities) of V and UNaV in relation to plasma ANP levels being significantly increased during WI. Plasma renin activity decreased only during WI and was significantly correlated with both V and UNaV only during WI. Plasma and urinary arginine vasopressin levels were unchanged during WI. Infusion of ANP prevented the usual decline of hematocrit that occurs during WI. These results support the view that the renal sensitivity to ANP is augmented during the plasma volume expansion of WI. In addition, ANP may attenuate the transcapillary fluid shift that occurs during WI.

Water immersion (WI) increases plasma volume in awake dogs. The contribution of lymph flow to this fluid shift was studied in six splenectomized conscious dogs with a side fistula of the thoracic duct. Lymph flow, hematocrit (Hct), and plasma (CP) and lymph (CL) protein concentration were measured during 60 min in air and 120 min of WI (37 degrees C). Lymph flow in air averaged 0.96 +/- 1.0 (SE) ml/min. Lymph flow tended to decrease immediately in WI and was maintained at a level averaging 0.66 ml/min. CP/CL did not change significantly, whereas Hct fell significantly by 1.51 +/- 0.2% (Hct units) at 40 min of WI. Urine flow increased significantly to a maximum value of 1.5 +/- 0.5 ml/min at 40-60 min of WI compared with a mean value in air of 0.3 +/- 0.1 ml/min. The Hct and urine flow responses indicate that fluid shifted into the intravascular space during WI. Since lymph flow tended to decrease, the fluid shift in WI occurs across the capillary wall and not via lymphatic channels.

Hypoxia alters the relationship of aldosterone secretion to plasma renin activity. The potential role plasma electrolytes play in this modification is not clear. This study analyzed the interrelationships among renin, aldosterone, vasopressin (ADH), and plasma electrolytes during 96 h of normobaric hypoxia. Eight ewes were exposed, in discrete experiments, to hypocapnic hypoxia [arterial O2 tension (PaO2) 37-42 mmHg, arterial CO2 tension (PaCO2) 26-28 mmHg] and eucapnic hypoxia (PaO2 40-43 mmHg, PaCO2 28-31 mmHg) by N2 dilution in an environmental chamber. Urine output (24 h) was measured, and arterial plasma samples were collected during the normoxic control period and at 24-h intervals of hypoxia. Plasma Na+, K+, renin, and ADH levels did not change from the normoxic values during either hypocapnic or eucapnic hypoxia. However, urinary aldosterone excretion [critical significance (alpha) less than 0.046] and K+ excretion (alpha less than 0.046) decreased markedly during each type of hypoxia. All sheep developed a pronounced negative K+ balance by 96 h of hypoxia. These data suggest that plasma K+ concentration is preserved by movement of K+ out of the intracellular compartment; this change in K+ distribution may inhibit aldosterone secretion during hypoxia.

Blood volume (51Cr-erythrocyte dilution method), hematocrit, and arterial (Pa), central venous (Pv), plasma colloid osmotic (IIpl), and interstitial fluid hydrostatic (Pcps, Guyton's capsule method) pressures were measured continuously to determine the kinetics of the transvascular fluid shift during 100 min of water immersion (WI) at 37 degrees C in six splenectomized dogs. Urine flow increased by 180% above control levels (P less than 0.05) by 30 min of WI. Plasma volume (PV) started to increase at 5 min of WI and rose by 7.2% (P less than 0.05) above control levels by 35 min of WI, and then it decreased gradually. PV returned to control levels immediately after WI. Plasma protein concentration and IIpl decreased significantly by 0.2 g/100 ml and 1.2 mmHg, respectively, at 35 min of WI, while plasma osmolality and Na+ concentration were constant. Pa and Pv increased (P less than 0.05) by 25 and 12 mmHg, respectively. Mean capillary pressure, which was calculated from Pa, Pv, and an estimated pre-to-postcapillary resistance ratio of 5-12, increased by 13-14 mmHg while Pcps increased (P less than 0.05) by 17 and 26 mmHg at upper hindlimb and lower forelimb, respectively. The changes in mean capillary pressure and IIpl tend to promote capillary filtration in WI; however, the greater elevation of Pcps more than offsets these forces and leads to a net transvascular shift into the plasma compartment.

The effect of SITS (4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene) on the transport of organic ions in the rabbit kidney cortical slice was studied. SITS at a concentration of 10(-4) to 10(-3) M significantly decreased the slice-to-medium (S/M) concentration ratio of the organic anions p-aminohippurate (PAH), and 2,4,5-trichlorophenoxyacetate, but had no significant effect on that of the organic cation tetraethylammonium. The S/M ratio of PAH decreased to 0.52 +/- 0.03 (SE) in the presence of 10(-3) M SITS. The inhibition of PAH uptake caused by SITS was reversed in the presence of 0.5% bovine serum albumin in the medium. SITS at a concentration of 10(-4) M had no significant effect on the efflux of PAH. However, there was a small increase in PAH efflux at a concentration of 10(-3) M SITS. A Lineweaver-Burk analysis of the data indicates that SITS competitively inhibits PAH uptake and that SITS has a Ki value of 2.3 X 10(-4) M. SITS had no effect on the tissue water content, [14C]inulin space, or intracellular Na and K concentrations. It is suggested that the primary effect of SITS is to inhibit the entry of organic anions from the medium into the cell across the basolateral membrane.

The renal transport of N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine), a potential photoaffinity label, was studied using the rabbit renal cortical slice and the isolated perfused rat kidney. NAP-taurine inhibited the slice accumulation of PAH in a dose-dependent manner (ID50 = 2.5 X 10(-5) M). It accumulated to a steady-state slice-to-medium concentration ratio of 14. However, NAP-taurine was not toxic to the tissue, as it did not influence the accumulation of the organic cation tetraethylammonium. NAP-taurine transport was saturable and its accumulation was inhibited by the metabolic inhibitors PAH, probenecid, 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS), ouabain, and the absence of sodium. Kinetic studies showed that the Km for NAP-taurine is 3.5 X 10(-5) M, and also that PAH competitively inhibits NAP-taurine influx with a Ki of 1.2 X 10(-3) M. Experiments with the rat isolated perfused kidney gave the NAP-taurine-to-inulin clearance ratio of approximately 5, indicating net tubular secretion. DIDS significantly reduced this clearance ratio to 0.8. The results suggest NAP-taurine is handled by the kidney in a manner analogous to PAH and may thus be useful as a photoaffinity label for the renal organic anion transport system.

The present investigation was undertaken to test the hypothesis that a circadian variation in the atrial natriuretic factor (ANF) response is responsible for the nocturnal inhibition of renal responses to head-out immersion (HOI). Plasma ANF responses to a 3-h HOI (water temperature 34.5 +/- 0.5 degrees C) were studied during day (1000-1300) and night (2400-0300) in six hydropenic male human subjects. In agreement with the previous observations, the renal responses, especially the diuresis, to HOI were attenuated at night compared with the day; furthermore, plasma renin activity decreased to the same low level during HOI at both day and night. Plasma ANF during time control periods was 30-40 pg/ml without showing any circadian variation. Moreover, plasma ANF showed a similar twofold increase within 1 h of HOI and was maintained at this elevated level throughout the 3-h HOI period in both the daytime and the nighttime series. On termination of HOI, plasma ANF decreased linearly to the pre-HOI level within 1 h. Hematocrit during time control periods was higher during the day compared with the night (P less than 0.05). Although HOI appears to induce a transient increase in plasma volume (as indicated by decreases in hematocrit) during the 1 h of HOI, the magnitude of the decrease in the latter parameters was not different between day and night. It is concluded that nocturnal inhibition of renal responses to HOI cannot be fully accounted for by circadian differences in the ANF and fluid shift response to HOI.

Water immersion (WI) causes an increase in plasma volume in humans and dogs. To determine the mechanism for this fluid movement, the transmission of external water hydrostatic pressure to the interstitial and vascular compartments was studied in six conscious dogs. Systemic arterial, central venous, peripheral arterial (ulnar artery) and venous (cephalic vein), pleural, intra-abdominal, and interstitial fluid hydrostatic (by Guyton's capsule and wick catheter method) pressures and external reference water pressure were measured at three different levels of WI: 1) extremities only, 2) midchest, and 3) midcervical levels at 37 degrees C. There was a significant linear relationship between interstitial fluid hydrostatic pressure (X) and external water pressure (Y): (Y = 0.86X + 1.4, r = 0.93 by Guyton's capsule; Y = 0.85X + 2.4, r = 0.93 by wick catheter. However, vascular pressures did not change when dogs were immersed at the level of the extremities. These pressures increased only during WI at the midchest and midcervical levels. Therefore the pressure gradient that develops between the interstitial and intravascular compartments is probably the major reason for the transcapillary fluid shift during WI.

The relationship between Na-K-ATPase activity and p-aminohippurate (PAH) transport was examined in renal cortical slices of mixed-breed rabbits by using ouabain to vary the level of Na-K-ATPase activity. Ouabain increased passive PAH uptake, measured in the presence of probenecid. Slice homogenate Na-K-ATPase activity was inhibited by 50% with about 2 X 10(-6) M ouabain. When the concentration was 10(-5) M or above, active PAH uptake was inhibited. Lower concentrations (10(-7) and 10(-6) M) of ouabain stimulated active PAH uptake. Acetate stimulated PAH uptake when medium ouabain was less than 10(-4) M. Ouabain, 10(-5) M and higher, caused intracellular K+ to fall and Na+ to rise. The changes in active PAH uptake observed with ouabain correlated much better with changes in intracellular K+ or Na+ levels (P less than 0.01) than with changes in slice homogenate Na-K-ATPase activity. Vmax for active PAH uptake decreased with 5 X 10(-5) M ouabain, and tended to increase with 10(-7) M ouabain. Ouabain did not alter Km for active PAH uptake. The data support the hypothesis that there is a functional link between active PAH transport and Na-K-ATPase activity. This linkage may be an indirect one mediated by changes in intracellular cation concentrations.

We determined the regional blood flow responses to head-out water immersion (WI) in intact (INT) and cardiac-denervated (CD) conscious dogs. Immersing dogs in thermoneutral water (37 degrees C) in the quadruped position for 100 min resulted in significant increases in cardiac output (Qco) above control values by 38.7% in the INT dogs and 39.2% in the CD dogs (P less than 0.01). Arterial pressure increased by 32 and 34.7% in the INT and CD groups, respectively, during WI, with no significant changes occurring in the calculated total peripheral resistance. Regional blood flow responses were measured with 15-microns radiolabeled microspheres. Flows in the INT and CD groups increased significantly to the heart (40, 38%), skin (93, 96%), fat (79, 83%), diaphragm (44, 48%), and intercostal muscles (58, 55%), whereas there were no changes in renal cortical blood flows during WI. Total brain blood flows did not change significantly on immersion; however, blood flows in both INT and CD animals were increased to the cerebellum (19, 22%), but a significant decrease in pituitary flow (52%) was observed only in the CD group during WI. Gastrointestinal tissue flows increased only during early WI in both INT (45%) and CD (47%) animals. However, blood flows to the skeletal muscles increased only during late WI in the INT (53%) and CD (47%) groups. There were no significant differences between the INT and CD groups. Rectal temperatures and systemic O2 consumption (VO2) were unchanged during WI in both groups of animals. These observations indicate that WI leads to a sustained elevation of Qco accompanied by selective increases in regional tissue perfusion that may be accounted for in some tissues by an increase in metabolic demand or by local heating responses and produces a time-dependent redistribution of blood flow away from the gastrointestinal tissues toward skeletal muscle tissues, which may be due to a partial uncoupling of the normal Q/VO2 relationship. This may be caused by thermal or central neurohumoral mechanisms. These regional circulatory responses are not dependent on the presence of the cardiac nerves.

The circulatory, renal, and hormonal responses during 100 min of thermoneutral (37 degrees C) head-out water immersion (WI) were investigated in conscious intact (INT) and cardiac-denervated (CD) dogs. In the INT group, both left and right atrial and aortic transmural distending pressures and left ventricular contractile performance (LV dP/dtmax) increased, and total peripheral resistance remained unchanged. Cardiac output (QCO) increased in association with an increase in heart rate and LV dP/dtmax. Urine flow (V), sodium excretion (UNaV), and osmolal clearance (Cosmol) all increased, whereas glomerular filtration rate, as indicated by creatinine clearance (CCr), remained constant. The diuresis and natriuresis occurred in the absence of any significant changes in plasma levels of antidiuretic hormone (ADH), aldosterone, or plasma renin activity. The CD animals showed a similar hemodynamic response except that the increase in QCO was now associated with an increase in stroke volume and no change in heart rate or LV dP/dtmax. Although the increase in V was similar in both magnitude and time course of the INT animals, there was no significant change in UNaV. Thus there is a striking change in the character of the response of the denervated group in that the natriuresis is abolished and, instead, a water diuresis occurred. Free water clearance increased, but no significant changes in Cosmol or CCr were observed. In addition plasma ADH levels significantly declined during WI in the CD group. These data indicate that cardiac receptors are important in determining the nature of the renal response to WI.

The present investigation compares brush-border (BBM) and basolateral membrane (BLM) vesicles in terms of purity, function, appearance on sodium dodecyl sulfate (SDS) gels, and labeling pattern by use of N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonic acid (NAP-taurine), a photoaffinity analogue of p-aminohippurate (PAH). Both BLM and BBM were vesicular by demonstration of PAH uptake into an osmotically active space and had probenecid-inhibitable uptake of PAH. Time courses for uptake were similar. 250 microM NAP-taurine resulted in a 35% inhibition of PAH uptake in BLM but it did not significantly effect PAH uptake into BBM. The latter was affected by 1 mM NAP-taurine. A comparison of Coomassie blue SDS gels of BLM and BBM showed markedly different staining patterns. A major band at approximately 52,000 daltons was more intensely stained in BLM than BBM. Major bands at approximately 40,000 and approximately 80,000 were stained more heavily in BBM than BLM. A minor protein at 26,000 in the BLM did not appear in BBM. An irreversible inhibition of PAH uptake in BLM was observed after photolysis in the presence of NAP-taurine. This was associated with the labeling of four protein bands on SDS polyacrylamide gels. In contrast no labeling was observed in BBM.

Two stilbene derivates that had been used to covalently label the Cl- carrier in the erythrocyte were investigated for reactivity with the renal organic anion system. These compounds, 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and (4,4'-diisothiocyano)-dihydrostilbene-2,2'-disulfonate (H2DIDS), were found to be potent inhibitors (Ki congruent to 35 microM) of p-aminohippurate (PAH) transport in the renal cortical slice without affecting tetraethylammonium (TEA) transport or tissue viability. During renal clearance studies performed in the perfused kidney, DIDS decreased the PAH/inulin clearance ratio to congruent to 1. When the possible renal transport of [3H]H2DIDS was investigated, the renal slice transport or binding of [3H]H2DIDS reached a slice-to-medium ratio of congruent to 6, and this accumulation was decreased by probenecid. In perfused kidney experiments, the [3H]H2DIDS/inulin clearance ratio was congruent to 0.8. Since probenecid reduced this clearance ratio to congruent to 0.5, there was the possibility that H2DIDS underwent tubular secretion. In conclusion, DIDS and H2DIDS interacted with the renal organic anion transport system, which indicated that these compounds were possible probes for this transport system.

The NA-K-ATPase of toad skin was characteristically sensitive to Na, K, and ATP. It was not affected by amiloride, vasopressin, cAMP, and thyroxine, but stimulated by insulin. Ouabain, a potent inhibitor at 37 degrees C, did not inhibit the enzyme activity significantly at 23 degrees C. The optimal pH for the enzyme activity increased as temperature decreased. However, the optimal OH-/H+ ratio of the medium remained constant at 16 regardless of temperature. The Km for ATP remained unchanged between 37 and 8 degrees C if the OH-/H+ ratio was held constant at 16, but increased as temperature decreased if the pH of the medium was held constant at 7.4. The enzyme activity showed no appreciable variation between 37 and 20 degrees C with a constant OH-/H+ ratio of 16, whereas it decreased logarithmically at a constant pH of 7.4 over the same temperature range. These results indicate the presence of a typical Na-K-ATPase system in toad skin and that the enzyme is in the most active catalytic state at a fixed level of OH-/H+ ratio in the medium regardless of incubation temperature.

The potential role of atrial natriuretic factor (ANF) in the renal response to head-out water immersion (WI) was studied. Five female mongrel dogs, trained to stand for 100 min in air followed by 100 min of thermoneutral WI at 37 degrees C or 200 min in air (timed control, TC), were chronically instrumented with arterial and venous catheters. The animals were hydrated with a volume of 0.45% NaCl solution equivalent to 2% of their body weight. Prehydration levels of arterial ANF were 243 +/- 15 (SE), and venous ANF levels were 211 +/- 21 pg/ml. WI resulted in an increase in urine flow from 0.7 +/- 0.1 ml/min to a peak flow of 2.2 +/- 0.3 ml/min (P less than 0.05). On immersion, plasma venous and arterial ANF levels increased significantly by 29 and 21% from the preimmersion values of 183 +/- 14 and 222 +/- 20 pg/ml, respectively. The arterial-venous difference for plasma ANF was maintained at 35 +/- 14 pg/ml (P less than 0.05) during WI; therefore venous sampling may suffice as a measure of circulating ANF levels. Sodium excretion increased linearly during WI to a peak value of 228 +/- 32 mu eq/min from a base line of 52 +/- 12 mu eq/min (P less than 0.05). These data indicate that peripheral tissues extract ANF and that WI is a physiological stimulus for the release of ANF. However, the time course and magnitude of the changes in plasma ANF and urine sodium excretion during WI are not comparable, and other mechanisms are likely responsible for the WI natriuresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular fluid volume (ECF, [125I]iothalamate space), blood volume (BV, 51Cr-labeled erythrocyte space), and hematocrit were measured continuously to study the kinetics of fluid movements between intracellular, interstitial, and plasma compartments during water immersion (WI) at 38 degrees C in seven splenectomized and acutely nephrectomized dogs. ECF and plasma volume (PV) increased linearly during WI by 10 +/- 2 ml/kg (4% of initial ECF volume, P less than 0.05) and 12 +/- 2 ml/kg (33% of initial PV, P less than 0.05), respectively, above the control level by 120 min of WI. We estimate that 83% of the fluid entering the intravascular compartment is derived from the intracellular space at 120 min of WI. The results of this study indicate that WI leads to a sustained fluid movement of intracellular fluid toward the intravascular compartment. The increase in interstitial hydrostatic pressure (wick method) by 28.5 mmHg from the control level at 5 min of WI in response to the external water pressure exceeds the increase in mean capillary pressure by 10-11 mmHg relative to the control level. We postulate that this negative hydrostatic pressure gradient across the capillary wall leads to an increase in PV during WI.

The influence of head-out water immersion (WI) (37 degrees C) on baroreflex control of heart rate was studied in five trained, instrumented, conscious dogs. Arterial pressure was raised and lowered using occluder cuffs implanted around the aorta and inferior vena cava. Function curves relating transmural systolic arterial pressure (TSAP = systolic arterial pressure-pleural pressure) to heart rate (HR) were constructed to compare responses in air and during WI. The resting TSAP in air [142 +/- 8 mmHg (mean +/- SE) at 78 +/- 6 beats/min] increased significantly during WI (161 +/- 9 mmHg at 109 +/- 9 beats/min). During WI, the saturation TSAP at the bradycardia plateau of the relation increased significantly, by 19 mmHg, whereas the average gain (slope) of the relation decreased significantly, from -1.426 to -0.752 beats.min-1.mmHg-1. Therefore, WI elicits both a resetting and a decrease of the average gain of the TSAP-HR relation. The heart rate range increased during WI as well. After cholinergic blockade with atropine, WI did not elicit a resetting of the relation and the change in average gain was abolished. However, after beta 1-blockade with metoprolol, the resting TSAP increased significantly during WI and resetting persisted, but the decrease of average gain was abolished. Therefore, the alteration of the TSAP-HR relation in WI is achieved via a modulation of both adrenergic and cholinergic regulation of HR.