Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A.

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38 degrees C, or the cellular uptake of isotope during a similar incubation was measured. The influx rate of untreated thymocytes was: 2.3-10(-12) moles cm-2-s-1 and efflux rate: 1.8-10(-12) moles cm-2-s-1. When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 mug/ml, but concentrations as low as 0.75 mug/ml were effective. Hydrocortisone resistant thymocytes responded at least was well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times. Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl-:43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present. Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A. The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, alpha-methyl-D-mannoside.     

Unidirectional sodium and potassium fluxes through the sodium channel of squid giant axons.

Unidirectional 22Na-traced sodium influx or 42K-traced potassium efflux across the membranes of voltage-clamped squid giant axons was measured at various membrane potentials under bi-ionic conditions. Tetrodotoxin almost entirely eliminated the extra K+ efflux induced by short repetitive depolarizations in the presence of tetraethylammonium or 3,4-diaminopyridine. A method of determining the voltage dependence of the unidirectional flux through voltage-gated channels is described. This technique was used to obtain the unidirectional flux-voltage relation for the sodium channel in bi-ionic and single-ion conditions. It allows the determination of the unidirectional flux at the zero-current potential which, for influx, was found to be approximately 20% of the value measured 80 mV negative to the zero-current potential. The unidirectional flux ratio under bi-ionic conditions was also measured and the flux ratio exponent found to average 1.15 with an external sodium and an internal potassium solution. A three-barrier, two-site, multi-occupancy model previously obtained for other conditions was found to predict a similar non-unity average for the flux ratio exponent. It is also shown that some single-occupancy models can predict non-unity values for the flux ratio exponent in bi-ionic conditions.     

Unidirectional K+ fluxes through recombinant Shaker potassium channels expressed in single Xenopus oocytes

We describe a method to evaluate the ratio of ionic fluxes through recombinant channels expressed in a single Xenopus oocyte. A potassium channel encoded by the Drosophila Shaker gene tested by this method exhibited flux ratios far from those expected for independent ion movement. At a fixed extracellular concentration of 25 mM K+, this channel showed single-file diffusion with an Ussing flux-ratio exponent, n', of 3.4 at a membrane potential of -30 mV. There was an apparent, small voltage dependence of this parameter with n' values of 2.4 at -15 and -5 mV. These results indicate that the pore in these channels can simultaneously accommodate at least four K+ ions. If each of these K+ ions is in contact with two water molecules, the minimum length of the pore is 24 A.     

Unidirectional water and sodium fluxes and respiratory metabolism in the African lung-fish,Protopterus annectens

Summary Simultaneous measurements have been made of the efflux rates of tritiated water and radiosodium from the lung-fishProtopterus annectens under conditions in which the fish can either use its gills, its lungs or both for respiration. Tritiated water efflux rate was similar (43–44% h^–1) when the fish was in oxygenated water with or without access to air but decreased slightly in deoxygenated water. An efflux rate constant of 44% h^–1 obtained in oxygenated water is similar to those of other inactive fish in fresh water. The efflux rate constant for sodium was 0.84% h^–1 and total sodium flux was comparable to that of fresh-water fish. The decrease in both tritiated water and sodium efflux rates in deoxygenated water are explained in terms of diversion of blood flow away from the functional gills to the lungs. Oxygen consumption was highest (38.7 ml kg^–1 h^–1) in deoxygenated water and lowest when the fish was out of water. Despite the reduced functional significance of the gill for respiration, it is still as permeable to water and electrolytes as that of fresh-water teleosts.This work was supported by a travel grant from the Commonwealth Universities Interchange Scheme of the British Council and laboratory facilities of the Department of Biological Sciences, University of Lancaster     

Unidirectional Na+, Ca2+, and K+ fluxes through the bovine rod outer segment Na-Ca-K exchanger

The properties of the Na-Ca exchanger in the plasma membrane of rod outer segments isolated from bovine retinas (ROS) were studied. Unidirectional Ca2+, Na+, and K+ fluxes were measured with radioisotopes and atomic absorption spectroscopy. We measured K+ fluxes associated with the Ca-Ca self-exchange mode of the Na-Ca exchanger to corroborate our previous conclusion that the ROS Na-Ca exchanger differs from Na-Ca exchangers in other tissues by its ability to transport K+ (Schnetkamp, P. P. M., Basu, D. K. & Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-C157). The Na-Ca-K exchanger was the only functional cation transporter in the plasma membrane of bovine ROS with an upper limit of a flux of 10(5) cations/ROS/s or a current of 0.01 pA contributed by other cation channels, pumps, or carriers; cation fluxes via the Na-Ca-K exchanger amounted to 5 x 10(6) cations/ROS/s or a current of 1 pA. Ca2+ efflux via the forward mode of the Na-Ca-K exchanger did not operate with a fixed single stoichiometry. 1) The Na/Ca coupling ratio was increased from three to four when ionophores were added that could provide electrical compensation for the inward Na-Ca exchange current. 2) The K/Ca coupling ratio could vary by at least 2-fold as a function of the external Na+ and K+ concentration. The results are interpreted in terms of a model that can account for the variable Ca/K coupling ratio: we conclude that the Ca2+ site of the exchanger can translocate independent of translocation of the K+ site, whereas translocation of the K+ site requires occupation of the Ca2+ site, but not its translocation. The results are discussed with respect to the physiological role of Na-Ca-K exchange in rod photoreceptors.     

Unidirectional calcium and nucleotide fluxes in sarcoplasmic reticulum. I. Interpretation of flux ratios for different reaction schemes.

The relation between unidirectional calcium and nucleotide fluxes was examined for different ATPase reaction schemes of the sarcoplasmic reticulum. The schemes considered differed in the order of sorption and desorption of calcium, ATP, and ADP. The results suggest that the theoretical relation between calcium and nucleotide fluxes depends on the reaction scheme and that experimental measurements can distinguish among them. The results obtained are generally valid and do not depend on assumptions of equilibrium or pseudoequilibrium between intermediate states of the pump.     

Unidirectional fluxes in saturated single-file pores of biological and artificial membranes I. Pores containing no more than one vacancy

A theoretical treatment of isotope fluxes in filled single-file pores is given. The treatment is confined to pores permitting only one vacancy at a time. Tracer flux, unidirectional fluxes and net flux are calculated. The exponent n which is obtained by representing the ratio of unidirectional fluxes as a power of the electrochemical activity-ratio proves to be closely related with m, the number of sites per pore. The minimum and maximum values of n are m?1 and m. It is shown that measurement of n provides sufficient information to determine m if the energetic properties of sites and barriers do not change too much with varying concentrations. Unlike the unidirectional fluxes, the net fluxes yield no direct information about the number of sites. The concentration dependence of the net fluxes, however, can be used to discriminate between the two limiting cases, n = m?1 and n = m. It is interesting with regard to the instantaneous potassium currents of nerve membranes that a suitable choice of the energetic parameters of a filled pore leads to a quasi-linearity of the net flux-voltage curves, which is only slightly affected by variation of the external concentration.     

Unidirectional fluxes in saturated single-file pores of biological and artificial membranes. II. Asymptotic behavior at high degrees of saturation

A rate-theory analysis of isotope fluxes in filled single-file pores is given. It is assumed that the relation between the activities in solution and the fluxes can be described by the asymptotic laws for high activities. Only pore states containing no more than two vacancies need be taken into account. The flux-ratio exponent n, which is obtained by representing the ratio of unidirectional fluxes as a power of the electrochemical activity ratio, is found to provide information about m, the number of sites per pore. The general relation between n and m, for m ≥ 4, is m ? 3 ≤ n ≤ m ?1. For m = 2 or 3 the minimum value is 1. The theoretical results are discussed with respect to the internal structure of gramicidin pores in artificial bilayer membranes.     

Microcirculatory hematocrit and blood flow

Direct measurements from many laboratories indicate that the oxygen tension in skeletal muscle is significantly less than in the large veins draining these tissues. Harris (1986) has proposed that because of the parallel anatomic arrangement of large arterioles and venules in skeletal muscle, a counter-current exchange between these vessels can occur. He theorized that diffusion of O2 between arteriole and venule would lower the PO2 in the blood as it enters capillaries and result in a decreased tissue PO2 and an increase in large vein PO2. Calculations (Appendix) show that the amount of O2 transferred between arteriole and venule is inadequate to account for this difference in PO2 between tissue and veins due to the small surface area that is involved. It is well documented that the microcirculatory hematocrit ranges between 20 and 50% of that in the supply vessels. The reduced hematocrit lowers the oxygen content in these vessels and results in a low oxygen tension in the surrounding tissue. True arteriovenous shunts are not present in most skeletal muscles, but 15-20% of the microvessels represent thoroughfare or preferential flow channels. It is suggested that these vessels contain a greater than normal hematocrit to account for a conservation of red cell mass across the microcirculation. Furthermore, it is shown that the hematocrit in the preferential flow channels is an inverse function of the flow rate for any level of the microcirculatory hematocrit. The increased hematocrit raises the flow resistance in these vessels which reduces flow further and represents a positive feedback condition which may contribute to the intermittent and uneven flow patterns which are present within the microcirculation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unidirectional fluxes of short-chain fatty acids across segments of the large intestine in pig,sheep and pony compared with guinea pig

Unidirectional fluxes of short-chain fatty acids across pig, sheep and pony caecum, proximal and distal colon were studied under short-circuit current conditions in Ussing chambers. Findings are compared with results from guinea pig. Marked species differences are apparent; highest mucosal-to-serosal fluxes of acetate, propionate and butyrate were seen in guinea pig, lower values in pig and smallest fluxes in sheep and pony. Segmental differences between caecum, proximal and distal colon exist mainly in guinea pig and are less developed in pig, sheep and pony. Inhibition of Na⁺/H⁺ exchange by amiloride added to the mucosal solution decreased the mucosal-to-serosal fluxes of short-chain fatty acids clearly in guinea pig caecum and proximal colon, and very little in distal colon. This effect was somewhat less pronounced in pig caecum and distal colon, in caecum and distal colon of sheep and caecum of the pony. In pig, sheep and pony proximal colon and pony distal colon no significant inhibition was observed. Inhibition of the K⁺-H⁺ ATPase by addition of ouabain to the mucosal solution diminished mucosal-to-serosal fluxes of short-chain fatty acids in the guinea pig distal colon extensively. No comparable inhibition was seen in any of the other segments in the animals studied.     

Tracer Diffusion and Unidirectional Fluxes

Available experimental data have been utilized to examine the effects of cross-coefficients on tracer diffusion and on the estimation of unidirectional fluxes from observations on tracer flow. In free solution or in a nonselective membrane, the interaction between the flows of tracer and the unlabelled substance are small at concentrations of biological interest for the nonelectrolytes urea, alanine, and β-alanine, and for sodium and chloride ions. Under these conditions, measurement of tracer flow can be used to predict flow of the bulk substance to an accuracy of a few per cent.     

Effects of reducing uterine blood flow on fetal blood flow distribution and oxygen delivery.

We examined the effect of graded reduction in uterine blood flow on distribution of cardiac output and oxygen delivery to fetal organs and venous blood flow patterns in 9 fetal sheep using the radionuclide-labeled microsphere technique. We reduced uterine blood flow in two steps, decreasing fetal oxygen delivery to 70% and 50% of normal, and compared the results with those from a similar study from our laboratory on graded umbilical cord compression. With 50% reduction in fetal oxygen delivery, blood flow and the fraction of the cardiac output distributed to the brain, heart, and adrenal gland increased and that to the lungs, carcass, skin, and scalp decreased. Oxygen delivery to the brain and myocardium was maintained, while that to the adrenal doubled, and that to the brain stem increased transiently. The decrease in oxygen delivery to both carcass and lower body segment correlated linearly with oxygen consumption (P less than 0.001). The proportion of umbilical venous blood passing through the ductus venosus increased from 44.6% to 53% (P less than 0.05). The preferential distribution of ductus venosus blood flow through the foramen ovale to the heart and brain increased, but that to the upper carcass decreased so that ductus venosus-derived blood flow to the upper body did not change. Hence, the oxygen delivered to the brain from the ductus venosus was maintained, and that to the heart increased 54% even though ductus venosus-derived oxygen delivery to the upper body fell 34%. Abdominal inferior vena caval blood flow and its contribution to cardiac output decreased, but the proportion of the abdominal inferior vena caval blood distributed through the foramen ovale also increased from 23.0 to 30.9%. However, the actual amount of inferior vena caval blood passing through the foramen ovale did not change. There was a 70% fall in oxygen delivery to the upper body segment from the inferior vena cava. A greater portion of superior vena caval blood was also shunted through the foramen ovale to the upper body, but the actual amounts of blood and oxygen delivered to the upper body from this source were small. Thus, graded reduction of uterine blood flow causes a redistribution of fetal oxygen delivery and of venous flow patterns, which is clearly different from that observed previously during graded umbilical cord occlusion.