An integrative, in situ approach to examining K+ flux in resting skeletal muscle

The contributions of Na+/K+-ATPase, K+ channels, and the NaK2Cl cotransporter (NKCC) to total and unidirectional K+ flux were determined in mammalian skeletal muscle at rest. Rat hindlimbs were perfused in situ via the femoral artery with a bovine erythrocyte perfusion medium that contained either 86Rb or 42K, or both simultaneously, to determine differences in ability to trace unidirectional K+ flux in the absence and presence of K+-flux inhibitors. In most experiments, the unidirectional flux of K+ into skeletal muscle (JinK) measured using 86Rb was 8–10% lower than JinK measured using 42K. Ouabain (5 mM) was used to inhibit Na+/K+-ATPase activity, 0.06 mM bumetanide to inhibit NKCC activity, 1 mM tetracaine or 0.5 mM barium to block K+ channels, and 0.05 mM glybenclamide (GLY) to block ATP-sensitive K+ (KATP) channels. In controls, JinK remained unchanged at 0.31 ± 0.03 µmol·g–1·min–1 during 55 min of perfusion. The ouabain-sensitive Na+/K+-ATPase contributed to 50 ± 2% of basal JinK, K+ channels to 47 ± 2%, and the NKCC to 12 ± 1%. GLY had minimal effect on JinK, and both GLY and barium inhibited unidirectional efflux of K+ (JoutK) from the cell through K+ channels. Combined ouabain and tetracaine reduced JinK by 55 ± 2%, while the combination of ouabain, tetracaine, and bumetanide reduced JinK by 67 ± 2%, suggesting that other K+-flux pathways may be recruited because the combined drug effects on inhibiting JinK were not additive. The main conclusions are that the NKCC accounted for about 12% of JinK, and that KATP channels accounted for nearly all of the JoutK, in resting skeletal muscle in situ.Key words: sodium potassium chloride cotransporter, NKCC, Na+/K+-ATPase, potassium channels, potassium transport, in situ rat hindlimb.

Arterio–venous differences to study macronutrient metabolism: introduction and overview

The arterio-venous difference technique is now well established in the study of organ and tissue metabolism. This technique requires samples to be obtained of the arterial blood supplying and the venous drainage from a tissue, together with a measurement of the blood flow through the tissue. The technique is most appropriate when the arterial concentration and tissue metabolism of a substance are constant, and when the blood flow is stable. If these criteria are not satisfied, care is needed in the interpretation of the results obtained. It should be recognized that the arterio-venous difference technique only measures the net exchange of a substance with the tissue, and that tracers are needed if unidirectional flux needs to be estimated. The other factors which must be borne in mind when intending to use this technique are the transit times of blood and the substance of interest through a tissue, the volume of distribution of the substance in the tissue, and the possibility that the venous samples obtained are derived from a mixture of different tissues.

ATP decreases acutely and reversibly transport through the paracellular pathway in human cervical cells

We studied the effect of ATP on transepithelial transport through the paracellular pathway in human cervical cells. Transepithelial conductance and transepithelial permeability (determined from the measurements of unidirectional flux of inert molecules) were measured in Caski cells grown on permeable support. Transepithelial conductance was 55.9 +/- 17.7 mS/cm2 and permeability was 12.5 +/- 2.7 x 10(-6) cm/s for a 0.51-kDa probe. Addition of ATP to the medium decreased acutely and reversibly the conductance and the permeability to probes between 0.18 and 10 kDa by 23-31% in a dose-related fashion; the 50% effective concentration was 1 microM, with a maximal effect at 5-10 microM extracellular ATP. The ATP effect was observed regardless of the pressure gradient across the epithelium. These results indicate that extracellular ATP in micromolar concentrations decreases acutely and reversibly the permeability through the paracellular pathway in cervical cells, possibly by affecting the permeability of the tight junctions and the resistance of the intercellular space. On the basis of these data, we speculate that ATP may play a role in the regulation of solutes and fluid transport across the cervical epithelium in vivo.

pH-sensitive anion exchanger in rat lacrimal acinar cells

Basolateral membranes from rat lacrimal acinar cells contain Na(+)-H+ and Cl(-)-HCO3- antiport activities [Invest. Ophthalmol. Visual Sci. 28: 1726-1729, 1989; Am. J. Physiol. 255 (Gastrointest. Liver Physiol. 18): G367-G373, 1988]. This study evaluated factors involved in coupling ion fluxes through these antiporters. 22Na+ flux into acini isolated from rat exorbital glands was 94 +/- 6 nmol.mg-1.min-1, and it was accelerated threefold by 10(-5) M carbachol; neither resting nor stimulated influx was affected by bumetanide. It is, therefore, likely that a portion of the carbachol-dependent Na+ influx is mediated by Na(+)-H+ antiporters. 36Cl- flux into Cl(-)-loaded, unstimulated acini was 275 +/- 21 nmol.mg-1.min-1; Cl- flux into HCO3(-)-loaded acini was 204 +/- 2; Cl- flux into acini loaded with both Cl- and HCO3- was 253 +/- 32; and influx in the absence of exchangeable intracellular anions was 176 +/- 13. Therefore, Cl(-)-Cl- self-exchange represented the major component of anion exchanger-mediated Cl- flux into resting cells. As pHi was increased above 7.2 by potassium-nigericin pH clamping, Cl- fluxes into Cl(-)- and HCO3(-)-containing acini, but not into Cl(-)-depleted acini, were significantly accelerated. SITS completely abolished the pHi-activated increment of Cl(-)-Cl- exchange. Carbachol increased Cl- unidirectional flux into Cl(-)-loaded cells by 25% (P less than 0.1), apparently as a result of Na(+)-H+ antiporter-mediated cytoplasmic alkalinization.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic AMP effects on chloride transport and carbonic anhydrase activity in frog skin

Unidirectional (36Cl) chloride fluxes across isolated and short-circuited frog skin were measured, with both sides bathed in low chloride solution. Transepithelial chloride influx was inhibited by exogenous cAMP as well as by substances enhancing its cellular concentration, such as epinephrine, isoproterenol, and 3-isobutyl-1-methylxanthine (IBMX). Epinephrine and isoproterenol addition resulted in an increase of transepithelial chloride outflux, but exogenous cAMP or IBMX had no significant effect on this unidirectional flux. Phenylephrine had no significant effect on influx or outflux. Carbonic anhydrase (CA) activity in extracts obtained from frog skin epithelium was inhibited by pretreatment with IBMX at 4–5 °C and prolonged exposure to cAMP at freezing point. cAMP or IBMX alone had no significant effects on CA activity. This catalytic activity was chloride insensitive and was abolished by 0.1 μM. acetazolamide. Results suggest a [Formula: see text] exchange inhibition by cAMP via carbonic anhydrase inactivation. Chloride outflux stimulation by β-adrenergic agonists does not seem to depend solely on an increase in cAMP concentration.Key words: frog skin, chloride transport, exogenous and induced cAMP effects, carbonic anhydrase inhibition.

A unidirectional water flux model of fruit growth

A mathematical model of fruit growth has been developed based upon a unidirectional flux of solution into the fruit. The model relies entirely upon the physical process of diffusion and water evaporation driven by radiant energy and does not invoke any requirement for metabolic inputs by the developing fruit. The model will "grow" fruit of different varieties of different species to the correct size in the correct time. Key words: Lycopersicon esculentum, tomato, Prunus avium, cherry, fruit growth, phloem, translocation, transpiration, model.