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AbstractBackground/Aims: The aim of this study was to elucidate what kind of plasma proteins would change their urinary excretions when the glomerular filtration rate (GFR) was increased. Methods: We measured urinary excretions of three plasma proteins with different molecular radii (MR) and isoelectric points (pI): albumin, orosomucoid (OM) and transferrin (Tf), after acute protein loading in healthy subjects. Results: Urinary excretion of OM with more anioic charge and smaller MR than albumin, and Tf with more cationic charge and slightly larger molecular weight than albumin, significantly increased in parallel with increased creatinine clearances after acute protein loading. These renal responses returned to basal levels 9 h after protein ingestion. In contrast, increases in urinary excretion of albumin were not observed. Conclusion: Because these findings could not be explained by changes in either size or charge selectivity of shunt pores in the glomerular capillary wall, it is suggested that urinary excretion of albumin may have a special property that distinguishes it from other plasma proteins and may be a less sensitive marker to reflect changes in renal hemodynamics than the other plasma proteins.Copyright © 2005 S. Karger AG, Basel

1. The inside‐out configuration of the patch‐clamp method was used to study the effects of MgATP, free ATP and Mg2+ on single ATP‐sensitive K+ channel currents in rat pancreatic beta‐cells. 2. Magnesium ions caused a marked reduction of channel activity: 5 mM‐free Mg2+ produced a 50% reduction in the activity of inward currents recorded at ‐60 mV in symmetrical K+ concentrations. 3. Inhibition of channel activity by MgATP does not involve phosphorylation as both free ATP (i.e. ATP in the absence of divalent cations) and non‐hydrolysable ATP analogues were effective inhibitors. 4. Magnesium ions produced a striking reduction in the ability of ATP (total) to inhibit channel activity. When channel activity was plotted as a function of the total ATP concentration, the Ki for channel inhibition was 4 microM in Mg2(+)‐free solution, compared to a Ki of 26 microM in the presence of 2 mM‐Mg2+. The shape of the relationship between channel activity and the total ATP concentration was not changed by Mg2+. When channel activity was plotted as a function of the free ATP concentration, however, Mg2+ had little effect on Ki. This suggests that free ATP is the more potent inhibitor of channel activity and that MgATP has little inhibitory effect. 5. ATP analogues that dissociate only as far as the tribasic form were also able to inhibit channel activity. This suggests that both ATP4‐ and ATPH3‐ can block the channel. 6. Like ATP, ADP was more effective at inhibiting channel activity in the absence of Mg2+, that is as the free base. The non‐hydrolysable ATP analogues AMP‐PNP and AMP‐PCP, however, were more effective in the presence of Mg2+. 7. It is suggested that (1) the potency of inhibition is related to the amount of negative charge carried by the ion and (2) the intracellular concentration of free ATP will be an important modulator of channel activity in the intact beta‐cell.

A class of K channels in cardiac muscle is reversibly blocked by intracellular adenosine 5'‐triphosphate (ATP). The characteristics of this K channel were studied by recording single‐channel currents in ventricular cells isolated enzymatically from guinea‐pig heart. The reversal potential of single‐channel currents agreed well with the K equilibrium potential. Blockers of other K channels, such as tetraethylammonium and 4‐aminopyridine, decreased the mean open time of the channel. The chord conductance increased as the 0.24th power of the K concentration on the outer surface of the membrane, and showed a marked inward‐going rectification on strong depolarizations. The degree of rectification was larger with increasing Na concentration on the inner side of the membrane. The kinetics of the channel were almost voltage independent, but depended on the concentration of intracellular ATP. The conductance of the channel was not affected by ATP. When channel kinetics were examined in the presence of ATP, the distribution of open times and closed times was fitted well with a sum of two exponential components. When ATP concentration was increased, the time constants obtained from the open‐time histogram decreased and those from the closed‐time histogram increased, resulting in a decrease of the open‐state probability. The channel was blocked by ATP, adenosine 5'‐diphosphate,5'‐adenylylimidodiphosphate, guanosine 5'‐triphosphate and uridine 5'‐triphosphate, but not by adenosine 5'‐monophosphate, creatine phosphate, creatine or adenosine. Plots of the open‐state probability versus the ATP concentration revealed Michaelis‐Menten saturation kinetics with strong co‐operativity of multiple receptor sites (Hill coefficient 3‐4, concentration of half‐saturation 0.5 mM). It was concluded that this K channel has three or four receptor sites selective for triphosphate nucleotide on the inner surface of the membrane, and that the channel is blocked through the binding of agonists to the receptors.

The patch‐clamp method was applied to single atrioventricular (a.v.) node cells of the rabbit heart to study the characteristics of the K+ channel. When the electrode contained 5.4 mM‐K+, depolarizations of the cell‐attached patch membrane induced outward single channel currents characterized by burst‐like openings; the open‐state probability increased from 0.005‐0.01 at ‐40 mV to 0.07‐0.1 at +20 mV of membrane potential. The reversal potentials of the current at K+ concentrations of 5.4, 20 and 130 mM in the electrode agreed with those given by the Nernst equation, indicating that this channel is selective for K+ ions. The slope conductance of the channel decreased beyond 60‐90 mV positive to the reversal potential (inward‐going rectification). The conductance near the reversal potential increased with increasing K+ concentrations on either side of the membrane: from 31‐32 pS at 5.4 mM‐K+ to 41‐42 pS at 20 mM‐K+ on the outside, and from 19 pS at 90 mM‐K+ to 29.3 pS at 130 mM‐K+ on the inside. Superfusion of the cell with 5.4 mM‐CN‐, glucose‐free Tyrode markedly increased the number of channel openings in the cell‐attached patch. In the inside‐out patch, application of 1 mM‐adenosine‐5'‐triphosphate (ATP) at the inner surface of the patch membrane blocked reversibly the channel activity, while 1 mM‐adenosine‐5'‐diphosphate (ADP) failed to block it. The conductance and kinetics of the channel were not modified by increasing the Ca2+ concentration from 10(‐8) M to 5 X 10(‐6) M on the inner side of the membrane, while a further increase in Ca2+ to 10(‐4) M decreased the open‐state probability. The probability density for the open time fitted well with an exponential distribution (time constant of 5.4 ms at 60 mV positive to the resting potential), while that for the closed time was separated into a fast and a slow component (time constants of 4.0 and 132.0 ms, respectively). The time constant of the slow component decreased significantly with depolarization in some preparations. However, neither the time constant of the fast component of the closed‐time histogram nor that of the open‐time histogram was voltage‐dependent.

1. The patch‐clamp method has been used to study the selectivity of single ATP‐sensitive potassium channels in excised membrane patches from dissociated rat pancreatic beta‐cells. 2. In symmetrical K+ concentrations the current‐voltage relation of this channel showed slight inward rectification. The K+ permeability coefficient was 1.05 x 10(‐13) cm3/s ([K+]o = 140 mM; 20 degrees C) in the inside‐out patch and somewhat smaller when measured under the same conditions in the outside‐out configuration (0.86 x 10(‐13) cm3/s). 3. When intracellular Rb+ replaced K+, inward K+ currents were unaffected but the outward currents carried by Rb+ were substantially smaller. The extent of the reduction in the outward currents depended on the internal Rb+/K+ ratio and increased as [Rb+]i was raised. Both inward and outward Rb+ currents were blocked by 1 mM‐ATP. No currents were measurable in symmetrical 140 mM‐Rb+ solutions. 4. With 140 mM [K+]o and 140 mM [Rb+]i the single‐channel current‐voltage relation reversed at +8 mV and the potential at which the variance of the ATP‐sensitive current was least was shifted by 6 mV to more positive potentials. These data suggest a PRb/PK ratio of around 0.7. 5. Outward Rb+ currents were reduced at all potentials in inside‐out patches exposed to 107 mM‐Rb+ solution intracellularly and 5 mM‐K+ externally. 6. Partial replacement of external K+ with Rb+ substantially reduced the inward currents recorded from outside‐out patches and also decreased outward K+ currents. 7. In outside‐out patches, the addition of 1 mM‐Rb+ to the external solution produced a block of inward K+ currents that initially increased and then decreased again with hyperpolarization. This suggests that the Rb+ block of K+ currents is voltage dependent and that Rb+ acts as a permeant blocker of K+ currents. 8. The sodium permeability of the channel, relative to that of potassium, was 0.39 for internal Na+ and 0.007 for external Na+ ions. 9. We conclude that Rb+ serves as an acceptable tracer for K+ in efflux studies when changes in K+ flux through ATP‐sensitive K+ channels are of interest but that the magnitude of such fluxes will be considerably underestimated.