Dependent 86Rb Research Articles

Parameter Estimation for Models of NH4+ Transport by the Renal Na‐K‐2Cl Cotransporter

An optimization algorithm was used to compute parameter values for kinetic models of NH4+ transport by the renal Na‐K‐2Cl cotransporter (NKCC2). The optimization method finds model off‐binding and translocation rate constants by minimizing the difference between model unidirectional fluxes and published data on NH4+‐ dependent 86Rb+ uptake by the NKCC2 cotransporter in transfected Xenopus oocytes. By using different symmetry assumptions, which reduces the number of parameters, five different models were optimized. The computed parameters for each model yielded fluxes consistent with the experimental curves, and the fittings of the five models exhibited residual errors that were not significantly different from each other. However, a systematic exploration of the parameter space revealed that, in some models, different parameter sets yield essentially identical fits to the data. With the identified parameters, the NKCC2 models show K+/NH4+ exchange, and enhancement of Na+ uptake by NH4+ binding when external [K+] is low. Further, for the A, B, and F isoforms, the optimization method identified parameter sets that differ only in off‐binding affinities, a result that is consistent with a published mutagenesis analysis of the molecular and structural bases for differences in 86Rb+ uptake between isoforms.This research was supported in part by NIH grants SC1GM084744 and DK042091.

Modulation of Na+,K+-ATPase activity is of importance for RVD.

This study was performed to examine the role of Na+,K+-ATPase activity for the adaptive response to cell swelling induced by hypoosmoticity, i.e. the regulatory volume decrease (RVD). The studies were performed on COS-7 cells transfected with rat Na+,K+-ATPase. To study changes in cell volume, cells were loaded with the fluorescent dye calcein and the intensity of the dye, following exposure to a hypoosmotic medium, was recorded with confocal microscopy. Ouabain-mediated inhibition of Na+,K+-ATPase resulted in a dose dependent decrease in the rate of RVD. Total 86Rb+ uptake as well as ouabain dependent 86Rb+ uptake, used as an index of Na+,K+-ATPase dependent K+ uptake, was significantly increased during the first 2 min following exposure to hypoosmoticity. Since protein kinase C (PKC) plays an important role in the modulation of RVD, a study was carried out on COS-7 cells expressing rat Na+,K+-ATPase, where Ser23 in the catalytic alpha1 subunit of rat Na+,K+-ATPase had been mutated to Ala (S23A), abolishing a known PKC phosphorylation site. Cells expressing S23A rat Na+,K+-ATPase exhibited a significantly lower rate of RVD and showed no increase in 86Rb+ uptake during RVD. Taken together, these results suggest that a PKC-mediated transient increase in Na+,K+-ATPase activity plays an important role in RVD.

Molecular, functional, and genomic characterization of human KCC2, the neuronal K–Cl cotransporter

The expression level of the neuronal-specific K–Cl cotransporter KCC2 (SLC12A5) is a major determinant of whether neurons will respond to GABA with a depolarizing, excitatory response or a hyperpolarizing, inhibitory response. In view of the potential role in human neuronal excitability we have characterized the hKCC2 cDNA and gene. The 5.9 kb hKCC2 transcript is specific to brain, and is induced during in vitro differentiation of NT2 teratocarcinoma cells into neuronal NT2-N cells. The 24-exon SLC12A5 gene is on human chromosome 20q13, and contains a polymorphic dinucleotide repeat within intron 1 near a potential binding site for neuron-restrictive silencing factor. Expression of hKCC2 cRNA in Xenopus laevis oocytes results in significant Cl −-dependent 86Rb + uptake under isotonic conditions; cell swelling under hypotonic conditions causes a 20-fold activation, which is blocked by the protein phosphatase inhibitor calyculin-A. In contrast, oocytes expressing mouse KCC4 do not mediate isotonic K–Cl cotransport but express much higher absolute transport activity than KCC2 oocytes under hypotonic conditions. Initial and steady state kinetics of hKCC2-injected oocytes were performed in both isotonic and hypotonic conditions, revealing K ms for K + and Cl − of 9.3±1.8 mM and 6.8±0.9 mM, respectively; both affinities are significantly higher than KCC1 and KCC4. The K m for Cl − is close to the intracellular Cl − activity of mature neurons, as befits a neuronal efflux mechanism.

Volume-sensitive KCI cotransport associated with human cervical carcinogenesis.

This study investigates the volume-sensitive KCI cotransporter (KCC) in various types of human cervical epithelial cell, testing the hypothesis that cervical malignancy is accompanied by differential expression of volume-sensitive KCC. Normal human cervical epithelial cells have KCCs which are quiescent in normal physiological conditions and are relatively refractory to hypotonic stress. By contrast, cervical cancer cells have KCCs which are also nearly quiescent in normal physiological conditions but high transport rates are observed in response to hypotonic challenge. Using isoform-specific primers, mRNA transcripts of KCC1, KCC3 and KCC4 were identified by reverse transcriptase polymerase chain reaction (RT-PCR) in several types of cervical cell, and confirmed by digestion with specific restriction endonucleases. By semiquantitative RT-PCR with beta-actin as the internal standard, the results indicate that cervical carcinogenesis is accompanied by the up-regulation of mRNA transcripts in KCC1, KCC3 and KCC4. [(Dihydroindenyl)oxy] alkanoic acid (DIOA), a KCC inhibitor, blocked both the regulatory volume decrease (RVD) process and volume-sensitive 86Rb+ efflux from cervical cancer cells in a dose-dependent manner. The volume-sensitive 86Rb+ efflux from cervical cancer cells was also blocked by two protein phosphatase inhibitors, calyculin A and okadaic acid, with IC50 values of 0.8 and 6 nM, respectively. Conversely, protein kinase inhibitors, chelerythrine and staurosporine, increased Cl- dependent 86Rb+ efflux. NEM (1 mM) led to a fivefold stimulation of 86Rb+ efflux which was totally Cl- dependent in cervical cancer cells. Hypotonicity could not stimulate any further 86Rb+ efflux after NEM treatment. These results indicate that the volume-sensitive KCC in cervical cancer cells plays a significant role in volume regulation and that the activities are modulated by a phosphorylation cascade. Taken together with our previous studies, we suggest the volume-regulatory ion channels and the co-transport systems work synergistically for volume regulation in human cervical cancer cells.

Secretagogue-induced 86Rb+ efflux from bovine parotid is HCO3- dependent.

Muscarinic agonist (carbachol)-induced K+ loss from a bovine parotid mince was studied using 86Rb+ as a K+ marker. In contrast to our previous studies with the rat parotid [Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G111-G118, 1991] in which both Cl(-)-dependent and HCO3(-)-dependent components of carbachol-induced 86Rb+ efflux were observed, no significant evidence for Cl(-)-dependent 86Rb+ loss was detected in the bovine parotid. HCO3(-)-dependent agonist-induced 86Rb+ loss was blunted by K+ and Cl- channel blockers and by removal of extracellular Ca2+, consistent with the hypothesis that this 86Rb+ loss occurs via a Ca(2+)-activated K+ channel and that this cation loss serves to electrically balance the concomitant loss of HCO3- via an electrically conductive pathway, presumably an apical anion channel. Acetate, formate, and propionate could substitute for HCO3-. Interpreted in terms of current models of salivary fluid secretion, which hypothesize that the production of fluid is secondary to anion secretion accompanied by an electrically coupled K+ loss, these results indicate that salivary production in the bovine parotid is driven almost exclusively by acinar HCO3- secretion.

Electric organ polyamines and their effects on the acetylcholine receptor.

1. The electric organ of Torpedo nobiliana contained putrescine (PUT), spermidine (SPD), spermine (SPM), and cadaverine (CAD). Traces of acetylated SPD and SPM were occasionaly seen. 2. Upon fractionation of the tissue by differential centrifugation, the polyamines (PA) were found predominantly in the soluble fraction. The postsynaptic membrane fraction, containing a high concentration of acetylcholine receptor (AChR), was proportionally enriched in SPM. The molar ratio of SPM to AChR was approximately two in these membranes. 3. The effect of exogeneous PA on AChR function was studied by two methods: carbamoylcholine (CCh)-dependent 86Rb+ influx into receptor-rich membrane vesicles and [alpha-125I]bungarotoxin (Bgt) binding to the AChR. 4. SPM inhibited both ion influx and the rate of Bgt binding at concentrations above 1 mM, and therefore it appears to act as a competitive antagonist of the AChR. 5. At submicromolar concentrations, and only after preincubation with the receptor-rich membrane, SPM and PUT increased the ion influx by about 20% over control values. 6. Preincubation with 100 nM SPM did not affect the equilibrium binding of iodinated toxin or the rate of toxin binding, and therefore SPM was not uncovering new receptors. 7. By measuring the initial rate of toxin binding after different periods of preincubation with 1 microM CCh, the rate of the slow phase of receptor desensitization was determined. This rate was not changed by 100 nM SPM. 8. Although these results suggest that at low concentrations SPM is a positive modulator of the AChR, the precise mechanism of action is not determined yet.

Pharmacological characterization of nicorandil by 86Rb efflux and isometric vasorelaxation studies in vascular smooth muscle.

Nicorandil and cromakalim were found to stimulate 86Rb efflux (a marker of K+ ions) from resting preparations of rabbit aorta. This action was suppressed by 10(-5) M glibenclamide, an antagonist of K(+)-channel openers in vascular smooth muscle. Through intracellular production of cyclic GMP, and subsequent suppression of cellular Ca2+ activation, nitrovasodilators interfere indirectly with the activation of Ca(2+)-dependent ion channels. 8-Bromo-cyclic GMP and sodium nitroprusside antagonized the Ca(2+)-dependent 86Rb efflux induced by 3 x 10(-7) M norepinephrine. When nicorandil and cromakalim were investigated in the same experimental setup in the presence of glibenclamide to suppress stimulation of K+ channels, only nicorandil also suppressed the norepinephrine-induced increase of the 86Rb efflux. These results confirm that nicorandil acts as both an opener of K+ channels and a nitrovasodilator. Nicorandil relaxed helical strips from rabbit aorta contracted by 10(-7) M norepinephrine, but in contrast to the relaxant action of cromakalim, this response was not antagonized by the use of glibenclamide, indicating a greater importance of the nitrovasodilator mechanism than of the K(+)-channel-opening activity for relaxation in this tissue. However, when the nitrate-like action of nicorandil was suppressed by 10(-5) M methylene blue, the K(+)-channel-opening activity was unmasked on addition of 10(-4) M glibenclamide at high concentrations of nicorandil.

Mechanism of secretagogue-induced HCO3- and Cl- loss from rat parotid acini

The Cl(-)- and HCO3(-)-dependent components of muscarinic agonist (carbachol)-induced K+ loss from a rat parotid mince were studied using 86Rb+ as a K+ marker. Both components of 86Rb+ loss were blunted by K+ and Cl- channel blockers and by removal of extracellular Ca2+, consistent with the hypothesis that 86Rb+ loss occurs via a Ca(2+)-activated K+ channel and that this cation loss serves to electrically balance a concomitant loss of the corresponding anion via one or more conductive pathways (channels). Two tissue "pools" of 86Rb+ were observed, a carbachol-sensitive pool and a carbachol-insensitive pool (approximately 70 and approximately 30% of the total 86Rb+ content, respectively). There was no evidence for a time-dependent desensitization of the muscarinic response of the carbachol-sensitive pool. Cl(-)-dependent 86Rb+ loss was not affected by HCO3- addition, suggesting that both Cl- and HCO3- secretion are accompanied by 86Rb+ loss from the same pool and thus occur from the same cells. HCO3(-)-dependent 86Rb+ loss was not enhanced by lowering the extracellular Na+ concentration, indicating that the HCO3- exit pathway is not a Na(+)-HCO3- symport. The data are consistent with the postulate that Cl- and HCO3- are secreted by rat parotid acinar cells via the same or very similar conductive transport pathways in response to muscarinic stimulation.

RP 49356: inhibitory effects of ionic and secretory events in rat pancreatic islets

Rp 49356, a ‘K + channel opener’, inhibited 86Rb outflow, 45Ca outflow and insulin release from glucose-stimulated islets perifused in the presence of Ca 2+. The drug lost its capacity to reduce 86Rb outflow in islets exposed to glucose-free or Ca 2+-free medium. These observations suggest that RP 49356 might exhibit antagonistic actions on B-cell Ca 2+ channels. This will, in turn. reduce glucose-stimulated insulin output and Ca 2+-dependent 86Rb outflow.

Altered plasma membrane ion permeability in mercury-induced cell injury: Studies in hepatocytes of elasmobranch Raja erinacea

The effects of HgCl 2, CH 3HgCl, p-chloromercuribenzene sulfonate (PCMBS), and CdCl 2 on plasma membrane and cell metabolic functions of skate ( Raja erinacea) hepatocytes in suspension culture were assessed by measuring (a) the rates of Na +-dependent and -independent l-[ 14C]alanine uptake, (b) Na +-dependent 86Rb + uptake, a measure of NaK-ATPase activity, (c) 86Rb + efflux, a measure of K + permeability, (d) the difference between the 3H 2O and [ 14C]inulin distribution spaces, a measure of intracellular water volume, (e) cellular ATP concentrations, and (f) glutathione (GSH) and glutathione disulfide (GSSG) levels. The initial rates of l-alanine and 86Rb + uptake were inhibited by each of these metals in the following order: HgCl 2 > CH 3HgCl > PCMBS > CdCl 2. Inorganic mercury significantly inhibited the initial rates of Na +-dependent l-alanine and 86Rb uptakes at a concentration of 10 μ m, whereas 100 μ m produced nearly complete inhibition. These efects were dose-dependent, immediate (observed after <5 min of incubation with the metal), and persistent. Mercuric chloride also impaired volume regulatory mechanisms in skate hepatocytes: cells treated with 50 μ m HgCl 2 swelled slowly over a 60-min interval to volumes nearly double those of control cells. In addition, HgCl 2 prevented the normal volume regulatory decrease observed after swelling the hepatocytes in hypotonic media. Mercuric chloride (5–50 μ m) produced a rapid initial loss of a large fraction of intracellular 86Rb, followed by a slower rate of release of the remaining isotope. These effects were prevented if GSH was added with, but not following HgCl 2. In contrast, dithiothreitol, a more permeable thiol, both prevented and even partially reversed the effects of mercury. Mercuric chloride (10 μ m) had no effect on cellular ATP, GSH, or GSSG levels for up to 4 hr incubation. These findings indicate that 86Rb + (K +) efflux is a sensitive indicator of mercury toxicity, and are consistent with the hypothesis that the plasma membrane is a primary target for mercury's effects. A change in membrane permeability to K + would dissipate transmembrane electrochemical gradients, and may contribute to the apparent inhibition of transport processes energized by these gradients, such as Na +-alanine cotransport, and volume regulatory mechanisms.

Properties of the apamin-sensitive Ca2+-activated K+ channel in PC12 pheochromocytoma cells which hyper-produce the apamin receptor.

Undifferentiated PC12 cell produce high levels of apamin receptors (measured with 125I-apamin) after 7 days in culture. These levels are at least 50 times higher than those found in other cellular types which are also known to have apamin receptors and apamin-sensitive Ca2+-activated K+ channels in their membranes. Treatment of undifferentiated PC12 cells with nerve growth factor maintains these cells in a state having a low level (10 times less after 7 days of culture) of apamin receptors. Ca2+ injection into PC12 cells with the calcium ionophore A23187 has been used to monitor the activity of the Ca2+-activated K+ channel following 86Rb+ efflux. A large component of this Ca2+-activated 86Rb+ efflux is inhibited by apamin. Half-maximum inhibition by apamin of both 86Rb+ efflux and 125I-apamin binding was observed at 240 pM apamin. Another component of 86Rb+ efflux is due to another type of Ca2+-activated K+ channel which is resistant to apamin and sensitive to tetraethylammonium. The Ca2+ channel activator Bay K8644 also triggers an apamin-sensitive Ca2+-dependent 86Rb+ efflux. Bay K8644 has been used to analyze the internal Ca2+ concentration dependence of the apamin-sensitive channel activity. Under normal conditions, the internal Ca2+ concentration is 109 +/- 17 nM, and the apamin-sensitive channel is not activated. The channel is fully activated at an internal Ca2+ concentration of 320 +/- 20 nM.

A calmodulin dependent Ca 2+-activated K + channel in the adipocyte plasma membrane

Increased membrane permeability (conductance) that is specific for K + and directly activated by Ca 2+ ions, has been identified in isolated adipocyte plasma membranes using the K + analogue, 86Rb +. Activation of these K + conductance pathways (channels) by free Ca 2+ was concentration dependent with a half-maximal effect occurring at 32 ± 4 nM free Ca 2+ (n = 7). Addition of calmodulin further enhanced the Ca 2+ activating effect on 86Rb + uptake (K + channel activity). Ca 2+-dependent 86Rb + uptake was inhibited by tetraethylammonium ion and low pH. It is concluded that the adipocyte plasma membrane possesses K + channels that are activated by Ca 2+ and amplified by calmodulin.

The role of calmodulin on Ca 2+-dependent K + transport regulation in the human red cell

Several lipophilic calmodulin antagonists (phenotiazines, butyrophenones and diphenylbutylpiperidines) inhibited Ca 2+-induced loss of KCl from human red cells. However, the K i values for this effect did not bear good correlation with the K i values reported for well-known calmodulin-dependent systems. In addition, the inhibition was strongly dependent on the haematocrit and valinomycin-induced KCl fluxes were also affected. Added calmodulin did not have any effect on Ca 2+-dependent 86Rb uptake by inside-out vesicles derived from red cell membranes whereas stimulation of Ca 2+-dependent ATPase was apparent. Lipophilic anticalmodulins at high doses had all kinds of effects on 86Rb uptake by inside-out vesicles: increase, decrease or no change of the fraction of activated vesicles reached at submaximal Ca 2+ concentrations, with or without modification of the relative rate of 86Rb uptake. The hydrophylic compound 48/80 decreased the fraction of activated vesicles reached at submaximal Ca 2+ concentrations without affecting the relative rate of 86Rb uptake, but this effect took place only at concentrations 10-fold higher than the reported K i for calmodulin-dependent systems. These results suggest that Ca 2+-dependent K + channels of red cells are not regulated by calmodulin.

Increased membrane atpase and K + transport rates in streptococcusfaecalis induced by K + restriction during growth

Summary The membrane ATPase in Streptococcus faecalis increased 1.5 – 2.1 fold when K+ in the growth medium was lowered to levels insufficient for the maintenance of optimal growth. Two other membrane enzymes tested, p-nitrophenylphosphatase and NADH dehydrogenase, did not increase. Cells with the induced increase in ATPase also exhibited increased rates of glycolysis dependent 86Rb+ uptake and net K+ uptake. The findings support the view that the membrane ATPase in S. faecalis functions in the active transport of K+ and that the organism responds adaptively to lack of K+ during growth by producing additional ATPase.