1,1′-Diethyl-2,2′-cyanine (decynium22) potently inhibits the renal transport of organic cations

Summary The excretion of cationic compounds by renal proximal tubule cells involves at least two distinct transporters: the basolateral type which transports organic cations from the plasma into the proximal tubule cell, and the apical type which secretes the organic cations into the lumen of the tubule. However, potent inhibitors were known for neither type of transporter. Here we introduce a compound, decynium22, that potently, competitively, and selectively inhibits the apical type of the renal organic cation transporter.The transport of the prototypical organic cation ¹⁴C-tetraethylammonium through the apical plasma membrane of clonal proximal tubule cells (LLC-PK₁) was used as experimental system. Initial rates of ¹⁴C-tetraethylammonium transport into LLC-PK₁ cells were saturable, the K_m and V_max being 27 mol/l and 200 pmol/(mg protein · min), respectively. Decynium 22 competitively and potently inhibited ¹⁴C-tetraethylammonium transport (K_i = 5.6 nmol/l). Moreover, the effect of decynium22 on basolateral to apical directed transepithelial transport of ¹⁴C-tetraethylammonium through a confluent monolayer of LLC-PK₁ cells was determined. Decynium22 (30 nmol/l) applied to the apical medium, reduced transepithelial transport by 76% and increased intracellular accumulation of ¹⁴C-tetraethylammonium 1.5-fold. In contrast, application of 30 nmol/l decynium22 to the basolateral medium failed to affect transepithelial transport and intracellular accumulation of ¹⁴C-tetraethylammonium.Decynium22 is the most potent inhibitor of the renal transport of organic cations known so far. With decynium22 it is now possible to distinguish precisely between a decynium22-sensitive apical type and a decynium22-resistant basolateral type of renal organic cation transporter in renal proximal tubule cells.Supported by the Deutsche Forschungsgemeinschaft (SFB176) and the Universitätsbund WürzburgCorrespondence to E. Schömig at the above address     

Kinetic Analysis of Tetraethylammonium Transport in the Kidney Epithelial Cell Line,LLC-PK1

Purpose. The aims of this study were to establish a kinetic means of analyzing the membrane transport of organic cations in renal epithelial cells, and to simultaneously evaluate drug interactions in apical and basolateral membranes. Methods. Tetraethylammonium (TEA) transport was measured using LLC-PK₁, cell monolayers grown on microporous membrane filters. After incubating the cells with unlabeled TEA or other drugs, apical or basolateral medium was changed to that containing labeled TEA, and transcellular transport and cellular accumulation were measured. Clearance from apical medium to cells (CL₁₂), cells to apical medium (CL₂₁), cells to basolateral medium (CL₂₃) and basolateral medium to cells (CL₃₂) were calculated based on a three compartment model. Results. TEA was accumulated progressively in the monolayers from the basolateral side and was transported unidirectionally to the apical side. CL₃₂ was greater than CL₁₂ and CL₂₃ was greater than CL₂₁. Therefore, the rate limiting step of TEA transport from the basolateral to the apical medium was the cell-to-apical step. Co-incubation of TEA with procainamide decreased the transport parameters of TEA, CL₁₂, CL₂₁ and CL₃₂, whereas that with levofloxacin decreased only CL₁₂ and CL₂₁, not affecting the parameters in basolateral membranes. Conclusions. Using a simple model, we analyzed the transport of organic cation in kidney epithelial cell line, LLC-PK₁ This method can be useful for the analysis of cation transport and drug interactions in the apical and basolateral membranes of renal tubules.     

Cadmium perturbs calcium homeostasis in rat osteosarcoma (ros 17/2.8) cells; a possible role for protein kinase c

The mechanism of the toxic effects of Cd²⁺ on bone cell function is not completely understood at this time. This study was designed to characterize the effect of Cd²⁺ on Ca²⁺ metabolism in ROS 17/2.8 cells. Cells were labeled with ⁴⁵Ca (1.87 mM Ca) for 20 h in the presence of 0.01, 0.1, or 1.0 μM Cd²⁺ and kinetic parameters were determined from ⁴⁵a efflux curves. Three kinetic compartments described the intracellular metabolism of ⁴⁵Ca. Cd²⁺ (0.01 μM) caused an approximate 9 × increase in Ca²⁺ flux across the plasma membrane and a decrease in the most rapidly exchanging intracellular Ca²¹ compartment (S₁). However, there was no change in total cell Ca²⁺, indicating an increased cycling of Ca²⁺ across the plasma membrane. Flux between S₁, and the intermediate Ca²⁺ compartment (83) was also increased and S₂ increased significantly. All Cd²⁺ induced changes in Ca²⁺ homeostasis were obliterated by concurrent treatment with 0.1 μM calphostin C (CC), a potent protein kinase C (PKC) inhibitor. This data suggests that Cd²⁺ perturbs Ca²⁺ metabolism via a PKC dependent process.     

Inhibition of the liberation of arachidonic acid by cadmium ions in rabbit alveolar macrophages

The effects of CdCl₂ on the liberation of arachidonic acid (20∶4) from membrane phospholipids of A23187-stimulated rabbit alveolar macrophages and on the activity of phospholipase A₂ (PLA₂) in a cytosolic fraction were studied. Alveolar macrophages were prelabeled with [³H]arachidonic acid (20∶4) and then treated with A23187. This treatment resulted in a remarkable increase in the liberation of [³H]20∶4 from their phospholipids. Exposure of cells to Cd²⁺ inhibited the liberation of [³H]20∶4 in a dose-dependent manner. Liberation of [³H]20∶4 from cell lipids was calcium dependent and the inhibitory effect of Cd²⁺ competed with the stimulatory effect of Ca²⁺. When Ca²⁺ was removed from the incubation medium, Cd²⁺ did not influence the liberation of [³H]20∶4. Entry of⁴⁵Ca²⁺ into cells was enhanced by treatment of A23187. However, Cd²⁺ did not influence the cellular uptake of⁴⁵Ca²⁺. Treatment with A23187 markedly enhanced entry of¹⁰⁹Cd²⁺ into cells. The effect of Cd²⁺ on the activity of phospholipase A₂ was determined with 1-palmitoyl-2-[¹⁴C]arachidonoyl-sn-glycero-3-phosphocholine as substrate. Calcium-dependent activation of PLA₂ was observed and Cd²⁺ inhibited activation in a dose-dependent manner. These results suggest that exposure of alveolar macrophages to Cd²⁺ causes a reduction in the rate of liberation of 20∶4 from cell lipids, as a possible result of the inhibition of PLA₂ activity by Cd²⁺.     

Cadmium‐induced Cytosolic Ca2+ Elevation and Subsequent Apoptosis in Renal Tubular Cells

Abstract: Cadmium (Cd²⁺) is an industrial and environmental metal. The effect of Cd²⁺ on intracellular free‐Ca²⁺ levels ([Ca²⁺]_i) and viability in Madin Darby canine kidney cells was explored. Cd²⁺increased [Ca²⁺]_i in a concentration‐dependent manner with an EC₅₀ of 85 µM. Cd²⁺‐induced Mn²⁺ entry demonstrated Ca²⁺ influx. Removal of extracellular Ca²⁺ decreased the [Ca²⁺]_i signal by 60%. The [Ca²⁺]_i signal was inhibited by La³⁺ but not by L‐type Ca²⁺ channel blockers. In Ca²⁺‐free medium, Cd²⁺‐induced [Ca²⁺]_i signal was abolished by pre‐treatment with 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺pump inhibitor) and 2 µM carbonylcyanide m‐chlorophenylhydrazone (CCCP; a mitochondrial uncoupler). Cd²⁺‐induced Ca²⁺ release was not altered by inhibition of phospholipase C. At concentrations between 10 and 100 µM, Cd²⁺killed cells in a concentration‐dependent manner. The cytotoxic effect of 100 µM Cd²⁺was reversed by pre‐chelating cytosolic Ca²⁺with BAPTA. Cd²⁺‐induced apoptosis was demonstrated by propidium iodide. Collectively, this study shows that Cd²⁺ induced a [Ca²⁺]_i increase in Madin Darby canine kidney cells via evoking Ca²⁺ entry through non‐selective Ca²⁺ channels, and releasing stored Ca²⁺ from endoplasmic reticulum and mitochondria in a phospholipase C‐independent manner.     

Stimulation of the frequency of MEPPs at the frog neuromuscular junction by extracellular EGTA

Summary Exposure of the frog neuromuscular junction at 17° C or 23° C to salines with low [Ca²⁺], buffered with EGTA, causes mepp frequency to fall after 4–6 min to about 20% of the control rate. Results obtained in the presence of verapamil suggest that this fall is a consequence of a lower Ca²⁺-influx coupled with the action of the extracellular EGTA in promoting Ca²⁺-efflux from the terminals. These findings confirm the suggestion that [Ca²⁺]_i has a major role in determining mepp frequency.At 13° C, the fall in mepp frequency after addition of EGTA is preceded by a transient (1–2 min) rise in mepp rate which is not present at 17° C or in the presence of verapamil. This transient acceleration in spontaneous release is believed to be because EGTA promotes the emptying of a Ca²⁺-reservoir on or beneath the inner face of the membrane, thus causing a rapid Ca²⁺-efflux via the Ca²⁺-sensitive sites that trigger exocytosis of transmitter.The significance of the sensitivity of the response to temperature is discussed. The suppressive effect of higher temperatures can be reversed to some extent by hyperosmotic salines, an effect that may reflect the action of hypertonicity on the plasmalemma. It is concluded that the characteristics of the release system may change markedly at about 16° C.Ca²⁺-EGTA buffers are widely used; it is suggested that extracellular EGTA can also modify [Ca²⁺]_i in cellular systems.     

Characterization of the transport of uracil across Caco-2 and LLC-PK1 cell monolayers

Purpose. The purpose of this study was to characterize the transport of uracil, a pyrimidine nucleobase, in Caco-2 and LLC-PK₁ cells. Methods. Caco-2 and LLC-PK₁ cells were grown to confluency on a permeable polycarbonate membrane insert to permit transport and uptake experiments after the loading of uracil on either the apical or basolateral side. Results. The vectorial transport of uracil in both directions was saturable with comparable K_m and V_max in Caco-2 cell monolayers, probably because of a Na⁺-independent transport system located on the basolateral membrane. In LLC-PK₁ cell monolayers, two distinct transport systems, namely a Na⁺-dependent and a Na⁺-independent, were functional in the apical to basolateral (A-B) transport of uracil. The first system was saturable with a K_m value of 3.67 ± 0.40 M, a V_max of 11.31 ± 0.91 pmol/cm²/min, and a Na⁺:uracil coupling stoichiometry of 1.28 ± 0.20. The second system was Na⁺ independent and satuable with a low affinity (K_m, 50.37 ± 9.61 M) and V_max (16.01 ± 4.48 pmol/cm²/min). The two transport systems appeared to be located on the apical membrane. Conclusion. The mechanism of uracil transport differs depending on cell lines; a Na⁺-independent system on the basolateral membrane in Caco-2 cells and both Na⁺-dependent and Na⁺-independent systems on the apical membrane in LLC-PK₁ cells seem to be responsible for the difference.     

Calmodulin-mediated cadmium inhibition of phosphodiesterase activity,in vitro

Ion-stripped bovine brain calmodulin (CaM) binds 4 moles Cd²⁺ as well as 4 moles Ca²⁺ per mole protein, with similar affinity; in the presence of 1 mM Mg²⁺ the molar binding ratio of CaM for Ca²⁺ decreased to 3, the apparent K_0.5 for Ca²⁺ nearly doubled, but the binding characteristics of CaM for Cd²⁺ were not changed. Saturating concentrations Ca²⁺ did not affect the molar binding ratio of CaM for Cd²⁺, but increased the apparent K_0.5 for Cd²⁺; vice versa, saturating concentrations Cd²⁺ decreased the molar binding ratio for Ca²⁺ to 2 without affecting the apparent K_0.5 for Ca²⁺.CaM-independent phosphodiesterase (PDE) activity was inhibited at [Cd²⁺]>10^–5 M. Cd²⁺-CaM as well as Ca²⁺-CaM activated PDE. However, the Cd²⁺-CaM complex is less effective than the Ca²⁺-CaM complex in stimulating CaM-dependent enzyme activities. Cd²⁺ inhibits Ca²⁺- and CaM-dependent PDE in a competitive way. Introduction of Cd²⁺ in a medium containing Ca²⁺ and CaM may, therefore, result in a reduction of CaM-dependent enzyme stimulation.By its interference with Ca²⁺- and CaM- dependent PDE activity, Cd²⁺ could upset the catabolic pathway of cellular cyclic nucleotide metabolism.     

Identification of a B2-bradykinin receptor linked to phospholipase C and inhibition of dopamine stimulated cyclic AMP accumulation in the human astrocytoma cell line D384

Summary We have examined the activation of a phospholipase C signal transduction pathway by a B₂-bradykinin receptor in the human astrocytoma cell line D384 and how this influences D1-dopamine receptor stimulated cyclic AMP accumulation. Addition of bradykinin to D384 cells resulted in a concentration-dependent (10^–11 – 10^–6 M) increase in the accumulation of [³H]inositol phosphates and a similar concentration-dependent transient increase in specific [³H]-phorbol-12,13-dibutyrate binding which is indicative of translocation of protein kinase C from the cytosol to the membrane. Changes in intracellular Ca²⁺ of single cells, measured using the fluorescent indicator dye fura-2, indicated that bradykinin produced a rapid, but transient, increase in intracellular calcium. The Ca²⁺ response was largely independent of extracellular Ca²⁺ supporting the idea that receptor activation leads to mobilization of Ca²⁺ from intracellular stores. However, extracellular Ca²⁺ was required for a response to a rechallenge with bradykinin. The bradykinin B₂-receptor agonist kallidin increased cytosolic Ca ²⁺ in a similar manner to bradykinin. The Ca²⁺ response to bradykinin could be partially reduced in the presence of the B₂-receptor antagonist [d-Arg¹⁰-Hyp,d-Phe⁷,-(2-Thienyl)-Ala⁵,⁸]-bradykinin, whereas the B₁-receptor agonists (Des-Arg⁹]-bradykinin and [Des-Arg¹⁰]-kallidin were ineffective. Bradykinin was also found to attenuate dopamine stimulated cyclic AMP accumulation in D384 cells, at similar concentrations previously observed to stimulate the phospholipase C signal transduction pathway, in the presence of the phosphodiesterase inhibitor, rolipram. In contrast, no attenuation was observed in the presence of the phosphodiesterase inhibitor 1-isobutyl 3-methylxanthine, although the level of dopamine stimulated cyclic AMP observed was lower than in the presence of rolipram. Furthermore, the effect of bradykinin could be mimicked by a calcium ionophore, but not a phorbol ester. These data suggest that D384 cells express a B₂ bradykinin receptor coupled to polyphosphoinositide-specific phospholipase C. Activation of this receptor results in elevated levels of cytosolic Ca²⁺ and to a reduction of D1-dopamine receptor stimulated cyclic AMP accumulation. Correspondence to A. J. Balmforth at the above address     

Transepithelial Transport of Aliphatic Carboxylic Acids Studied in Madin Darby Canine Kidney (MDCK) Cell Monolayers

Transport of ¹⁴C-labeled acetic, propionic (PA), butyric, valeric, heptanoic (HA), and octanoic (OA) acids across the Madin Darby canine kidney (MDCK) epithelial cell monolayer grown on a porous polycarbonate membrane was studied in Hanks' balanced salt solution (HBSS) at 37°C in both apical-to-basolateral and basolateral-to-apical directions. At micromolar concentrations of solutes, metabolic decomposition was significant as evidenced by [¹⁴C]CO₂ production during the OA transport. The apparent permeability (Pe) indicates that as lipophilicity increases, diffusion across the unstirred boundary layer becomes rate limiting. In support of this notion, transport of OA and HA was enhanced by agitation, showed an activation energy of 3.7 kcal/mol for OA, and resulted in identical Pe values for both transport directions. Analysis of Pe changes with varying alkyl chain length resulted in a G of –0.68 ± 0.09 kcal/mol for –CH₂-group transfer from an aqueous phase to the MDCK cells. When the intercellular tight junctions were opened by the divalent chelator EGTA in Ca²⁺/Mg²⁺ -free HBSS, transport of the fluid-phase marker Lucifer yellow greatly increased because of paracellular leakage. PA transport also showed a significant increase, but OA transport was independent of EGTA. Although albumin also undergoes paracellular transport in the presence of EGTA and OA binds strongly to albumin, OA transport in EGTA solution was unchanged by albumin. These observations indicate that transmembrane transport is the major mechanism for lipophilic substances. The present study, together with earlier work on the transport of polar substances, shows that the MDCK cell monolayer is an excellent model of the transepithelial transport barrier.     

Side-specific effects by cadmium exposure: Apical and basolateral treatment in a coculture model of the blood-air barrier

Cadmium (Cd²⁺) is a widespread environmental pollutant, which is associated with a wide variety of cytotoxic and metabolic effects. Recent studies showed that intoxication with the heavy metal most importantly targets the integrity of the epithelial barrier.In our study, the lung epithelial cell line, NCI H441, was cultured with the endothelial cell line, ISO-HAS-1, as a bilayer on a 24-well HTS-Transwell® filter plate. This coculture model was exposed to various concentrations of CdCl₂.The transepithelial electrical resistance decreased on the apical side only after treatment with high Cd²⁺ concentrations after 48 h. By contrast, a breakdown of TER to less than 5% of baseline could be observed much earlier (after 24 h) when Cd²⁺ was administered from the basal side. Observations of cell layer fragmentation and widening of intercellular spaces confirmed the barrier breakdown only for the basolaterally treated samples. Furthermore, the cytotoxicity and release of proinflammatory markers was enhanced if samples were exposed to Cd²⁺ from the basal side compared to treatment from the apical side. Moreover, we could demonstrate that a high concentration of Ca²⁺ could prevent the barrier-disrupting effect of Cd²⁺.In conclusion, the exposure of Cd²⁺ to cocultures of lung cells caused a decrease in TER, major morphological changes, a reduction of cell viability and an increase of cytokine release, but the effects markedly differed between the two modes of exposure. Therefore, our results suggest that intact epithelial TJs may play a major role in protecting the air-blood barrier from inhaled Cd²⁺.     

Extracellular calcium-sensing receptor mediates human bronchial epithelial wound repair

The airway epithelium routinely undergoes damage that requires repair to restore epithelial barrier integrity. Cell migration followed by proliferation are necessary steps to achieve epithelial repair. Calcium-sensing receptor (CaSR) is implicated in cell migration and proliferation processes. Thus we hypothesized that CaSR mediates lung epithelial wound repair. We detected CaSR expression in human lung and in well-differentiated human bronchial epithelial cells (HBEC). To test the CaSR functionality, HBEC loaded with fura-2 were stimulated with extracellular Ca²⁺ ([Ca²⁺]_out) which resulted in a concentration-dependent intracellular Ca²⁺ ([Ca²⁺]_i) increase (potency ∼ 5.6 mM [Ca²⁺]_out). Furthermore, increasing [Ca²⁺]_out induced phosphorylation of the extracellular signal-regulated kinase (ERK1/2) which was blocked by siRNA-CaSR and the specific inhibitor of CaSR, NPS2390.Epithelial repair after mechanical injury of differentiated HBEC was a process dependent of [Ca²⁺]_out since it accelerated wound repair and HBEC proliferation being highest at 5 mM [Ca²⁺]_out. Furthermore, U73122 (an inhibitor of phospholipase C (PLC)) and PD 98059 (an inhibitor of ERK1/2) as well as siRNA-CaSR and NPS2390 partially inhibited wound repair and HBEC proliferation. On the other hand, mechanical injury produced an [Ca²⁺]_i wave propagation that was partially inhibited by siRNA-CaSR, NPS2390 and the extracellular Ca²⁺ chelator EGTA, which suggest a link of CaSR between cell-cell communication and wound repair in differentiated HBEC. Our data, for the first time, shows that CaSR plays an important role in airway epithelial repair, which may help to develop novel regenerative therapeutics allowing the rapid repair of lung damaged epithelium.     

Effects of Cd2+, Pb2+ and CH3Hg+ on high voltage-activated calcium currents in pheochromocytoma (PC12) cells: potency,reversibility, interactions with extracellular Ca2+ and mechanisms of block

Effects of the neurotoxic heavy metals Cd²⁺, Pb²⁺ and CH₃Hg⁺ on current carried by Ca²⁺ ions (I_Ca) through high-voltage activated Ca²⁺ channels in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells were examined to characterize possible differences in the mechanism of action of these metals on Ca²⁺ channel function. Specifically, the potency and reversibility of effect on I_Ca by each metal was examined, as well as the relationship between extracellular [Ca²⁺] and potency of block of I_Ca by Cd²⁺ and Pb²⁺. In addition, the effect of each of these metals on Ca²⁺ channels when applied to the intracellular side of the membrane was also examined. When extracellular solution contained 20, 10 or 5 mM Ca²⁺, the estimated IC₅₀ values (total metal concentration) for block of I_Ca were 15, 10, and 6.5 μM for Cd²⁺ and 7.5, 2.0 and 1.1 μM for Pb²⁺, respectively. CH₃Hg⁺ (1–10 μM) blocked I_Ca (20 mM Ca²⁺) in a time- and concentration-dependent manner. When cells were washed with metal-free solutions, block of I_Ca by Cd²⁺ was reversed rapidly, whereas block by Pb²⁺ was reversed only partially, and block of I_Ca by CH₃Hg⁺ was not reversed. When Pb²⁺ and CH₃Hg⁺ treated cells were washed in metal-free solutions containing 50 μM d-penicillamine (DPEN), block of I_Ca by 10 μM Pb²⁺ was rapidly and completely reversed, whereas, block of I_Ca by 5 μM CH₃Hg⁺ was not reversed. Higher concentrations (500 μM) of 2,3-dimercapto-1-propane sulfonic acid (DMPS) did reverse partially the block of I_Ca by 5 and 10 μM CH₃Hg⁺. When Cd²⁺, Pb²⁺ or CH₃Hg⁺ was present in the intracellular solution, Ca²⁺ channel currents were significantly reduced. These results characterize effects of Cd²⁺ on Ca²⁺ channels and demonstrate that Cd²⁺, Pb²⁺ and CH₃Hg⁺ differ in their actions on Ca²⁺ channels.     

Studies on cadmium-induced myotonia in the mouse diaphragm

Summary The purpose of this investigation was to study the possible mechanism of the potentiation of the contractile response and myotonia caused by Cd²⁺ in the mouse diaphragm. Cd²⁺ increased both amplitude and duration of the contractile response to direct stimulation in either 0.25 mM Ca²⁺ Krebs or 2.5 mM Ca²⁺ Krebs containing the K⁺-channel blockers, 4-aminopyridine, uranyl nitrate or tetraethylammonium ion. High K⁺ and tetrodotoxin inhibited these effects of Cd²⁺. Electrophysiological studies revealed that only one or two action potentials were triggered by passing a short depolarizing current across the muscle fibre membrane in 0.25 mM Ca²⁺ Krebs, but in the presence of Cd²⁺, a train of action potentials (153 ± 21 Hz) which lasted for 0.7 ± 0.2 s was induced. Furthermore, Cd²⁺ triggered a train of action potentials evoked by a single extracellular direct stimulation on the muscle fibre in 2.5 mM Ca²⁺ Krebs solution containing either 4-aminopyridine or uranyl nitrate. The membrane depolarized during the repetitive firing and then repolarized immediately after the cessation of repetitive firing. Cd²⁺ (0.1 mM) increased the input resistance of the muscle fibre by 53 ± 7% and this effect was inhibited in low [CI^–]_o. These findings suggest that the contractile potentiation and myotonia induced by Cd²⁺ in the mouse diaphragm are mediated by lowering the Cl^– conductance of the membrane. Send offprint requests to S. Y. Lin-Shiau at the above address     

Biphasic increase in cytosolic free calcium induced by bradykinin and histamine in cultured tracheal smooth muscle cells: is the sustained phase artifactual?

The effects of bradykinin (BK) and histamine on intracellular Ca²⁺ ([Ca²⁺]i) were studied in fura-2-loaded guinea-pig tracheal smooth muscle cells in culture. BK, at 10 nM, and histamine, at 100 M, induced a rise in [Ca²⁺]i which was inhibited by the B2 antagonist Hoe 140 and by the H1 antagonist triprolidine, respectively. This rise in [Ca²⁺]_i is biphasic, consisting of a rapid transient phase followed by a sustained phase. The transient phase, induced by either BK or histamine, was strongly inhibited by thapsigargin, a microsomal Ca²⁺-ATPase inhibitor, usually used to deplete certain intracellular Ca²⁺-stores. Ni²⁺ (4 mM) did not affect the transient phase but abolished the sustained phase when cells were stimulated by BK, further supporting the fact that the transient phase was only dependent on intracellular Ca²⁺ pools. The sustained phase was partially (for BK) and completely (for histamine) inhibited by 30 M Mn²⁺. This effect could be completely reversed by the addition of DTPA, a cell-impermeant chelator of Mn²⁺, indicating that the Mn²⁺ exerted its effect extracellularly. The presence of 1 mM probenecid (an inhibitor of a membrane organic anion transporter that extrudes fura-2) drastically inhibited the sustained phase by more than 77010 for BK and 88010 for histamine. Our results suggest that the effects of BK and histamine on airway smooth muscle cells are mediated via bradykinin B2 receptors and histamine H1 receptors, respectively whose activation allows the rapid transient rise in [Ca²⁺]i from thapsigargin-sensitive intracellular Ca²⁺ pools. The sustained phase is proposed to be drastically influenced by an acceleration of fura-2 extrusion during the increase of [Ca²⁺]_i via a probenecid-sensitive mechanism.Abbreviations BK bradykinin - [Ca²⁺]_i cytosolic free calcium - CICR Ca²⁺-induced Ca²⁺ release - DMEM Dulbecco's modified Eagle's medium - DTPA diethylenetriaminepentaacetic acid - EGTA ethylene glycol-bis(b-aminoethylether) N,N,N,N-tetraacetic acid - FCS fetal calf serum - HEPES 2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethansulfonic acid - IP inositol phosphates - TG thapsigargin - TSMC tracheal smooth muscle cells Correspondence to: C. Bronner     

Functional characteristics of a renal H+/lipophilic cation antiport system in porcine LLC-PK1 cells and rats

We have recently found an H⁺/quinidine (a lipophilic cation, QND) antiport system in Madin-Darby canine kidney (MDCK) cells. The primary aim of the present study was to evaluate whether the H⁺/lipophilic cation antiport system is expressed in porcine LLC-PK₁ cells. That is, we investigated uptake and/or efflux of QND and another cation, bisoprolol, in LLC-PK₁ cells. In addition, we studied the renal clearance of bisoprolol in rats. Uptake of QND into LLC-PK₁ cells was decreased by acidification of the extracellular pH or alkalization of the intracellular pH. Cellular uptake of QND from the apical side was much greater than from the basolateral side. In addition, apical efflux of QND from LLC-PK₁ cells was increased by acidification of the extracellular pH. Furthermore, lipophilic cationic drugs significantly reduced uptake of bisoprolol in LLC-PK₁ cells. Renal clearance of bisoprolol in rats was approximately 7-fold higher than that of creatinine, and was markedly decreased by alkalization of the urine pH. The present study suggests that the H⁺/lipophilic cation antiport system is expressed in the apical membrane of LLC-PK₁ cells. Moreover, the H⁺/lipophilic cation antiport system may be responsible for renal tubular secretion of bisoprolol in rats.     

Vasopressin-induced activation of human blood platelets: prominent role of Mg2+

Summary Arginine-vasopressin (AVP) caused a marked shape change reaction and rise in [Ca²⁺]_i in human blood platelets only when the extracellular buffer contained Mg²⁺ or Ca²⁺. At physiological concentrations of the cations the potency of AVP was higher in the presence of Mg²⁺ than of Ca²⁺. The amplitude of the shape change reaction was also greater with Mg²⁺ than with Ca²⁺, although the [Ca²⁺]_i-rise was slightly more marked with extracellular Ca²⁺. The concentration of Mg²⁺ at which AVP showed half of its maximal effects was below the physiological plasma level of the cation, whereas the corresponding value for Ca²⁺ was higher. Addition of Ca²⁺ to the Mg²⁺ containing medium did not further enhance the action of AVP on platelet shape. In platelet-rich plasma the potency and efficacy of AVP in causing a shape change were similar in the presence and absence of EGTA, whereas with EDTA in the medium AVP had no effect. In conclusion, Mg²⁺ has an essential physiological role in AVP-induced platelet activation, which is brought about partly by release of intracellular calcium and partly by some other intracellular mechanism.     

Endothelium-derived bradykinin is responsible for the increase in calcium produced by angiotensin-converting enzyme inhibitors in human endothelial cells

Summary The effects of angiotensin-converting enzyme (ACE) inhibitors on intracellular calcium concentration ([Ca²⁺]_i) were examined under resting conditions and after stimulation with bradykinin in cultured human umbilical vein endothelial cells. The ACE inhibitors ramiprilat and enalaprilat (0.3 M) enhanced the increase in [Ca²⁺]_i elicited by bradykinin (3 nM) and also caused an increase in resting [Ca²⁺]_i when given alone. This increase in resting [Ca²⁺]_i was long-lasting and accompanied by an increased formation of nitric oxide, as assessed by a N^G-nitro-l-arginine-sensitive cyclic GMP accumulation in the cells. Both increases in resting [Ca²⁺]_i and nitric oxide production by ACE inhibitors were inhibited by preincubation of the cells with the B₂-receptor antagonist Hoe 140. These data indicate that ACE inhibitors are able to unmask a release of bradykinin from cultured human endothelial cells. This endothelium-derived bradykinin can exert an autocrine function by stimulating endothelial B₂-receptors with a subsequent increase in [Ca²⁺]_i and nitric oxide formation. Send offprint requests to R. Busse at the above address     

Use of ion channel blockers in the exploration of possible mechanisms involved in the myopathy of diabetic mice

Summary Changes in the muscle contractions of the phrenic nerve-diaphragm preparation from the diabetic mouse were investigated by means of K⁺- and Cl^–-channel blockers and the Ca²⁺-mobilizing agent, selenite. The K⁺-channel blockers (UO₂ ²⁺ and 4-aminopyridine) cooperated synergistically with the Cl^–-channel blockers (Cd²⁺ and 9-anthracenecarboxylic acid) in increasing normal muscle contraction as described previously, but failed to induce this effect in the diaphragm of the diabetic mouse. Treatment with a Cl^–-channel blocker alone in 0.25 mmol/1 Ca²⁺ Krebs solution induced a myotonic activity accompanied by stimulus-bound repetitive action potential firings. This effect was also diminished in the diaphragm from diabetic mice. The membrane potential of the muscle cells in the diaphragm of the diabetic mouse was slightly but significantly decreased. The membrane input resistance was also increased and was refractory to being further increased by either a Cl^–-channel blocker or a low Cl^–-medium. Furthermore, the membrane chloride conductance was found to be decreased, but the membrane K+ conductance remained unchanged in the muscle from diabetic mice. These changes of membrane properties in the muscles from diabetic mice were shown to be similar to those induced by either Cl^–-channel blockers or a low Cl^–-medium. In addition, the combined treatment of the diaphragm from diabetic mice with Cd²⁺ Plus UO₂ ²⁺ in 0.25 mmol/l Ca²⁺ Krebs solution and then stepwise replenishment of Ca²⁺ led to a greater restoration of muscle contractions at a lower cumulative Ca²⁺ concentration than that was found with the normal diaphragm. The sustained muscle contracture of the mouse diaphragm induced by U0₂ ²⁺ plus selenite was partially inhibited in the diaphragm from diabetic mice, indicating that the Ca²⁺ mobilizing mechanism of the diaphragm of the diabetic mouse was also altered. All of these observations obtained with the diaphragm of the diabetic mouse can be attributed to the diabetic state, because most of them could be normalized by insulin administration in vivo. Therefore, it is concluded that diabetes-induced changes of sarcolemmal ion channels and ion transporters may cause inhibition of muscle contraction and eventually lead to diabetic myopathy. Correspondence to: S. Y. Lin-Shiau at the above address     

Calcium mobilization induced by endothelins and sarafotoxin in cultured canine tracheal smooth muscle cells

Endothelins (ETs)- and sarafotoxin (S6b)-induced rises in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were monitored in cultured canine tracheal smooth muscle cells by using a fluorescent Ca²⁺ indicator fura-2. ET-1, ET-2, ET-3 and S6b elicited an initial transient peak and followed by a sustained elevation of [Ca²⁺]_i, with half-maximal effect (EC₅₀) of 18, 20, 38 and 21 nM, respectively. BQ-123, an ET_A receptor antagonist, had a high affinity to block the rise in [Ca2⁺]_i response to ET-1, ET-2, and S6b, as well as a low affinity for ET-3. Removal of external Ca²⁺ by addition of EGTA during the sustained phase, caused a rapid decline in [Ca²⁺]_i to the resting level. In the absence of external Ca²⁺, only an initial transient peak of [Ca²⁺]_i was seen, the sustained elevation of [Ca²⁺]. could then be evoked by addition of 1.8 mM Ca²⁺. Ca²⁺ influx was required for the changes of [Ca²⁺]_i, since the Ca²⁺-channel blockers, diltiazem, verapamil, and Ni²⁺, decreased both the initial and sustained elevation of [Ca²⁺]_i response to these peptides. ETs exhibited homologous desensitization of the Ca²⁺ response, but partial heterologous desensitization of the Ca²⁺ response mediated by carbachol to different extents. In contrast, ETs did not desensitize the Ca²⁺ response induced by ATP or vice versa. These data demonstrate that the initial detectable increase in [Ca2⁺]_i stimulated by these peptides is due to the activation of ET_A receptors and subsequently the release of Ca²⁺ from internal stores, whereas the contribution of external Ca²⁺ follows and partially involves a diltiazem- and verapamil-sensitive process. There is a cross-regulation among ETs and other receptor-coupling signal transduction pathways through PI hydrolysis in canine tracheal smooth muscle cells. Correspondence to: C. Mao Yang at the above address