Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.     

Single-channel analysis of a potassium inward rectifier in myocytes of newborn rat heart

Summary Unitary K⁺ currents in single cells isolated from ventricular muscle of newborn rat hearts were measured in response to different potentials and [K] _o . TheI/V curves were linear for potentials more negative than the zero-current voltage: especially in high [K] _o (150nm KCl), no clear outward currents could be detected indicating a drastic rectification in the inward direction. The channel is mainly selective to K⁺ but Na⁺ ions are also carried (P _Na/P _K=0.056). The channel conductance is proportional to the square root of [K] _o but Na⁺ ions seem to have a facilitatory effect on _K, the single-channel conductance. The channel activity, measured asP _o, i.e. the probability to find the channel in open state, decreased as the membrane was hyperpolarized. This behavior was tentatively explained by an inactivation process as the membrane becomes more negative. The rate constants of the transitions between the different states were calculated according to a C–O–C model. A control of the gating process by permeant ion K⁺ was postulated, based on the increase of one of the rate constants from the closed to the open state with [K] _o . Finally, the macroscopicI/V curves calculated fromP _o and i, the unit current, were found to be characteristic of a ion-blocked inward rectifier.     

Effects of saponin extracts from Solanum elaeagnifotium fruits on ion uptake by clover seedlings

Solanum elaeagnifolium Cav. fruits contain high concentrations of steroidal saponins. Treatment of 3-day-old clover seedlings with aqueous fruit extracts modified Ca²⁺ uptake without significantly altering K⁺ and H₂PO₄⁻ uptake. The extracts increased Ca²⁺ uptake in the concentration range of 0.2 to 20 m M Ca²⁺. Uptake curves could be represented by two phases. In the lower phase (0.2-1.0 m M Ca²⁺), this change could be related to an increase in V_max. Pretreatment of seedlings with saponin extracts significantly reduced ATP-dependent Ca²⁺ uptake and Ca²⁺-dependent ATPase activity in a fraction isolated from root homogenates by centrifugation at 1500 g for 15 min. Saponins purified from S. eleagnifolium extracts by thin-layer chromatography modified in vitro the Ca²⁺-ATPase activity of this fraction, indicating that the steroid may act directly on Ca²⁺ transport across membranes.     

Effects of monovalent cations on red cell shape and size

Human erythrocytes were incubated in isotonic solutions of different monovalent cations. The apparent size of the red cells measured on scanning electron microscopic pictures decreases in the order Li⁺>Na⁺=K⁺>Rb⁺. These differences in size are abolished after pretreatment with trypsin, which removes a large part of the charges associated with membrane glycoproteins. Shape alterations are also observed. Normal biconcave shapes are visible after Na⁺ or K⁺ incubation, whereas Li⁺ leads to flabby, flattened cells with a certain tendency to crenation, and Rb⁺ causes more pronounced biconcavity with a certain tendency to cupping. The overall effects of pretreatment with trypsin are similar to those of Li⁺. Our results provide evidence that the electrostatic repulsion of glycoproteins and other charged membrane components may play an essential role in maintaining red cell shape.     

脯氨酸对缺钾植株的保护作用

在缺钾培养条件下,高粱苗地上部单位鲜重含钾量由于地上部鲜重的增加而下降。从高梁苗钾缺乏症出现的时间、频率和程度来看,Pro培养似乎有利于缓和或减轻缺钾对植物造成的伤害。在同样含钾量情况下,以Ca(NO_3)_2为氮源的苗受到缺钾伤害要重于以Pro为氮源的苗受到的伤害,其钾缺乏症植株的百分数增加值与含钾量降低值之比为0.26,以Pro为氮源的缺钾营养液培养的苗为0.09,即下降同样量的钾前者苗的钾缺乏症的增加要大大地超过后者。Pro并不减轻在缺钾情况下叶绿素含量的下降。     

Control of K+ conductance by cholecystokinin and Ca2+ in single pancreatic acinar cells studied by the patch-clamp technique

Summary The effect of cholecystokinin (CCK) and internal Ca²⁺ on outward K⁺ current in isolated pig pancreatic acinar cells has been investigated using the patch-clamp method for whole-cell current recording under voltage-clamp conditions. CCK (2 × 10^–10 M) applied to the bath evoked a marked increase in the outward K⁺ current associated with depolarizing voltage steps, and this effect was fully reversible and acutely dependent on the presence of external Ca²⁺. When strongly buffered Ca²⁺-EGTA solutions were used inside the cells CCK failed to evoke an effect. Increasing the internal Ca²⁺ concentration ([Ca²⁺] _i ) from 5 × 10^–10 M to 10^–7 and 5 × 10^–7 M mimicked the effect of CCK. It would appear therefore that CCK controls K⁺ conductance in the acinar cells via changes in the internal free ionized Ca²⁺ concentration.     

Regulation of the basolateral potassium conductance of theNecturus proximal tubule

Summary Two methods, the measurement of the response of the basolateral membrane potential (V _bl) of proximal tubule cells ofNecturus to step changes in basolateral K⁺ concentration, and cellular cable analysis, were used to assess the changes in basolateral potassium conductance (G _K) caused by a variety of maneuvers. The effects of some of these maneuvers on intracellular K⁺ activity (a _K ⁱ ) were also evaluated using double-barreled ion-selective electrodes. Perfusion with 0mm K⁺ basolateral solution for 15 min followed by 45 min of 1mm K⁺ solution resulted in a fall in basolateral potassium (apparent) transference number (t _K),V _bl anda _K ⁱ . Results of cable analysis showed that total basolateral resistance,R _b , rose. The electrophysiological effects of additional manipulations, known to inhibit net sodium reabsorption across the proximal tubular epithelium ofNecturus, were also investigated. Ouabain caused a fall int _K accompanied by large decreases ina _K ⁱ andV _bl. Lowering luminal sodium caused a fall int _K and a small reduction inV _bl. Selective reduction of peritubular sodium, a maneuver that has been shown to block sodium transport from lumen to peritubular fluid, also resulted in a significant decrease int _K. These results suggest thatG _K varies directly with rate of transport of the sodium pump, irrespective of the mechanism of change in pump turnover.Part of this material has been presented at the 10th International Conference on Biological Membranes (Cohen & Giebisch, 1984).     

Current-voltage relations of the basolateral membrane in tight amphibian epithelia: Use of nystatin to depolarize the apical membrane

Summary Exposure of the mucosal side of toad(Bufo bufo) urinary bladder and frog(Rana ridibunda) skin to the polyene ionophore nystatin, resulted in stable preparations in which the apical resistance was negligible compared to the basolateral resistance. The preparations support passive K currents in both directions and an amiloride-insensitive Na current in the apicalserosal direction which is blocked by ouabain. The nystatintreated toad bladder was used to study the electrical properties of the basolateral membrane by means of current-voltage curves recorded transepithelially. The K current showed strong rectification at cellular potentials negative with respect to the interstitial space. The ouabain-sensitive current increased with membrane voltage at negative voltages but saturated above+20 mV.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

Tetracyclines,verapamil and nifedipine induce callose deposition at specific cell sites in Riella helicophylla

The Ca²⁺ indicator 7-chlorotetracycline has been shown to bind to a pore complex on both outer surfaces of all non-meristematic cells in the unistratose thallus of Riella (chlorotetracycline-binding surface region=CSR; Grotha, 1983, Planta 158, 473–481). Prolonged treatment of the thallus with 7-chlorotetracycline, 5-hydroxytetracycline, verapamil and desmethoxyverapamil induces the deposition of callose at the same region. The influence of various treatments on verapamil-induced CSR-callose was measured in situ by microfluorometry of aniline-blue-stained material. Callose deposition is maximal at 10^-4M verapamil or 5·10^-5M desmethoxyverapamil with 2·10^-4M Ca²⁺ or Mg²⁺ in the medium. The reaction is completely inhibited at pH 5.5 and is optimal between pH 6.5 and 7.5. The production of CSR-callose is absolutely light-dependent with callose being first visible after 30 min of light. La³⁺, ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid and amiprophosmethyl, antagonists of Ca²⁺ functions, and 2-deoxy-D-glucose suppress the verapamil induction of CSR-callose. Furthermore the ionophores A 23187, valinomycin and monensin effectively block the reaction. The deposition of CSR-callose is diminished at increasing external osmolarity and is abolished at osmotic values that stimulate plasmolysis-callose. Wounding causes the formation of wound-callose but inhibits the induction of CSR-callose in cells of the wound edge. Nifedipine increases or prolongs callose synthesis in cell plates. The Ca²⁺-channel blocker diltiazem is completely ineffective. It is suggested as a working hypothesis that verapamil-induced CSR-callose synthesis is caused by a local change in membrane permeability, possibly as a consequence of the opening of Ca²⁺ channels being involved in Golgi-vesicle mediated exocytosis (A. Kramer and H. Lehmann, 1986, Ber. Dtsch. Bot. Ges. 99, 111–121).Abbreviations APM amiprophosmethyl - APW artificial pond water - CSR chlorotetracycline-binding surface region - CTC 7-chlorotetracycline - DDG 2-deoxy-D-glucose - EGTA ethylone glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid - OTC 5-hydroxytetracycline - Pipes 1,4-piperazinediethane sulfonic acid Dedicated to Professor Luise Stange on the occasion of her 60^th birthday