Carlos Ojeda

The steady state TTX-sensitive ("window") sodium current in cardiac Purkinje fibres

Voltage clamp experiments on isolated sheep Purkinje fibres showed an increase of the steady state outward membrane current, over the potential range −65 mV to −15 mV, in the presence of tetrodotoxin (TTX, 3 · 10−5 M). This “window” current is considered to be the steady state component of the fast sodium current (INa), resulting from the crossover of the activation and inactivation curves which govern the opening of the sodium channel.TTX had no significant effect on the reversal potential, activation curve, kinetics or instantaneous I–V relationship of the pacemaker currentIK2.The window found in these experiments extends to potentials well into the range of the action potential plateau. Consequently small changes of the steady stateINa might have large effects on the action potential duration. The effects of TTX and local anaesthetics are discussed in this context.

Osteoclast spreading kinetics are correlated with an oscillatory activation of a calcium-dependent potassium current

Cell movement and spreading involve calcium-dependent processes and ionic channel activation. During bone resorption, osteoclasts alternate between spread, motile and resorptive phases. We investigated whether the electrical membrane properties of osteoclasts were linked to their membrane morphological changes. Rabbit osteoclasts were recorded by time-lapse videomicroscopy performed simultaneously with patch-clamp whole cell and single channel recordings. Original image analysis methods were developed and used to demonstrate for the first time an oscillatory activation of a spontaneous membrane current in osteoclasts, which is directly correlated to the membrane movement rate. This current was identified as a calcium-dependent potassium current (IKCa) that is sensitive to both charybdotoxin and apamin and was generated by a channel with unitary conductance of approximately 25±2 pS. Blockade of this current also decreased osteoclast spreading and inhibited bone resorption in vitro, demonstrating a physiological role for this current in osteoclast activity. These results establish for the first time a temporal correlation between lamellipodia formation kinetics and spontaneous peaks of IKCa, which are both involved in the control of osteoclast spreading and bone resorption.

The effect of calcium channel blockers on blood platelet function, especially calcium uptake

The effects of organic and inorganic calcium antagonists on washed platelets from rat and human have been studied. Platelet aggregation was assessed by turbidimetry. Endogenous serotonin release was measured on the same sample by means of electrochemically treated carbon fiber electrodes. The organic calcium antagonist, nitrendipine, and the inorganic calcium channel blockers (Co2+, Mn2+, Cd2+, La3+) drastically inhibited rat and human platelet aggregation induced by thrombin, ADP or adrenaline in the presence of 0.32 mM Ca2+. In our conditions, the thrombin-induced release of endogenous serotonin was found to be external Ca2+-dependent and completely inhibited by 20 microM nitrendipine or 1 mM Cd2+. In addition, Ba2+ or Sr2+ ions can be substituted for Ca2+ to bring about platelet aggregation as well as endogenous serotonin secretion. In Ba2+ or Sr2+-containing media, rat platelet aggregation and/or serotonin secretion can be inhibited by either nitrendipine or Cd2+. Finally, we have also studied the thrombin- and external Ca2+-dependence of radiolabeled calcium uptake by rat platelets. We found that the thrombin-induced 45Ca uptake was inhibited by either 18 microM nitrendipine or 1 mM Cd2+. These results provide strong evidence for the existence of an influx of divalent cations (Ca2+, Sr2+, Ba2+) triggering platelet function. They also suggest, although they do not prove, that the translocation of these cations occurs through an agonist-operated channel as proposed by Hallam and Rink (FEBS Lett. 186 (1986) 175-179).

Action of salicylate ions on the electrical properties of sheep cardiac Purkinje fibres.

1. In sheep Purkinje fibre preparations, salicylate ions produce reversible changes in resting potential and in action potential duration. In most preparations these effects resemble those produced by ‐ow extracellular K concentration: the resting potential first increases and then decreases, the action potential is prolonged and eventually, low potential oscillations occur in the plateau range. In a few preparations, action potential shortening occurs. 2. The threshold current for initiating action potentials by an intracellular electrode is reversibly increased by salicylate. 3. The activation curve, soo(Em), for the pace‐maker K current, iK2, shifted in a hyperpolarizing direction. The magnitude of the shift is about ‐5 mV in 5 mM‐salicylate and ‐30 mV in 50 mM‐salicylate. 4. The apparent reversal potential for iK2 is shifted in a negative direction. The magnitude of this shift at a given salicylate concent;ation varies with the K concentration. In an extracellular K concentration of 2.7 mM an average shift of ‐18 mV occurs in 10 mM‐salicylate; in 8 mM, the average shift is only ‐1 mV. 5. It is proposed that most of these effects may be produced by an increase in surface negative potential produced by the binding of salicylate to the cell membrane. This would produce the hyperpolarizing shift of activation curves for ionic current and, by increasing surface K activity, may lead to stimulation of the Na‐K pump to produce an increase in the K gradient across the cell membrane.

Kinetic analysis of the delayed outward currents in frog atrium. Existence of two types of preparation

1. The outward currents underlying delayed rectification have been studied in frog auricular trabeculae with the double sucrose gap method.

Block by Cs of K currentiK1 and of carbachol induced K currentiCch in frog atrium

Abstract1. In frog atrium, Cs ions block both the inward rectifieriK1 and the carbachol induced K currentiCch. 2. BothiK1 andiCch display a high affinity for Cs with a K0.5 of 4×10−5 M forik1 and of 8×10−5 M foriCch atV=−50 mV. 3. Block of bothiK1 andiCch is strongly voltage dependent. When fitted by the block model of Woodhull (1973), δ is >1 for the two currents. 4. From these similarities, action of Cch on frog atrium K permeability could be interpreted as a modification ofiK1.

Functional Expression of the L-Type Calcium Channel in Mice Skeletal Muscle during Prenatal Myogenesis

The densities of skeletal muscle intramembrane charge movement and macroscopic L-type Ca(2+) current have been shown to increase during prenatal development. In the present work, the electrophysiological characteristics of L-type Ca(2+) channels were analyzed over the embryonic period E14 to E19 using the whole-cell and cell-attached procedures. At the macroscopic level, the whole-cell L-type Ca(2+) conductance increased 100% between E14 and E19. This enhancement was accompanied by a small negative shift of the voltage dependence and a marked acceleration of the inactivation kinetics. At the single-channel level, the unitary conductance decreased significantly from 13.2 +/- 0.1 pS (n = 8) at E14 to 10.7 +/- 0.3 pS (n = 7) at E18 and the open probability was multiplied by 2. No significant change of the density of functional channels was observed during the same period. In contrast to the density of intramembrane charge movement, which, under the same conditions, has been shown to increase between 16 and 19 days, L-type Ca(2+) channels properties change mostly between 14 and 16 days. Taken together, these results suggest that the two functions of the dihydropyridine receptor are carried by two different proteins which could be differentially regulated by subunit composition and/or degree of phosphorylation.

Activation by angiotensin II of Ca(2+)-dependent K+ and Cl- currents in zona fasciculata cells of bovine adrenal gland.

Abstract. The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These effects were abolished by Losartan (10−5m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method, a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau current. In Cl−-free solution, the outward current reversed at −78.5 mV, a value close to EK. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K+-free solution, the transient outward current, sensitive to Cl− channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than ECl. Its magnitude changed in the same direction as the driving force for Cl−. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca2+-free solution (47% of cells) and was reversibly blocked by Cd2+ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca2+ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin II is due to Ca2+-dependent K+ and Cl− currents. These two membrane currents are co-activated in response to an internal increase of [Ca2+]i originating from intra- and extracellular stores.

Platelet aggregation and endogenous 5-ht secretion in presence of ca2+, sr2+ and ba2+. effects of calcium antagonists

Washed rat platelets aggregation and endogenous serotonin release were studied after thrombin stimulation in the presence of different concentrations of Ca2+, Sr2+ or Ba2+. The extent of platelet aggregation and release was found to depend upon the external concentration of these cations. For all of them, an optimum concentration could be defined. Higher concentrations were shown to inhibit both aggregation and release. Efficiency to support thrombin-induced aggregation was in the order Ca2+ greater than Sr2+ greater than Ba2+. Complete inhibition of aggregation and release induced by thrombin was obtained after a 30 second preincubation with 38 uM nitrendipine, 1 mM Cd2+ or 1 mM Mn2+. Inhibition was obtained in the presence of Ca2+, Sr2+ or Ba2+. These results are consistent with the hypothesis that Sr2+ and Ba2+ are able to support platelet activation acting as Ca2+ substitutes. Following thrombin stimulation, they could penetrate the platelets and mimick a rise in cytoplasmic Ca2+.

Two types of pharmacologically distinct Ca(2+) currents with voltage-dependent similarities in zona fasciculata cells isolated from bovine adrenal gland.

Abstract. Voltage-activated Ca2+ currents, in zona fasciculata cells isolated from calf adrenal gland, were characterized using perforated patch-clamp recording. In control solution (Ca2+: 2.5 mm) a transient inward current was followed, in 40% of the cells, by a sustained one. In 20 mm Ba2+, 61% of the cells displayed an inward current, which consisted of transient and sustained components. The other cells produced either a sustained or a transient inward current. These different patterns were dependent upon time in culture. Current-voltage relationships show that both the transient and sustained components activated, peaked and reversed at similar potentials: −40, 0 and +60 mV, respectively. The two components, fully inactivated at −10 mV, were separated by double-pulse protocols from different holding potentials where the transient component could be inactivated or reactivated. The decaying phase of the sustained component was fitted by a double exponential (time constants: 1.9 and 20 sec at +10 mV); that of the transient component was fitted by a single exponential (time constant: 19 msec at +10 mV). Steady-state activation and inactivation curves of the two components were superimposed. Their half activation and inactivation potentials were similar, about −15 and −34 mV, respectively. The sustained component was larger in Ba2+ than in Sr2+ and Ca2+. Ni2+ (20 μm) selectively blocked the transient component while Cd2+ (10 μm) selectively blocked the sustained one. (±)Bay K 8644 (0.5 μm) increased the sustained component and nitrendipine (0.5–1 μm) blocked it selectively. The sustained component was inhibited by calciseptine (1 μm). Both components were unaffected by ω-conotoxin GVIA and MVIIC (0.5 μm). These results show that two distinct populations of Ca2+ channels coexist in this cell type. Although the voltage dependence of their activation and inactivation are comparable, these two components of the inward current are similar to T- and L-type currents described in other cells.