InsP3-dependent Ca2+ oscillations linked to activation of voltage-dependent H+ conductance in Rana esculenta oocytes.

In normal medium supplemented with 10 mM tetraethylammonium chloride (TEACl), membrane depolarizations of immature Rana esculenta oocytes elicited an oscillatory outward current associated with a voltage-dependent H+ current (IH+). The voltage threshold of these oscillations was 22 +/- 5 (n = 10). The oscillations were blocked by intracellular injection of ethylene glycol-O,O'-bis-(2-acetaminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), by application of 1 mM of 4-acetamido-4'-isocyanatostilbene-2,2'-disulfonic acid (SITS), by caffeine (1 mM), and by the intracellular injection of heparin, suggesting that they arose from calcium release from inositol trisphosphate (InsP3)-sensitive stores, monitored by a calcium-dependent chloride current (IClCa2+). The oscillations were independent of the external calcium concentration, and the depolarizations did not affect the InsP3 level. Ni2+, a IH+ inhibitor, blocked the oscillations. Extracellular alkalinization, which lowered the voltage threshold of IH+ and increased its amplitude, also lowered the voltage threshold of the oscillations and increased their amplitude, whereas extracellular acidification produced opposite effects. We suggest that the oscillations are linked to activation of IH+ through a pH-dependent sensitization of InsP3 receptors.

Interaction between Ca2+ release from inositol trisphosphate sensitive stores and Ca2+ entry through neuronal Ca2+ channels expressed in Xenopus oocyte.

Rat cerebellar RNA injected into Xenopus oocytes leads to the expression of putative P-type voltage-dependent Ca2+ channels (VDCCs). The monitoring of intracellular Ca2+ variations by recording the Ca2+ dependent chloride current in voltage clamped oocytes indicates that activation of these Ca2+ channels by depolarization gives rise to two distinct components of cytosolic Ca2+ elevation. If the early component (T1) can be directly attributed to the Ca2+ entry through VDCCs, the second delayed one (T2) is related to a Ca2+ release from InsP3 sensitive stores activated following Ca2+ entry. Modifications of cytosolic Ca2+ by direct injection of Ca2+ into oocytes or by increasing the Ca2+ influx through VDCCs suggest that the Ca2+ release from intracellular InsP3 sensitive stores can be modulated in a differential manner. Namely, discrete elevations of cytosolic Ca2+ switch on the Ca2+ release whereas higher Ca2+ concentrations dampen the release. These results suggest a functional coupling between P-type VDCCs and InsP3 receptors.

Ins(1,4,5)P3 formation and fluctuating chloride current response induced by external ATP in Xenopus oocytes injected with embryonic guinea pig brain mRNA.

In voltage-clamped Xenopus oocytes injected with embryonic guinea pig mRNA, effective concentrations of extracellular ATP elicited an inward fluctuating current. This current, carried by Cl-ions, was mainly dependent upon liberation of Ca2+ ions from stores as demonstrated by experiments using intracellular EGTA loading and TMB-8 superfusion. Neomycin inhibited these fluctuating currents indicating that the transplanted purinoceptor is linked to phospholipase C activity and triggers Ins(1,4,5)P3 formation. Ins(1,4,5)P3 production evoked by external ATP was clearly demonstrated by directly measuring the water-soluble Ins(1,4,5)P3 level in injected oocytes. Finally, it is suggested that the ATP effect was mediated by a Ca2+ release from Ins(1,4,5)P3 sensitive pools since heparin blocked the ATP responsiveness. The acquired purinoceptor may be made apparent to a P2 subtype since ATP and ADP were equipotent in eliciting Cl- current while AMP and Adenosine were ineffective in injected oocytes.

Endogenous DHP-sensitive Ca(2+) channels in Pleurodeles oocytes.

The double electrode voltage-clamp technique was used to study voltage-dependent Ca(2+) channels in Pleurodeles oocytes. From a holding potential of -80 mV, Ba-current (IBa) (recorded in Cl-free solution, Ba(2+ = 40 mM) activated at -36.7 +/- 4 mV, peaked at -11.6 +/- 4 mV and reversed at 55 +/- 7 mV (n = 24). This current activated slowly (rise time was 0.98 +/- 0.2 s;n = 14 at -10 mV) and was not inactivated. Cadmium (Cd(2+), 500 microM) completely inhibited I(Ba). The effect of Cd(2+) was dose-dependent (EC(50) = 37 +/- 5 microM; n = 5). Moreover, IBa was insensitive to omega-conotoxin (10 microM) but interestingly this I(Ba) displayed dihydropyridine (DHP) sensitivity. Bay K 8644 (5 microM), a DHP activator, increased the peak current amplitude in a dose-dependent manner (EC(50) = 5.9 +/- 0.6 microM; n = 10) and shifted the threshold and the maximum of current/voltage relationship towards negative potentials by -10 mV. Nifedipine (5 microM), a DHP antagonist, decreased I(Ba) by 80% at HP of -80 mV (EC(50) = 1.2 +/- 0.2 microM; n = 6). We concluded that Pleurodeles oocytes possess High-Voltage Activated Ca(2+) channels with properties similar to L-type Ca(2+) channels.

Is inward calcium current in crayfish muscle membrane constituted of one or two components?

Two inward currents were observed in crayfish muscle membrane during depolarization steps by the method described by Adrian et al. (1970). Under voltage clamp conditions, hyperpolarization steps elicited a large current (leak current If), associated with an inward voltage dependent current. This inward current was inhibited by niflumic acid (NA), a drug known to block Cl---HCO-3 exchange (Cousin et Motais 1982; Brûlè et al. 1983b). Dynamic outward currents triggered by depolarizing steps were inhibited to a great extent by TEA, the not inhibited portion disappearing when procaine (2 mmol/l) was added to external solution. In the presence of TEA, procaine and NA, it was thus possible to dissect the regenerative calcium current (ICa) into two components: a "fast component" (ICa1) and a "slow component" (ICa2). The reversal potential of ICa was 65 mV (for [Ca]0 = 2.8 mmol/l), and [Ca]i could be calculated to be 1.6 X 10(-5) mol/l. This value of [Ca]i is the same as calculated from values reported by Hencek and Zachar (1977). ICa1 was triggered at a threshold membrane potential of -45 mV and ICa2 at -30 mV. Moreover, the inactivation kinetics for ICa1 was faster than that for ICa2. Our results are in perfect agreement with those obtained by Zahradník and Zachar (1982) who postulated two populations of calcium channels.

Possible effects of membrane polarization on calcium uptake by and release from sarcoplasmic reticulum vesicles.

Rates of calcium uptake by and calcium release from sarcoplasmic reticulum vesicles isolated from skeletal muscle of the crab seem to depend on membrane potential generated by potassium (K) and chloride (Cl) gradients. This does not appear to be due to an effect of the ions themselves since media of different ionic compositions leading to the same membrane potential, also lead to the same ATP hydrolysis and the same Ca uptake by SR vesicles. From a large positive intravesicular potential (conditions termed "normal" in this paper), membrane depolarization of passively Ca loaded vesicles, produced by changes in K and Cl concentrations in the media, resulted in: i) decrease in rate of calcium uptake; ii) decrease in calcium loading; iii) increase in rate of calcium release despite a decrease in the driving force for calcium ions. Moreover, the addition of caffeine (5 mmol/l) to the different polarization media resulted in a increase in calcium release.

A special scanner speeds orders for this lab and its satellites.

The authors' laboratory integrated optical mark readers with its existing mainframe computer system to streamline order processing--and improved staff morale as well.

[The effect of cations on the contraction of myofibrils in the striated muscles of crabs]. / Effets des cations sur la contraction des myofilaments de la fibre striée de crabe

Myofilaments have been prepared from crab legs according to a technique derived from that of F.J. Julian (1971). Tension variations have been recorded while the isolated fiber was submitted to the action of different saline solutions. As expected, it could be confirmed that the tension mainly depends on the content in Ca++ and Mg++ ions and in ATP. Our results have also shown that monovalent ions (Na+ and K+) when added separately, produce an important tension increase wheras their simultaneous addition or withdrawal modifies the tension only slightly.

[Inward currents of crab striated muscle fiber measured by the voltage clamp technique with microelectrodes]. / Le courant entrant de la fibre musculaire striée de crabe mis en évidence par la technique du potentiel imposé avec micro-électrodes

Membrane currents during depolarisation steps were determined by a method in which three electrodes were inserted near the end of the crab's striated muscular fibre. The value of the reversal potential for the early current appears to be consistent with the calcium equilibrium potential. This early transient inward current is abolished by manganese. The value of about +120mV is consistent with the level of internal free Ca++ ions, as generally tested by different authors.

A voltage-dependent and pH-sensitive proton current in Rana esculenta oocytes.

Voltage clamp technique was used to study macroscopic ionic currents in Rana esculenta oocytes. Depolarization steps led to the activation of a single type of outward current (Iout) when contaminant potassium and calcium-dependent chloride currents were pharmacologically inhibited. The voltage threshold of Iout activation was 10 mV and this current, which did not inactivate, presented a deactivation the time constant of 73 +/- 21 msec (n = 26) corresponding to a membrane voltage of -60 mV. Its reversal potential (Erev) was dependent on the magnitude of the depolarization and also on pulse duration. These changes in Erev were thought to reflect intracellular ion depletion occurring during activation of the remaining outward current. Furthermore, the activation threshold of Iout was clearly affected by modifications in extracellular and intracellular H+ concentrations. Indeed, intracellular alkalinization (evoked by external application of ammonium chloride) or extracellular acidification induced a rightward shift in the activation threshold while intracellular acidification (evoked by external application of sodium acetate) or extracellular alkalinization shifted this threshold toward a more negative value. Lastly, Iout was dramatically reduced by divalent cations such as Cd2+, Ni2+ or Zn2+ and was strongly decreased by 4 Aminopyridine (4-AP), well-known H+ current antagonists already described in many cell types. Therefore, it was suggested that the outward current was prominently carried by H+ ions, which may play a key role in the regulation of intracellular pH and subsequent pH dependent processes in Rana oocyte.

[Inhibition, by an amphiphilic substance, niflumic acid, of the inward rectification of the crustacean muscle fiber]. / Inhibition par une substance amphiphile, l'acide niflumique, de la rectification dans le sens entrant de la fibre musculaire de Crustacé.

Agents such as TEA+ or CS+ ions, these last ions instead of K+ ions in poor K extracellular solution, known to reduce or abolish the inwardly rectifying channel in many preparations produced no effect in crayfish muscle membrane By contrast, poor Cl extracellular solution (Cl- ions were replaced by CH3OSO3- ions) blocked the inward current activated by hyperpolarizing pulses and produced an increase of the resting potential. Niflumic acid is a agent which inhibited the inward going rectification of the crayfish muscle membrane. Apparent dissociation constant of niflumic acid with membrane sites was equal to about 6 X 10(-8) M; this value corresponds to that given by Cousin & Motais (1979) concerning translocation of Cl- ions in the membrane of red cells. Activation of the inward going rectification in the crayfish membrane is responsible of an inward current carried by Cl- ions.

Two components of contraction in guinea pig papillary muscle.

Biphasic contractions were produced in guinea pig papillary muscle by inducing partial depolarization in a K+ -rich solution (22 mM) containing 10(-6) M isoproterenol. However, when the same conditions were applied to frog and rat, monophasic contractions were obtained. In the case of guinea pig, an increase in the beating frequency produced an increase in amplitude of the first component and a reduction of the second, while in frog and rat, only a decrease in the amplitude of contractions was recorded. Caffeine (10(-3) M) eliminated the first component and increased the second in guinea pig, while in the case of rat and frog it decreased the amplitude of contractions. Procaine (10(-3) M) suppressed the first component and decreased the second one. The contraction in frog appears to be similar to the second component of contraction in guinea pig, while in rat, the contraction is comparable with the first component in guinea pig. It is suggested that the calcium ions which activate the two components of contraction in guinea pig under the given experimental conditions may arise from two different sources.

[Modification of the affinity of actomyosin calcium sites by anions]. / Modification de l'affinité des sites calciques de l'actomyosine par les anions.

ATP hydrolysis-pCa and isometric tension-pCa relationships were determined for isolated myofibrils under a variety of ionic conditions i.e. in presence of Cl- ions (normal conditions), in presence of CH3COO- or CH3OSO3- (ions replacing all or a part of Cl- ions). In all ionic conditions, these relationships have a S-shape. For each ionic condition, the relationships between isometric tension or ATP hydrolysis and pCa were parallel, but the isometric tension-pCa was shifted towards a smaller value of pCa. The relation between ATP hydrolysis and pCa has been analysed by means of the enzymatic reaction described by Michaelis-Menten. The analysis of isometric tension-pCa relationship can be also described by using the consecutive scheme of reaction for the co-operative action of two Ca2+ ions in the process of tension activation given by Ashley & Moisescu (1977) for barnacle myofibrils. The dissociation constant (k1) equal to the one that we determined for the ATP hydrolysis probably corresponds to the Ca2+ binding site of troponin (M1). This association (M1-Ca) would lead to the phosphorylation of the second site (M2) localized on myosin, which can bind a Ca2+ ion. In presence of CH3COO-, ATP hydrolysis (V) was increased with no change of the affinity of the first site for the Ca2+ ion. At the opposite, V did not change but the affinity of M1 site was decreased in presence of CH3OSO3-. CH3OSO3- must be considered as a competitive inhibitor whereas CH3COO- must be considered as an activator ion of the ATP hydrolysis reaction. In all cases, the relationship between isometric tension and ATP hydrolysis was hyperbolic.

[Effects of hypertonic solutions on the excitation-contraction coupling of the skeletal muscle fibre of the crab. II. Ultrastructural aspects (author's transl)]. / Effets de l'hypertonie sur le couplage excitation-contraction de la fibre musculaire squelettique de crabe. II. Aspects ultrastructuraux.

Experiments were performed to investigate the modifications of electrical activity and the loss of contraction by examination of the ultrastructure. These alterations were obtained with a control physiological solution (artificial sea water, ASW) which was made hypertonic by adding sucrose, glycerol, choline chloride or urea. 1. Under these conditions, the ultrastructural results show that: a) all those substances induce a modification of the coupling junction (either diad or triad). This modification can explain the inhibition of the mechanical phenomenon; b) sucrose (Figs. 6C and 6D) and glycerol (Figs. 4A and 4B) induce alterations of the surface membrane and of the tubular system (less important with glycerol). They can explain the decrease of the resting potential and the abolishment or the modification of the action potential; c) in contrast, choline chloride (Figs. 7A to 7E) and urea (Figs. 9A to 9F) only modify the organisation of the coupling junction. The action potential is maintained. 2. A short exposure of the fibre in an isotonic sucrose solution (Figs. 5A to 5D) deeply modifies the tubular system, particularly, in cutting it off from the sarcolemma. 3. Choline chloride and urea produce similar changes in the ultrastructure but as urea is not ionized, it can be added to the ASW to investigate the electrical phenomenon which cannot be modified by contraction.

Regulation of endogenous Ca2+ channels by cyclic AMP and cyclic GMP-dependent protein kinases in Pleurodeles oocytes.

The effects of cyclic AMP (cAMP) and cyclic GMP (cGMP) on dihydropyridine sensitive Ca2+ channels were investigated under voltage-clamp in defolliculated Pleurodeles oocytes. Intracellular injection of cAMP or extracellular application of the permeable cAMP analogue (8-Bromo cAMP, 8Br-cAMP) decreased the Ba current (IBa). This effect on IBa was blocked by the injection of protein kinase A inhibitor. Similar results were found upon internal application of the catalytic subunit of protein kinase A. In contrast, the injection of cGMP or perfusion of 8Br-cGMP increased IBa amplitude. The increase of IBa by 8Br-cGMP was blocked by the injection of the selective inhibitor of protein kinase G (KT5823). These results support the hypothesis that the basal Ba current amplitude of Pleurodeles oocytes is under the control of Protein Kinases A (PKA) and G (PKG) activity. This regulation of Ca2+ channels by the second messengers, and particularly by cAMP may reflect an important step in the maturation processus of Pleurodeles oocytes.

Calcium channels and excitation-contraction coupling in cardiac cells. I. Two components of contraction in guinea-pig papillary muscle.

Biphasic contractions have been obtained in guinea-pig papillary muscle by inducing partial depolarization in K+-rich solution (17 mM) containing 0.3 microM isoproterenol; whereas in guinea-pig atria, the same conditions led to monophasic contractions corresponding to the first component of contraction in papillary muscle. The relationships between the amplitude of the two components of the biphasic contraction and the resting membrane potential were sigmoidal curves. The first component of contraction was inactivated for membrane potentials less positive than those for the second component. In Na+-low solution (25 mM), biphasic contraction became monophasic subsequent to the loss of the second component, but tetraethylammonium unmasked the second component of contraction. The relationship between the amplitude of the first component of contraction and the logarithm of extracellular Ca2+ concentration was complex, whereas for the second component it was linear. When Ca2+ ions were replaced by Sr2+ ions, only the second component of contraction was observed. It is suggested that the first component of contraction may be triggered by a Ca2+ release from sarcoplasmic reticulum, induced by the fast inward Ca2+ current and (or) by the depolarization. The second component of contraction may be due to a direct activation of contractile proteins by Ca2+ entering the cell along with the slow inward Ca2+ current and diffusing through the sarcoplasm. These results do not exclude the existence of a third "tonic" component, which could possibly be mixed with the second component of contraction.

Calcium channels and excitation-contraction coupling in cardiac cells. II. A pharmacological study of the biphasic contraction in guinea-pig papillary muscle.

Biphasic contractions were obtained in guinea-pig papillary muscle by inducing partial depolarization in K+-rich solution (17 mM) in the presence of 0.3 microM isoproterenol. Mn2+ ions inhibited the two components of contraction in a similar way. Nifedipine and particularly Cd2+ ions specifically inhibited the second component of contraction. Isoproterenol and BAY K 8644 markedly increased the amplitude of the second component (P2) of contraction. Nevertheless, a moderate positive inotropic effect of isoproterenol was found on the first component (P1) of contraction when excitability was restored by 0.2 mM Ba instead of isoproterenol. Acetylcholine and hypoxia decreased the amplitude of the second component of contraction to a greater extent. In the presence of digoxin or Na+-free solution, P1 was strongly increased. When sarcoplasmic reticular function was hindered by 1mM caffeine or in the presence of Ca2+-free Sr2+ solution, digoxin always induced a negative inotropic effect on P2. Inversely in these conditions the transient positive inotropic effect of Na+-free solution was strongly reduced. These results are consistent with the hypothesis that the late component of contraction is triggered by the slow inward Ca2+ current and that the early component is due to Ca2+ release from the sarcoplasmic reticulum.