Charge Dependent Distribution of Endogenous Proteins within Vitellogenic Ovarian Follicles of Actias luna

In vitellogenic ovarian follicles of Actis luna, internal Ca(2+) activity currents create an electrical gradient which influences the distribution of charged macromolecules between nurse cells and oocyte. We show that, between oocyte and nurse cells, there is an ionic gradient of 1-12 mV with the nurse cells being more electronegative than the oocyte by an average 3.5+/-0.2 mV(s.e.)(p<0.001). As previously reported for another saturniid, Hyalophora cecropia, the transbridge ionic gradient of luna: (1) is focused across the intercellular bridges, (2) is abolished by 200 &mgr;M vanadate and (3) includes a [Ca(2+)](i) gradient. Endogenous soluble proteins collected from control and from vanadate treated populations of nurse cells and oocytes were separated by two-dimensional (2-D) gel electrophoresis and visualized with sliver stain. Densitometric analysis showed that 14 out of the 19 acidic proteins and six of the eight basic proteins studied, changed their oocyte-to-nurse cell distribution in consort with change in the transbridge ionic gradient. This suggests that a transbridge ionic gradient may be, at least within the saturniidae, a method for maintaining different molecular concentrations in nurse cells compared to oocytes. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Ion physiology of vitellogenic follicles

The ion physiology of vitellogenic follicles from a lepidopteran (Hyalophora cecropia) and a hemipteran (Rhodnius prolixus) are compared. Similarities that can be expected to occur in vitellogenic follicles of many other insects include: (1) gap junctions, which unite the cells of a follicle into an integrated electrical system, (2) transmembrane K⁺ and H⁺ gradients that account for over 60% of follicular membrane potentials, (3) absence of a Cl⁻ potential, (but the opening of channels to this anion when vitellogenesis terminates in H. cecropia), (4) an electrogenic proton pump that supplements follicular membrane potentials, (5) Ca²⁺ action potentials evoked by injecting depolarizing currents into oocytes, and (6) the use of osmotic pressure to control epithelial patency. Differences include: a Na⁺/K⁺-ATPase that accounts for about 20% of the follicular resting potential in R. prolixus but is absent from H. cecropia, and an intrafollicular Ca²⁺ current that moves from oocyte to nurse cells through cytoplasmic bridges in H. cecropia. Evidence is also summarized for two promising mechanisms that require further substantiation: (1) transmission via gap junctions of a follicle cell product that promotes endocytosis in the oocyte; and (2) transport of the proton pump back and forth between cell surface and endosomes as the membrane that carries it recycles through successive rounds of vitellogenin uptake.     

Demonstration of a voltage dependent calcium current in follicles of the cockroach Nauphoeta cinerea

Summary

The electrical properties of vitellogenic cockroach follicles have been studied using a two-electrode voltage-clamp technique. The resting potential of freshly isolated follicles was usually between ?30 mV and ?45 mV. A voltage-dependent inward current could be activated when the follicles were placed in a medium containing high concentrations of either Ca²⁺ or Ba²⁺. Maximal current amplitudes were 0.8μA in a medium containing 30 mM Ca²⁺ and 2 μA in 30 mM Ba²⁺. Maximal amplitudes could be elicited with depolarizing pulses to about +15 mV. The steady-state inactivation curve indicated half maximal current at a holding potential of about ?20 mV. Co²⁺, Ni²⁺ and Mn²⁺ inhibited the divalent cation currents in a concentration dependent manner. Half maximal inhibition was observed at about 3 mM concentration for each of the inhibitory cations.     

Gap junctions in ovarian follicles of Drosophila melanogaster: inhibition and promotion of dye-coupling between oocyte and follicle cells

The analysis of chimeras has shown that communication between germ-line and soma cells plays an important role during Drosophila oogenesis. We have therefore investigated the intercellular exchange of the fluorescent tracer molecule, Lucifer yellow, pressure-injected into the oocyte of vitellogenic follicles of Drosophila. The dye reached the nurse cells via cytoplasmic bridges and entered, via gap junctions, the somatic follicle cells covering the oocyte. The percentage of follicles showing dye-coupling between oocyte and follicle cells was found to increase with the developmental stage up to stage 11, but depended also on the status of oogenesis, i.e., the stage-spectrum, in the respective ovary. During late stage 10B and stage 11, dye-coupling was restricted to the follicle cells covering the anterior pole of the oocyte. No dye-coupling was observed from stage 12 onwards. During prolonged incubation in vitro, the dye was found to move from the follicle cells back into the oocyte; this process was suppressable with dinitrophenol. Dyecoupling was inhibited when prolonged in vitro incubation preceded the dye-injection. Moreover, dye-coupling was inhibited with acidic pH, low [K⁺], high intracellular [Ca²⁺], octanol, dinitrophenol, and NaN₃, but not with retinoic acid, basic pH, or high extracellular [Ca²⁺]. Dyecoupling was stimulated with a juvenile hormone analogue and with 20-hydroxyecdysone. Thus, gap junctions between oocyte and follicle cells may play an important role in intercellular communication during oogenesis. We discuss the significance of our findings with regard to the electrophysiological properties of the follicles, and to the coordinated activities of the different cell types during follicle development and during the establishment of polarity in the follicle.     

Neutral carrier ion-selective microelectrodes for measurement of intracellular free calcium

This paper describes the making and testing of calcium-selective microelectrodes for measurement of intracellular [free Ca²⁺] levels. Pipettes of tip outer diameter down to 0.4 μm were siliconized by a novel and easy method of vapor treatment. The tips were filled with a sensor mixture using the neutral ligand and solvent of Oehme et al. (Oehme, M., Kessler, M. and Simon, W. (1976) Chimia (Aarau) 30, 204–206) but with very hydrophobic cations replacing Na⁺ in the salt component. This change improved electrode stability and greatly reduced hysteresis in the responses to changing [Ca²⁺] levels. Lowering the Ca²⁺ concentration in the internal electrolyte also increased electrode lifetime.Electrodes showed a Nernstian response to [Ca²⁺] down to 1 μM free concentration in 0.1 M KCl, and usually a useful response to below 100 nM Ca²⁺. Selectivity for Ca²⁺ over Mg²⁺ and H⁺ was sufficiently high that typical free intracellular levels of Mg²⁺ and H⁺ caused negligible interference. Selectivity for Ca²⁺ over Na⁺ was adequate for cells with 10^?2 M free Na⁺, but higher levels could raise significantly the detection limit for Ca²⁺. Preliminary measurements of [free Ca²⁺] have been made in frog skeletal muscle, ferret ventricular myocardium, and early embryos of Xenopus laevis. Relative merits of Ca²⁺ microelectrodes and optical indicators are discussed.     

Existence,properties, and functional expression of “Maxi-K”-type,Ca2+-activated K+ channels in short-term cultured hepatocytes

A large-conductance, Ca²⁺-activated K⁺ channel was identified and characterized in embryonic chick hepatocytes using the patch-electrode voltage-clamp technique. The channel conductance was 213 pS in excised patches bathed in symmetrical 145 mM KCl and 1 mM Ca²⁺. Current-voltage relationships were linear with high K⁺ on both sides of the membrane but showed constant field rectification as the K⁺ gradient was increased. The reversal potential shifted 58 mV per 10-fold change in the ratio of external to internal K⁺. Channel openings occurred at potentials higher than +50 mV in cell-attached patches. The open probability × voltage relationship shifted to more negative potentials in excised, inside-out patches exposed to a solution containing high Ca²⁺. The voltage sensitivity of the channel was not significantly affected by changes in internal Ca²⁺ concentration. Conversely, channel gating, reflected in the half-activation potential, shifted 118 mV per 10-fold change in internal Ca²⁺ at concentrations less than ∼2 μM, although at higher Ca²⁺, this parameter was Ca²⁺ insensitive. Channel open probability in cell-attached patches increased significantly following exposure of the cells to either the Ca²⁺ ionophore A-23187 or L-alanine, a cell-volume modulator. Channel density increased with time spent in culture from no observations in 10-hr cells, through 13 and 80% of patches in 24-and 48-hr cultured cells, respectively. The implications of delayed functional expression for ion channel studies in acutely dissociated cells is discussed. J. Cell. Physiol. 171:87–94, 1997. © 1997 Wiley-Liss, Inc.     

Fractional contribution of major ions to the membrane potential of Drosophila melanogaster oocytes

In ovarian follicles of Drosophila melanogaster, ion substitution experiments revealed that K⁺ is the greatest contributor (68%) in setting oocyte steady‐state potential (E_m), while Mg²⁺ and a metabolic component account for the rest. Because of the intense use made of Drosophila ovarian follicles in many lines of research, it is important to know how changes in the surrounding medium, particularly in major diffusible ions, may affect the physiology of the cells. The contributions made to the Drosophila oocyte membrane potential (E_m) by [Na⁺]_o, [K⁺]_o, [Mg²⁺]_o, [Ca²⁺]_o, [Cl^?]_o, and pH (protons) were determined by substitutions made to the composition of the incubation medium. Only K⁺ and Mg²⁺ were found to participate in setting the level of E_m. In follicles subjected to changes in external pH from the normal 7.3 to either pH 6 or pH 8, E_m changed rapidly by about 6 mV, but within 8 min had returned to the original E_m. Approximately half of all follicles exposed to reduced [Cl^?]_o showed no change in E_m, and these all had input resistances of 330 kΩ or greater. The remaining follicles had smaller input resistances, and these first depolarized by about 5 mV. Over several minutes, their input resistances increased and they repolarized to a value more electronegative than their value prior to reduction in [Cl^?]_o. Together, K⁺ and Mg²⁺ accounted for up to 87% of measured steady‐state potential. Treatment with sodium azide, ammonium vanadate, or chilling revealed a metabolically driven component that could account for the remaining 13%. © 2009 Wiley Periodicals, Inc.     

Vitellogenesis in the cockroach Nauphoeta cinerea: Separation of two classes of ovarian binding sites and calcium effects on binding and uptake

Two classes of vitellogenin binding sites with K_d-values of 7.3 nM and 290 nM were observed in follicle-membrane preparations of the cockroach Nauphoeta cinerea using a membrane-binding assay at pH 8. Separation of follicle cells and basal laminae from oocyte membranes prior to binding studies showed that the fraction consisting of follicle cells and basal laminae (FC/BL) contained high-affinity binding sites for vitellogenin (K_d=16.6 nM), whereas loweraffinity binding sites (K_d=200 nM) were found in the oocyte membrane fraction. The concentration of Ca²⁺ had a distinct effect on vitellogenin binding and uptake: maximal binding to the oocyte membrane fraction was observed at 0.3 mM Ca²⁺ and to the FC/BL fraction at 10 mM, whereas uptake of vitellogenin by oocytes in vitro was highest at 4 mM Ca²⁺. The calcium ionophore A23187 decreased vitellogenin uptake. This effect of A23187 could be counteracted by the calcium chelator Quin2. A hypothetical model for the uptake of vitellogenin into follicles of Nauphoeta cinerea is suggested.     

Thyroxine influence on different ion-specific adenosine triphosphatase activities in fat body of Bombyx mori during development

The effects of thyroxine on the activity of different ATPases (Na⁺-K⁺, Ca²⁺, and Mg²⁺) in fat body cells of the silkworm, Bombyx mori, were investigated during different developmental stages. In both sexes the maximum enzyme activity was observed in the fat body cells of day 7 last instar larva (the day before spinning). Na⁺-K⁺, Ca²⁺-, and Mg²⁺-ATPase activity in the fat body markedly declined after pupation and continued to decrease in day 1 adults. Injection of thyroxine (T4) at doses of 1.0 and 2.0 μg/g during fifth instar significantly elevated all ATPase activities in the larval, pupal, and adult stages in both sexes. At a dose of 0.5 μg/g, T4 had no effect on day 2 fifth instar larva, although it increased the ATPase activity at the other stages investigated. A higher dose (3.0 μg/g) caused a significant reduction in enzyme activity in all stages with the exception of day 2 fifth instar larva. Thus, the repression of enzyme activity with the higher dose and the elevation of enzyme activity with the lower dose establish the biphasic nature of T4 action on the ATPase system in fat body cells of the silkworm. Arch. Insect Biochem. Physiol. 37:191–196, 1998. © 1998 Wiley-Liss, Inc.     

Measurements of intracellular ionized calcium in squid giant axons using calcium-selective electrodes

Ca²⁺-selective electrodes have been used to measure free intracellular Ca²⁺ concentrations in squid giant axons. Electrodes made of glass cannulas of about 20 μm in diameter, plugged with a poly(vinyl chloride) gelled sensor were used to impale the axons axially. They showed a Nernstian response to Ca²⁺ down to about 3 μM in solutions containing 0.3 M K⁺ and 0.025 M Na⁺. Sub-Nernstian but useful responses were obtained up to pCa 8. The electrodes showed adequate selectivity to Ca²⁺ over Mg²⁺, H⁺, K⁺ and Na⁺. To calibrate them properly, a set of standard solutions were prepared using different Ca²⁺ buffers (EGTA, HEEDTA, nitrilotriacetic acid) after carefully characterizing their apparent Ca²⁺ association constants under conditions resembling the axoplasmic environment. In fresh axons incubated in artificial seawater containing 4 mM Ca²⁺, the mean resting intracellular ionized calcium concentration was 0.106 μM ($\text{n = 15}$). The Ca²⁺-electrodes were used to investigate effects of different experimental procedures on the [Ca²⁺]_i. The main conclusions are: (i) intact axons can extrude calcium ions at low [Ca²⁺]_i levels by a process independent of external Na⁺; (ii) poisoned axons can extrude calcium ions at high levels of [Ca²⁺]_i by an external Na⁺-dependent process. The level of free intracellular Ca attained at these latter conditions is about an order to magnitude greater than the resting physiological value.     

Inactivation of calcium channels in vascular smooth muscle myocytes

Many of the structural domains involved in Ca²⁺ channel (CACN) inactivation are also involved in determining their sensitivity to antagonist inhibition. We hypothesize that differences in inactivation properties and their structural determinants may suggest candidate domains as targets for the development of novel, selective antagonists. The characteristics of Ca²⁺ current (I_Ca) inactivation, steady-state inactivation (SSIN), and recovery from inactivation were studied in freshly dispersed smooth muscle cells from rabbit portal vein (RPV) using whole-cell, voltage-clamp methods. The time course of inactivation could be represented by two time constants. Increasing I_Ca by increasing [Ca²⁺]_o or with more negative holding potentials decreased both time constants. With Sr²⁺, Ba²⁺, or Na⁺ as the charge carrier, I_Ca inactivation was also represented by two time constants, both of which were larger than those found with Ca²⁺. With Ca²⁺, Sr²⁺, or Ba²⁺ as the charge carrier, both time constants had minimum values near the voltage associated with maximum current. When Na⁺ (140 mM) was the charge carrier, voltages for I_max (−20 mV) or τ_min (o mV) did not correspond. SSIN of I_Ca had a half-maximum voltage of −32±4 mV for Ca²⁺, −43 mV±5 mV for Sr²⁺, −41±5 mV for Ba²⁺, and −68±6 mV for Na⁺. The slope factor for SSIN per e-fold voltage change was 6.5±0.2 mV for Ca²⁺, 6.8±0.3 for Sr²⁺, and 6.6±0.2 for Ba²⁺, representing four equivalent charges. When Na⁺ or Li⁺ was the charge carrier, the slope factor was 13.5±0.7 mV, representing two equivalent charges. For I_Ca in rat left ventricular (rLV) myocytes, there was no difference in the slope factor of SSIN for Ca²⁺ and Na⁺. The rate of recovery of I_Ca from inactivation varied inversely with recovery voltage and was independent of the charge carrier. These results suggest that inactivation of I_Ca in PV myocytes possess an intrinsic voltage dependence that is modified by Ca²⁺. For RPV but not rLV I_Ca, the charge of the permeating ion confers the voltage-dependency of SSIN.     

Lysophosphatidylcholine induces Ca2+-independent cellular injury attenuated by d-propranolol in rat cardiomyocytes

In isolated rat cardiomyocytes, exogenous lysophosphatidylcholine (LPC) (15 μM) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) from 72 ± 5 to 3042 ± 431 nM accompanied by cell injury as indicated by the hypercontracture of the cells and the increase in creatine phosphokinase (CPK) release. In order to understand whether the cell injury induced by LPC was a consequence of the elevation of [Ca²⁺]_i, the effect of LPC was examined in the Ca²⁺-free solution containing EGTA. Under the Ca²⁺ -free conditions, LPC did not increase [Ca²⁺]_i, whereas it still inflicted injury on the cells in terms of cell-shape change and CPK release to the same degree as that under the Ca²⁺-present condition. Addition of ryanodine (10 μM) failed to prevent the changes in cell-shape and CPK release induced by LPC under both Ca²⁺-free and Ca²⁺ -present conditions. Preincubation of the myocytes with d-propranolol (50 μM) inhibited the LPC-induced changes in cell-shape and CPK release under both Ca²⁺ -free and Ca²⁺ -present conditions (p < 0.05). Our study provides clear evidence that the cellular injury induced by LPC could be independent of the increase in [Ca²⁺]_i, and the Ca²⁺ -independent cellular injury induced by LPC could be attenuated by d-propranolol, although the mechanism remains unknown.     

The electrical changes accompanying fertilization and cortical vesicle secretion in the medaka egg

The electrical properties of the egg of the medaka, Oryzias latipes, were studied before, during, and after fertilization. The resting potential of the unfertilized egg averaged ?39 ± 9 mV in Yamamoto's Ringers (Y. Ringers), but 20% of the values were between ?50 and ?60 mV. Fertilization triggers a small depolarization of 4 ± 3 mV in 10% Y. Ringers with an average duration of 20 ± 10 sec. The amplitude of this depolarization is independent of [Na⁺]_o, [Ca²⁺]_o, and [Cl^?]_o, so it appears to be due to a nonspecific leak triggered by sperm-egg fusion. The depolarization is followed by a longer hyperpolarizing phase with an average amplitude of 31 ± 12 mV. Recovery from this hyperpolarization has a fast phase lasting 155 ± 18 sec, followed by a slower phase which reaches a steady average membrane potential of ?19 ± 1 mV by 9 min after fertilization. The membrane resistance falls 10-fold during the first 2 min after fertilization, from 40 (1520 kΩ-cm²) to 3 MΩ. This is largely due to an increase in the K⁺ conductance. At the peak of the hyperpolarization, the membrane potential exhibits a 28 mV/decade [K⁺]_o dependence and a 6 mV/decade [Na⁺]_o dependence. The membrane resistance slowly recovers over the next 8 min to a value about 30% larger than before fertilization. The relation of current vs voltage was linear before, during, and after fertilization and indicated a reversal potential of ?98 ± 20 mV for the hyperpolarization peak. The egg's capacitance averaged 0.04 ± 0.01 μF (0.9 μF/cm²) before fertilization and approximately doubles within 90 sec after fertilization. It then decreases over a 9-min period, reaching a value 25% smaller than before fertilization.     

Fast Na+ channels and slow Ca2+ current in smooth muscle from pregnant rat uterus

Summary Smooth muscle cells normally do not possess fast Na²⁺ channels, but inward current is carried through two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Using whole-cell voltage clamp of single smooth muscle cells isolated from the longitudinal layer of 18-day pregnant rat uterus, depolarizing pusles, applied from a holding potential of –90 mV, evoked two types of inward current, fast and slow [8]. The fast inward current decayed within 30 ms, depended on [Na]₀, and was inhibited by TTX (K_0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]₀, and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na²⁺ channel current, and that the slow inward current is a Ca²⁺ channel current was not evident. Thus, the ion channels which generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na⁺ channels and dihudropuridine-sensitive slow Ca²⁺ channels. The number of fast Na⁺ channels increased during gestation [9]. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells which possess fast Na²⁺ channels, and it suggested that the fast Na⁺ current may be involved in spread of excitation. The Ca²⁺ channel current density also was higher during the latter half of gestation. These results indicate that the fast Na⁺ channels and Ca²⁺ slow channels in myometrium become more numerous as term approaches, and may facilitate parturition. Isoproterenol (beta-agonist) did not affect either I_Ca(s) or I_Na(f), whereas Mg²⁺ (K_0.5 of 12 mM) and nifedipine (K_0.5 of 3.3 nM) depressed I_Ca(s). Oxytocin had no effect on I_Na(f) and actually depressed I_Ca(s) to a small extect. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of I_Ca(s), whereas that of Mg²⁺ can be so explained. The stimulating action of oxytocin on uterine contractions is not due to stimulation of I_Ca(s).     

Membrane potential-dependent calcium transport in right-side-out plasma membrane vesicles from Zea mays L. roots

Right-side-out plasma membrane vesicles isolated from Zea mays roots were used to study membrane potential (ΔΨ)-dependent Ca²⁺ transport. Membrane potentials were imposed on the vesicles using either K⁺ concentration gradients and valinomycin or SCN concentration gradients, and the size of the imposed ΔΨ was measured with [¹⁴C]tetraphenylphosphonium. Uptake of ⁴⁵Ca²⁺ into the vesicles was stimulated by inside-negative ΔΨ. The rate of transport increased to a maximum at a ΔΨ of about -80 mV and then declined at more negative ΔΨ. When extravesicular Ca²⁺ concentration was varied, uptake was maximal in the range 100–200 μM Ca²⁺. Neither dihydropyridine nor phenylalkylamine Ca²⁺ channel blockers had any effect on Ca²⁺ uptake but 30 μM ruthenium red was completely inhibitory with half maximal inhibition at 10–15 μM ruthenium red. Calcium transport was also inhibited by inorganic cations. Zn²⁺, Gd³⁺ and Mg²⁺ inhibited by a maximum of 30% while La³⁺, Nd³⁺ and Mn²⁺ inhibited by 70%. The inhibitory effects of La³⁺ and Gd³⁺ were additive. Lanthanum-insensitive Ca²⁺ five Ca²⁺ transport was totally inhibited by 80 μM Gd³⁺ and showed maximum activity at a ΔΨ of -60 mV, with less uptake at both higher and lower ΔΨ. Lanthanum and Gd³⁺ also inhibited Ca²⁺ uptake into protoplasts isolated from Zea roots and their individual and combined effects were similar in extent to those observed with plasma membrane vesicles. It is concluded that maize root plasma membrane contains two Ca²⁺-permeable channels that can be distinguished by their susceptibility to inhibition by La³⁺ and Gd³⁺. Both are inhibited by ruthenium red but not by other organic Ca²⁺ channel blockers.     

Oxytocin induced a biphasic increase in the intracellular Ca2+ concentration of porcine myometrial cells: Participation of a pertussis toxin—insensitive G-protein,inositol 1,4,5-trisphosphate—sensitive Ca2+ pool,and Ca2+ channels

This study investigated the underlying mechanisms of oxytocin (OT)-induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in acutely dispersed myometrial cells from prepartum sows. A dosedependent increase in [Ca²⁺]_i was induced by OT (0.1 nM to 1 μM) in the presence and absence of extracellular Ca²⁺ ([Ca²⁺]_e). [Ca²⁺]_i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca²⁺]_e. However, in the absence of [Ca²⁺]_e, the [Ca²⁺]_i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca²⁺ ionophore. Administration of OT (1 μM) for 15 sec increased inositol 1,4,5-trisphosphate (IP₃) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 μg/ml) for 2 hr failed to alter the OT-induced increase in [Ca²⁺]_i and IP₃ formation. U-73122 (30 nM to 3 μM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca²⁺]_i by OT dose dependently. U-73122 (3 μM) also abolished the OT-induced IP₃ formation. Thapsigargin (2 μM), an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, did not increase [Ca²⁺]_i. However, it did time-dependently inhibit the OT-induced increase in [Ca²⁺]_i. Nimodipine (1 μM), a Voltage-dependent Ca²⁺ channel (VDCC) blocker, inhibited the OT-induced plateau by 26%. La³⁺ (1 μM), a nonspecific Ca²⁺ channel blocker, abrogated the OT-induced plateau. In whole-cell patch-clamp studies used to evaluate VDCC activities, OT (0.1 μM) increased Ca²⁺ Current (I_ca) by 40% with no apparent changes in the current-voltage relationship. The OT-induced increase in I_ca reached the maximum in 5 min, and the increase was abolished by nimodipine (1 μM). These results suggested that (1) activation of OT receptors in porcine myometrium evokes a cascade in the PTX-insensitive G-protein–PLC-IP₃ signal transduction, resulting in an increase in [Ca²⁺]_i; (2) the OT-induced increase in [Ca²⁺]_i is characterized by a biphasic pattern, in which the spike is predominately contributed by the intracellular Ca²⁺ release from the IP₃-sensitive pool, and to a lesser extent by Ca²⁺ influx, whereas the plateau is from increased Ca²⁺ influx; and (3) the influx is via VDCC and receptor-operated Ca²⁺ channels. © 1995 Wiley-Liss, Inc.     

Electrophysiological characterization of voltage-gated Na+ current expressed in the highly metastatic Mat-LyLu cell line of rat prostate cancer

Voltage-gated Na⁺ channels, classically associated with impulse conduction in excitable tissues, are also found in a variety of epithelial cell types where their possible functions are not known so well. We have previously reported expression of a voltage-gated Na⁺ channel specifically in the highly metastatic Mat-LyLu rat prostate cancer cell line; blockage of the current with tetrodotoxin (TTX) significantly reduced the invasiveness of the cells in vitro, suggesting that the channel may have a functional role in metastasis. The aim of the present study was to characterize this current using the whole-cell patch clamp recording technique, and compare it to Na⁺ currents found in various other tissues. The inward current of the Mat-LyLu cells was abolished completely, but reversibly, in Na⁺-free solution, confirming that Na⁺ was indeed the permeant ion. Activation occurred at −40 mV and currents reached a maximal amplitude at around 6 mV. Boltzmann fits to current activation and steady-state inactivation revealed that the currents were half activated at about −15 mV and half inactivated at −80 mV. Both current inactivation and recovery from inactivation followed a double-exponential time course with fast and slow components. The Na⁺ currents were highly sensitive to block by TTX (IC₅₀ ≃ 18 nM), whilst 1 μM μ-conotoxin GIIIA mostly had no effect. 100 μM Cd²⁺ also had no effect on the current, whilst 2.5 mM Cd²⁺, Mn²⁺, and Co²⁺ each caused a depolarizing shift in activation and a reduction in peak conductance of around 20%. In conclusion, the Na⁺ channel expressed in the highly metastatic Mat-LyLu cell line appeared to have electrophysiological and pharmacological properties of TTX-sensitive channels. Further work is needed, however, to elucidate the exact nature of the channel protein and the mechanism(s) of its involvement in cellular invasiveness. J. Cell. Physiol. 175:50–58, 1998. © 1998 Wiley-Liss, Inc.     

Effect of hydrocortisone and thyroxine on ATPase activities of neuronal and glial cell lines in culture

The effect of hydrocortisone and thyroxine, on the activities of Ca²⁺-and Mg²⁺-ATPase was studied in cultured neuronal (clone M1) and glial (clones NN and C6) cell lines. For M1 and NN cells an increase in Ca²⁺-and Mg²⁺-ecto-ATPase activity was found when the cells were cultured during 4–6 days in presence of hydrocortisone or together with thyroxine. In the same conditions, a decrease in Ca²⁺-and Mg²⁺-ecto-ATPase activity was found for the C6 cells. In C6 cells the effect of hormones was more pronounced for the Mg²⁺-than for the Ca²⁺-ecto-ATPase activity. The observed decrease may be related to the tumoral origin of the C6 cells. The activity of (Na⁺, K⁺)-ATPase in all three cell lines increased in presence of hydrocortisone or together with thyroxine when the cells were cultured during 4–6 days, in presence of the hormones, whereas the total Mg²⁺-ATPase activity increased only after 6 days of treatment. Thyroxine alone has very few effect either on Ca²⁺-and Mg²⁺-ecto-ATPase, or on (Na⁺, K⁺)-and total Mg²⁺-ATPase activity. These observations are interpreted to indicate that hormones may modulate or induce enzymatic activities involved in active transport phenomena in nervous tissue.     

Multi-target novel neuroprotective compound ITH33/IQM9.21 inhibits calcium entry, calcium signals and exocytosis

Compound ITH33/IQM9.21 (ITH/IQM) belongs to a new family of l-glutamic acid derivatives with antioxidant and neuroprotective properties on in vitro and in vivo models of stroke. Because neuronal damage after brain ischemia is tightly linked to excess Ca²⁺ entry and neuronal Ca²⁺ overload, we have investigated whether compound ITH/IQM antagonises the elevations of the cytosolic Ca²⁺ concentrations ([Ca²⁺]_c) and the ensuing exocytotic responses triggered by depolarisation of bovine chromaffin cells. In fluo-4-loaded cell populations, ITH/IQM reduced the K⁺-evoked [Ca²⁺]_c transients with an IC₅₀ of 5.31 μM. At 10 μM, the compound decreased the amplitude and area of the Ca²⁺ transient elicited by challenging single fura-2-loaded cells with high K⁺, by 40% and 80%, respectively. This concentration also caused a blockade of K⁺-induced catecholamine release at the single-cell level (78%) and cell populations (55%). These effects are likely due to blockade of the whole-cell inward Ca²⁺ currents (IC₅₀ = 6.52 μM). At 10 μM, ITH/IQM also inhibited the Ca²⁺-dependent outward K⁺ current, leaving untouched the voltage-dependent component of I_K. The inward Na⁺ current was unaffected. Inhibition of depolarisation-elicited Ca²⁺ entry, [Ca²⁺]_c elevation and exocytosis could contribute to the neuroprotective effects of ITH/IQM in vulnerable neurons undergoing depolarisation during brain ischemia.