scholarly journals 279 PLASMA MEMBRANE ELECTRICAL PROPERTIES AND INTRACELLULAR CALCIUM STORES IN IMMATURE AND IN VITRO-MATURED ADULT AND JUVENILE SHEEP OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 290 ◽  
Author(s):  
R. Boni ◽  
N. Cocchia ◽  
F. Silvestre ◽  
G. Tortora ◽  
R. Lorizio ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Electrical Properties ◽  
Intracellular Calcium ◽  
In Vitro Maturation ◽  
Calcium Currents ◽  
Resting Potential ◽  
Calcium Stores ◽  
Ca2 Channels

The low developmental efficiency recorded in juvenile oocytes represents, besides its technological relevance, an opportunity for increasing the knowledge of mechanisms regulating developmental competence in the oocytes. To analyze the biological reasons that make an adult oocyte different from a juvenile one, we monitored membrane electrical properties, i.e. resting potential, steady-state conductance and calcium currents, and calcium stores in these two oocyte types both at immature (GV) stage and after in vitro maturation (MII). Ovaries of cycling ewes and 40-day-old lambs were collected at abattoir and transported at 30°C. Cumulus-oocyte complexes (COC) were recovered by mincing. In vitro maturation was carried out in TCM199 supplemented with 10% fetal calf serum, 10 IU/mL of LH, 0.1 IU/mL of FSH, and 1 mg/mN of 17β-estradiol at 39.0°C in 5% CO2 for 24 h. Zona pellucida of immature and in vitro-matured oocytes was removed after incubation for 1–1.5 min in 0.5% (w/v) protease solution. Zona-free oocytes were placed in Ham F10 at 38.5°C and voltage clamped by standard techniques (Tosti et al. 2002 Reproduction 124, 835–846). After obtaining a giga-seal, the patch was ruptured. The permeability of the plasma membrane was verified by applying depolarizing and hyperpolarizing voltage steps of 10 mV and 500 ms before and at the peak current to generate the voltage-dependent currents. The voltage clamp was set at −80 and −30 mV to differentiate the Ca2+ current components, i.e. L-type Ca2+ channels. For intracellular calcium determinations, oocytes were placed in Ham F10 and injected with the 0.5 mM calcium green dextran (Mr 10,000). Ca2+ stores were evoked by the addition of 5 μM Ca2+ ionophore, monitored using a computer-controlled photo-multiplier system, and measured as relative fluorescence units (RFU) by normalizing fluorescence against baseline fluorescence. In lamb and ewe, differences in electrical features and calcium dynamics between GV (n = 36 and 17) and MII (n = 42 and 32) oocytes were tested by ANOVA and expressed as mean ± SEM. Resting potential was higher at MII than GV stages (−15.2 ± 0.9 vs. −12.1 ± 1.1 mV, respectively; P < 0.02) but it did not differ between animal age. GV stage and ewe showed either a higher steady-state conductance (25.4 ± 0.2 vs. 11.7 ± 0.2 nS and 21.7 ± 0.2 vs. 15.4 ± 0.2 nS, respectively; P < 0.01) or L-type Ca2+ channels (9.7 ± 1.4 vs. 2.7 ± 1.3 pA and 9.2 ± 1.5 vs. 3.2 ± 1.1 pA, respectively; P < 0.01). No differences were found between resting potential peaks yielded after Ca2+ ionophore exposure but a higher ion activation current was found in lamb oocytes (489 ± 56 vs. 300 ± 73 pA; P < 0.05). Ca2+ stores did not differ between animal age but they were larger at MII than at GV stage (0.70 ± 0.07 vs. 0.44 ± 0.07 RFU; P < 0.01). These results supply further information on both reproductive biology in ovine species and the physiology of oocytes collected from juvenile and adult individuals. This work was supported by Italian Ministry of University and Research (MIUR) COFIN 2002 Project.

scholarly journals Agonist-induced Ca2+ influx in human neutrophils is secondary to the emptying of intracellular calcium stores

1991 ◽  
Vol 277 (1) ◽  
pp. 73-79 ◽  
Author(s):  
M Montero ◽  
J Alvarez ◽  
J Garcia-Sancho
Keyword(s):  
Plasma Membrane ◽  
Intracellular Calcium ◽  
Time Course ◽  
Human Neutrophils ◽  
Calcium Stores ◽  
Free Medium ◽  
Prolonged Incubation ◽  
Intracellular Calcium Stores ◽  
Cytochrome P 450 ◽  
In Cells

Emptying of the intracellular calcium stores of human neutrophils, by prolonged incubation in Ca(2+)-free medium, by treatment with low concentrations of the Ca2+ inophore ionomycin, or by activation with cell agonists, increased the plasma-membrane permeability to Ca2+ and Mn2+. The chemotactic peptide formylmethionyl-leucyl-phenylalanine and the natural agonists platelet-activating factor and leukotriene B4 released different amounts of calcium from the stores and induced Ca2+ (Mn2+) uptake, the rate of which correlated inversely with the amount of calcium left in the stores. The increased Mn2+ uptake induced by these agonists was persistent in cells incubated in Ca(2+)-free medium, but returned to basal levels in cells incubated in Ca(2+)-containing medium, with the same time course as the refilling of the calcium stores. The calcium-stores-regulated Mn2+ influx, including that induced by agonists, was prevented by cytochrome P-450 inhibitors. We propose that agonist-induced Ca2+ (Mn2+) influx in human neutrophils is secondary to the emptying of the intracellular stores which, in turn, activates plasma-membrane Ca2+ channels by a mechanism involving microsomal cytochrome P-450, similar to that described previously in thymocytes [Alvarez, Montero & Garcia-Sancho (1991) Biochem. J. 274, 193-197].

Appearance and evolution of calcium currents and contraction during the early post-fusional stages of rat skeletal muscle cells developing in primary culture

Development ◽  
1993 ◽  
Vol 117 (3) ◽  
pp. 1153-1161
Author(s):  
C. Cognard ◽  
B. Constantin ◽  
M. Rivet-Bastide ◽  
N. Imbert ◽  
C. Besse ◽  
...  
Keyword(s):  
Primary Culture ◽  
Contractile Activity ◽  
Primary Cultures ◽  
Calcium Currents ◽  
Calcium Stores ◽  
Developmental Evolution ◽  
Time To Peak ◽  
Mechanical And Electrical Properties ◽  
Developing Muscle

Primary cultures from enzymatically dissociated satellite cells of newborn rat skeletal muscles enabled developmental in vitro studies of mechanical and electrical properties during the first steps of myogenesis. The present work focused on the appearance, evolution and roles of two types of calcium currents (ICa,T and ICa,L) and of depolarization-induced contractile activity during the early stages of muscle cell development in primary culture. Prefusional mononucleated cells (myoblasts), young myotubes of 1 day (with less than 10 nuclei) or 2–3 days (more than 9 nuclei) and myoballs from 4–6, 7–9, 10–12 and 13–16 days cultures were patch-clamped (whole-cell configuration), and calcium currents and contraction simultaneously recorded. Sodium but not calcium currents could be recorded at the myoblast stage. In young myotubes (1 day), ICa,L was present with high incidence as compared to ICa,T, which was poorly expressed. Contractile responses appeared at the next stage (2-3 days) while the occurrence of ICa,T progressively increased. This developmental evolution of the calcium currents and contraction expression was accompanied by some changes in their characteristics: the ICa,T/ICa,L amplitudes ratio progressively increased and the time-to-peak of contraction progressively decreased with the age of myoballs. Physiological functions for calcium currents in developing muscle are suggested and discussed: ICa,T, which is transiently expressed, could be involved in the pacemaker-like activity while ICa,L could serve as an early contraction triggering mechanism and/or initially to fill and then to maintain the intracellular calcium stores.

Cytosolic Ca2+ dynamics in hamster ascending thin limb of Henle's loop

1995 ◽  
Vol 268 (6) ◽  
pp. F1148-F1153 ◽  
Author(s):  
N. Takahashi ◽  
Y. Kondo ◽  
O. Ito ◽  
Y. Igarashi ◽  
K. Omata ◽  
...  
Keyword(s):  
Steady State ◽  
Intracellular Calcium ◽  
State Level ◽  
Steady State Level ◽  
Subsequent Reduction ◽  
Fura 2 ◽  
Henle's Loop ◽  
K Concentration ◽  
Thin Limb

Intracellular calcium plays an important role in the regulation of Cl- reabsorption in the ascending thin limb of Henle's loop (ATL). To elucidate the cytosolic Ca2+ dynamics in the ATL, intracellular Ca2+ concentration activity ([Ca2+]i) was measured in the in vitro microperfused hamster ATL using fura 2. Basal [Ca2+]i was 89.1 +/- 7.3 nM (n = 9 tubules). Removal of Ca2+ from the peritubular solution decreased [Ca2+]i from 89.1 +/- 7.3 to 64.1 +/- 7.1 nM in 2 min (n = 9, P < 0.05), whereas [Ca2+]i did not change after removal of Ca2+ from the luminal solution. Addition of 1 mM NaCN to the bath increased [Ca2+]i. This effect was completely abolished by the elimination of ambient Ca2+. Trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) in the bath reversibly increased [Ca2+]i, whereas addition of 1 mM ouabain to the bath decreased [Ca2+]i. Rates of changes in [Ca2+]i after removal and replacement of basolateral Ca2+ were not affected by removal of Na+, K+, or Cl- from the bath, whereas nicardipine decreased these parameters. Increasing bath K+ from 5 to 100 mM decreased [Ca2+]i from 69.3 +/- 5.8 to 50.8 +/- 5.0 nM in 1 min (n = 6, P < 0.05). Subsequent reduction of K+ from 100 to 5 mM increased [Ca2+]i to 174.0 +/- 30.8 nM in 1 min, followed by a gradual decrease in [Ca2+]i to a steady-state level of 74.2 +/- 8.0 nM in 2 min. Changes in basolateral K+ concentration did not affect [Ca2+]i in the absence of ambient Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Calcium efflux and cycling across the synaptosomal plasma membrane

1985 ◽  
Vol 226 (1) ◽  
pp. 225-231 ◽  
Author(s):  
R Snelling ◽  
D Nicholls
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Potential Gradient ◽  
Electrochemical Potential ◽  
Electrochemical Gradient ◽  
Calcium Efflux ◽  
Normal Medium ◽  
Voltage Dependent ◽  
Synaptosomal Plasma Membrane ◽  
Ca2 Channels

Ca2+ efflux from intact synaptosomes is investigated. Net efflux can be induced by returning synaptosomes from media with elevated Ca2+ or high pH to a normal medium. Net Ca2+ efflux is accelerated when the Na+ electrochemical potential gradient is collapsed by veratridine plus ouabain. Under steady-state conditions at 30 degrees C, Ca2+ cycles across the plasma membrane at 0.38 nmol . min-1 . mg-1 of protein. Exchange is increased by 145% by veratridine plus ouabain, both influx and efflux being increased. Increased influx is probably due to activation of voltage-dependent Ca2+ channels, since it is abolished by verapamil. The results indicate that, at least under conditions of low Na+ electrochemical gradient, some pathway other than a Na+/Ca2+ exchange must operate in the plasma membrane to expel Ca2+.

scholarly journals Dexamethasone stimulates store-operated calcium entry and protein degradation in cultured L6 myotubes through a phospholipase A2-dependent mechanism

2010 ◽  
Vol 298 (5) ◽  
pp. C1127-C1139 ◽  
Author(s):  
Kiyoshi Itagaki ◽  
Michael Menconi ◽  
Bozena Antoniu ◽  
Qin Zhang ◽  
Patricia Gonnella ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Protein Degradation ◽  
Intracellular Calcium ◽  
Muscle Wasting ◽  
Muscle Protein ◽  
Calcium Entry ◽  
Calcium Stores ◽  
Fluorescence Measurements ◽  
Store Operated Calcium Entry

Muscle wasting in various catabolic conditions is at least in part regulated by glucocorticoids. Increased calcium levels have been reported in atrophying muscle. Mechanisms regulating calcium homeostasis in muscle wasting, in particular the role of glucocorticoids, are poorly understood. Here we tested the hypothesis that glucocorticoids increase intracellular calcium concentrations in skeletal muscle and stimulate store-operated calcium entry (SOCE) and that these effects of glucocorticoids may at least in part be responsible for glucocorticoid-induced protein degradation. Treatment of cultured myotubes with dexamethasone, a frequently used in vitro model of muscle wasting, resulted in increased intracellular calcium concentrations determined by fura-2 AM fluorescence measurements. When SOCE was measured by using calcium “add-back” to muscle cells after depletion of intracellular calcium stores, results showed that SOCE was increased 15–25% by dexamethasone and that this response to dexamethasone was inhibited by the store-operated calcium channel blocker BTP2. Dexamethasone treatment stimulated the activity of calcium-independent phospholipase A2(iPLA2), and dexamethasone-induced increase in SOCE was reduced by the iPLA2inhibitor bromoenol lactone (BEL). In additional experiments, treatment of myotubes with the store-operated calcium channel inhibitor gadolinium ion or BEL reduced dexamethasone-induced increase in protein degradation. Taken together, the results suggest that glucocorticoids increase calcium concentrations in myocytes and stimulate iPLA2-dependent SOCE and that glucocorticoid-induced muscle protein degradation may at least in part be regulated by increased iPLA2activity, SOCE, and cellular calcium levels.

scholarly journals Cat Heart Muscle in Vitro

1962 ◽  
Vol 46 (2) ◽  
pp. 189-199 ◽  
Author(s):  
Ernest Page
Keyword(s):  
Steady State ◽  
Heart Muscle ◽  
Potential Difference ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Papillary Muscles ◽  
K Concentration ◽  
Cat Heart ◽  
External K

The steady state transmembrane resting potential difference (Vm) has been measured in quiescent papillary muscles. Vm was determined as a function of the external K concentration in Cl and SO4 solutions and compared with the K equilibrium potential. Other measurements were made after replacement of external Na by choline, K by Rb and Cs, and Cl by SO4, CH3SO4, and NO3. Effects on Vm of albumin, temperature, and variation in internal K concentration are described.

Effect of procaine on electrical properties of squid axon membrane

1959 ◽  
Vol 196 (5) ◽  
pp. 1071-1078 ◽  
Author(s):  
Robert E. Taylor
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Electrical Properties ◽  
Potassium Conductance ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Squid Axon ◽  
Voltage Step ◽  
Axon Membrane ◽  
Rate Of Development

Procaine (0.025–0.1%; pH 7.9) caused a reduction in the amount and rate of development of the early transient (sodium) and late steady state (potassium) currents which occur during a depolarizing voltage step applied to the excised, voltage clamped squid axon. Consistent results were obtained by holding the membrane potential at a hyperpolarized value prior to the applied step. No effect was seen on the resting potential, on the sodium equilibrium potential, or on the proportion of the sodium carrying system which was ‘inactive’ at any membrane potential. The blocking action of procaine is a result of the inhibition by the drug of the sodium carrying system. The effect of procaine on the potassium conductance is such as to oppose the blocking action.

scholarly journals The Calcium Binding Loops of the Cytosolic Phospholipase A2 C2 Domain Specify Targeting to Golgi and ER in Live Cells

2004 ◽  
Vol 15 (1) ◽  
pp. 371-383 ◽  
Author(s):  
John H. Evans ◽  
Stefan H. Gerber ◽  
Diana Murray ◽  
Christina C. Leslie
Keyword(s):  
Plasma Membrane ◽  
Intracellular Calcium ◽  
Calcium Binding ◽  
Structural Information ◽  
Lipid Binding ◽  
Live Cells ◽  
C2 Domain ◽  
Cytosolic Phospholipase

Translocation of cytosolic phospholipase A2 (cPLA2) to Golgi and ER in response to intracellular calcium mobilization is regulated by its calcium-dependent lipid-binding, or C2, domain. Although well studied in vitro, the biochemical characteristics of the cPLA2C2 domain offer no predictive value in determining its intracellular targeting. To understand the molecular basis for cPLA2C2 targeting in vivo, the intracellular targets of the synaptotagmin 1 C2A (Syt1C2A) and protein kinase Cα C2 (PKCαC2) domains were identified in Madin-Darby canine kidney cells and compared with that of hybrid C2 domains containing the calcium binding loops from cPLA2C2 on Syt1C2A and PKCαC2 domain backbones. In response to an intracellular calcium increase, PKCαC2 targeted plasma membrane regions rich in phosphatidylinositol-4,5-bisphosphate, and Syt1C2A displayed a biphasic targeting pattern, first targeting phosphatidylinositol-4,5-bisphosphate-rich regions in the plasma membrane and then the trans-Golgi network. In contrast, the Syt1C2A/cPLA2C2 and PKCαC2/cPLA2C2 hybrids targeted Golgi/ER and colocalized with cPLA2C2. The electrostatic properties of these hybrids suggested that the membrane binding mechanism was similar to cPLA2C2, but not PKCαC2 or Syt1C2A. These results suggest that primarily calcium binding loops 1 and 3 encode structural information specifying Golgi/ER targeting of cPLA2C2 and the hybrid domains.

Visual adaptation modulates a potassium conductance in retinular cells of the crayfish

2000 ◽  
Vol 17 (3) ◽  
pp. 353-368 ◽  
Author(s):  
C.S. MILLER ◽  
R.M. GLANTZ
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Light Exposure ◽  
Potassium Conductance ◽  
Visual Sensitivity ◽  
Calcium Currents ◽  
Resting Potential ◽  
Delayed Rectifier ◽  
Dependent Change ◽  
Retinular Cells

Crayfish photoreceptors exhibit a voltage-dependent potassium conductance, GK, that is generally similar to the delayed rectifier channel described in neurons and other arthropod retinular cells. GK activation (i.e. the apparent threshold, Vth) occurs near the resting potential and GK is substantially reduced by 25 mM extracellular tetraethylammonium (TEA) and by intracellular Cs+ injections. Light exposure, sufficient to reduce visual sensitivity 100-fold, increases Vth (shifts it in the depolarizing direction) by about 20 mV. The light-dependent change in Vth does not depend upon the corresponding increase (depolarization) of the steady-state membrane potential nor does it depend upon inward calcium currents. Vth is slightly influenced by fluctuations in Ko associated with the light-elicited currents. During light exposure Ko (measured with K+-sensitive electrodes) increases by 2.1 mM (equivalent to an 8 mV increase in EK). This increase in EK makes only a modest contribution to the light-dependent change in Vth as determined by perfusion with high potassium salines. Intracellular calcium injections increase Vth by 10 to 20 mV and reduce visual sensitivity by 5- to 10-fold. The results imply that during exposure to high levels of illumination, K+ currents at the steady-state membrane potential are diminished by a calcium-dependent change in GK gating and, to a smaller degree, by a reduced K+ concentration gradient. It is notable that Ca2+ appears to inhibit both GK and the light-elicited conductance from both inside and outside the plasma membrane. As a consequence of the light-dependent change in Vth, GK makes only modest contributions to the changes in sensitivity and speed normally associated with light adaption. These functions are regulated by the transduction pathway and are revealed at the resting potential in the time course and magnitude of the light-elicited currents.

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