Change in the activity of calcium ions during illumination of a suspension of visual cell outer segment fragments]

Changes in the activity of calcium ions in the medium containing outer fragments suspension of bovine eye retina rods have been studied by the method of calcium-selective electrodes. Illumination of the suspension increases calcium ion activity in the incubation medium. Photoinduced yield of calcium ions depends on Ca+2 concentration: it equals 0.11+/-0.015 M Ca2+/1m rodopsin in the medium containing 0.1 mM CaCl2 and 0.046+/-0.002Ca2+/1M rodopsin in the medium containing 0.05 mM CaCl2. In the medium containing more than 10(-4) M CaCl2 both an increase and a decrease of Ca2+ ions have been observed.     

Cooperative phenomena in the membrane potential of parathyroid cells induced by divalent cations

Membrane potentials of mouse parathyroid cells were measured by means of the intracellular microelectrode method. The membrane potential in external Krebs solution containing 2.5 mM of Ca++ was -23.6 +/- 0.4 mV (mean +/- standard error of mean). The low concentration of Ca++ (1.0 mM) caused hyperpolarization of the membrane potential to -61.7 +/- 0.8 mV. The membrane potential was proportional to the logarithm of the concentration of K ion in the solution of low Ca ion. The concentration of external Na+, C1- and HPO4-- had no effect on the membrane potential. The sigmoidal transition of membrane potentials was induced by the change of Ca ion concentration in the range from 2.5 to 1.0 mM. The change of the membrane potentials in low Ca ion is originated from increase in potassium permeability of the cell membrane. The similar sigmoidal changes of the membrane potentials were observed in the solution containing 4 to 3 mM of Sr ion. The Mg and Ba ion showed smaller effect on the membrane potential. The Goldman equation was extended to divalent ions. Appling the extended membrane potential equation, ratios of the permeability coefficients were obtained as follows: PK/PCa = 0.067 for 2.5 mM Ca++, 0.33 for 1.0 mM Ca++; PK/PSr = 0.08 for 4 mM Sr++ and 0.4 for 3 mM Sr++; PK/PMg = 0.5; PK/PBa = 0.67 for all range of concentration. The Hill constants of Sr ion and Ca ion were 20; the relationship between Sr ion and Ca ion was competitive. The Hill constants of Mg and Ba ion were 1 each. The Hill constant of Ca ion was depend of the temperature; nmax = 20 at 36 degrees C, n = 9 at 27 degrees C, n = 2 at 22 degrees C. The enthalpy of Ca-binding reaction was obtained from the Van't Hoff plot as 0.58 kcal. The activation energies of the K+ permeability increase were obtained from the Arrhenius plots as 3.3 kcal and 4 kcal. The difference, 0.7 kcal, corresponds to the enthalpy change of this reaction, of which value is close to that of the Ca-binding reaction.     

Slow membrane potential changes in skeletal muscle induced in Cl(-)-free medium.

1. Conventional microelectrode techniques were used to measure simultaneous changes in membrane potential (Vm) and conductance (Gm) induced by single electrical stimuli in muscles bathed in Cl(-)-free solution containing 40 mM of tetraethylammonium (TEA+). 2. Stimulation induced slow transient depolarizations (slow response) accompanied by increased calcium conductance, while the potassium conductance was first elevated and later reduced. 3. Stepwise elevation of [K+]0 from 2.5 to 5 or 10 mM during the slow response evoked an abrupt repolarization of 42.3 +/- 8.9 mV (n = 4; p less than 0.001), and 24.8 +/- 3.5 +/- mV (n = 5; p less than 0.001), respectively, while Gm was increased to 1.45 +/- 0.25-fold (n = 5; p less than 0.05). Neither the slow response nor K(+)-induced changes in Vm or Gm were sensitive to tetrodotoxin (3 microM), however, nifedipine (10 microM) abolised the slow response. 4. It was concluded that beyond the increase of calcium conductance, the ionic conductance of the inward rectifier K+ channel was reduced during the slow response, which could be restored by the elevation of [K+]0. The results suggest the possible contribution of these mechanisms to the electrical instability of myotonic muscles. Potential therapeutic consequences are discussed.     

Ion concentration-dependence of rat cardiac unitary L-type calcium channel conductance

Little is known about the native properties of unitary cardiac L-type calcium currents (i(Ca)) measured with physiological calcium (Ca) ion concentration, and their role in excitation-contraction (E-C) coupling. Our goal was to chart the concentration-dependence of unitary conductance (gamma) to physiological Ca concentration and compare it to barium ion (Ba) conductance in the absence of agonists. In isolated, K-depolarized rat myocytes, i(Ca) amplitudes were measured using cell-attached patches with 2 to 70 mM Ca or 2 to 105 mM Ba in the pipette. At 0 mV, 2 mM of Ca produced 0.12 pA, and 2 mM of Ba produced 0.19 pA unitary currents. Unitary conductance was described by a Langmuir isotherm relationship with a maximum gammaCa of 5.3 +/- 0.2 pS (n = 15), and gammaBa of 15 +/- 1 pS (n = 27). The concentration producing half-maximal gamma, Kd(gamma), was not different between Ca (1.7 +/- 0.3 mM) and Ba (1.9 +/- 0.4 mM). We found that quasi-physiological concentrations of Ca produced currents that were as easily resolvable as those obtained with the traditionally used higher concentrations. This study leads to future work on the molecular basis of E-C coupling with a physiological concentration of Ca ions permeating the Ca channel.     

Calcium specificity of the antigen-induced channels in rat basophilic leukemia cells

Ion channels, activated upon IgE-Fc epsilon receptor aggregation by specific antigen, were studied in micropipet-supported lipid bilayers. These bilayers were reconstituted with purified IgE-Fc epsilon receptor complex and the intact 110-kDa channel-forming protein, both isolated from plasma membranes of rat basophilic leukemia cells (line RBL-2H3). In order to identify the current carrier through these ion channels and to determine their ion selectivity, we investigated the currents flowing through the IgE-Fc epsilon receptor gated channels in the presence of a gradient of Ca2+ ions. Thus, the solution in which the micropipet-supported bilayer was immersed contained 1.8 mM CaCl2, while the interior of the micropipet contained 0.1 microM Ca2+ (buffered with EGTA). Both solutions also contained 150 mM of a monovalent cation chloride salt (either K+ or Na+). The currents induced upon specific aggregation of the IgE (by either antigen or anti-IgE antibodies) were examined over a range of potentials imposed on the bilayer. The type of conductance event most frequently observed under the employed experimental conditions was a channel that has a slope conductance of 3 pS and a reversal potential practically identical with the calculated value for the reversal potential of calcium (134 +/- 11 mV in the presence of sodium, 125 +/- 13 mV in the presence of potassium). These results indicate that this channel is highly selective for calcium against the monovalent cations sodium and potassium. This same channel has a conductance of 4-5 pS in the presence of symmetrical solutions containing only 100 mM CaCl2 and 8 pS in the presence of 0.5 M NaCl with no calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potassium current activated by depolarization of dissociated neurons from adult guinea pig hippocampus

Currents were generated by depolarizing pulses in voltage-clamped, dissociated neurons from the CA1 region of adult guinea pig hippocampus in solutions containing 1 microm tetrodotoxin. When the extracellular potassium concentration was 100 mM, the currents reversed at -8.1 +/- 1.6 mV (n = 5), close to the calculated potassium equilibrium potential of -7 mV. The currents were depressed by 30 mM tetraethylammonium in the extracellular solution but were unaffected by 4-aminopyridine at concentrations of 0.5 or 1 mM. It was concluded that the currents were depolarization-activated potassium currents. Instantaneous current-voltage curves were nonlinear but could be fitted by a Goldman-Hodgkin-Katz equation with PNa/PK = 0.04. Conductance-voltage curves could be described by a Boltzmann-type equation: the average maximum conductance was 65.2 +/- 15.7 nS (n = 9) and the potential at which gK was half-maximal was -4.8 +/- 3.9 mV (mean +/- 1 SEM, n = 10). The relationship between the null potential and the extracellular potassium concentration was nonlinear and could be fitted by a Goldman-Hodgkin-Katz equation with PNa/PK = 0.04. The rising phase of potassium currents and the decay of tail currents could be fitted with exponentials with single time constants that varied with membrane potential. Potassium currents inactivated to a steady level with a time constant of approximately 450 ms that did not vary with potential. The currents were depressed by substituting cobalt or cadmium for extracellular calcium but similar effects were not obtained by substituting magnesium for calcium.     

Inositol 1,4,5-trisphosphate increases myoplasmic [Ca2+] in isolated muscle fibers. Depolarization enhances its effects

Inositol 1,4,5-trisphosphate (InsP3) has been proposed as an intracellular messenger which mobilizes calcium from the sarcoplasmic reticulum, during excitation-contraction coupling in skeletal muscle. We have measured the myoplasmic free calcium concentration ([Ca2+]i) by means of calcium selective microelectrodes in intact fibers isolated from Leptodactylus insularis microinjected with InsP3. In muscle fibers bathed in normal Ringer, the mean resting [Ca2+]i was 0.11 +/- 0.01 microM (M +/- SEM, n = 30). The microinjection of 0.3, 0.5 and 1 microM InsP3 induced transient increments in the [Ca2+]i to 0.35 +/- 0.02 microM (n = 9), to 0.53 +/- 0.03 microM (n = 11) and 0.94 +/- 0.06 microM (n = 10) respectively. Microinjection of 0.3, 0.5 and 1 microM InsP3 in muscle fibers incubated in low Ca2+ solution induced increments in [Ca2+]i similar to those observed in fibers bathed with normal Ringer. The microinjection of 0.3, 0.5 and 1 microM InsP3 in muscle fibers partially depolarized with 10 mM [K+]o induced transient enhancements of the resting [Ca2+]i that were greater than the transients observed in the normally polarized muscle. In partially depolarized fibers microinjected with 0.3, 0.5 and 1 microM InsP3, the [Ca2+]i was changed to 1.45 +/- 0.14 microM (n = 20), to 3.37 +/- 0.34 microM (n = 7) and to 7.43 +/- 0.70 microM (n = 6) respectively. In all partially depolarized fibers these increments in [Ca2+]i were associated with local contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single alpha-subunit isoform of about 110 kDa. The (Na+, K+)-ATPase hydrolyzed ATP with Michaelis Menten kinetics with K5, = 165+/-5 microM and Vmax = 686.1+/-24.7 U mg(-1). Stimulation by potassium (K0.5 = 2.4+/-0.1 mM) and magnesium ions (K0.5 = 0.76+/-0.03 mM) also obeyed Michaelis-Menten kinetics, while that by sodium ions (K0.5 = 6.0+/-0.2 mM) exhibited site site interactions (n = 1.6). Ouabain (K0.5 = 61.6+/-2.8 microM) and vanadate (K0.5 = 3.2+/-0.1 microM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A. These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium.     

Bicarbonate and ammonia changes in brain during spreading depression

An alkaline, followed by an acid-going transient, characterizes acid-base changes in the interstitial space during spreading depression in a variety of brain structures. In rat, such changes are associated with a significant rise in brain lactate content. How brain proton buffers behave during spreading depression is unknown. Techniques to significantly improve the response time of gas permeable membrane semimicroelectrodes for carbon dioxide and ammonia are reported. Measurements with such electrodes, when coupled to measurements of hydrogen ion concentration (from microelectrodes), permit rapid changes to be determined in bicarbonate concentration or ammonia and ammonium ion concentration, respectively. Bicarbonate concentration fell from 30 +/- 1 (n = 16) to 14 +/- 1 mM (n = 16) during spreading depression. On the other hand, ammonia concentration rose from 2.3 +/- 0.1 to 4.4 +/- 0.3 microM (n = 17) while ammonium ion concentration rose from 116 +/- 11 (n = 17) to 382 +/- 30 microM (n = 17) during spreading depression. Bicarbonate changes probably reflect titration of brain bicarbonate stores by accumulated lactic acid. Similar physicochemical changes do not explain the rise in ammonia and ammonium ion concentrations. Instead, elevation of the latter can only result from an increase in ammonia content of the interstitial space.     

Regulation of cytosolic sodium ion activity in frog sartorius

To explore the regulation of cytosolic sodium ion activity in the frog sartorius, we used Na(+)-selective microelectrodes to monitor intracellular sodium ion activity in situations of lowering external sodium concentration and elevating external potassium concentration. Reductions of 20%, 40%, 60% and 80% in extracellular sodium concentration produced slight but statistically insignificant changes in the membrane potential of the muscle. However, cytosolic sodium ion activity decreased significantly from 10.0 +/- 1.1 mM to 7.8 +/- 1.1 mM, 7.1 +/- 1.4 mM, 6.5 +/- 1.2 mM and 5.9 +/- 1.1 mM, respectively. In addition, elevation of the external potassium concentration from 2 mM to 12 mM, 32 mM and 62 mM caused respective stepwise depolarization of membrane potential from -87.2 +/- 1.6 mV to -62.4 +/- 3.6 mV, -45.4 +/- 3.0 mV, -27.2 +/- 1.8 mV. Under these conditions, the cytosolic sodium ion activity decreased from 10.5 +/- 1.4 mM to 7.3 +/- 1.6 mM, 6.4 +/- 1.1 mM and 5.2 +/- 0.8 mM, respectively. The results illustrate that the net sodium flux is out of cell either in the reduction of sodium chemical gradient or in the potassium depolarization across the cell membrane.     

Modulation by ammonium ions of gill microsomal (Na+,K+)-ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion

The modulation by Na(+), K(+), NH(4)(+) and ATP of the (Na(+),K(+))-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V=35.4+/-2.1 Umg(-1) and K(0.5)=54.0+/-3.6 nM, obeying cooperative kinetics (n(H)=3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis-Menten kinetics with K(M)=55.0+/-3.0 microM and V=271.5+/-17.2 Umg(-1). This is the first demonstration of a crustacean (Na(+),K(+))-ATPase with two ATP hydrolyzing sites. Stimulation by sodium (K(0.5)=5.80+/-0.30 mM), magnesium (K(0.5)=0.48+/-0.02 mM) and potassium ions (K(0.5)=1.61+/-0.06 mM) exhibited site-site interactions, while that by ammonium ions obeyed Michaelis-Menten kinetics (K(M)=4.61+/-0.27 mM). Ouabain (K(I)=147.2+/-7.microM) and orthovanadate (K(I)=11.2+/-0.6 microM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Na(+),K(+))-ATPase activity by ammonium ions, and the excretion of NH(4)(+) in benthic crabs are discussed.     

Mineralocorticosteroid receptor of the chick intestine. Oligomeric structure and transformation

The binding of [3H]aldosterone in the chick intestine cytosol was analyzed in terms of affinity and specificity. In this tissue, aldosterone binds to the mineralocorticosteroid receptor, with a high affinity (Kd approximately 0.3 nM) and low capacity (approximately 50 fmol/mg protein), and to the glucocorticosteroid receptor. The selective labeling of the mineralocorticosteroid receptor was achieved by incubating the cytosol with [3H]aldosterone in the presence of RU 486. This synthetic steroid completely inhibited the binding of [3H]aldosterone to the glucocorticosteroid receptor and did not bind to the mineralocorticosteroid receptor. The oligomeric structure of the mineralocorticosteroid receptor was studied by using BF4, a monoclonal antibody which reacts with the 90-kDa heat shock protein (hsp 90), a nonhormone-binding component of nontransformed steroid receptors. The mineralocorticosteroid receptor sedimented at 8.5 +/- 0.4 S (n = 8) in a 15-40% glycerol gradient. This peak was shifted to 11.2 +/- 0.6 S (n = 5) after incubation with BF4, indicating that, in the cytosol, hsp 90 was associated with the mineralocorticosteroid receptor. Dissociation of the complex was observed on gradients containing 0.4 M KCl, as judged by the absence of displacement by BF4 of the 4.3 +/- 0.4 S (n = 10) peak. The effect of molybdate and tungstate ions, and of dimethyl pimelimidate, an irreversible cross-linking agent, on the stability of the hsp 90-receptor complex was investigated. Complexes recovered in the presence of 20 mM molybdate ions dissociated on gradients containing 0.4 M KCl (5.2 +/- 0.6 S (n = 4), whereas complexes prepared in the presence of 20 mM tungstate ions sedimented at 8.5 +/- 0.4 S (n = 7). Similarly, complexes prepared in the presence of molybdate ions dissociated during high pressure liquid chromatography (HPLC) gel filtration analysis performed in 0.4 M KCl (RS (Stokes radius) = 3.9 +/- 0.5 nm (n = 3) versus 7.3 +/- 0.2 nm (n = 3) in the presence of 20 mM molybdate ions), whereas complexes prepared in the presence of tungstate ions did not dissociate (RS = 6.9 +/- 0.2 nm (n = 3]. As observed for the tungstate-stabilized receptor, the cross-linked receptor dissociated neither on gradient containing 0.4 M KCl (9.5 +/- 0.1 S (n = 3] nor during HPLC performed in 0.4 M KCl (RS = 6.5 +/- 0.3 (n = 4]. Furthermore, the cross-linked receptor was more resistant to the inactivating effect of urea on aldosterone binding than the noncross-linked receptor prepared in the presence of either molybdate or tungstate ions.     

Internal Na+ and Mg2+ blockade of DRK1 (Kv2.1) potassium channels expressed in Xenopus oocytes. Inward rectification of a delayed rectifier

Delayed rectifier potassium channels were expressed in the membrane of Xenopus oocytes by injection of rat brain DRK1 (Kv2.1) cRNA, and currents were measured in cell-attached and inside-out patch configurations. In intact cells the current-voltage relationship displayed inward going rectification at potentials > +100 mV. Rectification was abolished by excision of membrane patches into solutions containing no Mg2+ or Na+ ions, but was restored by introducing Mg2+ or Na+ ions into the bath solution. At +50 mV, half- maximum blocking concentrations for Mg2+ and Na+ were 4.8 +/- 2.5 mM (n = 6) and 26 +/- 4 mM (n = 3) respectively. Increasing extracellular potassium concentration reduced the degree of rectification of intact cells. It is concluded that inward going rectification resulting from voltage-dependent block by internal cations can be observed with normally outwardly rectifying DRK1 channels.     

Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca++

Gap junctional conductance (gj) between cardiac ventricular myocyte pairs is rapidly, substantially, and reversibly reduced by sarcoplasmic acidification with CO2 when extracellular calcium activity is near physiological levels (1.0 mM CaCl2 added; 470 microM Ca++). Intracellular calcium concentration (Cai), measured by fura-2 fluorescence in cell suspensions, was 148 +/- 39 nM (+/- SEM, n = 6) and intracellular pH (pHi), measured with intracellular ion-selective microelectrodes, was 7.05 +/- 0.02 (n = 5) in cell pair preparations bathed in medium equilibrated with air. Cai increased to 515 +/- 12 nM (n = 6) and pHi decreased to 5.9-6.0 in medium equilibrated with 100% CO2. In air-equilibrated low-calcium medium (no added CaCl2; 2-5 microM Ca++), Cai was 61 +/- 9 nM (n = 13) at pHi 7.1. Cai increased to only 243 +/- 42 nM (n = 9) at pHi 6.0 in CO2-equilibrated low-calcium medium. Junctional conductance, in most cell pairs, was not substantially reduced by acidification to pHi 5.9-6.0 in low-calcium medium. Cell pairs could still be electrically uncoupled reversibly by the addition of 100 microM octanol, an agent which does not significantly affect Cai. In low-calcium low-sodium medium (choline substitution for all but 13 mM sodium), acidification with CO2 increased Cai to 425 +/- 35 nM (n = 11) at pHi 5.9-6.0 and gj was reduced to near zero. Junctional conductance could also be reduced to near zero at pHi 6.0 in low-calcium medium containing the calcium ionophore, A23187. The addition of the calcium ionophore did not uncouple cell pairs in the absence of acidification. In contrast, acidification did not substantially reduce gj when intracellular calcium was low. Increasing intracellular calcium did not appreciably reduce gj at pHi 7.0. These results suggest that, although other factors may play a role, H+ and Ca++ act synergistically to decrease gj.     

Coupling between transmembrane calcium transport and membrane potential in retinal rod discs

Changes in the transmembrane potential of bovine rod discs were studied by use of the potential-sensitive fluorescence probes diS-C3-(5) and diBA-C4-(5). The disc membrane was shown to be impermeable to potassium ions. Their concentration in the disc is as high as 2.1 +/- 0.3 mM. The permeability of the disc membrane to Ca2+ was shown to be selective. The accumulation and release of Ca2+ were found to be accompanied by the generation of inside positive and inside negative transmembrane potentials, respectively. The uptake of Ca2+ in the discs may operate against the concentration gradient of the ion. The value of the potential developed is directly proportional to the logarithm of free Ca2+ concentration in the medium (delta phi m = 11.2 +/- 1.6 mV at 4.85 microM Ca2+fr). The accumulation of Ca2+ is decreased by sodium ions and totally inhibited by monensin. This indicates that a Na-Ca exchange process participates in Ca2+ uptake of photoreceptor discs.     

K+-selective microelectrode study of internally dialyzed squid giant axons.

Intracellular potassium activity, (aK)i, and axoplasmic K+ concentration, [K+]i, were measured by means of K+-selective microelectrodes and atomic absorption spectroscopy, respectively, in squid giant axons dialyzed with K+-free dialysis solution and bathed in K+-free artificial sea water. (aK)i measurements indicated that axoplasmic free K+ could be depleted by dialysis, whereas [K+]i measurements on axoplasm extruded from these axons suggest substantial retention of K+ (15.5 +/- 1.7 mmol/kg axoplasm K+; n = 9). In comparison, [K+]i in axoplasm extruded from freshly dissected axons was 330 +/- 16 mmol/kg axoplasm (n = 6). These data suggest that approximately 5% of the axoplasmic K+ ions are not easily removed by dialysis and that these ions are either bound to macromolecular sites or sequestered into membrane-enclosed organelles.     

The effect of oxygen tension on porcine embryonic development is dependent on embryo type

Reducing oxygen concentration from atmospheric levels during in vitro culture generally, but not invariably, improves embryonic development across a range of species. Since the few published reports of such an action in the pig are contradictory--perhaps a consequence of the derivation of the embryos prior to culture--a study was performed to examine the effect of O2 tension during culture on three different types of porcine embryos, namely: in vivo flushed embryos, and in vitro matured oocytes either fertilized in vitro or parthenogenetically activated. In vivo embryos (n=208) were flushed at the 2-8 cell stage. Cumulus oocyte complexes (COCs) destined for IVF or parthenogenetic activation were derived from 2 to 6 mm, post-pubertal ovarian follicles and matured for 48 h in TCM-199. Parthenogenones were generated by activating denuded oocytes (n=573) with 10 mM calcium ionophore, followed by 2 mM DMAP prior to culture. The IVF embryos (n=971) were produced by fertilizing COCs (day 0) with fresh ejaculated semen in modified tris-based medium for 6 h before cumulus removal. All embryos were cultured in BECM-3 containing 12 mg/mL fatty-acid-free BSA up to day 4, followed by BECM-3 supplemented with 10% calf serum until day 7. The gas environment for IVM/IVF was 5% CO2 in air, while that for IVC was either 5% CO2 in air or 5% O2, 5% CO2 and 90% N2. Low O2 tension increased both day 7 blastocyst rates (high versus low O2, respectively; 9.3+/-2.9%: 26/280; 23.9+/-4.2%: 71/293; P<0.001) and total cell numbers (39.3+/-2.9, n=24 versus 61.2+/-7.7, n=61; P=0.01) of parthenogenetically activated embryos. In contrast, such a treatment neither affected blastocyst rates (89.3+/-6.9 versus 87.8+/-7.5) nor cell numbers (87.4+/-4.5 versus 87.7+/-4.8) of in vivo flushed embryos. The effect of reduced O2 concentration on IVF embryos was intermediate, since only cell numbers were improved (69.8+/-3.5, range=17-204, n=49; 88.5+/-5.8, range=28-216; n=66; P<0.01), equivalent to that recorded in in vivo flushed embryos. However, blastocyst rates were unaffected (10.7+/-1.4%: 51/486; 12.9+/-2.2%: 67/485). The effect, when present, of reducing O2 concentration from 20 to 5% was beneficial for pig in vitro embryonic development. The responses are apparently dependent on firstly, the manner by which the embryonic cell cycle is activated and secondly, the derivation of the tissue prior to placement into culture, if the observed resilience of in vivo embryos is independent of treatment duration.     

Colorimetric determination of chloride in biological samples by using mercuric nitrate and diphenylcarbazone

A colorimetic method is outlined for the determination of the chloride ion in biological samples (blood serum, plasma, and urine). The present method is based on the quantitative reduction of free mercuric ions by chloride ions. Chloride ions form an indissociable complex with mercuric ions. The remaining free mercuric ions form a purple complex with diphenylcarbazone with an absorption maximum at 550 nm. The reduction of color intensity at 550 nm is directly proportional to chloride concentration in the sample. The linear concentration range in the final reaction mixture was 0–100 μM with a correlation coefficient of −0.9997. The coefficient of variation for the 50 μM chloride ion in the final reaction mixture was 0.9% (n=6). The analyzed value of chloride concentration in the human control serum Accutrol™ Normal (Sigma) was 101±4 mM (mean±SD, n=12). The certified value of chloride in Accutrol Normal by Sigma is 102 mM, with a mean in the range 91–113 mM. This method was applied to the measurement of urinary chloride excretion in experimental rats. During 16-h urine collection, no food was given and rats had free access to purified water. The urinary excretion rate of chloride was 23.6±9.3 μmol/h (mean±SD, n=8) and 126.2±28.0 μmol/h (n=8) for rats fed a normal diet (2.6 g NaCl/kg diet) and a high-salt diet (82.6 g NaCl/kg diet) for 70 d prior to urine collection, respectively. This method is appropriate for low concentrations of chloride in samples or when sample volume is limiting, as in many animal studies such as metabolic urine collection from rats. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of the products that may also be suitable.     

Relation of Ca2+ efflux from the heart to the concentration of Ca-channel blockers

Dependence of calcium ion efflux from the heart ventricles on the concentration of verapamil, fenigidine and nicardipine was studied in frogs, using Ca-sensitive electrodes. The effect of sodium and potassium ions was also investigated. It was established that dependence of calcium ion efflux (delta Ca2+) on the concentration of Ca-antagonists (B) may be expressed by the following formula: (Formula: see text). With cellular membrane depolarization 50 mM KCl, none of the blockers (10(-5) M) caused Ca2+ efflux from ventricular cells. Analogous phenomenon was noted in low-sodium solution (60 mM).     

Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo

The intracellular magnesium and calcium ion concentrations of in vivo-developed 2-cell hamster embryos were measured using ratiometric fluorometry. Intracellular magnesium and calcium ion concentrations were found to be 0.369 ± 0.011 mM and 129.3 ± 7.5 nM respectively. Culture of 1-cell hamster embryos for 24 hr to the 2-cell stage in control medium containing 0.5 mM magnesium and 2.0 mM calcium resulted in approximately a threefold increase to 343.5 ± 8.0 nM in intracellular calcium ion concentration, while magnesium ion levels were not altered (0.355 ± 0.007 mM). Increasing medium magnesium concentrations to 2.0 mM significantly increased intracellular magnesium ion concentrations of cultured 2-cell embryos with a concomitant reduction in intracellular calcium ion concentrations. Furthermore, increasing the medium magnesium concentration to 2.0 mM significantly increased development of 1-cell embryos collected at either 3 or 9 hr post-egg activation to the morula/blastocyst and blastocyst stages. Resultant blastocysts had an increased total cell number and increased development of the inner cell mass. Most important, however, culture with 2.0 mM magnesium increased the fetal potential of cultured 1-cells twofold. Therefore, because highest rates of development were observed in a medium that resulted in reduced intracellular calcium ion concentrations, it appears that altered calcium homeostasis is associated with impaired developmental competence of 1-cell embryos in culture. Mol. Reprod. Dev. 50:443–450, 1998. © 1998 Wiley-Liss, Inc.