Free Extracellular Medium Research Articles

Effects of endothelin on villous trophoblast differentiation and free intracellular calcium

Trophoblast differentiation is a complex process involving interactions of cytotrophoblastic cells with their evolutive milieu. The trophoblastic layer differentiates from the fusion of cytotrophoblastic cells into the syncytiotrophoblast which expresses specific hormonal productions. The intracellular and extracellular signals which trigger and mediate the fusion of cytotrophoblastic cells still need to be investigated. Regarding their endocrine and paracrine activities, endothelins (ETs) are considered as peptide hormones and growth factors. ET A and ET B endothelin receptors subtypes are expressed on the trophoblast and the biosynthesis of ET by the syncytiotrophoblast (ST) was previously demonstrated. The exact mechanisms whereby endothelins cause biological effects after binding to trophoblastic cell membrane receptors have never been investigated. Furthermore, little is known regarding Ca ++ homeostasis. Therefore, by means of microspectrofluorimetry, we have measured the effects of ETs on cytosolic [Ca ++ ]. To monitor the ET −1 action on trophoblast differentiation, βhCG secretion, hCS expression, proliferative cell nuclear antigen and desmoplakin immunostainings were used. The presence of ET −1 (10 −8 M) for 3 days partially inhibited ST formation as confirmed by desmoplakin immunostaining. Interestingly, PCNA (Proliferative Cell Nuclear Antigen) immunostaining was detected in cytotrophoblastic cell aggregates cultured in the presence of 10 −8 M Et −1 , whereas it was absent in control conditions. In parallel, βhCG release and hCS expression were significantly reduced by ET −1 . Moreover, our study demonstrated that ET −1 could induce a biphasic increase of [Ca ++ ] i : an initial transient rise sometimes followed by a sustained plateau higher than the basal level. Endothelin responses are mainly mediated by ET β receptors. The persistence of the rapid transient [Ca ++ ] i rise in Ca ++ free extracellular medium together with patch clamp studies argue for the release of intracellular Ca ++ store rather than Ca ++ entry through voltage-sensitive channels in the response to endothelin stimulation. We conclude that endothelin impairs human trophoblast differentiation and behaves as a typical calcium mobilizing peptide in trophoblast.

Characterization of volume-sensitive taurine- and Cl(-)-permeable channels.

Volume-sensitive Cl- channels [ICl(vol)] were studied using taurine efflux and patch-clamp experiments in 9HTEo- human tracheal cells. Cells were stimulated with the Ca(2+)- elevating agents ATP and ionomycin in isotonic medium or in hypotonic solutions. ATP (100 microM) or ionomycin (1 microM) and hypotonic shock produced a synergic effect. Indeed, the resulting taurine efflux was much higher than the sum of the single effects elicited by ATP, ionomycin, or hypotonic medium. The taurine release elicited by hypotonic shock and the potentiation by ATP and ionomycin were markedly inhibited by using a Ca(2+)-free extracellular medium and by incubating the cells with the membrane-permeable 1,2-bis(2-amino- phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester chelating agent. Patch-clamp experiments confirmed the role of Ca2+ on ICl(vol) channels. Swelling-induced taurine efflux was inhibited by reactive blue 2, suramin, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid. Patch-clamp experiments demonstrated that these compounds shift the voltage-dependent inactivation of ICl(vol) channels toward more negative values. This study indicates that the sensitivity of ICl(vol) to cell volume changes is modulated by intracellular Ca2+ and that purinergic receptor antagonists represent a new class of CI- channel blockers.

Long-term GABAA receptor activation increases [Ca2+]i in single lactotrophs.

Prolactin (PRL) release by pituitary lactotrophs is inhibited by gamma-aminobutyric acid (GABA). We have investigated the effect of long-lasting activation of GABAA receptors on membrane potential and cytosolic free calcium concentration ([Ca2+]i) in single identified lactotrophs. Membrane potential was recorded using the perforated patch-clamp technique and [Ca2+]i using indo 1 as a fluorescent Ca2+ probe. When cells were bathed in muscimol (10 microM) for 30 min, [Ca2+]i unexpectedly increased in 53% of the lactotrophs. This was due to a Ca2+ influx, since it was inhibited by Ca(2+)-free extracellular medium or by Ca2+ channel blockers such as the dihydropyridine PN 200-110. In cells incubated in muscimol, wash of muscimol from the cell membrane potential and reduced [Ca2+]i to the levels found in control cells. This effect was mimicked by picrotoxin, a GABA-operated Cl- channel blocker, thus supporting the involvement of a muscimol-induced Cl- flux. Conversely, under similar experimental conditions, static assays of PRL release revealed an inhibition of release by muscimol, unaffected by the dihydropyridine PN 200-110. Our observations suggest that GABAA, receptors may not regulate the process of exocytosis within the Ca(2+)- regulated steps.

Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells.

The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of approximately 30% in suspended (i.e., round) cells and approximately 60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with BAPTA-AM and by depletion of Cl-, indicating the role of calcium-regulated K+ and Cl- efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (approximately 0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na(+)-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl(-)-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl- that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi.

Multiple pathways for ATP-induced intracellular calcium elevation in pheochromocytoma (PC12) cells.

Extracellular ATP excites neurons in both the peripheral and central nervous system. To elucidate the mechanisms involved, we used spectrofluorometric analysis to study the pathways by which extracellular ATP elevates the intracellular Ca2+ concentration ([Ca2+]i) in individual, fura-2-loaded, rat pheochromocytoma PC12 cells. ATP (> 1 microM) increased [Ca2+]i. The ATP effect on [Ca2+]i was completely abolished by a nominally Ca(2+)-free extracellular medium, which indicates that the ATP-induced increase in [Ca2+]i was due to an influx of extracellular Ca2+. We next applied specific blockers of voltage-dependent Ca2+ channels and used experimental protocols with depolarizing external K(+)-rich solutions. Our results show that ATP induces influx of extracellular Ca2+ through (a) dihydropyridine-sensitive (Ln-type) Ca2+ channels, (b) Ca(2+)-permeable, voltage-independent, Cd(2+)-insensitive cation channels, and (c) an as yet unidentified, voltage-dependent, Cd(2+)-sensitive Ca2+ influx system.

Effect of thiamine on the processes responsible for acetylcholine secretion in the frog neuromuscular synapses

The effect of vitamin B1 (thiamine, 10−10–10−3 M) on direct (transmitter secretion) and recurrent (resynthesis of the transmitter and its storage in synaptic vesicles) processes of acetylcholine (ACh) secretion was studied in the frog neuromuscular junction. In Ca2+-containing extracellular medium, the facilitatory effects of thiamine and α-latrotoxin (an increase in the frequency of miniature end-plate potentials, MEPP) were additive, regardless of the sequence of their application. After partial exhaustion of the synaptic vesicle stores caused by α-latrotoxin (2 nM) in Ca2+-free extracellular medium, thiamine accelerated the Ca2+-induced recovery of the ACh secretion. In the presence of thiamine, there were two phases in the dependence of quantum content of an end-plate potential (EPP) on stimulation frequency, which are typical of the effects of Sr2+ and Ba2+ on the ACh secretion. Under conditions of depression and postdepression recovery, the effect of thiamine on the time course of the changes in EPP amplitude was similar to that produced by Ba2+. Possible mechanisms of the effects of vitamin B1 on the processes responsible for the ACh secretion and the dependence of the MEPP frequency on the concentrations of thiamine and thiamine diphosphate are discussed in light of the above results.

Manganese Uptake and Efflux in Cultured Rat Astrocytes

Astrocytes play a central role in manganese (Mn) regulation in the CNS. Using primary astrocyte cultures from neonatal rat brains, these studies demonstrate a specific high-affinity transport system for Mn2+. Saturation kinetics are clearly indicated by both 1/v versus 1/s plots (Km = 0.30 +/- 0.03 microM; Vmax = 0.30 +/- 0.02 nmol/mg of protein/min) and plots of v versus [s]. Several divalent cations (Co2+, Zn2+, and Pb2+) failed to inhibit the initial rate of 54Mn2+ uptake. In contrast, extracellular Ca2+ at 10 microM decreased 54Mn2+ uptake. Exchange with extracellular Mn2+ was not obligatory for the efflux of 54Mn2+ into extracellular medium because efflux occurred into Mn(2+)-free extracellular medium, but efflux of 54Mn2+ was enhanced when astrocytes were equilibrated in the presence of unlabeled Mn2+. Efflux of 54Mn2+ was biphasic with both a rapid and a slow component. Efflux was most rapid during the first 10 min of incubation, with 27.5 +/- 2.2% of 54Mn2+ transported extracellularly, and 37.2 +/- 1.2% of preloaded 54Mn2+ was retained by the astrocytes at 120 min. These studies show, for the first time, that mammalian astrocytes can transport Mn via a specific transport system.

Indirect inhibitory effect of succinylcholine on acetylcholine-activated channel activities and its modulation by external Ca2+ in mouse skeletal muscles.

1. The effect of extracellular calcium on single acetylcholine (ACh)-activated channel activities when desensitizing concentrations of succinylcholine (SuCh) were applied to the surrounding endplate membrane was investigated by the cell-attached patch-clamp technique at endplates of single skeletal muscle (flexor digitorum brevis) fibres of adult mice. 2. Bath-applied SuCh (0.1-3 microM, in 2.5 mM Ca2+) increased in a concentration-dependent manner the mean open time of ACh-activated channel currents recorded at membrane potentials which cancelled the SuCh-induced depolarizations. 3. In the presence of 0.5 and 2.5 mM external Ca2+, SuCh (3 microM) applied outside the patch pipette prolonged the mean open time of ACh-activated channel currents in a time-dependent manner (by 45% and 52%, respectively), and simultaneously significantly decreased the single channel conductance (by 14% and 10%, respectively). These SuCh-induced effects did not occur in a nominally Ca(2+)-free extracellular medium. 4. Under the same conditions, SuCh (3 microM) augmented the time-dependent decline in the opening frequency of ACh-activated channel currents obtained in nominally Ca(2+)-free medium. 5. These results suggest that external calcium ions act to modulate nicotinic ACh receptor channel activity, and accelerate desensitization of the receptor.

Muscarinic receptor hyperpolarizes cochlear hair cells of chick by activating Ca(2+)‐activated K+ channels.

1. Electrical responses to extracellularly applied acetylcholine (ACh) and to intracellularly introduced substances were studied in isolated short and tall hair cells from the chick cochlear organ by a whole-cell voltage clamp technique using a patch electrode. These cells were isolated without using proteolytic enzymes. 2. Short hair cells generated a transient outward current at -50 mV in normal saline in response to puff-applied 100 microM-ACh, when the patch electrode was filled with a 160 mM-K+ and 100 microM-EGTA-based intracellular medium. The amplitude was 317.1 +/- 97.1 pA (n = 32). When ACh was applied ionophoretically, the outward current was generated with a delay of about 10 ms. 3. The amplitude of ACh-induced current was dose dependent with a KD of 19 microM and a Hill coefficient of 1.6 when measured at -50 mV. 4. The ACh (100 microM)-induced current was suppressed by 1 microM-atropine. ACh-induced current was generated in a Ca(2+)-free extracellular medium; however, the second ACh puff in the Ca(2+)-free medium generated a much reduced response. ACh-induced current was suppressed reversibly by 100 microM-quinine. 5. Intracellular injections of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), inositol 1,4,5-trisphosphate (IP3) or Ca2+ (1 microM) via the patch pipette activated outward currents at -50 mV. 6. When the internal medium with strong Ca(2+)-buffering capacity (5 mM-EGTA) was used, the ACh-induced current was reduced to 39.3 +/- 6.8 pA (n = 4) at -50 mV (12.3% of the response in the low-EGTA medium). 7. The reversal potential of the ACh-induced current was -85.7 +/- 4.2 mV (n = 3) in normal saline containing 5 mM-K+. The reversal potential was dependent on the extracellular K+ concentration ([K+]o) and was shifted 57 mV by a 10-fold increase in [K+]o at room temperature (20-25 degrees C). 8. These results (points 4-7) indicate that ACh induces a K+ conductance by releasing Ca2+ intracellularly, probably by activating the pathway of muscarine receptor, G-protein and IP3. 9. Channel activities were recorded using cell-attached patch electrodes. Channel activities were rarely observed when ACh was applied to the extra-patch membrane, while robust channel activities were observed when ACh was included in the patch pipette medium. It is therefore suggested that Ca(2+)-activated K+ channels exist in the membrane in close vicinity to muscarinic receptor molecules and intracellular Ca2+ release sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Measurement of mitochondrial and non-mitochondrial Ca 2+ in isolated intact hepatocytes: a critical re-evaluation of the use of mitochondrial inhibitors

Isolated rat hepatocytes treated with mitochondrial inhibitors FCCP or antimycin A release discrete amounts of Ca 2+ in a Ca 2+-free extracellular medium as revealed by changes in the absorbance of the Ca 2+ indicator arsenazo III. The process is completed in 2 min and the amount of Ca 2+ released is not affected by the type of the mitochondrial poison employed. The subsequent treatment with the cation ionophore A23187 causes a further release of Ca 2+ that does not appear related to the specificity of the previous treatment with FCCP or antimycin A. Both FCCP and antimycin A cause a progressive loss of cellular ATP associated with a decrease in the ATP/ADP ratio from 6 to 2-1.5. However, this decrease does not significantly prevent 45Ca 2+ accumulation in isolated liver microsomes. Moreover, the decrease of the ATP/ADP ratio to 1, does not promote a significant release of 45Ca 2+ from 45Ca 2+-preloaded microsomes. Finally, experiments with Fura-2-loaded hepatocytes reveal that agents specifically releasing Ca 2+ from non-mitochondrial stores (vasopressin and 2,5-di- tert-butyl-1-4-benzohydroquinone) are still able to increase the cytosolic Ca 2+ concentration in FCCP-treated cells. Taken together, these findings demonstrate that, in freshly isolated hepatocytes, FCCP specifically releases Ca 2+ from mitochondrial stores without significantly affecting active Ca 2+ sequestration in other cellular pools. For these reasons, FCCP can be used to release and quantitate mitochondrial Ca 2+ in liver cells.

Muscarinic agonists and ATP increase the intracellular Ca2+ concentration in chick cochlear hair cells.

1. Cholinergic muscarinic agonists applied by the pressure puff method increased intracellular Ca2+ concentration in Fura-2-loaded hair cells. The Ca2+ response outlasted the agonist application. 2. The Ca2+ response induced by acetylcholine (ACh) was ACh dose dependent with a KD of 200 microM. Desensitization was negligible, and almost identical Ca2+ responses were observed when two ACh puffs were separated by 150 s. The response was blocked by d-tubocurarine (dTC). The KD of dTC blocking was 500 microM when 100 microM-ACh induced the Ca2+ response. 3. The amplitude of the ACh-induced Ca2+ responses were potentiated to 3 times the control by incubation with calcitonin gene-related peptide (CGRP; 0.1-1 microM). CGRP did not affect the resting Ca2+ concentration. Glycine (100 microM) potentiated the ACh response to 1.4 times the control, and also increased the resting Ca2+ concentration slightly. 4. The ACh-induced Ca2+ response was suppressed by atropine. It was induced in Ca2(+)-free extracellular medium, and in Ca2(+)-free medium desensitization to a second ACh stimulation was significant. The amplitude of the second Ca2+ response was 44% of the first when two ACh puffs were separated by 117 s in Ca2+ free medium. 5. Muscarine and carbamylcholine induced similar Ca2+ responses, with KD values of 130 microM for muscarine and 340 microM for carbamylcholine. Desensitization of Ca2+ responses was negligible in both agonists. 6. ATP co-exists with ACh in some presynaptic nerve terminals (Burnstock, 1981). Puff-applied ATP (100 microM) generated a Ca2+ response with a rapid rising phase and a following slow phase. In Ca2(+)-free medium the rapid phase disappeared and only the slow phase was observed. The rapid phase is due to the influx of Ca2+ ions and the slow phase is due to a release of Ca2+ ions from an intracellular reservoir. Under voltage clamp ATP induced a fast inward current and a following slow outward current. 7. Nicotine, adenosine, glycine, GABA, glutamate and bradykinin did not induce Ca2+ responses in the hair cell. 8. ACh induced hyperpolarization of the hair cell membrane under current clamp, most probably by the activation of Ca2+ activated K+ conductance. Therefore, a cholinergic muscarinic receptor may mediate the inhibitory effects of efferent innervation observed in hair cells.

Acetylcholine regulation of nicotinic receptor channels through a putative G protein in chick myotubes.

1. Single-channel currents induced by acetylcholine (ACh) were recorded from unstriated and non-innervated embryonic chick myotubes using the cell-attached patch-clamp technique. 2. ACh applied to the non-patched membrane decreased both channel opening probability and conductance. These ACh-induced effects occurred also when the non-patched membrane was exposed to nominally Ca2+-free extracellular medium, but were absent when it was treated with curare. 3. ACh-induced membrane current recorded under whole-cell patch-clamp conditions decreased in amplitude and time course when myotubes were intracellularly loaded with guanosine-5'-O-(3-thiotriphosphate) GTP gamma S), but not with guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) or cyclic adenosine-5'-monophosphate (cyclic AMP). Internal perfusion of GTP gamma S affected the ACh-induced openings in a similar manner to the non-patch ACh application. 4. These results suggest that ACh, in addition to its direct effect, acts indirectly on the nicotinic receptor channels by delivering an intracellular messenger and through the activation of a putative G protein.

Ionic movements through light-sensitive channels of toad rods.

Electrical photoresponses of rods in the isolated toad retina were recorded during ionic manipulations of the Na+-free extracellular medium. In the presence of a concentration of external Ca2+ above 10(-5) M, voltage photoresponses were observed only in the presence of external Na+ or Li+. When external Ca2+ was reduced below 10(-6) M, voltage photoresponses of normal polarity could be detected even in the absence of Na+ or Li+, but in the presence of external Mg2+. In the presence of normal extracellular Ca2+ hyperpolarizing photoresponses were observed even in the absence of Na+ or Li+, provided small amounts of phosphodiesterase inhibitors (IBMX, RO 20-1724, papaverine, caffeine, theophylline) were added to the perfusate. Responses obtained in low-Na+ IBMX solutions required the presence of millimolar amounts of a variety of divalent cations, among which Mn2+ and Ba2+ were the most effective. When the concentration of both external Ca2+ and Mg2+ was reduced to micromolar amounts, depolarizing photoresponses were observed. In these conditions measurements with radioactive tracers showed a light-modulated efflux of 42K+ or 86Rb+. The light-modulated 42K+ or 86Rb+ efflux was halved by 2 X 6 mM-external K+ and was completely blocked when K+ was raised above 10 mM. These results show that ionic movements through light-sensitive channels are controlled by Ca2+ and Mg2+ and possibly also be the intracellular level of cyclic nucleotides. Moreover, the movement of ions through the light-sensitive channel, does not obey the independence principle.