Activation of volume-regulated Cl(-) channels by ACh and ATP in Xenopus follicles.
(33/803)
Osmolarity-dependent ionic currents from follicle-enclosed Xenopus oocytes (follicles) were studied using electrophysiological techniques. Whole follicle currents were monitored using a two-electrode voltage clamp and single-channel activity was measured using the patch-clamp technique. In follicles held at -60 mV two chloride currents were activated in external hyposmotic solutions. One was the habitual volume-regulated current elicited by external hyposmolarity (ICl,swell), and the second was a slow and smooth current (Sin) generated by ACh or ATP application. In follicles, the permeability ratios for different anions with respect to Cl- were similar for both ICl,swell and Sin, with a sequence of: SCN- > I- > Br- >= NO3- >= Cl- > gluconate >= cyclamate > acetate > SO42-. Extracellular ATP blocked the outward component of Sin. Also, extracellular pH modulated the inactivation kinetics of Sin elicited by ACh; e.g. inactivation at +80 mV was approximately 100 % slower at pH 8.0 compared with that at pH 6.0. Lanthanides inhibited ICl, swell and Sin. La3+ completely inhibited ICl,swell with a half-maximal inhibitory concentration (IC50) of 17 +/- 1.9 microM, while Sin was blocked up to 55 % with an apparent IC50 of 36 +/- 2.6 microM. Patch-clamp recordings in follicular cells showed that hyposmotic challenge opened inward single-channel currents. The single channel conductance (4.7 +/- 0.4 pS) had a linear current-voltage relationship with a reversal membrane potential close to -20 mV. This single-channel activity was increased by application of ACh or ATP. The ICl,swell generation was not affected by pirenzepine or metoctramine, and did not affect the purinergic activation of the chloride current named Fin. Thus, ICl,swell was not generated via neurotransmitters released during cellular swelling. All together, equal discrimination for different anions, similar modulatory effects by extracellular pH, the blocking effects by ATP and La3+, and the same single-channel activity, strongly suggest that ICl,swell and Sin currents depend on the opening of the same type or a closely related class of volume-regulated chloride channels. (+info)
Multiple action sites of flufenamate on ion transport across the rat distal colon.
(34/803)
The antisecretory effects of flufenamate in the rat distal colon were investigated with the Ussing-chamber and the patch-clamp method as well as by measurements of the intracellular Ca(2+) concentration using fura-2-loaded isolated crypts. Flufenamate (5.10(-4) mol l(-1)) suppressed the short-circuit current (Isc) induced by carbachol (5.10(-5) mol l(-1)), forskolin (5.10(-6) mol l(-1)) and the Isc induced by the membrane-permeable analogue of cyclic AMP, CPT - cyclic AMP (10(-4) mol l(-1)). Indomethacin (10(-6) - 10(-4) mol l(-1)) did not mimic the effect of flufenamate, indicating that the antisecretory effect of flufenamate is not related to the inhibition of the cyclo-oxygenase. When the basolateral membrane was depolarized by a high K(+) concentration and a Cl(-) current was induced by a mucosally directed Cl(-) gradient, the forskolin-stimulated Cl(-) current was blocked by flufenamate, indicating an inhibition of the cyclic AMP-stimulated apical Cl(-) conductance. When the apical membrane was permeabilized by the ionophore, nystatin, flufenamate decreased the basolateral K(+) conductance and inhibited the Na(+) - K(+)-ATPase. Patch-clamp experiments revealed a variable effect of flufenamate on membrane currents. In seven out of 11 crypt cells the drug induced an increase of the K(+) current, whereas in the remaining four cells an inhibition was observed. Experiments with fura-2-loaded isolated crypts indicated that flufenamate increased the basal as well as the carbachol-stimulated intracellular Ca(2+) concentration. These results demonstrate that flufenamate possesses multiple action sites in the rat colon: The apical Cl(-) conductance, basolateral K(+) conductances and the Na(+) - K(+)-ATPase. (+info)
Characteristics of the Ca(2+)-dependent inhibition of cyclic AMP accumulation by histamine and thapsigargin in human U373 MG astrocytoma cells.
(35/803)
1. Histamine, acting on H(1)-receptors, caused a Ca(2+)-dependent inhibition of forskolin- and isoprenaline-induced cyclic AMP accumulation in monolayers of human U373 MG cells (IC(50) 1.3+/-0.3 microM, maximum inhibition 66+/-3%). The inhibition was not reversed by the protein kinase inhibitor K-252A. 2. Thapsigargin also inhibited cyclic AMP accumulation (IC(50) 6.0+/-0.3 nM, maximum inhibition 72+/-1%). In the absence of extracellular Ca(2+) 5 microM thapsigargin caused only a 12+/-2% inhibition of cyclic AMP accumulation. 3. The inhibitory effect of 100 nM thapsigargin on forskolin-stimulated cyclic AMP accumulation was blocked by La(3+) (best-fit maximum inhibition 81+/-4%, IC(50) 125+/-8 nM). In contrast, the inhibitory action of 10 microM histamine was much less sensitive to reversal by 1 microM La(3+) (33+/-5% reversal, compared with 78+/-6% reversal of the inhibition by thapsigargin measured concurrently). However, in the presence of both thapsigargin and histamine the inhibition of cyclic AMP accumulation was reversed by 1 microM La(3+) to the same extent as the inhibition by thapsigargin alone. 4.++Thapsigargin (5 microM)+1 microM La(3+) caused only a 20+/-1% inhibition of histamine-stimulated phosphoinositide hydrolysis. 5. There was no indication from measurement of intracellular Ca(2+) of any persistent La(3+)-insensitive Ca(2+) entry component activated by histamine. 6. The results provide evidence that Ca(2+) entry is required for the inhibition by histamine and thapsigargin of drug-induced cyclic AMP accumulation in U373 MG astrocytoma cells. The differential sensitivity of the inhibitory action of the two agents to block by La(3+) suggests that more than one pathway of Ca(2+) entry is involved. (+info)
Manometric changes during retrograde biliary infusion in mice.
(36/803)
The manometric, ultrastructural, radiographic, and physiological consequences of retrograde biliary infusion were determined in normostatic and cholestatic mice. Intraluminal biliary pressure changed as a function of infusion volume, rate, and viscosity. Higher rates of constant infusion resulted in higher peak intraluminal biliary pressures. The pattern of pressure changes observed was consistent with biliary ductular and/or canalicular filling followed by leakage at a threshold pressure. Retrograde infusion with significant elevations in pressure led to paracellular leakage of lanthanum chloride, radiopaque dye, and [(14)C]sucrose with rapid systemic redistribution via sinusoidal and subsequent hepatic venous drainage. Chronic extrahepatic bile duct obstruction resulted in significantly smaller peak intrabiliary pressures and lower levels of paracellular leakage. These findings indicate that under both normostatic and cholestatic conditions elevated intrabiliary volumes/pressures result in an acute pressure-dependent physical opening of tight junctions, permitting the movement of infusate from the intrabiliary space into the subepithelial tissue compartment. Control of intraluminal pressure may potentially permit the selective delivery of macromolecules >18-20 A in diameter to specific histological compartments. (+info)
Single amino acid mutations in transmembrane domain 5 confer to the plasma membrane Ca2+ pump properties typical of the Ca2+ pump of endo(sarco)plasmic reticulum.
(37/803)
Conserved residues in some of the transmembrane domains are proposed to mediate ion translocation by P-type pumps. The plasma membrane Ca(2+) pump (PMCA) lacks 2 of these residues in transmembrane domains (TM) 5 and 8. In particular, a glutamic acid (Glu-771) residue in TM5, which is proposed to be involved in the binding and transport of Ca(2+) by the sarcoplasmic reticulum Ca(2+) pump (SERCA), is replaced by an alanine (Ala-854) in the PMCA pump. Ala-854 has been mutated to Glu, Asp, or Gln; Glu-975 in TM8, which is an Ala in the SERCA pump, has been mutated to Gln, Asp, or Ala. The mutants have been expressed in three cell systems, with or without the help of viruses. When expressed in large amounts in Sf9 cells, the mutated pumps were isolated and analyzed in the purified state. Two of the three TM8 mutants were correctly delivered to the plasma membrane and were active. All the TM5 mutants were retained in the endoplasmic reticulum; two of them (A854Q and A854E) retained activity. Their properties (La(3+) sensitivity and decay of the phosphorylated intermediate, higher cooperativity of Ca(2+) binding with a Hill's coefficient approaching 2) differed from those of the expressed wild type PMCA pump, and resembled those of the SERCA pump. (+info)
A calcium ionophore stimulating the secretion of catecholamines from the cat adrenal.
(38/803)
1. Experiments were performed on perfused cat adrenal glands to examine the effect of a calcium ionophore A-23187 in the secretion of catecholamines. 2. Ionophore (1-10 muM) caused a dose-dependent release of catecholamines and the output was about 100-fold greater at 10 mum than at 1 mum. 3. Release of catecholamines by the ionophore was dependent on the calcium concentration of the perfusion medium. Omission of calcium blocked the response to the ionophore while excess calcium facilitated it. 4. Magnesium antagonized the secretory response to the ionophore. Excess calcium overcame the inhibitory effect of magnesium. 5. The ionophore did not modify release of catecholamines by induced splanchnic nerve stimulation. 6. The results suggest that the ionophore, like depolarization, introduces calcium into the chromaffin cell to cause release of catecholamines. (+info)
Store depletion and store-operated Ca2+ current in human prostate cancer LNCaP cells: involvement in apoptosis.
(39/803)
1. In the present study, we investigated the mechanisms involved in the induction of apoptosis by the Ca2+-ATPase inhibitor thapsigargin (TG), in androgen-sensitive human prostate cancer LNCaP cells. 2. Exposure of fura-2-loaded LNCaP cells to TG in the presence of extracellular calcium produced an increase in intracellular Ca2+, the first phase of which was associated with depletion of intracellular stores and the second one with consecutive extracellular Ca2+ entry through plasma membrane, store-operated Ca2+ channels (SOCs). 3. For the first time we have identified and characterized the SOC-mediated membrane current (Istore) in prostate cells using whole-cell, cell-attached, and perforated patch-clamp techniques, combined with fura-2 microspectrofluorimetric and Ca2+-imaging measurements. 4. Istore in LNCaP cells lacked voltage-dependent gating and displayed an inwardly rectifying current-voltage relationship. The unitary conductance of SOCs with 80 mM Ca2+ as a charge carrier was estimated at 3.2 +/- 0.4 pS. The channel has a high selectivity for Ca2+ over monovalent cations and is inhibited by Ni2+ (0.5-3 mM) and La3+ (1 microM). 5. Treatment of LNCaP cells with TG (0.1 microM) induced apoptosis as judged from morphological changes. Decreasing extracellular free Ca2+ to 200 nM or adding 0.5 mM Ni2+ enhanced TG-induced apoptosis. 6. The ability of TG to induce apoptosis was not reduced by loading the cells with intracellular Ca2+ chelator (BAPTA-AM). 7. These results indicate that in androgen-sensitive prostate cancer cells the depletion of intracellular Ca2+ stores may trigger apoptosis but that there is no requirement for the activation of store-activated Ca2+ current and sustained Ca2+ entry in induction and development of programmed cell death. (+info)
Inhibition of TRP3 channels by lanthanides. Block from the cytosolic side of the plasma membrane.
(40/803)
The lanthanide ions La(3+) and Gd(3+) block Ca(2+)-permeable cation channels and have been used as important tools to characterize channels of the transient receptor potential (TRP) family. However, widely different concentrations of La(3+) and Gd(3+) have reportedly been required for block of TRP3 channels in various expression systems. The present study provides a possible explanation for this discrepancy. After overexpression of TRP3 in Chinese hamster ovary cells, whole-cell currents through TRP3 were reversibly inhibited by La(3+) with an EC(50) of 4 microm. For comparison, the organic blocker SKF96365 required an EC(50) of 8 microm. Gd(3+) blocked with an EC(50) of 0.1 microm, but this block was slow in onset and was not reversible after wash-out. When the two lanthanides were added to the cytosolic side of inside-out patches, block was achieved with considerably lower concentrations (EC(50) for La(3+), 0.02 microm; EC(50) for Gd(3+), 0.02 microm). Uptake of La(3+) into the cytosol of Chinese hamster ovary cells was demonstrated with intracellular fura-2. We conclude that lanthanides block TRP3 more potently from the cytosolic than from the extracellular side of the plasma membrane and that uptake of lanthanides will largely affect the apparent EC(50) values after extracellular application. (+info)