Characterization of Ryanodine-sensitive Ca2+ Release from Microsomal Vesicles of Rat Parotid Acinar Cells: Regulation by Cyclic ADP-ribose

We have measured ryanodine (caffeine)-sensitive ⁴⁵Ca²⁺ release from isolated microsomal vesicles of endoplasmic reticulum prepared from rat parotid acinar cells. After a steady state of ATP-dependent ⁴⁵Ca²⁺ uptake, the addition of caffeine (40 mm), ryanodine (10∼500 μm) or an NAD⁺ metabolite, cyclic ADP-ribose (cADPR, 4 μm) released about 10% of the ⁴⁵Ca²⁺ that had been taken up. The ⁴⁵Ca²⁺ release was not inhibited by heparin, an antagonist of IP₃ receptor. The effects of caffeine, ryanodine and cADPR on ⁴⁵Ca²⁺ release were also tested in the presence of thapsigargin (TG), an inhibitor of microsomal Ca²⁺-ATPase. When caffeine (10∼40 mm), ryanodine (10 μm) or cADPR (1∼10 μm) was added in the medium with 100 nm TG, a significant ⁴⁵Ca²⁺ release was seen, while higher concentrations of ryanodine (>100 μm) did not cause any ⁴⁵Ca²⁺ release in the presence of TG. The initial rate of caffeine (40 mm)-induced ⁴⁵Ca²⁺ release was increased by a pretreatment with 10 μm ryanodine, whereas the caffeine-induced ⁴⁵Ca²⁺ release was strongly inhibited by the presence of a higher concentration (500 μm) of ryanodine. cADPR-induced ⁴⁵Ca²⁺ release was also inhibited by 500 μm ryanodine. Caffeine (40 mm)- or cADPR (4 μm)-induced ⁴⁵Ca²⁺ release was abolished by a presence of ruthenium red (50∼100 μm). The presence of a low concentration (0.5 μm) of cADPR shifted the dose-response curve of caffeine-induced ⁴⁵Ca²⁺ release to the left. These results indicate the presence of a ryanodine sensitive Ca²⁺ release mechanism in the endoplasmic reticulum of rat parotid acinar cells that is distinct from the IP₃-sensitive Ca²⁺ channel and is activated by caffeine, cADPR and a low concentration (10 μm) of ryanodine, but is inhibited by higher concentrations (>100 μm) of ryanodine and ruthenium red. The properties of the ryanodine-sensitive mechanism are similar to that of the ryanodine receptor as described in muscle cells. Received: 11 June 1996/Revised: 14 November 1996     

Effects of 2,3-Butanedione 2-Monoxime on Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Single channel and [³H]ryanodine binding measurements were performed to test for a direct functional interaction between 2,3-butanedione 2-monoxime (BDM) and the skeletal and cardiac muscle sarcoplasmic reticulum Ca²⁺ release channels (ryanodine receptors). Single channel measurements were carried out in symmetric 0.25 m KCl media using the planar lipid bilayer method. BDM (1–10 mm) activated suboptimally Ca²⁺-activated (0.5–1 μm free Ca²⁺) single, purified and native cardiac and skeletal release channels in a concentration-dependent manner by increasing the number of channel events without a change of single channel conductances. BDM activated the two channel isoforms when added to either side of the bilayer. At a maximally activating cytosolic Ca²⁺ concentration of 20 μm, BDM was without effect on the cardiac channel, whereas it inhibited skeletal channel activities with IC₅₀≈ 2.5 mm. In agreement with single channel measurements, high-affinity [³H]ryanodine binding to the two channel isoforms was increased in a concentration-dependent manner at ≤1 μm Ca²⁺. BDM was without a noticeable effect at low (≤0.01 μm) Ca²⁺ concentrations. At 20 μm Ca²⁺, BDM inhibited the skeletal but not cardiac channel. These results suggest that BDM regulates the Ca²⁺ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle in a concentration, Ca²⁺ and tissue-dependent manner. Received: 31 December 1998/Revised: 9 March 1999     

Differential desensitization of Ca2+ mobilization and vasoconstriction by ET(A) receptors in the gerbil spiral modiolar artery

Endothelins are known to be among the most potent endogenous vasoconstrictors. Vasoconstriction of the spiral modiolar artery, which supplies the cochlea, may be implicated in hearing loss and tinnitus. The purpose of the present study was to determine whether the spiral modiolar artery responds to endothelin, whether a change in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) mediates the response and which endothelin receptors are present. The vascular diameter and [Ca²⁺]_i were measured simultaneously by videomicroscopy and microfluorometry in the isolated spiral modiolar artery from the gerbil. ET-1 induced a transient [Ca²⁺]_i increase and a strong and long-lasting vasoconstriction. The transient [Ca²⁺]_i increase underwent rapid desensitization, was independent of extracellular Ca²⁺ and inhibited by the IP₃-receptor blocker (75 μm) 2-aminoethoxydiphenyl borate (2-APB) and by depletion of Ca²⁺ stores with 10⁻⁶ m thapsigargin. In contrast, the vasoconstriction displayed no comparable desensitization. The initial vasoconstriction was independent of extracellular Ca²⁺ but maintenance of the constriction depended on the presence of extracellular Ca²⁺. The half-maximal concentration values (EC ₅₀) for the agonists ET-1, ET-3 and sarafotoxin S6c were 0.8 nm, >10 nm and >100 nm, respectively. Affinity constants for the antagonists BQ-123 and BQ-788 were 24 nm and 77 nm, respectively. These observations demonstrate that ET-1 mediates a vasoconstriction of the gerbil spiral modiolar artery via ET_A receptors and an IP₃ receptor-mediated release of Ca²⁺ from thapsigargin-sensitive Ca²⁺ stores. The marked difference in desensitization between Ca²⁺ mobilization and vasoconstriction suggests that Ca²⁺ mobilization is not solely responsible for the vasoconstriction and that other signaling mechanisms must be present. Received: 4 January 2001/Revised: 23 April 2001     

A9 Fibroblasts Transfected with the m3 Muscarinic Receptor Clone Express a Ca2+ Channel Activated by Carbachol,GTP and GDP

Muscarinic m3 receptor-mediated changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_l) occur by activation of Ca²⁺ release channels present in the endoplasmic reticulum membrane and Ca²⁺ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation of a voltage-insensitive, cation-selective channel of low conductance (3.2 ± 0.6 pS; 25 mm Ca²⁺ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side), suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised inside-out membrane patches from nonstimulated A9m3 cells GTP (10 μm) and GDP (10 μm) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mm Ca²⁺ as charge carrier) respectively. In contrast, ATP (10 μm), UTP (10 μm) or CTP (10 μm) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the activation of a cation channel that conducts calcium. Received: 14 November 1996/Revised: 4 April 1997     

Regulatory Volume Decrease by SV40-transformed Rabbit Corneal Epithelial Cells Requires Ryanodine-sensitive Ca2+-induced Ca2+ Release

The relationship between relative cell volume and time-dependent changes in intracellular Ca²⁺ concentration ([Ca²⁺] _i ) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (τ _vr ) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca²⁺] _i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca²⁺] _i transients were inhibited by either: (a) Ca²⁺-free medium; (b) 5 mm Ni²⁺ (inhibitor of plasmalemma Ca²⁺ influx); (c) 10 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca²⁺ content); or (d) 100 μm ryanodine (inhibitor of Ca²⁺ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca²⁺ influx and the emptying of intracellular Ca²⁺ stores during the [Ca²⁺] _i transients, Mn²⁺ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn²⁺ permeability 6-fold. However, Mn²⁺ permeability remained unchanged during exposure to either: 1.100 μm ryanodine; 2.10 μm CPA and 100 μm ryanodine. This report for the first time documents the time dependence of the components of the [Ca²⁺] _i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca²⁺ release from an intracellular store leads to a subsequent increase in plasmalemma Ca²⁺ influx, and that both are required for cells to undergo RVD. Received: 7 November 1996/Revised: 6 January 1997     

Mechanotransduction in Colonic Smooth Muscle Cells

We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration ([Ca²⁺] _i ) with peak of 422.7 ± 43.8 nm above an average resting [Ca²⁺] _i of 104.8 ± 10.9 nm (n= 57) followed by both rapid and prolonged recovery phases. The peak [Ca²⁺] _i increase was reduced by 50% in the absence of extracellular Ca²⁺, while the prolonged [Ca²⁺] _i recovery was either abolished or reduced to ≤15% of control values. In contrast, no significant effect of gadolinium chloride (100 μm) or lanthanum chloride (25 μm) on either peak transient or prolonged [Ca²⁺] _i recovery was observed. Pretreatment of cells with thapsigargin (1 μm) resulted in a 25% reduction of the mechanically induced peak [Ca²⁺] _i response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca²⁺] _i transient peak. [Ca²⁺] _i transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca²⁺-free solution, or when Ca²⁺ stores were depleted by thapsigargin in Ca²⁺-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 μm) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect of cytochalasin D was reversible and did not prevent the [Ca²⁺] _i increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical link via actin filaments, between the plasma membrane and an internal Ca²⁺ store. Received: 24 March 1997/Revised: 21 July 1997     

Magnesium Inhibition of Ryanodine-Receptor Calcium Channels: Evidence for Two Independent Mechanisms

The gating of ryanodine receptor calcium release channels (RyRs) depends on myoplasmic Ca²⁺ and Mg²⁺ concentrations. RyRs from skeletal and cardiac muscle are activated by μm Ca²⁺ and inhibited by mm Ca²⁺ and Mg²⁺. ⁴⁵Ca²⁺ release from skeletal SR vesicles suggests two mechanisms for Mg²⁺-inhibition (Meissner, Darling & Eveleth, 1986, Biochemistry 25:236–244). The present study investigates the nature of these mechanisms using measurements of single-channel activity from cardiac- and skeletal RyRs incorporated into planar lipid bilayers. Our measurements of Mg²⁺- and Ca²⁺-dependent gating kinetics confirm that there are two mechanisms for Mg²⁺ inhibition (Type I and II inhibition) in skeletal and cardiac RyRs. The mechanisms operate concurrently, are independent and are associated with different parts of the channel protein. Mg²⁺ reduces P _o by competing with Ca²⁺ for the activation site (Type-I) or binding to more than one, and probably two low affinity inhibition sites which do not discriminate between Ca²⁺ and Mg²⁺ (Type-II). The relative contributions of the two inhibition mechanisms to the total Mg²⁺ effect depend on cytoplasmic [Ca²⁺] in such a way that Mg²⁺ inhibition has the properties of Types-I and II inhibition at low and high [Ca²⁺] respectively. Both mechanisms are equally important when [Ca²⁺] = 10 μm in cardiac RyRs or 1 μm in skeletal RyRs. We show that Type-I inhibition is not the sole mechanism responsible for Mg²⁺ inhibition, as is often assumed, and we discuss the physiological implications of this finding. Received: 1 January 1996/Revised: 14 November 1996     

Release of Calcium from Mitochondrial and Nonmitochondrial Intracellular Stores in Mouse Pancreatic Acinar Cells by Hydrogen Peroxide

In the present study we have studied how [Ca²⁺] _i is influenced by H₂O₂ in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H₂O₂ caused a slow sustained [Ca²⁺] _i increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H₂O₂ was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H₂O₂ to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H₂O₂-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H₂O₂ did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca²⁺] _i . In addition, H₂O₂ was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca²⁺] _i induced by H₂O₂ was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H₂O₂ releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H₂O₂ is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases. Received: 15 May 2000/Revised: 4 October 2000     

Chloride-induced Ca2+ Release from the Sarcoplasmic Reticulum of Chemically Skinned Rabbit Psoas Fibers and Isolated Vesicles of Terminal Cisternae

There is increasing evidence that Ca²⁺ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl⁻ on the release of Ca²⁺ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca²⁺ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca²⁺ release from isolated SR was measured by rapid filtration of vesicles passively loaded with ⁴⁵Ca²⁺. Ca²⁺ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca²⁺-loading solution that had propionate as the major anion. Controls using saponin (50 μg/ml), indicated that the release of Ca²⁺ was due to direct action of Cl⁻ on the SR rather than via depolarization of T-tubules. Procaine (10 mm) totally blocked Cl⁻- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na⁺] + [K⁺]) × [Cl⁻] product of 6487.69 mm ² and 12361.52 mm ², respectively, and blocked 60% of Ca²⁺ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl⁻ and caffeine together. The data from both preparations suggests that Cl⁻ induces a relatively small amount of Ca²⁺ release from the SR by activating receptors other than RYR-1. In addition, Cl⁻ may increase the Ca²⁺ sensitivity of RYR-1, which would then allow the small initial release of Ca²⁺ to facilitate further release of Ca²⁺ from the SR by Ca²⁺-induced Ca²⁺ release. Received: 6 February 1996/Revised: 17 July 1996     

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

This combined study of patch-clamp and intracellular Ca²⁺ ([Ca²⁺] _i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca²⁺-dependent Cl⁻ channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca²⁺] _i and activation of Cl⁻ currents. In contrast, intracellular application of Ca²⁺ (10 μm) only induced transient activation of Cl⁻ currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca²⁺ and to the blocker of I _CRAC, La³⁺ (10 μm), despite the fact that both maneuvers led to a decline in [Ca²⁺] _i . The InsP3-induced rise in Cl⁻ conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca²⁺ stores or by removal of Ca²⁺ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca²⁺-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca²⁺/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl⁻-conductance in 50% of the cells investigated without affecting [Ca²⁺] _i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca²⁺] _i and Cl⁻ conductance. In summary, elevation of [Ca] _i by InsP3 leads to activation of Cl⁻ channels involving cytosolic Ca²⁺ stores and Ca²⁺ influx from extracellular space. Tyrosine kinases are essential for the Ca²⁺-independent maintenance of this conductance. Received: 15 October 1998/Revised: 3 March 1999     

Kinetic Differences in the Phospholamban-Regulated Calcium Pump When Studied in Crude and Purified Cardiac Sarcoplasmic Reticulum Vesicles

Phospholamban (PLN) phosphorylation contributes largely to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. The mechanical effects of PLN phosphorylation on the heart are generally attributed solely to an increase in the apparent affinity of the Ca pump in the sarcoplasmic reticulum (SR) membranes for Ca²⁺ with little or no effect on V _max(Ca). In the present report, we compare the kinetic properties of the cardiac SR Ca pump in commonly studied crude microsomes with those of our recently developed preparation of light SR vesicles. We demonstrate that in crude microsomes, the increase in the apparent affinity of the pump for Ca²⁺ is larger, while the increase in V _max(Ca) is smaller, than in purified vesicles. The greater phosphorylation-induced increase in apparent Ca²⁺ affinity in crude microsomes may be further enhanced by an ATP-sensitive inhibitory effect of ruthenium red on the activity of the pump at subsaturating, but not saturating, Ca²⁺ concentrations as a result of a greater inhibition in unphosphorylated microsomes. Upon increasing the ATP concentration from 1 to 5 mm, an inhibition by 10 μm ruthenium red is eliminated in phosphorylated microsomes and reduced in control microsomes. Addition of the phosphoprotein phosphatase inhibitor okadaic acid produces a considerable increase in the phosphorylation-induced effects in both crude and purified microsomes. We conclude that the use of purified cardiac SR vesicles is critical for the demonstration of a major increase in V _max(Ca) in addition to an increase in the pump's apparent affinity for Ca²⁺ in response to phosphorylation of PLN by protein kinase A. Received: 20 May 1998/Revised: 13 November 1998     

Modulation of the Calmodulin-induced Inhibition of Sarcoplasmic Reticulum Calcium Release Channel (Ryanodine Receptor) by Sulfhydryl Oxidation in Single Channel Current Recordings and [3H]Ryanodine Binding

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [³H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (P _o ) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in P _o (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4′-DTDP to higher concentrations in single channel recordings and [³H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (P _o > 0.9) and 0.3 mm 4,4′-DTDP a submaximal (P _o = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4′-DTDP affected Ca-[¹²⁵I]calmodulin binding to HSR. 1 mm MgCl₂ reduced P _o from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (P _o > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel. Received: 22 July 1999/Revised: 15 November 1999     

Mechanosensitive Calcium Entry and Mobilization in Renal A6 Cells

Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca²⁺ signal in response to osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior to a transient increase in [Ca²⁺] _i and the cells remained swollen after [Ca²⁺] _i had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca²⁺] after a delay of 22 sec. Both the RVD and [Ca²⁺] _i response to hypotonicity were inhibited in a Ca²⁺-free bathing solution and by gadolinium (10 μm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa) inside a cell-attached patch-pipette induced a similar global increase in [Ca²⁺] _i as occurred after hypotonic shock. A stretch-sensitive [Ca²⁺] _i increase was also observed in a Ca²⁺-free bathing solution, provided the patch-pipette contained Ca²⁺. The mechanosensitive [Ca²⁺] _i response was by gadolinium (10 μm) or Ca²⁺-free pipette solutions, even when Ca²⁺ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca²⁺] _i response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful amplification system linked to intracellular Ca²⁺ release mechanisms. Received: 3 August 1998/Revised: 19 November 1998     

Sarcoplasmic Reticulum Ca2+ Release in Rat Slow- and Fast-Twitch Muscles

The same isoform of ryanodine receptor (RYR1) is expressed in both fast and slow mammalian skeletal muscles. However, differences in contractile activation and calcium release kinetics in intact and skinned fibers have been reported. In this work, intracellular Ca²⁺ transients were measured in soleus and extensor digitorum longus (EDL) single muscle fibers using mag-fura-2 (K _D for Ca²⁺= 49 μm) as Ca²⁺ fluorescent indicator. Fibers were voltage-clamped at V _h =−90 mV and sarcoplasmic reticulum calcium release was measured at the peak (a) and at the end (b) of 200 msec pulses at +10 mV. Values of a-b and b were assumed to correspond to Ca²⁺-gated and voltage-gated Ca²⁺ release, respectively. Ratios (b/a-b) in soleus and EDL fibers were 0.41 ± 0.05 and 1.01 ± 0.13 (n= 12), respectively. This result suggested that the proportion of dihydropyridine receptor (DHPR)-linked and unlinked RYRs is different in soleus and EDL muscle. The number of DHPR and RYR were determined by measuring high-affinity [³H]PN200-110 and [³H]ryanodine binding in soleus and EDL rat muscle homogenates. The B _max values corresponded to a PN200-110/ryanodine binding ratio of 0.34 ± 0.05 and 0.92 ± 0.11 for soleus and EDL muscles (n= 4–8), respectively. These data suggest that soleus muscle has a larger calcium-gated calcium release component and a larger proportion of DHPR-unlinked RYRs. Received: 31 August 1995/Revised: 25 January 1996     

On the Role of Calcium in the Regulatory Volume Decrease (RVD) Response in Ehrlich Mouse Ascites Tumor Cells

The putative role for Ca²⁺ entry and Ca²⁺ mobilization in the activation of the regulatory volume decrease (RVD) response has been assessed in Ehrlich cells. Following hypotonic exposure (50% osmolarity) there is: (i) no increase in cellular Ins(1,4,5)P₃ content, as measured in extracts from [2-³H]myoinositol-labeled cells, a finding at variance with earlier reports from our group; (ii) no evidence of Ca²⁺-signaling recorded in a suspension of fura-2-loaded cells; (iii) Ca²⁺-signaling in only about 6% of the single, fura-2-loaded cells at 1-mm Ca²⁺ (1% only at 0.1-mm Ca²⁺ and in Ca²⁺-free medium), as monitored by fluorescence-ratio imaging; (iv) no effect of removing external Ca²⁺ upon the volume-induced K⁺ loss; (v) no significant inhibition of the RVD response in cells loaded with the Ca²⁺ chelator BAPTA when the BAPTA-loading is performed in K⁺ equilibrium medium; (vi) an inhibition of the swelling-induced K⁺ loss (about 50%) at 1-mm Ba²⁺, but almost no effect of charybdotoxin (100 nm) or of clotrimazole (10 μm), reported inhibitors of the K⁺ loss induced by Ca²⁺-mobilizing agonists. Thus, Ca²⁺signaling by Ca²⁺ release or Ca²⁺ entry appears to play no role in the activation mechanism for the RVD response in Ehrlich cells. Received: 8 December 1996/Revised: 14 January 1997     

``Frustrated Exocytosis' — A Novel Phenomenon: Membrane Fusion without Contents Release, Followed by Detachment and Reattachment of Dense Core Vesicles in Paramecium Cells

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca²⁺] _i transient and exocytosis of dense core vesicles (``trichocysts') in Paramecium cells, when applied at usual concentrations (≤10 μm) in presence of extracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>] <sub>o</sub> = 50 μm). When [Ca<sup>2+</sup>] <sub>o</sub> is kept at 30 nm (<[Ca<sup>2+</sup>]<sup>rest</sup> <sub>i</sub> ), in about one third of the population of extrudable trichocysts docked at the cell membrane, FM1-43 induces membrane fusion, visible by FM1-43 fluorescence of the vesicle membrane. However, in this system extrusion of secretory contents cannot occur in absence of any sufficient Ca<sup>2+</sup> <sub>o</sub> . Upon readdition of Ca<sup>2+</sup> <sub>o</sub> or some other appropriate Me<sup>2+</sup> <sub>o</sub> at 90 μm, secretory contents can be released (complete exocytosis). Resulting ghosts formed in presence of Ca<sup>2+</sup>, Sr<sup>2+</sup> or Mn<sup>2+</sup> are vesicular, but when formed in presence of Mg<sup>2+</sup>, for reasons to be elucidated, they are tubular, though both types are endocytosed and lose their FM1-43 stain. In contrast, in presence of [Mg<sup>2+</sup>] <sub>o</sub> = 3 mm (which inhibits contents release), the exocytotic openings reseal and intact trichocysts with labeled membranes and with still condensed contents are detached from the cell surface (``frustrated exocytosis') within ∼15 min. They undergo cytoplasmic streaming and saltatory redocking, with a half-time of ∼35 min. During this time, the population of redocked trichocysts amenable to exocytosis upon a second stimulus increases with a half-time of ∼35 min. Therefore, acquirement of competence for exocytotic membrane fusion may occur with only a small delay after docking, and this maturation process may last only a short time. A similar number of trichocysts can be detached by merely increasing [Mg²⁺] _o to 3 mm, or by application of the anti-calmodulin drug, R21547 (calmidazolium). Essentially we show (i) requirement of calmodulin and appropriate [Me²⁺] to maintain docking sites in a functional state, (ii) requirement of Ca²⁺ _o or of some other Me²⁺ _o to drive membrane resealing during exo-endocytosis, (iii) requirement of an ``empty' signal to go to the regular endocytotic pathway (with fading fluorescence), and (iv) occurrence of a ``filled' signal for trichocysts to undergo detachment and redocking (with fluorescence) after ``frustrated exocytosis'. Received: 20 January 2000/Revised: 5 May 2000     

Calcium-Sensitive Nonselective Cation Channel Identified in the Epithelial Cells Isolated from the Endolymphatic Sac of Guinea Pigs

We identified a Ca²⁺-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 ± 1.3 pS (n= 8) in our standard solution. The relative ionic permeability of the channel was in the order K⁺= Na⁺ > Ca²⁺≫ Cl⁻. This channel was weakly voltage-dependent but was strongly activated by Ca²⁺ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca²⁺ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 μm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 μm), or ATP (1 mm), which increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), activated the channel at an estimated [Ca²⁺]_i from 0.6 μm to 10 μm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca²⁺ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na⁺ under certain pathological conditions that will increase [Ca²⁺]_i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells. Received: 24 October 2000/Revised: 10 April 2001     

Skeletal Muscle Ryanodine Receptor Channels Are Activated by the Fungal Metabolite, Gliotoxin

Interactions between the reactive disulfide fungal metabolite, gliotoxin (GTX), and rabbit skeletal ryanodine receptor (RyR) calcium release channels have been examined. RyRs in terminal cisternae vesicles formed a covalent complex with 100 μm ³⁵S-GTX, which was reversed by 1 mm dithiothreitol (DTT) or 1 mm glutathione. GTX (80–240 μm), added to either cytoplasmic (cis) or luminal (trans) solutions, increased the rate of Ca²⁺ release from SR vesicles and the frequency of opening of single RyR channels in lipid bilayers. Channel activation was reversed upon addition of 2 mm DTT to the cis solution, showing that the activation was due to an oxidation reaction (2 mm DTT added to the cis solution in the absence of GTX did not affect RyR activity). Furthermore, RyRs were not activated by trans GTX if the cis chamber contained DTT, suggesting that GTX oxidized a site in or near the membrane. In contrast to cis DTT, 2 mm DTT in the trans solution increased RyR activity when added either alone or with 200 μm trans GTX. The results suggest that (i) GTX increases RyR channel activity by oxidizing cysteine residues that are close to the membrane and located on RyR, or associated proteins, and (ii) a disulfide bridge or nitrosothiol, accessible only from the luminal solution, normally suppresses RyR channel activity. Some of the actions of GTX in altering Ca²⁺ homeostatsis might depend on its modification of RyR calcium channels. Received: 12 November 1999/Revised: 14 March 2000     

Inhibition by cAMP of Calcium-Activated Chloride Currents in Cultured Sertoli Cells from Immature Testis

We have characterized a Ca²⁺-dependent Cl⁻ current (Cl_Ca) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 μm free Ca²⁺, exhibited outward currents which were inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca²⁺ strongly reduced the currents indicating that they were Ca²⁺ dependent. With cells dialyzed with Cs⁺ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 μm ionomycin induced outward currents having properties of Ca²⁺-activated Cl⁻ currents. With ATP-free pipette solution, the magnitude of currents was not modified suggesting the direct control by Ca²⁺. By contrast, addition of 0.1 mm cAMP in the pipette solution or the superfusion of cells by a permeant analogue of cAMP strongly reduced the currents. These results may suggest that Cl_Ca is inhibited by cAMP-dependent protein kinase. Finally, our results do not agree with the model of primary fluid secretion by exocrine cells, but are in agreement with a hyperpolarizing effect of cAMP in primary culture of Sertoli cells and the release of a low Cl⁻ and bicarbonate-rich primary fluid by these cells. Received: 30 November 1998/Revised: 2 March 1999     

CFTR Regulation of Intracellular Calcium in Normal and Cystic Fibrosis Human Airway Epithelia

In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl⁻ secretion to secretagogues acting via cAMP. Using a Ca²⁺ imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca²⁺] _i via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase in [Ca²⁺] _i in normal (16HBE14o⁻ cell line and primary lung culture) and in cystic fibrosis (CFTE29o⁻ cell line) human airway epithelia. The potency order of nucleotides on [Ca²⁺] _i variation was UTP ≫ ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca²⁺] _i response could be mimicked by activation of CFTR with forskolin (20 μm) in a temperature-dependent manner. In 16HBE14o⁻ cells, the forskolin-induced [Ca²⁺] _i response increased with increasing temperature. In CFTE29o⁻ cells, forskolin had no effect on [Ca²⁺] _i at body temperature-forskolin-induced [Ca²⁺] _i response in CF cells could only be observed at low experimental temperature (14°C) or when cells were cultured at 26°C instead of 37°C. Pretreatment with CFTR channel blockers glibenclamide (100 μm) and DPC (100 μm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 μm), inhibited the forskolin [Ca²⁺] _i response. Together, these results demonstrate that once activated, CFTR regulates [Ca²⁺] _i by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia. Received: 3 April 2000/Revised: 30 June 2000