In Vitro Sensitivity of Torulopsis glabrata to Amphotericin B, 5-Fluorocytosine, and Clotrimazole (Bay 5097)

Thirty-five strains of Torulopsis glabrata were tested by a tube dilution method for their susceptibility to amphotericin B, 5-fluorocytosine, and clotrimazole (Bay 5097). Amphotericin B was the most active in vitro, inhibiting all strains at a concentration of 1 μg/ml and killing all strains at 2 μg/ml. 5-Fluorocytosine inhibited over 80% of strains at 0.24 μg/ml, but three strains required ≥7.8 μg/ml for killing. A concentration of 2 μg of clotrimazole per ml inhibited less than 50% of strains, and 8 μg/ml killed only 10% of strains. Most strains of T. glabrata were killed by therapeutically achievable concentrations of amphotericin B and 5-fluorocytosine, but not clotrimazole.     

Nitrate Reduction in a Sulfate-Reducing Bacterium, Desulfovibrio desulfuricans, Isolated from Rice Paddy Soil: Sulfide Inhibition, Kinetics, and Regulation

From the second-highest dilution in a most-probable-number dilution series with lactate and sulfate as substrates and rice paddy soil as the inoculum, a strain of Desulfovibrio desulfuricans was isolated. In addition to reducing sulfate, sulfite, and thiosulfate, the strain also reduced nitrate to ammonia. The latter process was studied in detail, since the ability to reduce nitrate was strongly influenced by the presence of sulfide. Sulfide inhibited both growth on nitrate and nitrate reduction. A 70% inhibition of the nitrate reduction rate was obtained at 127 μM sulfide, and growth was inhibited by 50% at approximately 320 μM sulfide and was not detectable above 700 μM sulfide. In contrast, sulfate reduction was not affected at concentrations of up to 5 mM. After growth with sulfate, an induction period of 2 to 4 days was needed before nitrate reduction started. When nitrate and sulfate were present simultaneously, only sulfate was reduced, except when sulfate was present at very low concentrations (4 μM). At higher sulfate concentrations (500 μM), nitrate reduction was temporarily halted. The affinity for nitrate uptake was extremely high (K_m = 0.05 μM) compared with that for sulfate uptake (K_m = 5 μM). Thus, at low nitrate concentrations this bacterium is favored relative to denitrifiers (K_m = 1.8 to 13.7 μM) or other nitrate ammonifiers (e.g., Clostridium spp. [K_m = 500 μM]).     

Assessment of In Vivo and In Vitro Genotoxicity of Glibenclamide in Eukaryotic Cells

Glibenclamide is an oral hypoglycemic drug commonly prescribed for the treatment of type 2 diabetes mellitus, whose anti-tumor activity has been recently described in several human cancer cells. The mutagenic potential of such an antidiabetic drug and its recombinogenic activity in eukaryotic cells were evaluated, the latter for the first time. The mutagenic potential of glibenclamide in therapeutically plasma (0.6 μM) and higher concentrations (10 μM, 100 μM, 240 μM and 480 μM) was assessed by the in vitro mammalian cell micronucleus test in human lymphocytes. Since the loss of heterozygosity arising from allelic recombination is an important biologically significant consequence of oxidative damage, the glibenclamide recombinogenic activity at 1 μM, 10 μM and 100 μM concentrations was evaluated by the in vivo homozygotization assay. Glibenclamide failed to alter the frequency of micronuclei between 0.6 μM and 480 μM concentrations and the cytokinesis block proliferation index between 0.6 μM and 240 μM concentrations. On the other hand, glibenclamide changed the cell-proliferation kinetics when used at 480 μM. In the homozygotization assay, the homozygotization indices for the analyzed markers were lower than 2.0 and demonstrated the lack of recombinogenic activity of glibenclamide. Data in the current study demonstrate that glibenclamide, in current experimental conditions, is devoid of significant genotoxic effects. This fact encourages further investigations on the use of this antidiabetic agent as a chemotherapeutic drug.     

Comparison of the in vivo and in vitro genotoxicity of glyphosate isopropylamine salt in three different organisms

There is considerable controversy with regard to the genotoxicity of glyphosate, with some reports stating that this compound is non-toxic for fish, birds and mammals. In this work, we used the comet assay to examine the genotoxicity of glyphosate isopropylamine (0.7, 7, 70 and 700 μM) in human lymphocytes, erythrocytes of Oreochromis niloticus and staminal nuclei of Tradescantia (4430) in vitro and in vivo. Cells, nuclei and fish that had and had not been exposed to 5 mM N-nitrosodiethylamine (NDEA) were used as positive and negative controls, respectively. Significant (p < 0.01) genetic damage was observed in vivo and in vitro in all cell types and organisms tested. Human lymphocytes and Tradescantia hairs showed lower genetic damage in vivo compared to in vitro, possibly because of efficient metabolization of the herbicide. In O. niloticus erythrocytes, significant (p < 0.001) genotoxicity was observed at ≥ 7 μM, whereas in vitro, glyphosphate was genotoxic in human lymphocytes and Tradescantia hairs at ≥ 0.7 μM. These results indicate that glyphosate is genotoxic in the cells and organisms studied at concentrations of 0.7–7 μM.     

Discovery of orally active chalcones as histone lysine specific demethylase 1 inhibitors for the treatment of leukaemia

Histone lysine specific demethylase 1 (LSD1) has emerged as an attractive molecule target for the discovery of potently anticancer drugs to treat leukaemia. In this study, a series of novel chalcone derivatives were designed, synthesised and evaluated for their inhibitory activities against LSD1 in vitro. Among all these compounds, D6 displayed the best LSD1 inhibitory activity with an IC₅₀ value of 0.14 μM. In the cellular level, compound D6 can induce the accumulation of H3K9me1/2 and inhibit cell proliferation by inactivating LSD1. It exhibited the potent antiproliferative activity with IC₅₀ values of 1.10 μM, 3.64 μM, 3.85 μM, 1.87 μM, 0.87 μM and 2.73 μM against HAL-01, KE-37, P30-OHK, SUP-B15, MOLT-4 and LC4-1 cells, respectively. Importantly, compound D6 significantly suppressed MOLT-4 xenograft tumour growth in vivo, indicating its great potential as an orally bioavailable candidate for leukaemia therapy.     

Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (K_s, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (K_d, ≤3 nM) and prothioconazole-desthio the lowest (K_d, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC₅₀s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC₁₀₀, >256 μg ml⁻¹). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC₁₀₀ [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml⁻¹) with the exception of clotrimazole, which was as potent as malachite green (MIC₁₀₀, ∼1 μg ml⁻¹). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC₅₀, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC₁₀₀, ∼1 to 2 μg ml⁻¹) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.     

In Vitro Susceptibility of Atypical Mycobacteria To Rifampin

Atypical mycobacteria (209 strains) were examined for susceptibility to rifampin by the proportion method by using Middlebrook 7H-10 agar. All strains of Mycobacterium kansasii and tap-water scotochromogens were inhibited by 0.25 to 1 μg of the drug per ml. Seventy-six per cent of M. scrofulaceum and 61% of M. intracellulare strains were susceptible to 4 μg/ml or less; 5% of the former and 8% of the latter were resistant to 16 μg/ml. All strains of M. gastri and M. triviale and most strains of M. terrae were sensitive to 1 to 4 μg/ml. Two strains of M. borstelense were both inhibited by 8 μg/ml. Nearly all strains of M. fortuitum were resistant to the drug. The results of this study suggest that rifampin may be a valuable agent for the treatment of many atypical mycobacterial infections.     

Identification of Propofol Binding Sites in a Nicotinic Acetylcholine Receptor with a Photoreactive Propofol Analog

Propofol, a widely used intravenous general anesthetic, acts at anesthetic concentrations as a positive allosteric modulator of γ-aminobutyric acid type A receptors and at higher concentration as an inhibitor of nicotinic acetylcholine receptors (nAChRs). Here, we characterize propofol binding sites in a muscle-type nAChR by use of a photoreactive analog of propofol, 2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol (AziPm). Based upon radioligand binding assays, AziPm stabilized the Torpedo nAChR in the resting state, whereas propofol stabilized the desensitized state. nAChR-rich membranes were photolabeled with [³H]AziPm, and labeled amino acids were identified by Edman degradation. [³H]AziPm binds at three sites within the nAChR transmembrane domain: (i) an intrasubunit site in the δ subunit helix bundle, photolabeling in the nAChR desensitized state (+agonist) δM2-18′ and two residues in δM1 (δPhe-232 and δCys-236); (ii) in the ion channel, photolabeling in the nAChR resting, closed channel state (−agonist) amino acids in the M2 helices (αM2-6′, βM2-6′ and -13′, and δM2-13′) that line the channel lumen (with photolabeling reduced by >90% in the desensitized state); and (iii) at the γ-α interface, photolabeling αM2-10′. Propofol enhanced [³H]AziPm photolabeling at αM2-10′. Propofol inhibited [³H]AziPm photolabeling within the δ subunit helix bundle at lower concentrations (IC₅₀ = 40 μm) than it inhibited ion channel photolabeling (IC₅₀ = 125 μm). These results identify for the first time a single intrasubunit propofol binding site in the nAChR transmembrane domain and suggest that this is the functionally relevant inhibitory binding site.     

Coagulansin-A has beneficial effects on the development of bovine embryos in?vitro via HSP70 induction

Coagulansin-A (withanolide) is the steroidal lactone obtained from Withania coagulans which belong to Solanaceae family. The present study investigated the effects of coagulansin-A on bovine oocyte maturation and embryo development in vitro. All these oocytes were aspirated from the ovaries obtained from Korean Hanwoo cows at a local abattoir. To determine whether coagulansin-A has beneficial effects on bovine oocyte maturation in vitro, 355 oocytes per group (control and treated) in seven replicates were subjected with different concentrations (1, 2.5, 5, 7.5 and 10 μM) of coagulansin-A. The coagulansin-A was added in the in vitro maturation (IVM) media followed by in vitro fertilization (IVF) and then in vitro culture (IVC). Only treatment with 5 μM coagulansin-A remarkably (P<0.05) improved embryos development (Day 8 blastocyst) having 27.30 and 40.01% for control and coagulansin-A treated groups respectively. Treatment with 5 μM coagulansin-A significantly induced activation of heat shock protein 70 (HSP70) (P<0.05). Immunofluorescence analysis revealed that 5 μM coagulansin-A treatment also significantly inhibited oxidative stress and inflammation during bovine embryo development in vitro by decreasing 8-oxoguanosine (8-OxoG) (P<0.05) and nuclear factor-κB (NF-κB) (P<0.05). The expressions of HSP70 and NF-κB were also conformed through real-time PCR (RT-PCR). Additionally, the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay confirmed that coagulansin-A treatment significantly improved the embryo quality and reduced bovine embryo DNA damage (P<0.05). The present study provides new information regarding the mechanisms by which coagulansin-A promotes bovine embryo development in vitro.     

Antischistosomal,antionchocercal and antitrypanosomal potentials of some Ghanaian traditional medicines and their constituents

BackgroundGhana is endemic for some neglected tropical diseases (NTDs) including schistosomiasis, onchocerciasis and lymphatic filariasis. The major intervention for these diseases is mass drug administration of a few repeatedly recycled drugs which is a cause for major concern due to reduced efficacy of the drugs and the emergence of drug resistance. Evidently, new treatments are needed urgently. Medicinal plants, on the other hand, have a reputable history as important sources of potent therapeutic agents in the treatment of various diseases among African populations, Ghana inclusively, and provide very useful starting points for the discovery of much-needed new or alternative drugs.Methodology/Principal findingsIn this study, extracts of fifteen traditional medicines used for treating various NTDs in local communities were screened in vitro for efficacy against schistosomiasis, onchocerciasis and African trypanosomiasis. Two extracts, NTD-B4-DCM and NTD-B7-DCM, prepared from traditional medicines used to treat schistosomiasis, displayed the highest activity (IC₅₀ = 30.5 μg/mL and 30.8 μg/mL, respectively) against Schistosoma mansoni adult worms. NTD-B2-DCM, also obtained from an antischistosomal remedy, was the most active against female and male adult Onchocera ochengi worms (IC₅₀ = 76.2 μg/mL and 76.7 μg/mL, respectively). Antitrypanosomal assay of the extracts against Trypanosoma brucei brucei gave the most promising results (IC₅₀ = 5.63 μg/mL to 18.71 μg/mL). Incidentally, NTD-B4-DCM and NTD-B2-DCM, also exhibited the greatest antitrypanosomal activities (IC₅₀ = 5.63 μg/mL and 7.12 μg/mL, respectively). Following the favourable outcome of the antitrypanosomal screening, this assay was selected for bioactivity-guided fractionation. NTD-B4-DCM, the most active extract, was fractionated and subsequent isolation of bioactive constituents led to an eupatoriochromene-rich oil (42.6%) which was 1.3-fold (IC₅₀ <0.0977 μg/mL) more active than the standard antitrypanosomal drug, diminazene aceturate (IC₅₀ = 0.13 μg/mL).Conclusion/SignificanceThese findings justify the use of traditional medicines and demonstrate their prospects towards NTDs drug discovery.     

Occurrence of Fusaproliferin and Beauvericin in Fusarium-Contaminated Livestock Feed in Iowa

Fusarium fungal contaminants and related mycotoxins were investigated in eight maize feed samples submitted to the Iowa State University Veterinary Diagnostic Laboratory. Fusarium moniliforme, F. proliferatum, and F. subglutinans were isolated from seven, eight, and five samples, respectively. These strains belonged to mating populations A, D, and E of the teleomorph Gibberella fujikuroi. Fusaproliferin was detected at concentrations of 0.1 to 30 μg/g in four samples, and beauvericin was detected (0.1 to 3.0 μg/g) in five samples. Fumonisins were detected in all eight samples (1.1 to 14 μg/g). Ten of 11 strains of F. proliferatum and all 12 strains of F. subglutinans isolated from the samples produced fusaproliferin in culture on whole maize kernels (4 to 350 and 100 to 1,000 μg/g, respectively). Nine F. proliferatum strains also produced beauvericin in culture (85 to 350 μg/g), but none of the F. subglutinans strains produced beauvericin. Fumonisin B₁ was produced by all nine F. moniliforme strains (50 to 2,000 μg/g) and by 10 of the F. proliferatum strains (1,000 to 2,000 μg/g). This is the first report of the natural occurrence of fusaproliferin outside Italy and of the natural occurrence of beauvericin in North America.     

Microbial Degradation of a Macrotetrolide Miticide in Soil

Macrotetrolide, a miticide consisting of tetranactin, trinactin, and dinactin, was readily biodegradable and hence did not accumulate in soil. [U-¹⁴C]macrotetrolide was rapidly degraded via its constituent hydroxycarboxylic acids to carbon dioxide and water. In culture media, however, the mixture was hydrolyzed to homononactic and nonactic acids by three strains of Bacillus sp. and two of Micrococcus sp. The latter strains were able to hydrolyze 500 μg of the antibiotic per ml within a few days and to grow in the presence of 4,000 μg of the antibiotic per ml. However, they were unable to assimilate the constituent acids which accumulated in the culture medium.     

Multiple shoot regeneration and alkaloid cerpegin accumulation in callus culture of Ceropegia juncea Roxb.

This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of “Soma” drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 μM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog’s) medium fortified with either IAA or NAA (0.5–2.0 μM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 μM 2,4-D+5 μM BA. However the maximum cerpegin content (470 μg/g dry weight) was recorded in dried callus derived on MS+10 μM IAA+5 μM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.Key words: In vitro propagation, Pyridone alkaloid, Cerpegin, Callus, Ceropegia juncea     

Immucillins Impair Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis Multiplication In Vitro

Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05–500 μM and, when needed, 0.01–50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.     

Validation of a human-serum-based in vitro growth method for drug screening on juvenile development stages of Schistosoma mansoni

BackgroundSchistosomiasis affects over 200 million people worldwide but only praziquantel is available for treatment and control. Drug discovery is often based on phenotypic drug screening, involving different parasite stages retrieved from infected mice. Aiming to reduce animal use, we validated an in vitro growth method for juvenile Schistosoma mansoni for the purpose of drug sensitivity assays.Methodology/Principal findingsWe compared inter–batch variability of serum, worm size and organ development, gender distribution, and drug sensitivity between in vitro and in vivo grown worms over different life stages. In vitro developed S. mansoni in Hybridoma medium supplemented with 20% human serum were similar in size as in vivo worms until 28 days of incubation (males 1.4 ± 0.2 mm, females 1.1 ± 0.5 mm long). qPCR analysis revealed similar gender distribution both on newly transformed schistosomula and worms grown for 21 days. Worms developed in vitro and in vivo were similarly sensitive to praziquantel from 7 to 35 days of development with the exception of 21 days of development, where a slightly lower activity was observed for the in vitro grown worms (IC₅₀: 0.54 μM in vitro, 0.14 μM in vivo 72 hours post-incubation). The evaluation of five additional drugs revealed a similar sensitivity on worms developed for 21 days, with the exception of mefloquine, where we observed a 10-fold lower sensitivity on in vitro developed schistosomes when compared to in vivo grown (IC₅₀: 4.43 μM in vitro, 0.48 μM in vivo).ConclusionA large number of juvenile S. mansoni worms can be grown in vitro, which show similar drug sensitivity, gender distribution, size and morphology as the worms recovered from rodents, supporting the use of this method in drug screening efforts.     

Aqueous and Alcoholic Extracts of Triphala and Their Active Compounds Chebulagic Acid and Chebulinic Acid Prevented Epithelial to Mesenchymal Transition in Retinal Pigment Epithelial Cells,by Inhibiting SMAD-3 Phosphorylation

Epithelial to Mesenchymal Transition (EMT) of the retinal pigment epithelium is involved in the pathogenesis of proliferative vitreoretinopathy (PVR) that often leads to retinal detachment. In this study, Triphala, an ayurvedic formulation and two of its active ingredients, namely chebulagic acid and chebulinic acid were evaluated for anti-EMT properties based on in vitro experiments in human retinal pigment epithelial cell line (ARPE-19) under TGFβ1 induced conditions. ARPE-19 cells were treated with TGFβ1 alone or co-treated with various concentrations of aqueous extract (AqE) (30 - 300 μg/ml); alcoholic extract (AlE) (50 - 500 μg/ml) of triphala and the active principles chebulagic acid (CA) and chebulinic acid (CI) (CA,CI: 50 - 200 μM). The expression of EMT markers namely MMP-2, αSMA, vimentin and the tight junction protein ZO-1 were evaluated by qPCR, western blot and immunofluorescence. The functional implications of EMT, namely migration and proliferation of cells were assessed by proliferation assay, scratch assay and transwell migration assay. AqE, AlE, CA and CI reduced the expression and activity of MMP-2 at an ED50 value of 100 μg/ml, 50 μg/ml, 100 μM and 100 μM, respectively. At these concentrations, a significant down-regulation of the expression of αSMA, vimentin and up-regulation of the expression of ZO-1 altered by TGFβ1 were observed. These concentrations also inhibited proliferation and migration of ARPE-19 cells induced by TGFβ1. EMT was found to be induced in ARPE-19 cells, through SMAD-3 phosphorylation and it was inhibited by AqE, AlE, CA and CI. Further studies in experimental animals are required to attribute therapeutic potential of these extracts and their active compounds, as an adjuvant therapy in the disease management of PVR.     

Effect of Sarcoplasmic Reticulum (SR) Calcium Content on SR Calcium Release Elicited by Small Voltage-Clamp Depolarizations in Frog Cut Skeletal Muscle Fibers Equilibrated with 20 mM EGTA

Cut muscle fibers from Rana temporaria (sarcomere length, 3.5–3.9 μm; 14–16°C) were mounted in a double Vaseline-gap chamber and equilibrated with an external solution that contained tetraethyl ammonium– gluconate and an internal solution that contained Cs as the principal cation, 20 mM EGTA, and 0 Ca. Fibers were stimulated with a voltage-clamp pulse protocol that consisted of pulses to −70, −65, −60, −45, and −20 mV, each separated by 400-ms periods at −90 mV. The change in total Ca that entered into the myoplasm (Δ[Ca_T]) and the Ca content of the SR ([Ca_SR]) were estimated with the EGTA/phenol red method (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259–336). Fibers were stimulated with the pulse protocol, usually every 5 min, so that the resting value of [Ca_SR] decreased from its initial value of 1,700–2,300 μM to values near or below 100 μM after 18–30 stimulations. Three main findings for the voltage pulses to −70, −65, and −60 mV are: (a) the depletion-corrected rate of Ca release (release permeability) showed little change when [Ca_SR] decreased from its highest level (>1,700 μM) to ∼1,000 μM; (b) as [Ca_SR] decreased below 1,000 μM, the release permeability increased to a maximum level when [Ca_SR] was near 300 μM that was on average about sevenfold larger than the values observed for [Ca_SR] > 1,000 μM; and (c) as [Ca_SR] decreased from ∼300 μM to <100 μM, the release permeability decreased, reaching half its maximum value when [Ca_SR] was ∼110 μM on average. It was concluded that finding b was likely due to a decrease in Ca inactivation, while finding c was likely due to a decrease in Ca-induced Ca release.