Evaluation of the cytotoxicity of the Bithionol - cisplatin combination in a panel of human ovarian cancer cell lines.

BACKGROUND: Combination drug therapy appears a promising approach to overcome drug resistance and reduce drug-related toxicities in ovarian cancer treatments. In this in vitro study, we evaluated the antitumor efficacy of cisplatin in combination with Bithionol (BT) against a panel of ovarian cancer cell lines with special focus on cisplatin-sensitive and cisplatin-resistant cell lines. The primary objectives of this study are to determine the nature of the interactions between BT and cisplatin and to understand the mechanism(s) of action of BT-cisplatin combination. METHODS: The cytotoxic effects of drugs either alone or in combination were evaluated using presto-blue assay. Cellular reactive oxygen species were measured by flow cytometry. Immunoblot analysis was carried out to investigate changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27. Luminescent and colorimetric assays were used to test caspases 3/7 and ATX activity. RESULTS: The efficacy of the BT-cisplatin combination depends upon the cell type and concentrations of cisplatin and BT. In cisplatin-sensitive cell lines, BT and cisplatin were mostly antagonistic except when used at low concentrations, where synergy was observed. In contrast, in cisplatin-resistant cells, BT-cisplatin combination treatment displayed synergistic effects at most of the drug ratios/concentrations. Our results further revealed that the synergistic interaction was linked to increased reactive oxygen species generation and apoptosis. Enhanced apoptosis was correlated with loss of pro-survival factors (XIAP, bcl-2, bcl-xL), expression of pro-apoptotic markers (caspases 3/7, PARP cleavage) and enhanced cell cycle regulators p21 and p27. CONCLUSION: In cisplatin-resistant cell lines, BT potentiated cisplatin-induced cytotoxicity at most drug ratios via enhanced ROS generation and modulation of key regulators of apoptosis. Low doses of BT and cisplatin enhanced efficiency of cisplatin treatment in all the ovarian cancer cell lines tested. Our results suggest that novel combinations such as BT and cisplatin might be an attractive therapeutic approach to enhance ovarian cancer chemosensitivity. Combining low doses of cisplatin with subtherapeutic doses of BT can ultimately lead to the development of an innovative combination therapy to reduce/prevent the side effects normally occurring when high doses of cisplatin are administered.

K201 (JTV519) is a Ca2+-Dependent Blocker of SERCA and a Partial Agonist of Ryanodine Receptors in Striated Muscle.

K201 (JTV-519) may prevent abnormal Ca(2+) leak from the sarcoplasmic reticulum (SR) in the ischemic heart and skeletal muscle (SkM) by stabilizing the ryanodine receptors (RyRs; RyR1 and RyR2, respectively). We tested direct modulation of the SR Ca(2+)-stimulated ATPase (SERCA) and RyRs by K201. In isolated cardiac and SkM SR microsomes, K201 slowed the rate of SR Ca(2+) loading, suggesting potential SERCA block and/or RyR agonism. K201 displayed Ca(2+)-dependent inhibition of SERCA-dependent ATPase activity, which was measured in microsomes incubated with 200, 2, and 0.25 µM Ca(2+) and with the half-maximal K201 inhibitory doses (IC50) estimated at 130, 19, and 9 µM (cardiac muscle) and 104, 13, and 5 µM (SkM SR). K201 (≥5 µM) increased RyR1-mediated Ca(2+) release from SkM microsomes. Maximal K201 doses at 80 µM produced ∼37% of the increase in SkM SR Ca(2+) release observed with the RyR agonist caffeine. K201 (≥5 µM) increased the open probability (Po) of very active ("high-activity") RyR1 of SkM reconstituted into bilayers, but it had no effect on "low-activity" channels. Likewise, K201 activated cardiac RyR2 under systolic Ca(2+) conditions (∼5 µM; channels at Po ∼0.3) but not under diastolic Ca(2+) conditions (∼100 nM; Po < 0.01). Thus, K201-induced the inhibition of SR Ca(2+) leak found in cell-system studies may relate to potentially potent SERCA block under resting Ca(2+) conditions. SERCA block likely produces mild SR depletion in normal conditions but could prevent SR Ca(2+) overload under pathologic conditions, thus precluding abnormal RyR-mediated Ca(2+) release.

Eudistomin D and penaresin derivatives as modulators of ryanodine receptor channels and sarcoplasmic reticulum Ca2+ ATPase in striated muscle.

Eudistomin D (EuD) and penaresin (Pen) derivatives are bioactive alkaloids from marine sponges found to induce Ca(2+) release from striated muscle sarcoplasmic reticulum (SR). Although these alkaloids are believed to affect ryanodine receptor (RyR) gating in a "caffeine-like" manner, no single-channel study confirmed this assumption. Here, EuD and MBED (9-methyl-7-bromoeudistomin D) were contrasted against caffeine on their ability to modulate the SR Ca(2+) loading/leak from cardiac and skeletal muscle SR microsomes as well as the function of RyRs in planar bilayers. The effects of these alkaloids on [(3)H]ryanodine binding and SR Ca(2+) ATPase (SERCA) activity were also tested. MBED (1-5 µM) fully mimicked maximal activating effects of caffeine (20 mM) on SR Ca(2+) leak. At the single-channel level, MBED mimicked the agonistic action of caffeine on cardiac RyR gating (i.e., stabilized long openings characteristic of "high-open-probability" mode). EuD was a partial agonist at the maximal doses tested. The tested Pen derivatives displayed mild to no agonism on RyRs, SR Ca(2+) leak, or [(3)H]ryanodine binding studies. Unlike caffeine, EuD and some Pen derivatives significantly inhibited SERCA at concentrations required to modulate RyRs. Instead, MBED's affinity for RyRs (EC50 ∼ 0.5 µM) was much larger than for SERCA (IC50 > 285 µM). In conclusion, MBED is a potent RyR agonist and, potentially, a better choice than caffeine for microsomal and cell studies due to its reported lack of effects on adenosine receptors and phosphodiesterases. As a high-affinity caffeine-like probe, MBED could also help identify the caffeine-binding site in RyRs.

Coupled gating of skeletal muscle ryanodine receptors is modulated by Ca2+, Mg2+, and ATP.

Coupled gating (synchronous openings and closures) of groups of skeletal muscle ryanodine receptors (RyR1), which mimics RyR1-mediated Ca(2+) release underlying Ca(2+) sparks, was first described by Marx et al. (Marx SO, Ondrias K, Marks AR. Science 281: 818-821, 1998). The nature of the RyR1-RyR1 interactions for coupled gating still needs to be characterized. Consequently, we defined planar lipid bilayer conditions where ∼25% of multichannel reconstitutions contain mixtures of coupled and independently gating RyR1. In ∼10% of the cases, all RyRs (2-10 channels; most frequently 3-4) gated in coupled fashion, allowing for quantification. Our results indicated that coupling required cytosolic solutions containing ATP/Mg(2+) and high (50 mM) luminal Ca(2+) (Ca(lum)) or Sr(2+) solutions. Bursts of coupled activity (events) started and ended abruptly, with all channels activating/deactivating within ∼300 µs. Coupled RyR1 were heterogeneous, where highly active RyR1 ("drivers") seemed open during the entire coupled event (P(o) = 1), while other RyR1s ("followers") displayed abundant flickering and smaller amplitude. Drivers mean open time increased with cytosolic Ca(2+) (Ca(cyt)) or caffeine, whereas followers flicker frequency was Ca(cyt) independent and more sensitive to inhibition by cytosolic Mg(2+). Coupled events were insensitive to varying lumen-to-cytosol Ca(2+) fluxes from ∼1 to 8 pA, which does not corroborate coupling of neighboring RyR1 by local Ca(2+)-induced Ca(2+) release. However, coupling requires specific Ca(lum) sites, as it was lost when Ca(lum) was replaced by luminal Ba(2+) or Mg(2+). In summary, coupled events reveal complex interactions among heterogeneous RyR1, differentially modulated by cytosolic ATP/Mg(2+), Ca(cyt), and Ca(lum,) which under cell-like ionic conditions may parallel synchronous RyR1 gating during Ca(2+) sparks.

Single ryanodine receptor channel basis of caffeine's action on Ca2+ sparks.

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca(2+) sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca(2+) level on both sides of the channel. Cytosolic Ca(2+) enhanced RyR2 caffeine affinity, whereas luminal Ca(2+) essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC(50) of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca(2+) spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca(2+) sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.

CGP-37157 inhibits the sarcoplasmic reticulum Ca²+ ATPase and activates ryanodine receptor channels in striated muscle.

7-Chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one [CGP-37157 (CGP)], a benzothiazepine derivative of clonazepam, is commonly used as a blocker of the mitochondrial Na+/Ca²+ exchanger. However, evidence suggests that CGP could also affect other targets, such as L-type Ca²+ channels and plasmalemma Na+/Ca²+ exchanger. Here, we tested the possibility of a direct modulation of ryanodine receptor channels (RyRs) and/or sarco/endoplasmic reticulum Ca²+-stimulated ATPase (SERCA) by CGP. In the presence of ruthenium red (inhibitor of RyRs), CGP decreased SERCA-mediated Ca²+ uptake of cardiac and skeletal sarcoplasmic reticulum (SR) microsomes (IC50 values of 6.6 and 9.9 µM, respectively). The CGP effects on SERCA activity correlated with a decreased V(max) of ATPase activity of SERCA-enriched skeletal SR fractions. CGP (≥ 5 µM) also increased RyR-mediated Ca²+ leak from skeletal SR microsomes. Planar bilayer studies confirmed that both cardiac and skeletal RyRs are directly activated by CGP (EC(50) values of 9.4 and 12.0 µM, respectively). In summary, we found that CGP inhibits SERCA and activates RyR channels. Hence, the action of CGP on cellular Ca²+ homeostasis reported in the literature of cardiac, skeletal muscle, and other nonmuscle systems requires further analysis to take into account the contribution of all CGP-sensitive Ca²+ transporters.

Assessment of acyl-CoA cholesterol acyltransferase (ACAT-1) role in ovarian cancer progression-An in vitro study.

Abnormal accumulation of acyl-CoA cholesterol acyltransferase-1 (ACAT-1) mediated cholesterol ester has been shown to contribute to cancer progression in various cancers including leukemia, glioma, breast, pancreatic and prostate cancers. However, the significance of ACAT-1 and cholesterol esters (CE) is relatively understudied in ovarian cancer. In this in vitro study, we assessed the expression and contribution of ACAT-1 in ovarian cancer progression. We observed a significant increase in the expression of ACAT-1 and CE levels in a panel of ovarian cancer cell lines (OC-314, SKOV-3 and IGROV-1) compared to primary ovarian epithelial cells (normal controls). To confirm the tumor promoting capacity of ACAT-1, we inhibited ACAT-1 expression and activity by treating our cell lines with an ACAT inhibitor, avasimibe, or by stable transfection with ACAT-1 specific short hairpin RNA (shRNA). We observed significant suppression of cell proliferation, migration and invasion in ACAT-1 knockdown ovarian cancer cell lines compared to their respective controls (cell lines transfected with scrambled shRNA). ACAT-1 inhibition enhanced apoptosis with a concurrent increase in caspases 3/7 activity and decreased mitochondrial membrane potential. Increased generation of reactive oxygen species (ROS) coupled with increased expression of p53 may be the mechanism(s) underlying pro-apoptotic action of ACAT-1 inhibition. Additionally, ACAT-1 inhibited ovarian cancer cell lines displayed enhanced chemosensitivity to cisplatin treatment. These results suggest ACAT-1 may be a potential new target for the treatment of ovarian cancer.

An association of intrapartum synthetic oxytocin dosing and the odds of developing autism.

LAY ABSTRACT: Oxytocin is a hormone naturally produced in the human body that can make the womb (uterus) contract during labor. Manufactured oxytocin is frequently given to mothers in labor to strengthen the contractions or in some cases to start labor. This study compared children with a diagnosis of autism and children without autism to see whether children with autism received more oxytocin during labor. The odds of a child having an autism diagnosis were significantly higher if the delivery was a first-time Cesarean section, if the mother had a body mass index of 35 or higher, or if the reason for delivery were a range of fetal problems that made delivery necessary. It was found that boys who were exposed to oxytocin for longer periods of time during labor and received higher total doses of oxytocin had significantly higher odds of developing autism. There were no significant associations of oxytocin dosing and autism noted in female children. As this is the first study to look at any relationship between the dose of oxytocin received during labor and the odds of developing autism, further study needs to be done to determine whether there is any cause and effect relationship. Thus, at this time, there is no recommended change in clinical practice.

Assessment of peritoneal microbial features and tumor marker levels as potential diagnostic tools for ovarian cancer.

Epithelial ovarian cancer (OC) is the most deadly cancer of the female reproductive system. To date, there is no effective screening method for early detection of OC and current diagnostic armamentarium may include sonographic grading of the tumor and analyzing serum levels of tumor markers, Cancer Antigen 125 (CA-125) and Human epididymis protein 4 (HE4). Microorganisms (bacterial, archaeal, and fungal cells) residing in mucosal tissues including the gastrointestinal and urogenital tracts can be altered by different disease states, and these shifts in microbial dynamics may help to diagnose disease states. We hypothesized that the peritoneal microbial environment was altered in patients with OC and that inclusion of selected peritoneal microbial features with current clinical features into prediction analyses will improve detection accuracy of patients with OC. Blood and peritoneal fluid were collected from consented patients that had sonography confirmed adnexal masses and were being seen at SIU School of Medicine Simmons Cancer Institute. Blood was processed and serum HE4 and CA-125 were measured. Peritoneal fluid was collected at the time of surgery and processed for Next Generation Sequencing (NGS) using 16S V4 exon bacterial primers and bioinformatics analyses. We found that patients with OC had a unique peritoneal microbial profile compared to patients with a benign mass. Using ensemble modeling and machine learning pathways, we identified 18 microbial features that were highly specific to OC pathology. Prediction analyses confirmed that inclusion of microbial features with serum tumor marker levels and control features (patient age and BMI) improved diagnostic accuracy compared to currently used models. We conclude that OC pathogenesis alters the peritoneal microbial environment and that these unique microbial features are important for accurate diagnosis of OC. Our study warrants further analyses of the importance of microbial features in regards to oncological diagnostics and possible prognostic and interventional medicine.

Ryanoids and imperatoxin affect the modulation of cardiac ryanodine receptors by dihydropyridine receptor Peptide A.

Ca(2+)-entry via L-type Ca(2+) channels (DHPR) is known to trigger ryanodine receptor (RyR)-mediated Ca(2+)-release from sarcoplasmic reticulum (SR). The mechanism that terminates SR Ca(2+) release is still unknown. Previous reports showed evidence of Ca(2+)-entry independent inhibition of Ca(2+) sparks by DHPR in cardiomyocytes. A peptide from the DHPR loop II-III (PepA) was reported to modulate isolated RyRs. We found that PepA induced voltage-dependent "flicker block" and transition to substates of fully-activated cardiac RyRs in planar bilayers. Substates had less voltage-dependence than block and did not represent occupancy of a ryanoid site. However, ryanoids stabilized PepA-induced events while PepA increased RyR2 affinity for ryanodol, which suggests cooperative interactions. Ryanodol stabilized Imperatoxin A (IpTx(A)) binding but when IpTx(A) bound first, it prevented ryanodol binding. Moreover, IpTx(A) and PepA excluded each other from their sites. This suggests that IpTx(A) generates a vestibular gate (either sterically or allosterically) that prevents access to the peptides and ryanodol binding sites. Inactivating gate moieties ("ball peptides") from K(+) and Na(+) channels (ShakerB and KIFMK, respectively) induced well resolved slow block and substates, which were sensitive to ryanoids and IpTx(A) and allowed, by comparison, better understanding of PepA action. The RyR2 appears to interact with PepA or ball peptides through a two-step mechanism, reminiscent of the inactivation of voltage-gated channels, which includes binding to outer (substates) and inner (block) vestibular regions in the channel conduction pathway. Our results open the possibility that "ball peptide-like" moieties in RyR2-interacting proteins could modulate SR Ca(2+) release in cells.

Evaluation of the cytotoxicity of the Bithionol-paclitaxel combination in a panel of human ovarian cancer cell lines.

Previously, Bithionol (BT) was shown to enhance the chemosensitivity of ovarian cancer cell lines to cisplatin treatment. In the present study, we focused on the anti-tumor potential of the BT-paclitaxel combination when added to a panel of ovarian cancer cell lines. This in vitro study aimed to 1) determine the optimum schedule for combination of BT and paclitaxel and 2) assess the nature and mechanism(s) underlying BT-paclitaxel interactions. The cytotoxic effects of both drugs either alone or in combination were assessed by presto-blue cell viability assay using six human ovarian cancer cell lines. Inhibitory concentrations to achieve 50% cell death (IC50) were determined for BT and paclitaxel in each cell line. Changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27 were determined via immunoblot. Luminescent and colorimetric assays were used to determine caspases 3/7 and autotaxin (ATX) activity. Cellular reactive oxygen species (ROS) were measured by flow cytometry. Our results show that the efficacy of the BT-paclitaxel combination depends upon the concentrations and sequence of addition of paclitaxel and BT. Pretreatment with BT followed by paclitaxel resulted in antagonistic interactions whereas synergistic interactions were observed when both drugs were added simultaneously or when cells were pretreated with paclitaxel followed by BT. Synergistic interactions between BT and paclitaxel were attributed to increased ROS generation and enhanced apoptosis. Decreased expression of pro-survival factors (XIAP, bcl-2, bcl-xL) and increased expression of pro-apoptotic factors (caspases 3/7, PARP cleavage) was observed. Additionally, increased expression of key cell cycle regulators p21 and p27 was observed. These results show that BT and paclitaxel interacted synergistically at most drug ratios which, however, was highly dependent on the sequence of the addition of drugs. Our results suggest that BT-paclitaxel combination therapy may be effective in sensitizing ovarian cancer cells to paclitaxel treatment, thus mitigating some of the toxic effects associated with high doses of paclitaxel.

Exposure to subcutaneously implanted uranium dioxide impairs bone formation.

The introduction of uranium particles into subcutaneous tissue is a risk that affects workers engaged in the extraction, purification, and manufacture of uranium, as well as soldiers who are wounded with uranium shrapnel. The authors evaluated the effect of an internal source of an insoluble form of uranium on bone. Uranium dioxide powder (0.125 gm/kg body weight) was implanted subcutaneously in rats. After 30 days, animals exposed to uranium weighed less than controls. Bone formation activity in endochondral ossification and bone growth were also lower in the experimental animals, as evidenced by histomorphometric and morphometric methods. This is the first study to report bone damage resulting from continuous, nonlethal exposure to an insoluble compound of uranium dioxide over a period of 30 days.

Modulation of cardiac ryanodine receptor channels by alkaline earth cations.

Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.

Voltage-dependent modulation of cardiac ryanodine receptors (RyR2) by protamine.

It has been reported that protamine (>10 microg/ml) blocks single skeletal RyR1 channels and inhibits RyR1-mediated Ca2+ release from sarcoplasmic reticulum microsomes. We extended these studies to cardiac RyR2 reconstituted into planar lipid bilayers. We found that protamine (0.02-20 microg/ml) added to the cytosolic surface of fully activated RyR2 affected channel activity in a voltage-dependent manner. At membrane voltage (V(m); SR lumen-cytosol) = 0 mV, protamine induced conductance transitions to several intermediate states (substates) as well as full block of RyR2. At V(m)>10 mV, the substate with the highest level of conductance was predominant. Increasing V(m) from 0 to +80 mV, decreased the number of transitions and residence of the channel in this substate. The drop in current amplitude (full opening to substate) had the same magnitude at 0 and +80 mV despite the approximately 3-fold increase in amplitude of the full opening. This is more similar to rectification of channel conductance induced by other polycations than to the action of selective conductance modifiers (ryanoids, imperatoxin). A distinctive effect of protamine (which might be shared with polylysines and histones but not with non-peptidic polycations) is the activation of RyR2 in the presence of nanomolar cytosolic Ca2+ and millimolar Mg2+ levels. Our results suggest that RyRs would be subject to dual modulation (activation and block) by polycationic domains of neighboring proteins via electrostatic interactions. Understanding these interactions could be important as such anomalies may be associated with the increased RyR2-mediated Ca2+ leak observed in cardiac diseases.

Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP.

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca(2+) release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs(+) solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca(2+) release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca(2+)) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca(2+) (channel agonist) and counteracted by Mg(2+) (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca(2+) release is enhanced by high ATP-low Mg(2+) in the cytosol and by increased SR Ca(2+) load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.

Effect of chronic inhibition of converting enzyme on proximal tubule acidification.

The acute effect of angiotensin-converting enzyme inhibition (ACEi) on proximal convoluted tubule (PCT) function is well documented. However, the effect of chronic treatment is less known. The aim of this work was to evaluate the effect of chronic ACEi on PCT acidification (J(HCO(3)(-))). Rats received enalapril (10 mg.kg(-1).day(-1), added to the drinking water) during 3 mo. Micropuncture experiments were performed to measure the effect of chronic ACEi on J(HCO(3)(-)). Nitric oxide (NO.) synthesis in kidney cortex homogenates was assessed by quantifying the conversion of [(14)C]-L-arginine to [(14)C]-L-citrulline. Western blot analysis was performed to determine the abundances of V-H(+)ATPase and NHE3 isoform of the Na(+)/H(+) exchanger in proximal brush-border membrane vesicles (BBMV). Enalapril treatment induced an approximately 50% increase in J(HCO(3)(-)). Luminal perfusion with ethyl-isopropyl amiloride (EIPA) 10(-4)M or bafilomycin 10(-6)M decreased J(HCO(3)(-)) by approximately 60% and approximately 30%, respectively, in both control and enalapril-treated rats. The effect of EIPA and bafilomycin on absolute J(HCO(3)(-)) was larger in enalapril-treated than in control rats. Acute inhibition of NO. synthesis with N(G)-nitro-L-arginine methyl ester abolished the enalapril-induced increase in J(HCO(3)(-)). Cortex homogenates from enalapril-treated rats displayed a 46% increase in nitric oxide synthase (NOS) activity compared with those from untreated animals. Enalapril treatment did not affect the abundances of NHE3 and V-H(+)ATPase in BBMV. Our results suggest that PCT acidification is increased during chronic ACEi probably due to an increase in NO. synthesis, which would stimulate Na(+)/H(+) exchange and electrogenic proton transport.

Ca2+ entry-independent effects of L-type Ca2+ channel modulators on Ca2+ sparks in ventricular myocytes.

During the cardiac action potential, Ca(2+) entry through dyhidropyridine receptor L-type Ca(2+) channels (DHPRs) activates ryanodine receptors (RyRs) Ca(2+)-release channels, resulting in massive Ca(2+) mobilization from the sarcoplasmic reticulum (SR). This global Ca(2+) release arises from spatiotemporal summation of many localized elementary Ca(2+)-release events, Ca(2+) sparks. We tested whether DHPRs modulate Ca(2+)sparks in a Ca(2+) entry-independent manner. Negative modulation by DHPR of RyRs via physical interactions is accepted in resting skeletal muscle but remains controversial in the heart. Ca(2+) sparks were studied in cat cardiac myocytes permeabilized with saponin or internally perfused via a patch pipette. Bathing and pipette solutions contained low Ca(2+) (100 nM). Under these conditions, Ca(2+) sparks were detected with a stable frequency of 3-5 sparks.s(-1).100 microm(-1). The DHPR blockers nifedipine, nimodipine, FS-2, and calciseptine decreased spark frequency, whereas the DHPR agonists Bay-K8644 and FPL-64176 increased it. None of these agents altered the spatiotemporal characteristics of Ca(2+) sparks. The DHPR modulators were also without effect on SR Ca(2+) load (caffeine-induced Ca(2+) transients) or sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) activity (Ca(2+) loading rates of isolated SR microsomes) and did not change cardiac RyR channel gating (planar lipid bilayer experiments). In summary, DHPR modulators affected spark frequency in the absence of DHPR-mediated Ca(2+) entry. This action could not be attributed to a direct action of DHPR modulators on SERCA or RyRs. Our results suggest that the activity of RyR Ca(2+)-release units in ventricular myocytes is modulated by Ca(2+) entry-independent conformational changes in neighboring DHPRs.