Repair of ochratoxin A-induced DNA damage and modulation of OTA-related genotoxicity by co-incubation with bile acids and methotrexatein vitro

The mycotoxin ochratoxin A (OTA) induced DNA strand breaks in porcine urinary bladder epithelial cells (PUBEC) and in Madin Darby canine kidney (MDCK) cells. A co-incubation with bile acids or methotrexate reduced or even prevented this adverse effect of OTAin vitro. The protective effect is possibly attributable to a decreased OTA uptake in cells, since bile acids and methotrexate are known to share common transport systems such as organic anion transporters (OAT) and/or organic anion transporting polypeptides (OATP) with the mycotoxin. OTA uptake in cells and its modulation can be one factor which determines the extent of adverse effects in different cell types. Another aspect of interest in this regard relates to repair of DNA damage: PUBEC cells are sensitive to OTA-induced damage which is more pronounced when DNA repair is blocked (by cytosine β-D-arabino-furanosid/hydroxyurea). On the other hand, when cells are kept in fresh (toxin-free) medium for 3 h, OTA-induced DNA damage decreased to control levels. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004     

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 μM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 μM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 μM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Combinatory effects of citrinin and ochratoxin A in immortalized human proximal tubule cells

Ochratoxin A (OTA) and citrinin (CIT) are two mycotoxins often occurring together in grains and cereals. Although both are nephrotoxic and can induce apoptosis, combination effects have not been examined up to now. Therefore, the aim of this study was to take a close look at the interactions of citrinin and OTA in cultured human proximal tubule-derived cells (IHKE cells). The cytotoxicity of both mycotoxins was studied, measuring the metabolic activity and the cell number. Furthermore, caspase 3-activation as a marker for apoptosis was examined for both mycotoxin alone and in combination. The results show that citrinin had an antagonistic effect on ochratoxin A induced caspase 3-activation in concentrations of 2.5 and 5 μmol/l. Higher concentrations (7.5 and 15 μmol/l) lead to additive effects, lower citrinin concentrations (0.25 and 1 μmol/l) did not show any effect at all. The observed decrease in caspase 3-activity was specific for the combination with OTA, since the combination of citrinin with cisplatin did not show any effect. Citrinin did not influence of the OTA-induced apoptosis when added two hours after applying ochratoxin A. Also the combination of both toxins decreased the uptake of OTA into the cells which might be an explanation for the antagonistic effect of citrinin in certain concentrations. However, the transport into cells can not be the only explanation. so further examinations are necessary. Presented at the 27^th Mykotoxin-Workshop. Dortmund, Germany, June 13–15, 2005.     

Study of NO-synthase activity and detection of NO-dependent antimicrobial effects in primary culture of frog urinary bladder epithelial cells

We have shown previously that endogenous nitric oxide (NO) in frog urinary bladders modulates the influence of arginine-vasotocin on the increase in the osmotic water permeability of the bladder epithelium. The goal of the present work was to obtain a primary culture of epithelial cells from a frog urinary bladder in order to study in vitro the cellular activity of NO-synthase (NOS) and its regulation. The best conditions for cultivating the cells turned out to be with the use of a modified L-15 medium containing 10% fetal bovine serum and gentamicin (40 μg/ml) at room temperature. Under these conditions, at least 50% of the cells preserved their viability for 8 days. The NOS activity was estimated from the accumulation of nitrites (NO ₂ ^? ) in the cultivation medium; the amount of NO ₂ ^? in the presence of nitro-L-arginine methyl ester (L-NAME), an inhibitor of all types of NOS, was considered unspecific and was subtracted from each value. The NO ₂ ^? accumulation was linear in time for three days of cultivation and was inhibited by 1400W, an inducible NOS (iNOS) inhibitor, or by 7-nitroindazole, an inhibitor of constitutive NOSs, at concentrations of 5–50 and 10–200 μM, respectively. One-day incubation of the cells in the medium with low doses of gentamicin (1 or 2 μg/ml) led to a marked increase in the amount of bacteria in the cultivation m5 mM edium identified as E. coliand Acinetobacter sp. An addition of 5 mM L-NAME to the cultivation medium increased the amount of the bacteria by 1.5-and 2.5-fold in the presence of 2 and 1 μg/ml gentamicin/ml, respectively. Thus, the epithelial cells of frog urinary bladders have the NO-dependent antibacterial effect, which seems to be the result of enhanced iNOS expression. The obtained data indicate that the primary culture of the epithelial cells from frog urinary bladders is a prospective model for studying the role of NO and the regulation of NOS activity, which is of particular interest for studying protective NO effects in epithelial tissues.     

Long-term culture of porcine bladder epithelial cells

Epithelial cells from normal pig bladders proliferated when cocultured with lethally irradiated feeder cells of the LA7 rat mammary tumor line. When the bladder cells and feeders were plated together at a confluent density, the bladder cells proliferated as the feeder cells died, resulting in a confluent culture of bladder cells. The bladder cells were successfully subcultured by plating with freshly irradiated LA7 feeder cells. In this way, bladder cells from five pigs were carried to confluency in passages 1, 4, 7, 7, and 13, amounting to at least 6, 18, 24, 26, and 45 doublings in culture, respectively, and none showed signs of slowed proliferation at the time of culture termination. Fibroblasts never became a prominent feature of these cultures, and their frequency was determined to be about 26 fibroblasts per 10(5) cells in passage 9. Pig bladder cells in 0.5% serum doubled in number in slightly over 3 d, whereas cells in 5.0% serum doubled in about 6 d. In fresh medium without feeder cells only minimal proliferation of bladder cells occurred. In LA7-conditioned medium the bladder cell numbers decreased, leading to the conclusion that the stimulus from LA7 cells is mechanically or physically transmitted. The bladder cells reacted with antibodies to keratins 7 and 18 but not to keratin 14 or vimentin. Tight junctions, visualized with an antibody to the ZO1 protein, connected all the cells to their neighbors. Most cells in passage 9 carried the diploid chromosome number of 38.     

Arginase activity in frog urinary bladder epithelial cells and its involvement in regulation of nitric oxide production

The activity of arginase converting arginine into ornithine and urea is of particular interest among many factors regulating NO production in the cells. It is known that by competing with NO-synthase for common substrate (arginine), arginase can affect NO synthesis. In the present work, properties of arginase from the common frog Rana temporaria L. urinary bladder epithelial cells containing the NO-synthase were characterized, and possible contribution of arginase to regulation of NO production by epithelial cells was studied. It has been shown that the enzyme has temperature optimum in the range of 55–60°C, K _M for arginine 23 mM, and V _max about 10 nmole urea/mg of protein/min, and its activity was efficiently inhibited by (S)-(2-boronoethyl)-L-cysteine (BEC), an inhibitor of arginase, at concentrations from 10^?6 to 10^?4 M. The comparison of arginase activity in various frog tissues revealed the following pattern: liver > kidney > brain > urinary bladder (epithelium) > heart > testis. The arginase activity in isolated urinary bladder epithelial cells was 3 times higher that in the intact urinary bladder wall. To evaluate the role of arginase in regulation of NO production, the epithelial cells were cultivated in the media L-15 or 199 containing different amounts of arginine; the concentration of NO₂ ^?, the stable NO metabolites, was de-termined in the cultural fluid after 18–20 h of cell incubation. The vast majority of the produced nitrites are associated with NOS activity, as L-NAME, the NO inhibitor, decreased their accumulation by 77.1% in the L-15 medium and by 80% in the 199 medium. BEC (10^?4 M) increased nitrite production by 18.0% ± 2.7% in the L-15 medium and by 24.4% ± 3.5% in the 199 medium (p < 0.05). The obtained data indicate a relatively high activity of arginase in the frog urinary bladder epithelium and its involvement in regulation of NO production.     

Assessment of ochratoxin A and tenuazonic acid in Canadian ice-wines

Twenty-six samples of commercial ice-wine made from late-harvested grapes were assayed for the mycotoxins ochratoxin A and tenuazonic acid. Canadian wines originated in British Columbia (18) and Ontario (8). For comparison two German wines from Hesse were also studied. Four additional samples of research ice-wine originating in were also studied. In all wine samples, assays using immuno-affinity chromatography and fluorescence liquid chromatography indicated ochratoxin A below 0.15 μg/L, the limit of determination of the method. Tenuazonic acid was determined by solidphase micro-extraction and liquid chromatography and was below the limit of determination (70 μg/L) in all samples. The European Union food tolerance limit for ochratoxin A in wine is 2 μg/L. A tolerance for tenuazonic acid has not yet been established.     

Effect of dietary sodium bicarbonate supplementation on the toxicokinetics of ochratoxin A in pigs

The mycotoxin ochratoxin A (OA) is regarded as a causative agent for endemic nephropathy in farm animals and humans. Reabsorption of OA along the nephron results from nonionic diffusion and by carrier-mediated mechanisms indicating that urine alkalinization may help to accelerate OA excretion and thus reduce its toxicity. The aim of the present study was to investigate the effect of a dietary sodium bicarbonate (NaHCO₃) supplementation as a means to increase urinary pH on the systemic availability and excretion of OA in pigs. Dietary supplementation of 2% NaHCO₃ increased urinary pH (5.7±0.2 to 8.3±0.1) and daily urine volume (1108±276 to 2479±912ml) significantly. The systemic availability of OA and its dechloro-analog Ochratoxin B (OB) in the NaHCO₃ group calculated as the area under the serum concentration-time curve (AUC) was reduced to 75 and 68%, respectively, of the control (P<0.05). This effect was mainly due to an accelerated elimination of OA and OB in the urine. The faster renal elimination might be explained by a reduced reabsorption of the ochratoxins by nonionic diffusion, and other H⁺-dependent mechanisms. Thus, urinary alkalinization might be an efficient means to partially reduce the toxic effects and carry-over of OA in pigs. Presented at the 26^th Mykotoxin Workshop in Herrsching, Germany, May 17–19, 2004     

Apoptosis in cultured renal epithelial cells caused by ochratoxin A

Apoptosis induction in renal epithelial cells caused by exposure to ochratoxin A (OTA) was studied. Nanomolar concentrations of OTA led to activation of caspase 3 with subsequent chromatin condensation and DNA ladder formation, both characteristic for cells undergoing apoptosis. Inhibition of the mitochondrial ATP/ADP carrier prevented OTA induced caspase activation showing that intact mitochondria are necessary for OTA induced apoptosis.     

Time course of vasopressin-induced formation of microvilli in granular cells of toad urinary bladder

Summary Vasopressin-induced transformation of ridges to microvilli on the surface of granular cells of toad urinary bladder occurs in conjunction with induced alterations in the water permeability of the luminal membrane. This study was designed to establish the relationship between the time course for induction of microvilli and the time course for induction of increased water permeability after vasopressin stimulation. Hemibladders were examined at 2.5, 5, 10, 20 and 30 min following exposure to 20 mU/ml of vasopressin and at 5, 10, 20, 30, 40, 50 and 60 min after washout of vasopressin. Within 2.5 min, vasopressin initiated complete transformation of ridges to microvilli on approximately 13% of the granular cells, while osmotic water flow (Jv) was 0.31±0.10 l·min^–1·cm^–2. Five minutes following vasopressin stimulation, microvilli were present on approximately 30% of granular cells andJv was 2.27±0.13 l·min^–1·cm^–2. At 10 minJv was maximum at 4.03±0.15 l·min^–1·cm^–2 and 50% of the granular cells were covered with microvilli. This percentage increased to 70% at 20 min and was maintained at 30 min, althoughJv decreased to 3.9±0.35 l·min^–1·cm^–2 at 30 min. Five minutes following vasopressin washout, ridges interspersed with microvilli reappeared asJv fell to 1.10±0.30 l·min^–1·cm^–2. At 10 min after vasopressin washout,Jv approached basal levels, but the reversal of microvilli to ridges remained incomplete. At 60 min after vasopressin washout, the granular cells had regained their original ridgelike surface structures. Thus, these studies establish a temporal relationship between the induction and reversibility of vasopressin-induced microvillous formation and alterations in the osmotic water permeability of the apical plasmalemma.     

Cultured porcine urinary bladder epithelial cells as a screening model for genotoxic effects of aromatic amines: characterisation and application of the cell culture model

Isolated epithelial cells from porcine urinary bladders were maintained in dividing long-term monolayer cultures, and were used as a model system for the urinary bladder in toxicological studies in vitro. To examine the state of differentiation during the culture period, the culture system was characterised morphologically by light and transmission electron microscopy and by immune fluorescence labelling with antibodies against cytokeratins 7,13 and pan. The cultured cells were identified as urothelial epithelium by their polarised structure, and by their expression of several uroepithelial specific morphological features, such as fusiform vesicles, tight junctions and an asymmetric apical cell membrane. Additionally, the cells were labelled with anti-cytokeratin 7,13 and pan antibodies, and negatively with anti-vimentin antibodies. The maintenance of suitable culture conditions was shown by the stable enzyme activities of (gamma-glutamyltranspeptidase, alkaline phosphatase and acid phosphatase over a culture period of 4 weeks. A good viability of the cultured cells under the chosen culture conditions was shown by the presence of low amounts of lactate dehydrogenase (< of = 5%) in the culture medium. The activities of the chosen marker enzymes for cell differentiation (gamma-glutamyltranspeptidase), lysosomes (acid phosphatase) and luminal membranes (alkaline phosphatase) were relatively stable over the observed culture period. Enzyme activities involved in metabolism of xenobiotics were determined, to define the ability for metabolism in cultured cells compared with bladder tissue in situ. Several constitutive phase I and II enzyme activities were found to be stable during the culture period, indicating that the cultured cells should be able to metabolise xenobiotics in a comparable manner to the urothelium in vivo. The cytotoxic effects of xenobiotics were investigated and IC50 values were determined by means of lactate dehydrogenase leakage and inhibition of neutral red uptake. The induction of sister chromatid exchanges was used as a parameter for the genotoxic effects of several xenobiotics. This cell culture system was found to be a very good screening system for the testing of substances that affect the bladder, especially aromatic amines.     

Penicillium verrucosum in feed of ochratoxin A positive swine herds

Ochratoxin A contamination of cereal feed grain was monitored during October 1989–September 1990 by analysis of blood samples from slaughter swine in Sweden. The detection of ochratoxin A in swine blood was used as a method to identify swine herds fed ochratoxin A contaminated feed. The contamination level of ochratoxin A in the blood of the positive herds was in the range 2–45 ng/ml with the mean concentration 5.2 ng/ml. Feed samples for mycological analysis were collected from both ochratoxin A positive herds (2 ng/ml blood) and ochratoxin A negative herds (<2 ng/ml blood). From the ochratoxin A positive herds and the ochratoxin A negative herds 22 and 21 feed samples were collected, respectively. No quantitative differences in mould content, as determined by colony forming units, were observed between the two groups. However, there were differences in the mycoflora. The incidence of storage fungi (Penicillium and Aspergillus spp.) was significantly higher (p < 0.05) in feed from ochratoxin A positive herds. Particularly, Penicillium verrucosum was found to be significantly more common (p < 0.001). Altogether 274 isolates were screened for their ability to produce ochratoxin A. Ochratoxin A producers were found only within P. verrucosum; 38% of the 63 isolates produced detectable amounts of ochratoxin A. Ochratoxin A producing isolates of P. verrucosum were found in 60% of the feed samples collected from ochratoxin A positive swine herds and in one sample (5% ) of the feed samples collected from the ochratoxin A negative herds.     

The effects of ochratoxin A on lipid peroxidation and antioxidant enzymes: a protective role of melatonin

Various studies indicate that the mycotoxin ochratoxin A (OTA) is a carcinogenic, genotoxic, teratogenic, immunotoxic, and nephrotoxic agent. In the present study, the activities of some enzymes in the serum and liver of rats with ochratoxicosis and the effects of melatonin on these enzymes were investigated. Rats were divided into three equal groups, each consisting of eight rats; control, OTA (289 microg/kg per day) and OTA + melatonin groups for this study. In the OTA treated group, the level of lipid peroxidation (LPO) and the activities of glutathione peroxidase (GSH-Px) were increased in the liver and serum in comparison with the control group. The activities of catalase (CAT) and superoxide dismutase (SOD) were significantly changed in the serum when compared with controls. Our results showed structural tissue damage in the liver in OTA-treated rats. Melatonin decreased the OTA-induced damage to support the antioxidant defense system and/or with free radical scavenger action.     

Transformation of the mycotoxin ochratoxin A in plants. 2. Time course and rates of degradation and metabolite production in cell-suspension cultures of different crop plants

Ochratoxin A, one of the most toxic mycotoxins, can be metabolized nearly completely by suspension cultures of various plant cells. The transformation products identified in this study were almost the same in the cell-suspension cultures of maize, carrot, tomato, potato, soybean, wheat and barley, but the quantitative distribution differed strongly depending on incubation time and species of plant-cell culture. The compounds were extracted with ethyl acetate and detected by reversed-phase HPLC with gradient elution. From the result it is supposed that besides ochratoxin A also ochratoxin derivatives may occur in food and feedstuff of plant origin.     

Biomonitoring of ochratoxin A in grain workers

Handling agricultural commodities such as grain can result in an inhalation of mycotoxin-containing dusts. Ochratoxin A (OTA) is particularly well suited for biomonitoring studies due to its long half-life in blood, and served as a marker toxin to investigate whether or not exposure to dusts in occupational contexts may result in elevated OTA blood serum levels. OTA analysis was performed for blood samples (n=61) obtained from a cohort of male workers employed at granaries of several grain handling companies in Germany. OTA was analyzed in plasma extracts by HPLC with fluorimetric detection; calibration curves were run for each batch of samples collected between July 2005 and March 2006, and the level of detection was 0.05 ng/ml plasma. The OTA plasma levels of the 61 grain workers ranged between 0.07 ng/ml and 0.75 ng/ml. The mean (0.28±0.13 ng/ml) and median (0.26 ng/ml) OTA value for this cohort was similar to average values previously reported for the German population. Our results gave no indication that OTA in excess of those originating from typical dietary sources was ingested by these workers. Although measurable OTA concentrations have been found in dust samples collected at the corresponding workplaces (Mayeret al, this issue), the biomonitoring data do not provide evidence for a significant inhalatory burden of OTA in grain workers. Since deoxynivalenol and zearalenone were also detected in the dust samples in concentrations much higher than that of OTA, additional research should try to assess the potential relevance of an inhalation exposure to these mycotoxins. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

The effects of pH and surfactants on the absorption and fluorescence properties of ochratoxin A and zearalenone

The pH and surfactant dependencies of the absorption and fluorescence properties of ochratoxin A (OTA) and zearalenone (ZEN), the main mycotoxins found as contaminants in foods and feeds, were evaluated. Three surfactants with different ionic properties were investigated, namely sodium dodecyl sulfate (SDS, anionic), Tween 20 (nonionic) and hexadecyltrimethylammonium bromide (CTAB, cationic). The results show that the effects of pH on the absorption wavelength maxima and fluorescence efficiencies of the mycotoxins, which are a consequence of the presence of acidic phenol and/or carboxyl containing fluorophores, are dependent on the ionic nature of the added surfactants. Specifically, the fluorescence responses to pH changes of OTA and ZEN are similar in the presence or absence of Tween 20 and SDS. By contrast, the pH‐dependent fluorescence properties of these mycotoxins are altered when CTAB is present in the solutions. Moreover, unlike OTA, ZEN in aqueous solution displays almost no fluorescence. However, fluorescence enhancement takes place when surfactants are present in aqueous solutions of this mycotoxin. The results of this study demonstrate that the different microenvironments, present in the organized micellar systems created by the individual surfactants, can be potentially employed to modulate the sensitivities and selectivities of the fluorescence detection of OTA or ZEN. Copyright © 2015 John Wiley & Sons, Ltd.     

Biodegradation of ochratoxin A by Aspergillus section Nigri species isolated from French grapes: a potential means of ochratoxin A decontamination in grape juices and musts

Ochratoxin A (OTA) is a very dangerous mycotoxin, the presence of which is often reported in different foods, as well as in beverages such as grapes, grape juices and wines. Detoxifying these products is therefore of prime importance in protecting consumer health, and biological approaches have been the most promising methods. In this report, 40 isolates representing the black apergilli species Aspergillus carbonarius, A. niger aggregate and A. japonicus, isolated on French grapes, were assessed for OTA degradation capacities in CZAPEK yeast extract broth (CYB) and in a synthetic grape juice medium (SGM) contaminated with OTA at 2 mg L(-1) (5 microM). It was clearly observed that in both media these fungi had the ability to degrade OTA to OTalpha (ochratoxinalpha). However, there were differences between the media used and species tested during OTA degradation. In SGM and CYB, 77% and 45% of the isolates, respectively were able to degrade more than 80% of the OTA. Despite a better growth on SGM, specific OTA degradation was higher on CYB for most of the isolates. Kinetic studies carried out on SGM with three black Aspergillus isolates all showed different OTA degradation rates. After 9 days of incubation, OTalpha had decreased, whereas an unknown compound appeared. A. niger could be the first interesting species for OTA detoxification processes, followed by A. japonicus.     

Development and characterization of cell lines of normal mouse bladder epithelial cells and 2-Acetylaminoflourene-induced urothelial carcinoma cells grown in monolayer tissue culture

Summary Normal urinary bladder epithelial cells and cells derived from 2-acetylaminofluorene-induced urothelial carcinomas from male Balb/c mice were grown in monolayer culture and were characterized. Cell lines of normal bladder epithelium were mononucleated, sheet-forming cells, with a modal chromosome number of 40. Bladder epithelial carcinoma cells induced by 2-acetylaminofluorene were polynucleate, relatively fast growing, grew in soft agar, demonstrated a higher cloning efficiency than normal cells and formed tumors when inoculated into syngeneic hosts. Differences in morphology were recorded by photomicrography using phase optics and scanning electron microscopy.     

Penicillium verrucosum in wheat and barley indicates presence of ochratoxin A

AIMS: The aims of this study were to isolate and identify ochratoxin A (OTA) producing fungi in cereals containing OTA and to determine the best selective and indicative medium for recovery of OTA producing fungi. METHODS AND RESULTS: Seventy-six wheat, barley and rye samples from Europe containing OTA and 17 samples without OTA were investigated using three different media, dichloran yeast sucrose agar (DYSG), dichloran rose bengal yeast extract sucrose agar (DRYES) and dichloran 18% glycerol agar (DG18). Hundred kernels were plated on each medium and the kind and number of fungal OTA producers were recorded as percentage of infestation. Penicillium verrucosum was the sole OTA producer found in cereals. The average percentage of infestation of P. verrucosum counts was recorded as 28.3% on DYSG, 10.3% on DRYES and 9.9% on DG18 on the OTA containing samples and 0.8% on DYSG, 0.4% on DRYES and 0.6% on DG18 for the samples without OTA. CONCLUSIONS: Penicillium verrucosum was the sole OTA producer in European cereals. Determination of P. verrucosum infestation and infection was best detected on DYSG after 7 days at 20 degrees C. The percentage of infestation of P. verrucosum found on DYSG and OTA content in cereals were correlated. More than 7% infestation of P. verrucosum indicated OTA contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed method could be used as a cereal quality control.     

Ontogeny of the ryanodine receptor in rabbit urinary bladder smooth muscle

Bladder smooth muscle contraction is mediated by both direct calcium entry through the cell membrane, and by calcium induced calcium release (CICR) from the sarcoplasmic reticulum (SR) storage sites. Ryanodine is a neutral plant alkaloid which binds to an ion channel located on the SR membrane. Its effects in cardiac skeletal muscle are well characterized where it inibits the efflux of intracellular calcium stores, and thus it serves as a negative inotrope. It has also been shown that in the develpping rabbit myocardium, there is a gradual increase in the expression of this ion channel. Little has been written about the expression and function of the ryanodine sensitive ion channel in smooth muscle. Recently we have shown that neonatal rabbit bladder smooth muscle is not very sensitive to ryanodine, while that from mature rabbits is extremely sensitive. This leads us to quantify the expression of the ryanodine sensitive ion channel. In this paper we demonstrate that the Kd values do not change to any significant degree with normal rabbit bladder development. However the Bmax values for 3 day, 2, 4, 6, and 8 week rabbit bladder smooth muscle are 7, 10, 15, 29, and 44 fmol specifically bound ryanodine/mg protein. The differences between the neonatal groups and the mature groups are significant (P<0.5). This increase in ryanodine sensitive ion channel expression with normal growth would suggest that with normal maturation, the bladder smooth muscle cell acquires an increased pool of sequestrered intracellular calcium. This would follow a similar pattern of development that has already been described in rabbit myocardium.