DOES NITRIC OXIDE ACT AS A SIGNALING MOLECULE FOR MORPHOGENESIS IN ANURAN INTESTINAL REMODELING?

Breitenbach Samantha, Gudipati Mary Anitha and Menon J.Department of Biology, William Paterson University of New Jersey

Wayne, NJ 07470

Conclusions Presently observed high levels of NO derived from mitochondria and

NOS in larval epithelium during critical period of remodeling might be involved in activation of caspases required for apoptosis (Schreiber et al., 2005). NO was seen in apoptotic as well as large healthy cells signifying the role of NO as a signaling molecule in morphogenetic events (proliferation, differentiation and apoptosis) depending upon the concentration and presence of other free radical species (Weller, 1999). Clusters of small healthy NO positive cells are seen in anterior part of the intestine, which might be adult primordial cells which proliferate more rapidly than posterior part of intestine (Ishizuya-Oka et al., 1997).

Hypotheses

This research was supported in part supported by Center for Research (CfR), and Dean, College of Science & Health, WPUNJ.

Acknowledgements

References

Nitric oxide may be responsible for induction and suppression of the apoptosis during intestinal remodeling.Mitochondria may be the source of Nitric Oxide production.

Introduction

Future Studies Correlate role of NO in apoptosis by TUNEL staining in whole mounts. Double staining for mitochondria (using specific fluorescent dye) and NO.

Anuran metamorphosis involves reorganization of the body plan which includes remarkable remodeling of the intestine under the influence of thyroid hormones. The process involves morphogenetic events such as apoptosis, cell proliferation, and cell differentiation.

Menon and Rozman (2007) have shown that free radicals such as superoxide, H2O2 play an important role in intestinal remodeling. NO is produced by oxidation of L-arginine catalyzed by the enzyme nitric oxide synthase, (NOS). NOS is one of the known enzymes identical to nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) which is used as a histochemical marker for NOS. Thyroid hormone is reported to enhance shortening of the tail directly by stimulating mitochondrial generation of ROS including NO and indirectly by regulating the expression of various genes (Kashiwagi et al, 1999). Present study is designed to study the involvement of NO and NADPH-d in inducing and suppressing apoptosis during intestinal remodeling.

Materials & Methods

NADPH-diaphorase histochemistry

ResultsStage 60

Stage 66

NADPH- diaphorase histochemistry was done according to the method of Wildling (2007), on whole mounts. Tissues were fixed in 4% (wt/vol) paraformaldehyde (in PBS, pH 7.4) at room temperature for 30 min, rinsed in 0.1 M PBS 3 x 5 min, rinsed in Tris-HCL buffer (50 mM, pH 8.0) and permeabilized for 30 min in Tris-HCL buffer containing 0.3% (vol/vol) Triton X-100.Specimens were then incubated in 0.25 mg/ml nitroblue tetrazolium (NBT), 1 mg/ml β-NADPH, and 0.3% Triton X-100 in 0.1 M Tris-HCL buffer ( pH 8.0) at room temperature for 50 min and rinsed in Tris-HCL buffer for 10 min. Some tissues were pre-exposed to 1.0 mM ι – NMMA – inhibitor of NOS. Whole mount specimens were photographed and then processed for paraffin sections. 5 µm thick serial sections were cut, deparaffinized, mounted and photographed.

NOS in tissue NADPH + NBT NBT (REDUCED) + NADP⁺

Localization of NO in epidermal cells

Stage 61/62 (Inhibitor LNMA)

Mitochondrial staining by Silver chloride at stage 61/62

Amchenkova et al., 1988. J. Cell Biol. 107, 481-495Benard et al., 2007. Journal of Cell Science 120, 838-848 Menon, J. and R. Rozman. 2007.Comp. Biochem. Physiol. C: 145:625-631. Niewkoop, P. D. and J. Faber 1967. “Normal Table of Xenopus Laevus.” North Holland Publishing Company. Amsterdam. Wildling, S. and Kerschbaum H. 2007. J Comp. Pgysiol. B: 177:401-411. Weller, R. 1999. Clin. Exp. Dermatol. 24:388-391. Wang Y. and Marsden, P. 1995. Adv. Pharmacol. 34:71-90. Kashiwagi, et al., 1999. Free Radical Biol. Med. 26(7/8), 1001-1009.www.medicinelab.org.il/website/FileLib/2FluorescentSolutionsinCellBiologyIC04-28-06.

NO in epidermal cells was visualized by using 4, 5 diaminofluoresein diacetate (DAF-2DA). DAF-2DA, a membrane permeable compound, diffuses into the cells, and is diacetylated by cytoplasmatic esterases to DAF-2, which is membrane impermeable, and, consequently, trapped within the cells. DAF-2 reacts with NO to form fluorescent triazolofluorescein.Intestinal pieces were dissected, rinsed in amphibian saline and incubated with 30 μM DAF-2DA (diluted in amphibian saline from a stock solution 8 mM in DMSO) for 30 min in the dark at room temperature.Specimens were rinsed in amphibian saline for 10 min and examined with confocal laser scanning microscope ( Zeiss LSM Meta 510) using an excitation of 488 nm (argon laser) and a band pass filter of 505-530 nm.Some dissected tadpole tissues were pre-exposed to 1.0 mM ι – NMMA diluted in amphibian saline for 30 min, co exposed to 30 μM DAF-2DA and 1.0 mM ι – NMMA for 30 min in the dark at room temperature, rinsed in amphibian saline for 10 min, and analyzed as described above.

Silver stainingIntestine pieces were washed in distilled water, immersed in 0.25% silver nitrate for 30 s in dark, washed in distilled water and exposed to daylight for 30 mins and photographed.

Stage 63

At stage 61/62, NADPH-d histochemistry as well as fluorescence staining for NO showed activity in larval epithelium including typhlosole (3a, 3b, 2c). There were clusters of NO positive cells in anterior part of the intestine (Figs. 2a, 2b, 2c, 2d) in the anterior part of the intestine. Additionally, there were bands (b) of NO positive cells from the outer epithelium in the anterior part of the intestine (Figs. 4a, 4b, 5a, 5b. In the posterior part of intestine, there is diffuse population of NO positive cells (6a, 6b, 7a, 7b) Some of them were apoptotic (ap) and some healthy (h). Tissues treated with inhibitor of NOS (ι -NMA) did not show any activity (Figs. 8). At stage 63, when intestinal remodeling is completed, there were diffuse cells positive for NO (Fig. 9) and no NO positive cells were observed at stage 66 in froglet (Fig. 10).

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8 10 Purple color – NADPH-diaphorase activity whole mounts (Figs. 2b, 3a)Bright green – NO positive cells Black & white – Bright field corresponding with NOSchematic – Fig. 2aScale bar – 100 µm

Tadpoles of Xenopus laevis were obtained from Xenopus I (Ann Arbor, Michigan), and staged according to Nieuwkoop and Faber (1967). They were maintained in aquaria in 3000 ml. tap water to which AmQuel water detoxifier in the concentration of 0.5ml/ gallon was added. The water was changed every second day and tadpoles were fed a tadpole diet. Tadpoles (stages 58, 60, 61-62, 63, 66) were anesthetized in trimethanesulphonate (200mg/l). Portions of the intestine (at the junction where bile and pancreatic duct opens) was removed and used for the following studies: - Localization of NO in epidermal cells. - NADPH-diaphorase activity for examining NOS activity. - Silver nitrate staining for mitochondria.

Explanation to Figures:

3b

3a

www.medicinelab.org.il/website/FileLib/2FluorescentSolutionsinCellBiologyIC04-28-06

Note: Black precipitates of silver showing presence of mitochondria Scale bar: 100 µmArrows: Granular localization of mitochondria

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Mitochondria exists in living cells as a large tubular assembly, extending throughout the cytosol (Amchenkova et al., 1988) and form a dynamic network which gets affected by reactive oxygen species (Benard et al., 2007). 60 61/62 63 6658