Biochemical and Molecular Action of Nutrients Nitric Oxide Synthase Inhibitor Attenuates Intestinal Damage Induced by Zinc Deficiency in Rats Li Cui, Yoji Takagi, Masafumi Wasa, Kinya Sando, Jesmine Khan and Akira Okada 1 Department of Pediatric Surgery, Osaka University Medical School, Suita, Osaka 565, Japan ABSTRACT A nitric oxide synthase (NOS) inhibitor, N G -nitro-L -arginine methyl ester (L-NAME), was given to zinc-deficient (ZD) rats to determine whether it prevents the intestinal damage usually observed under these conditions. Weanling male rats were given free access to a ZD diet (2 mg zinc/kg), whereas control rats including pair-fed (PF) and ad libitum consumption (AL) groups were given a zinc-supplemented (50.8 mg zinc/kg) diet for 4 wk. Half of the ZD rats received L-NAME (0.3 g/L in drinking water) for 3 wk starting at the wk 2 of the deficient period. Plasma zinc concentration in ZD rats was significantly lower (P  0.05) than that of AL and PF rats. Administration of L-NAME did not alter this concentration. Intestinal zinc concentration did not differ among groups. However, metallothionein-1 (MT-1) mRNA level was significantly lower in the intestine of ZD rats than in AL or PF rats. Treatment of ZD rats with L-NAME did not affect this level. Intestinal microvascular permeability evaluated by Evans blue showed significantly higher extravasation in ZD rats than in AL rats, whereas L-NAME administration inhibited the extravasation. Expression of inducible NOS mRNA was observed in intestine of ZD but not of AL or PF rats, and there was no significant difference between ZD rats, regardless of L-NAME treatment. The activity ratio of inducible NOS to total NOS in ZD rats not receiving L-NAME was significantly higher than that in AL rats or ZD rats treated with L-NAME (P  0.05). The number of apoptotic-positive and goblet cells in intestinal villi was significantly higher in ZD rats compared with AL or PF rats. L-NAME administration in ZD rats reversed this effect. These results indicate that inhibition of NOS ameliorates zinc deficiency-induced intestinal damage in rats. J. Nutr. 129: 792–798, 1999. KEY WORDS: ● nitric oxide synthase inhibitor ● apoptosis ● goblet cell hyperplasia ● zinc deficiency ● intestine ● rats Zinc is one of the essential trace elements in human and animals. Its deficiency may occur in various disorders or during artificial nutritional support. Zinc deficiency often develops with gastrointestinal manifestations, including diarrhea, ab- dominal pain, vomiting and fever (Okada et al. 1976), indi- cating that the intestine is one of the tissues most sensitive to zinc deficiency. Our previous study (Cui et al. 1996), consis- tent with other reports (Nobili et al. 1997, Southon et al. 1984, Vallee and Falchuk 1993), demonstrated that zinc de- ficiency induces changes in intestinal morphology, including decreases in villous height and crypt depth, inflammatory cell infiltration of the lamina propria and lesions of intestinal mucosa. Nitric oxide, a free radical gas, is an important regulatory factor in physiologic processes (Vallance and Moncada 1994). The intestine possesses both the calcium-dependent constitu- tive nitric oxide synthase (NOS) 2 and the calcium-indepen- dent inducible NOS (iNOS), which has been demonstrated under lipopolysaccharide stimulation (Tepperman et al. 1993). iNOS, when induced, produces a large amount of nitric oxide (Iadecola et al. 1995), resulting in a decrease in cellular viability and local intestinal damage (Tepperman et al. 1993). Recently, we reported that zinc-deficient (ZD) rats had an iNOS gene expressed in the intestine and that interleukin-1 treatment caused many fold enhancement in expression and induced diarrhea (Cui et al. 1997). Accordingly, we reasoned that nitric oxide produced by iNOS may play a role in the mechanisms of zinc deficiency–induced damage in the intes- tine. Evans blue, when injected into the circulation, binds within seconds to serum proteins (mainly albumin), forming a dissociable complex. An increase in local capillary permeabil- ity to macromolecules, caused by inflammation or other types of damage, will therefore be detected as an extravasation and deposition of the protein-Evans blue complex in the intersti- tial tissues (Lange et al. 1994). Evans blue leakage technique has been used to evaluate the role of NO in microvascular permeability of the skin (Lippe et al. 1993). Apoptosis is a physiologically essential mechanism of cell death that together with cell proliferation is responsible for the precise regulation of cell numbers for a variety of cell popula- tions during normal development (Raff et al. 1993, Steller 1 To whom correspondence should be addressed. 2 Abbreviations used: AL, ad libitum intake of the zinc-supplemented diet; DDW, double distilled water; GAPDH, glyceraldehyde-3-phosphate-dehydroge- nase; IL-1, interleukin-1; iNOS, inducible nitric oxide synthase; LNAME, N G -nitro- L-arginine methyl ester; MT, metallothionein; NOS, nitric oxide synthase; PF, pair-fed the zinc-supplemented diet; RT-PCR, reverse transcription-polymerase chain reaction; TUNEL, TdT-mediated dUTP-biotin nick end labeling; ZD, free access to the zinc-deficient diet. 0022-3166/99 $3.00 © 1999 American Society for Nutritional Sciences. Manuscript received 1 June 1998. Initial review completed 7 July 1998. 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