Introduction important role in mediating descending relaxation in the rat

Brain Research 883 2000 15–21 www.elsevier.com locate bres Research report Decreased expression of nitric oxide synthase in the colonic myenteric plexus of aged rats a , b a a,b Toku Takahashi , Ammar Qoubaitary , Chung Owyang , John W. Wiley a Department of Internal Medicine , The University of Michigan Medical Center, 6520 MSRB I, Box 0682, Ann Arbor, MI 48109, USA b VA Medical Centers , Ann Arbor, MI 48109, USA Accepted 15 August 2000 Abstract Nitric oxide NO is a major non-adrenergic, non-cholinergic NANC inhibitory neurotransmitter in the gastrointestinal tract. NO released from the myenteric plexus enhances colonic transit and facilitates propulsion of the colonic contents by mediating descending relaxation. Although it has been suggested that colonic transit delays with aging, the mechanism of delayed colonic transit in aging remains unclear. We hypothesized that advanced age is associated with decreased expression of neuronal NO synthase nNOS and concomitant reduction in synthesis of NO in the rat colon. We studied nNOS mRNA expression, nNOS-immunohistochemistry, nNOS-immunoblotting and NOS catalytic activity in the mid-colon obtained from young age 4–8 months and aged age 22–28 months Fisher F3443BNF1 rats. Western blot analysis of PGP 9.5, a generic neuronal marker, of the colonic tissues were employed to study whether the total number of neurons of the myenteric plexus is reduced with aging. The number of nNOS-immunoreactive cells and nNOS synthesis in the colonic myenteric plexus were significantly reduced in aged rats. In contrast, expression of PGP 9.5 in colonic tissues was not affected in aged rats. Northern blot analysis demonstrated that the expression of neuronal nNOS mRNA was significantly 3 reduced in the colonic tissues in aged rats. Basal and veratridine-induced release of L -[ H]citrulline were significantly decreased in colonic tissues from aged rats, compared to young rats. It is suggested that advanced age is associated with diminished gene expression of nNOS, nNOS synthesis and catalytic activity of NOS. This may explain the mechanism of delayed colonic transit observed in advanced age.  2000 Elsevier Science B.V. All rights reserved. Theme : Development and regeneration Topic : Aging process Keywords : Colonic transit; Fisher F3443BNF1 rats; Immunohistochemistry; Western blot; Northern blot

1. Introduction important role in mediating descending relaxation in the rat

colon, an essential component of the peristaltic reflex Nitric oxide NO is a major non-adrenergic, non- [13,14]. cholinergic NANC inhibitory neurotransmitter candidate In general, gastrointestinal function is relatively well in the gastrointestinal tract. NO is synthesized from L - preserved with aging [15]. However, colonic transit ap- arginine by NO synthase NOS. In the rat and human, pears to slow with aging [21]. Madsen has previously NOS-immunoreactive neuronal cell bodies and fibers have demonstrated that older subjects 55–74 years had slower been detected throughout the entire gastrointestinal tract colonic transit of radiolabeled plastic particles than young [1,4]. Stimulation of the myenteric plexus releases NO, subjects 21–27 years, while age did not affect gastric which in turn produces relaxation of the smooth muscle in emptying or small intestinal transit [21]. The mechanism the gastrointestinal tract [3,4,6,7]. NO appears to play an of delayed colonic transit in aging remains unclear. Previous studies on aging in animal and human subjects suggest that advanced age is associated with a significant Corresponding author. Present address: Department of Surgery, decline in the number of neurons in the myenteric plexus DUMC Box 3479, Duke University Medical Center, Durham, NC 27710, that is most apparent in the colon [11,12,29]. In contrast, USA. Tel.: 11-919-286-0411 Ext. 6542; fax: 11-919-286-1140. E-mail address : ttakahasduke.edu T. Takahashi. Johnson et al. observed no significant loss of neurons in 0006-8993 00 – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 8 6 7 - 5 16 T the small intestine of aged rats, compared to young rats months old, n524 BN3F344 F1 rats were used in this using the neuronal marker, PGP 9.5 [18]. In these latter study. studies, neuronal counts that were corrected for age-associ- ated changes in intestinal length and circumference [18]. 2.3. Catalytic activity of NOS A modest decrease in the contribution of nitrergic innervation to NANC-mediated relaxation was observed L -Citrulline and NO are produced in a 1:1 ratio from with advanced age in the rat intestine [30,33]. Few studies L -arginine by the action of NOS. Production of NO was have examined the expression of NO synthase NOS with 3 measured in colonic tissue preloaded with L -[ H]arginine advancing age. The number of NADPH diaphorase a 3 and expressed as amount of L -[ H]citrulline formed in the histochemical marker for NOS containing neurons-posi- tissue as described by Bredt and Snyder [5]. As previously tive neurons in the small intestine myenteric plexus of rats described [16], the longitudinal muscles with adherent decreased modestly with age [28]. In contrast, Belai et al. myenteric plexus LMMP preparations were obtained observed a significant increase in NADPH diaphorase- from the mid-colon in young and aged rats and incubated positive neurons with age in the myenteric plexus of the in a 1.5 ml organ bath for 30 min at 378C in the presence proximal colon, but not the ileum of the rat intestine [2]. of cofactors 1 mM NADPH, 10 mM FAD, 10 mM FMN, The discrepancies in these observations might be explained and 10 mM tetrahydrobiopterin. LMMP preparations were by the possibility that NADPH diaphorase is a non-specific 3 further incubated with [ H]arginine 3 mCi ml for 4 min marker for NOS containing neurons [35]. at 378C. Immediately following the stimulation of ver- Therefore, based on available literature it is not clear atridine 5 mM for 5 min, the reaction was stopped by whether advanced age is associated with a decrease in the flash freezing in liquid nitrogen. The samples were stored total number of myenteric neurons or preferentially in- 3 at 2808C for subsequent measurement of L -[ H]citrulline. volves a reduction in a subpopulation of myenteric neu- Muscle tissues were homogenized with a 1-ml straight wall rons. The goals of this study were 2-fold; 1 to examine grinder. After centrifugation at 3000 rpm for 10 min at whether advanced age is associated with a loss of myen- 48C, the supernatant in 10 TCA was sonicated for 5 min. teric neurons in the rat colon using the neuronal marker, After washing with water-saturated ethyl ether, the super- PGP 9.5, and 2 to examine whether advanced age is natant was applied to a Dowex AG50WX-8 resin column associated with decreased expression of NOS in the rat 1 3 Na form and L -[ H]citrulline was eluted with Hepes colon. buffer pH 5.5 and water, as previously reported [16]. 3 L -[ H]Citrulline in the effluent was measured by liquid 3 scintillation spectroscopy, and the production of L -[ H]cit-

2. Materials and methods