Sympathetic (Adrenergic) Innervation Modulates But Does Not Generate Basal Tone in the Internal Anal Sphincter Smooth Muscle

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tomy. In those with persistent OSA on repeat sleep study, CPAP via nasal mask was administered (n ⫽ 34). Follow-up serum alanine aminotransferase (ALT) levels were assessed at 5–12 months after effective treatment of OSA, which was confirmed with another sleep study. A diagnosis of presumed NAFLD was made essentially if serum alanine aminotransferase level was abnormal with serologic workup and liver imaging obtained in as many children as possible. Not surprisingly, compared with nonobese children, a significantly higher proportion of obese children had elevated aminotransferases (32% vs ⬍1%; P ⬍ .001), OSA (78% vs 62%; P ⬍ .001), insulin resistance, and dyslipidemia. Compared with children without OSA (n ⫽ 175), a greater proportion of children with OSA (n ⫽ 342) had elevated ALT (ie, presumed fatty liver; 13% vs 3%; P ⬍ .001) but it is noteworthy that mean levels of aspartate aminotransferase or ALT that did not differ between these 2 groups. In a subset analysis, of 142 children with obesity, 46 were deemed to have fatty liver based on elevated ALT, and the prevalence of OSA among these 46 obese children with fatty liver was significantly higher than 96 obese children without fatty liver (91% vs 72%; P ⬍ .01). The authors estimated an odds ratio of 4.11 (95% confidence interval, 1.25–14.95) for OSA among obese children with presumed fatty liver compared with obese children without presumed fatty liver, even after controlling for age, gender, and race. But noteworthy are differences in dyslipidemia and insulin resistance between these 2 groups, which were not controlled for in their multivariable analysis. All 42 obese children with OSA and presumed fatty liver underwent effective treatment for their OSA and serum ALT levels normalized in 32 of these children (P ⬍ .001, before vs after treatment) without significant change in their body weight, but not available are any changes to lipid profile or insulin sensitivity indices.

cant improvement in liver biochemistries in children with OSA and presumed fatty liver. This study is interesting; however, some methodologic issues require discussion. The authors make a diagnosis of presumed fatty liver based on any elevation in ALT on a single measurement. Baseline fluctuations in ALT are not uncommon, especially in an obese population; it is erroneous to make a diagnosis of presumed NAFLD based on a single reading. This is especially an issue if any value over the upper limit of normal was considered to be abnormal rather than 1.5 or 2 times upper limit of normal. Although it is hard to justify their definition of fatty liver based on a single ALT reading, it may be reasonable to use such abnormality as a surrogate measure for the potential presence of fatty liver. The noted association between presumed fatty liver and OSA was not controlled for important covariates, such as insulin resistance or dyslipidemia. Without such analyses, it is not possible to exclude an incidental nature of the statistically significant association noted between presumed fatty liver and OSA. The authors report that in the majority of children ALT levels normalized after OSA treatment, but the value of this observation is limited without an untreated comparator group. It was surprising that in this cohort of predominantly obese children there was no association between body mass index (BMI) and ALT values. This finding is at odds with many other studies, which have shown consistent relationship between BMI and ALT in children. In summary, this paper suggests that there may be an association between NAFLD and OSA in children, but it was not optimally designed to differentiate if the noted association merely reflects coexistence of 2 common disorders or a cause-and-effect relationship. Owing to multiple confounders involved in this patient population, this question may be best addressed in animal models of obesity and fatty liver.

Comment. Over the last several years, many investigators

VIJAY L. MISRA, MD NAGA CHALASANI, MD

have proposed a relationship between OSA and NAFLD, but it is unknown if their relationship is independent of obesity, insulin resistance, and dyslipidemia, which are common in both of these conditions. Several studies have attempted to look at this association in adults, but the study by Kheirandish-Gozal et al. published in Chest 2008 is the first of its kind in children. Notwithstanding many limitations of this study, the authors should be congratulated for exploring this issue in children. Although many of their observations are mere confirmation of previously reported findings, there are some novel observations contained within this paper. These include (1) a higher frequency of presumed fatty liver in children with OSA compared with children without OSA, (2) a higher frequency of OSA in obese children with fatty liver compared with obese children without fatty liver disease, and (3) after the correction of OSA, there was a signifi-

SYMPATHETIC (ADRENERGIC) INNERVATION MODULATES BUT DOES NOT GENERATE BASAL TONE IN THE INTERNAL ANAL SPHINCTER SMOOTH MUSCLE Cobine CA, Fong R, Hamilton R, et al. (Department of Physiology and Cell Biology, University of Nevada, Reno, Nevada.) Species differences in the actions of sympathetic nerves and noradrenaline in the internal anal sphincter. Neurogastroenterol Motil 2007;19:937– 945. The smooth muscle of the internal anal sphincter (IAS) performs 2 important functions: It maintains

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tone in the basal state and relaxes in response to the rectoanal inhibitory reflex (RAIR; Neurogastroenterol Motil 2005;17:50 –59; Handbook of physiology, alimentary canal. Vol. IV. 1968;2121–2139; N Engl J Med 2007;356:1648 –1655; Gastroenterology 2004;126:S14 – S22; Aliment Pharmacol Ther 2001;15:887– 898; Gastroenterology 2007;133:1069 –1074). The basal tone in the IAS is important for rectoanal continence and relaxation functions for the successful completion of the RAIR. An impairment in these events leads to serious consequences, such as rectoanal incontinence, certain forms of constipation, and other related gastrointestinal motility disorders. The IAS is innervated by the extrinsic (sympathetic and parasympathetic nerves), and the intrinsic or enteric nervous system (ENS; Aliment Pharmacol Ther 2001;15: 887– 898; The enteric nervous system. 2007;1–288). The sympathetic nerves reaching the IAS are postganglionic, whereas the parasympathetic nerves are preganglionic. The parasympathetics may connect with the postganglionic adrenergic, cholinergic, and noncholinergic nonadrenergic (NANC), and sympathetics may connect with the cholinergic and NANC neurons within the IAS. These connections constitute a part of the ENS. Some of the neurotransmitters for the postganglionic sympathetic fibers terminating in the IAS are noradrenaline (NA) and neuropeptide Y (Am J Physiol Gastrointest Liver Physiol 1990;258:G59 –G64; Regul Pept 1991;III:29 –35). NA by the activation of ␣-adrenoceptors (especially ␣-1 [␣1-AR]) causes increase in the sphincteric tone and inhibition in the adjoining nonsphincteric smooth muscles (Goodman & Gilman’s the pharmacological basis of therapeutics, 10th ed. 2001;115–153; Aliment Pharmacol Ther 2001; 15:887– 898; Neurogastroenterol Motil 2005;17:50 –59; J Clin Invest 1990;86:424 – 429). The parasympathetic postganglionic fibers are either excitatory (neurotransmitters [eg, acetylcholine/substance P]), or inhibitory (via nitric oxide [NO], vasoactive intestinal polypeptide, adenosine triphosphate (ATP), or related substances; Am J Physiol Gastrointest Liver Physiol 1992;262:G107–G112; J Clin Invest 1988;81:1146 –1153; Neurogastroenterol Motil 2005;17:50 –59; J Auton Nerv Syst 1999;19:29 –37). The role of ␣1-AR in the basal tone may be examined by evaluating the effect of electrical field stimulation (EFS) after the blockade of cholinergic and NANC effects, ␣1-AR agonists and antagonists, and neurotoxins in the isolated IAS smooth muscle strips. Such studies in humans and different species (Aliment Pharmacol Ther 2001;15:887– 898; Br J Surg 2008;92:1263–1269; Br J Surg 2007;94:894 –902; Neurogastroenterol Motil 2005;17:50 – 59; Gastroenterology 1983;84:409 – 417; Gastroenterology 1992;102:679 – 683; Gut 1993;34:689 – 693) have led to the conclusion that, although ␣1-AR activation exerts important excitatory modulation, it may not contribute significantly to the basal tone in the IAS.

GASTROENTEROLOGY Vol. 134, No. 7

Recent studies by Cobine et al (Neurogastroenterol Motil 2007;19:937–945) examined the role of ␣1-AR in the IAS using different animal species (monkeys, mice, and rabbits). The authors examined the effects of EFS, NA, and adrenergic inhibitors in the IAS tone in the isolated smooth muscle strips. In the presence of the nitric oxide synthase (NOS) inhibitor N(␻)-nitro-L-arginine (L-NA) and anticholinergic atropine EFS and NA caused contraction in the monkey IAS but relaxation in the murine and rabbit IAS. The contractile responses in the monkey IAS were converted into relaxation in the presence of ␣1-AR antagonist phentolamine and adrenergic depletor guanethidine. The NA-induced relaxation in monkey IAS was abolished by the ␤-AR antagonist propranolol. The authors concluded that in contrast to the murine and rabbit, the monkey IAS is functionally innervated by the excitatory sympathetic nerves that contribute significantly to the IAS tone. Accordingly, for the adrenergic effects in the IAS, the monkey may be a more appropriate animal model as compared with mice and rabbits. Comment. Cobine et al have used a brilliant approach to

determine the contribution of adrenergic nerves by carrying out studies in the presence of NOS inhibitor L-NA and anticholinergic atropine to alleviate any interference of the nitrergic inhibitory neurotransmission and muscarinic excitatory transmission, respectively. Their findings in different species are consistent with the bulk of the literature, although with some important differences. The IAS smooth muscles isolated from different species examined demonstrate the development of spontaneous tone. In such experimental settings, the smooth muscle tissues are devoid of any circulating neurohumoral substances as is the case during the in vivo settings. Such information by itself may not be interpreted as the tone being myogenic because in addition to the smooth muscle cells (SMC) the smooth muscle has a number of inputs from adrenergic, cholinergic and NANC neurons among others and their signal transduction machineries, in various proportions (Handbook of methods in gastrointestinal pharmacology 1996;189 –224). Because of such variability, it is conceivable that EFS causes variable effects in different species. Studies further show contraction of the monkey IAS, and relaxation in the murine and rabbit IAS in response to EFS in the presence of NOS inhibitor L-NA, and atropine. A majority of the literature, however, in different species including humans, mice, and rabbits shows variable responses to EFS under similar experimental conditions, either almost complete obliteration of the relaxation, or a brief after contraction (Gastroenterology 1992;102:679 – 683; Am J Physiol Gastrointest Liver Physiol 1993;265:G792–G798; J Pediatr Surg 1994;29: 294 –300; Am J Physiol Gastrointest Liver Physiol 2005;

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289:G291–G299; Gastroenterology 1997;112:1575–1585; J Pharmacol Exp Ther 1996;277:1376 –1382; Aliment Pharmacol Ther 2001;15:887– 898; Neurogastroenterol Motil 2005;17:50 –59). The observed differences in the monkey IAS in response to EFS may reflect variability in the degree and nature of NANC inhibitory (NO, VIP, ATP) and excitatory neurotransmissions (NA, acetylcholine, neuropeptide Y, and substance P; Am J Physiol Gastrointest Liver Physiol 1990;258:G59 –G64; Regul Pept 1991;III:29 –35; Neurosci Lett 1987;74:304 –308). The other possible differences may lie in the relative distribution of sympathetic nerves and their terminations, and contribution of ␣1-AR and ␣2-AR after sympathetic nerve stimulation. The latter provide inhibitory input to a number of myenteric neurotransmissions. In addition, whether appropriate stimulus parameters required to activate different structures in the ENS in different species, play a role for the final observed response, remains to be determined. The authors show important differences in the responses to NA in monkeys compared with mice and rabbits. The inhibitory effects of NA seem to involve activation of ␣1-AR, ␣2-AR, and ␤-AR in different proportions. The studies clearly point out that with regard to the adrenergic effects in the IAS, monkey and other species (but not mice and rabbits) may be similar to humans. The studies by no means minimize the importance of mice and rabbits for the other functional studies in the IAS. In this respect, mice (especially because of the availability of a number of specific knockouts) will continue to provide important information on the IAS regulation. In agreement with the literature, the authors also found that in all the species investigated, neither the ␣1-AR antagonist phentolamine nor the adrenergic depletor guanethidine had any significant effect on the basal tone in the IAS. In most of the IAS preparations from different species including humans (Gastroenterology 1983;84:409 – 417; Aliment Pharmacol Ther 2001;15:887– 898; Neurogastroenterol Motil 2005;17:50 –59) blockade of different neurohumoral influences, and the neurotoxin tetrodotoxin (which impairs all neural inputs) produced no significant effect on the basal tone. These significant findings suggest that sympathetic innervation via adrenergic activation may provide excitatory modulation, but does not generate the basal tone in the IAS, suggesting the tone to be mostly myogenic in origin. Understanding of the mechanism(s) responsible for the basal tone in the IAS are vital for the advances in the pathophysiology and therapy of debilitating rectoanal motility disorders, as pointed out. Adrenergic regulation of the IAS tone would have provided important rationale for some of these motility disorders with the use of ␣1-AR agonists (in the case of hypotensive IAS as in certain forms of rectoanal incontinence) and with ␣1-AR antagonist (for the hypertensive IAS as in hemorrhoids and

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recurrent anal fissures). However, these approaches have not been uniformly successful in treating such conditions (Aliment Pharmacol Ther 2001;15:887– 898; Br J Surg 2000;87:38 – 42; Int J Colorect Dis 2007;22:1319 – 1324; Colorectal Dis 2001;3:165–168). In addition, stimulation and denervation of the sympathetic nerves supplying the IAS have produced inconsistent results (Gastroenterology 1983;84:409 – 417; Br J Surg 1987;74: 668 – 670; Int J Colorect Dis 1991;3:90 –95). All data put together suggest that the basal tone in the IAS is largely because of the distinct properties of the SMC of the IAS. Such myogenic phenomenon responsible for the IAS tone is inherent to the SMC (Am J Physiol Gastrointest Liver Physiol 2005;289:G1164 –G1175; Am J Physiol Gastrointest Liver Physiol 2007;292:G1747–G1756) to the extent that one can bioengineer the tonic IAS in reverse from the isolated cells (Am J Physiol Gastrointest Liver Physiol 2005;289:G188 –G196). Although no single animal model (including monkeys) can substitute for human studies, basic studies in the IAS using different animal models have added significantly to our present knowledge of the pathophysiology of human anorectal motility disorders. In addition, differences in the effects of EFS and NA in mice and rabbits (vs monkeys) may not suggest the absence of adrenergic innervation and ␣1-AR in these species. The most valid conclusion from the present studies is that mice and rabbits may not be appropriate animal models to investigate the adrenergic effects in the IAS. Detailed studies to tease out different components of sympathetic pathway (mapping out adrenergic innervation, sympathetic stimulation, and ␣1-AR distribution patterns) in mice and rabbits IAS may provide plausible explanation for the observed differences in these animal species. Finally, regardless of the species, critical survey of the literature suggests that although sympathetic motor innervation (via adrenergic activation) provides excitatory modulation, it may not be major contributor for the basal tone in the IAS. SATISH RATTAN, DVM

Reply. Dr Rattan has maintained in several recent stud-

ies (eg, Am J Physiol Gastrointest Liver Physiol 2007;292: G1747–G1756) that tone in the internal anal sphincter (IAS) is generated and maintained by mechanisms intrinsic to the smooth muscle cells. He reiterates this point of view in his comments on our recent paper. Although we agree that there are indeed intrinsic, myogenic mechanisms contributing to setting the contractile state of the IAS, this view is likely to be too simplistic to explain the moment-to-moment regulation of tone that is critical for fecal continence in human patients. Studies of neural innervation in isolated muscle strips are useful because they allow one to examine the actions of sympathetic nerves when stimulated electrically. The limitation of this approach is that the sympathetic nerves are separated from their cell bodies, which are located in