Lack of benefit of laxatives as adjunctive therapy for functional nonretentive fecal soiling in children.

OBJECTIVES: To determine whether the combination of laxative treatment and biofeedback therapy (BF) is more effective for management of functional nonretentive fecal soiling than biofeedback therapy alone. STUDY DESIGN: In a prospective nonblinded study, 48 children were randomized in 2 groups: treatment with oral laxatives (LAX) and 5 sessions of BF (BF + LAX) or 5 sessions of BF alone (BF) during a treatment intervention period of 7 weeks. Biofeedback was performed with perfused manometry catheters and rectal balloon distension. Training focused on awareness of balloon distension and instruction in correct defecation dynamics. Successful treatment was defined as <1 encopresis episode per 2 weeks. RESULTS: At the end of the intervention period, the number of encopresis episodes was significantly decreased in both groups: from 7 (2 to 24) to 2 (0 to 17) in the BF group and from 7 (3 to 25) to 2 (0 to 14) in the BF + LAX group. However, children given BF alone had significantly higher success rates than children treated with BF and additional oral laxatives (44% to 11%). CONCLUSIONS: There is no additional effect of laxative treatment in functional nonretentive fecal soiling. Children treated with BF in combination with laxatives showed a significantly lower success percentage compared with those treated with BF alone. These results suggest that children with functional nonretentive fecal soiling should be treated differently from children with constipation and encopresis.

Evidence for VIP(1)/PACAP receptors in the afferent pathway mediating surgery-induced fundic relaxation in the rat.

We previously reported activation of an inhibitory adrenergic and a non-adrenergic non-cholinergic (NANC) pathway during abdominal surgery relaxing the rat gastric fundus. In the present study, we investigated the possible role of nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) in the NANC part of the surgery-induced fundic relaxation. The effect of the NO biosynthesis inhibitor N(G)-nitro-L-arginine (L-NOARG), the non-selective VIP receptor antagonist [D-p-Cl-Phe(6),Leu(17)]-VIP and the selective VIP(1) receptor antagonist [Acetyl-His(1),D-Phe(2),Lys(15),Arg(16), Leu(17)]-VIP was investigated on the non-adrenergic fundic relaxation induced by manipulation of the small intestine followed by resection of the caecum. Guanethidine partly reduced the manipulation-induced fundic relaxation. Addition of L-NOARG reduced this non-adrenergic component, whereas the non-selective VIP receptor antagonist had no significant effect. Combination of L-NOARG and the non-selective VIP antagonist however further reduced the relaxation to manipulation. The selective VIP(1) receptor antagonist reduced the mean and maximal relaxation induced by abdominal surgery in the presence of guanethidine. When combined with L-NOARG, the relaxation of the gastric fundus was almost completely abolished. The VIP(1) receptor antagonist alone had no significant effect on the mean and maximal relaxation, but enhanced recovery of fundic tone. In conclusion, as VIP(1) receptors are not present in the rat gastric fundus, these results suggest that the NANC inhibitory pathway activated during abdominal surgery involves VIP(1) receptors, most likely in the afferent limb. The inhibitory neurotransmitters released at the level of the gastric fundus smooth muscle are NO and a substance different from VIP.

Activation of an adrenergic and vagally-mediated NANC pathway in surgery-induced fundic relaxation in the rat.

The aim of this study was to pharmacologically characterize and investigate the possible contribution of adrenergic and nonadrenergic noncholinergic (NANC) pathways involved in the relaxation of the rat gastric fundus following abdominal surgery. Using an intragastric balloon, the effect of skin incision (SI), laparotomy (LT) and manipulation of the small intestine followed by caecal resection (M + R) on fundic pressure was evaluated. SI resulted in a brief relaxation of the gastric fundus abolished by guanethidine and blocked by hexamethonium and the combination of phentolamine, propranolol and atropine (PPA). LT induced a longer lasting relaxation which was abolished by guanethidine and hexamethonium. It was blocked by PPA and the combination of ganglionectomy and vagotomy, but unaffected by atropine, vagotomy or ganglionectomy. M + R induced a long-lasting relaxation which was only partly blocked by guanethidine or PPA, illustrating an inhibitory NANC component. Vagotomy combined with guanethidine completely abolished the relaxation following M + R, whereas it was significantly blocked by hexamethonium and the combination of ganglionectomy with vagotomy. These results indicate that SI, LT and M + R induce inhibition of fundic motility via an adrenergic mechanism. During M + R, an additional vagally mediated inhibitory NANC pathway is activated. Finally, we suggest that LT and M + R inhibit the gastric fundus via both a splanchnic and a vagal reflex pathway.

Nicotinic acetylcholine receptor blocking effect of guanethidine in the rat gastric fundus.

1 Guanethidine is commonly used as a drug to investigate adrenergic neurotransmission and, in combination with atropine, to realize non-adrenergic non-cholinergic (NANC) conditions. Previous studies suggested a nicotinic acetylcholine receptor blocking effect of guanethidine. Therefore, we investigated the effect of increasing concentrations of guanethidine (0.1-100 microM) on nicotine-induced relaxations of longitudinal muscle strips of rat gastric fundus. 2 In the presence of 1 microM atropine and 3 microM guanethidine, nicotine (30 microM) induces a fast and sustained relaxation which is partly inhibited by the nitric oxide synthase inhibitors Nomega-nitro-L-arginine (L-NOARG) and Nomega-nitro-L-arginine methyl ester (L-NAME) (both 30 and 100 microM). One microM tetrodotoxin (TTX) completely blocks this nicotine-induced relaxation. 3 High concentrations of guanethidine (> or =10 microM), but not adrenoceptor blockade by the alpha-adrenoceptor antagonist phentolamine in combination with the beta-adrenoceptor antagonist nadolol (both 3 microM), inhibit the nicotine-induced relaxation. 4 Guanethidine (0.1-100 microM) has no effect on relaxations induced by electrical field stimulation (EFS; 1-8 Hz), nitric oxide (NO; 0.01-1 microM), vasoactive intestinal polypeptide (VIP; 0.1-10 nM) or isoprenaline (1-10 nM). 5 We conclude that high concentrations of guanethidine (> or =10 microM) block nicotine-induced NANC relaxations of longitudinal muscle strips of the rat gastric fundus most likely at the level of the nicotinic acetylcholine receptor.

Expression of the smooth-muscle proteins alpha-smooth-muscle actin and calponin, and of the intermediate filament protein desmin are parameters of cardiomyocyte maturation in the prenatal rat heart.

BACKGROUND: Coexpression of alpha- and beta-myosin heavy chain (MHC) is a characteristic of the primary myocardial tube. To establish if the smooth-muscle proteins alpha-smooth-muscle actin (alpha-SMA) and calponin, and the intermediate filament protein, desmin, contribute to the specific functional properties of these early cardiomyocytes, we studied their spatiotemporal expression pattern. METHODS: Sections of prenatal and neonatal Wistar rats were stained with antibodies against alpha- and beta-MHC, alpha-SMA, calponin, and desmin. RESULTS: The expression of alpha-SMA and calponin in embryonic cardiomyocytes increases to reach its highest level at ED14. Subsequently, these proteins gradually disappear, beginning in the interventricular septum (IVS) and followed successively by the compact myocardium of the left ventricle, the right ventricle, and the central atrium. Expression of alpha-SMA persists longer in the ventricular conduction system, making it a convenient marker for the ventricular conduction system of the fetal rat. Desmin becomes expressed one day later than alpha-SMA, but also reaches its peak at ED14, suggesting that a relatively high concentration is required to form mature sarcomeres. CONCLUSIONS: The results indicate that alpha-SMA, calponin, and desmin are involved in the myofibrillar development in rat heart. The presence of spatiotemporal differences in the expression of these proteins reveals regional differences in the developmental timing of cardiomyocyte maturation. The maturation process extends from the compact myocardium in the IVS to the left and right ventricular free walls, whereas the atrioventricular junction, the ventricular trabeculae, and developing ventricular conduction system show a relatively slow maturation. Smooth-muscle proteins may contribute to the slow shortening speed that is characteristic of the embryonic myocardium.

Response of hepatic amino acid consumption to chronic metabolic acidosis.

In a previous paper, we showed that an inhibition of amino acid transport across the liver plasma membrane is responsible for the decrease in urea synthesis in acute metabolic acidosis. We have now studied the mechanism responsible for the decline in urea synthesis in chronic acidosis. Chronic metabolic acidosis and alkalosis were induced by feeding three groups of rats HCl, NH4Cl, and NaHCO3 (8 mmol/day) for 7 days. Amino acids and NH4+ were measured in portal vein, hepatic vein, and aortic plasma, and arteriovenous differences were calculated. The rates of urinary urea and NH4+ excretion were also determined. Hepatic amino acid consumption was lower in both HCl and NH4Cl acidosis compared with NaHCO3-fed rats. Glutamine release was not different in the three conditions. Because intrahepatic concentrations of amino acids and intracellular protein degradation were similar under these conditions, it can be concluded that at low blood pH amino acid catabolism may be inhibited and might explain the observed decrease in urea excretion in HCl, but not NH4Cl, acidosis; urea excretion was comparable in the NH4Cl and NaHCO3 groups presumably because the increased NH4+ load in the former group was processed, uninhibited, to urea. Amino acids not used by the liver in acidosis could account for the 25-fold increase in NH4+ excretion in HCl and NH4Cl compared with alkalosis (P < 0.05). These findings indicate that urea synthesis is decreased in chronic HCl acidosis. They show that urea synthesis is controlled in chronic, as in acute, acidosis by amino acid uptake by the liver and/or intrahepatic degradation and that the ornithine cycle per se has only minor control of acid-base homeostasis.

Changes in hepatic nitrogen balance in plasma concentrations of amino acids and hormones and in cell volume after overnight fasting in perinatal and adult rat.

Important regulatory factors of intrahepatic protein synthesis and proteolysis are amino acids, glucagon, insulin, and cell volume. We have investigated the changes in these factors with development and after an overnight fast and evaluated their contribution to changes in the hepatic nitrogen balance in vivo. In the fed state, glucagon levels were highest in suckling animals and gradually declined in older rats, whereas the concentration of insulin increased during development. The amino acid concentrations in liver and plasma declined during the suckling period to levels that in vitro are highly permissive for induction of autophagic proteolysis. In all age groups investigated, fasting was associated with a drop in hepatic protein content, together with a marked decrease in hepatocellular volume and insulin concentrations. On the other hand, glucagon concentrations and the concentration of many amino acids in plasma and liver responded to fasting with a pronounced decrease in perinatal and suckling animals, but this response had become blunted at weaning and had disappeared in adult animals. These findings suggest that insulin and/or hepatocellular volume are more likely candidates as short-term physiologic regulators of the hepatic nitrogen balance than are glucagon or amino acids. In glucose-supplemented fetuses, high levels of insulin could not compensate for a decreased hepatocellular volume in averting a catabolic state, suggesting that cell volume is the more important factor. Although our study cannot discriminate between the effects of fasting on protein synthesis and degradation, our findings show unequivocally that, for a rapid growth of the liver, suckling animals have to be fed around-the-clock.

Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes.

In rat hepatocytes, autophagy is known to be inhibited by amino acids. Insulin and cell swelling promote inhibition by amino acids. Each of the conditions leading to inhibition of autophagic proteolysis was found to be associated with phosphorylation of a 31-kDa protein that we identified as ribosomal protein S6. A combination of leucine, tyrosine, and phenylalanine, which efficiently inhibits autophagic proteolysis, was particularly effective in stimulating S6 phosphorylation. The relationship between the percentage inhibition of proteolysis and the degree of S6 phosphorylation was linear. Thus, inhibition of autophagy and phosphorylation of S6 are under the control of the same signal transduction pathway. Stimulation of S6 phosphorylation by the presence of amino acids was due to activation of S6 kinase and not to inhibition of S6 phosphatase. The inhibition by amino acids of both autophagic proteolysis and autophagic sequestration of electro-injected cytosolic [14C]sucrose was partially prevented by rapamycin, a compound known to inhibit activation of p70 S6 kinase. In addition, rapamycin partially inhibited the rate of protein synthesis. We conclude that the fluxes through the autophagic and protein synthetic pathways are regulated in an opposite manner by the degree to which S6 is phosphorylated. Possible mechanisms by which S6 phosphorylation can cause inhibition of autophagy are discussed.

Acute acidosis inhibits liver amino acid transport: no primary role for the urea cycle in acid-base balance.

To examine further the role of the liver in acid-base homeostasis, we studied hepatic amino acid uptake and urea synthesis in rats in vivo during acute acidosis and alkalosis, induced by infusion of 1.8 mmol of HCl or NaHCO3 over 3 h. Amino acids and NH4+ were measured in portal vein, hepatic vein, and aortic plasma, and arteriovenous differences of amino acids and urinary urea and NH4+ excretion were measured. In acidosis, urinary urea excretion was reduced 36% (P < 0.01), whereas urinary NH4+ excretion increased ninefold (P < 0.01), but the sum of urea and NH4+ excretion was unchanged. Total hepatic amino acid uptake, as determined from arteriovenous differences, was decreased by 63% (P < 0.01) in acidosis, with the major effect being noted with alanine and glycine. Only glutamine was released in both acidosis and alkalosis but was not significantly different in the two conditions. Since intracellular concentrations of readily transportable amino acids were not different at low pH despite accelerated protein degradation, these results indicate that hepatic amino acid transport was inhibited markedly and sufficiently to explain the observed decrease in urea synthesis. Total hepatic vein amino acid content was greater in acidosis than alkalosis (P < 0.01). Directly or indirectly, by conversion to glutamine elsewhere, these increased amino acids were degraded in kidney and accounted for the ninefold increase in urinary NH4+ excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell swelling and the sensitivity of autophagic proteolysis to inhibition by amino acids in isolated rat hepatocytes.

In the isolated perfused rat liver, autophagic proteolysis is inhibited by hypo-osmotic perfusion media [Häussinger, D., Hallbrucker, C., vom Dahl, S., Lang, F. & Gerok, W. (1990) Biochem. J. 272, 239-242]. Here we report that in isolated hepatocytes, incubated in the absence of amino acids to ensure maximal proteolytic flux, proteolysis was not inhibited by hypo-osmolarity while the synthesis of glycogen from glucose, a process known to be very sensitive to changes in cell volume [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959], was stimulated under identical conditions. However, in isolated hepatocytes, hypo-osmolarity increased the sensitivity of autophagic proteolysis to inhibition by low concentrations of amino acids. The anti-proteolytic effect of hypo-osmolarity in our experiments was not due to stimulation of amino-acid transport into the hepatocytes: neither the consumption of most amino acids, nor the rate of urea synthesis was appreciably affected by hypo-osmotic incubation conditions. In the course of these studies we also found that hypo-osmolarity increased the affinity of protein synthesis for amino acids. In the presence of amino acids the intracellular level of ATP was not much affected. However, because of cell swelling under these conditions the intracellular concentration of ATP decreased. It is proposed that a small part of the inhibition of proteolysis by amino acids may be due to this fall in ATP concentration.

Effects of intracellular amino acid concentrations, cyclic AMP, and dexamethasone on lysosomal proteolysis in primary cultures of perinatal rat hepatocytes.

We have studied factors regulating the rate of protein degradation in cultured hepatocytes obtained from 17-day-old fetal, 7-day-old suckling, and 20-day-old weanling rats. At all three stages of development 60-70% of protein degradation was sensitive to inhibition by amino acids and 3-methyladenine, an inhibitor of macroautophagy, indicating a major role of the lysosomes in proteolysis under these conditions. A combination of dibutyryl cyclic AMP and dexamethasone strongly stimulated proteolysis in hepatocytes from weanling, but not from fetal and suckling rats. The stimulatory effect of these compounds was eliminated at high amino acid concentrations in the culture medium. Cultured perinatal hepatocytes responded to exposure to dibutyryl cyclic AMP and dexamethasone by de novo synthesis of mRNA for carbamoyl-phosphate synthase and for phosphoenolpyruvate carboxykinase, demonstrating that the developmental change in the effect of dibutyryl cyclic AMP and dexamethasone on proteolysis was due to a developmental change in the regulation of proteolysis. An analysis of the changes in intracellular amino acid concentrations in response to variations in the extracellular amino acid concentrations at all three stages of development showed that of all amino acids that could be identified, only Ile, Leu, Lys, Phe, and Tyr are implicated as possible regulators of hepatic proteolysis. Dibutyryl cyclic AMP and dexamethasone did not affect the intracellular concentrations of these amino acids, showing that hormonal regulation of proteolysis is not mediated by changes in intracellular concentrations of these amino acids. It is concluded that the lack of sensitivity of the proteolytic system to catabolic hormones in the period around birth, combined with higher circulating plasma amino acid concentrations, are mechanisms contributing to the low rate of intrahepatic proteolysis in vivo in the perinatal period and thus to the rapid growth of the liver in this period.