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Centrally applied atrial natriuretic factor diminishes bile secretion in the rat
Regulatory Peptides 102 Ž2001. 127–133 www.elsevier.comrlocaterregpep
Centrally applied atrial natriuretic factor diminishes bile secretion in the rat Liliana G. Bianciotti a,) , Marcelo S. Vatta b, Cristina Vescina c , Valeria Trippodi c , Maria E. Sabbatini b, Belisario E. Fernandez a a
Catedra de Fisiopatologıa, UniÕersidad de Buenos Aires, Junın ´ ´ Facultad de Farmacia y Bioquımica, ´ ´ 956, 5 Piso, 1113 Buenos Aires, Argentina b Catedra de Fisiologıa, UniÕersidad de Buenos Aires, Buenos Aires, Argentina ´ ´ Facultad de Farmacia y Bioquımica, ´ c Catedra de Quımica Analıtica, Facultad de Farmacia y Bioquımica, UniÕersidad de Buenos Aires, Buenos Aires, Argentina ´ ´ ´ ´ Received 30 May 2001; received in revised form 1 August 2001; accepted 14 August 2001
Abstract Little is known about the role of centrally applied peptides in the regulation of bile secretion. We previously reported that the intravenous injection of atrial natriuretic factor ŽANF. reduces bile acid dependent flow without affecting portal venous pressure in the rat. In the present work, we studied the effects of centrally applied ANF on bile secretion and the possible pathways involved. Rats were cannulated in the brain lateral ventricle for the administration of 1, 10 and 100 ngrml ANF. After 1 week, the common bile duct was cannulated and bile samples were collected every 15 min for 60 min after the administration of ANF. The excretion rate of various biliary components was assessed. Bile secretion experiments were also performed after bilateral truncal vagotomy or atropine administration to evaluate the participation of a vagal pathway. In addition, the role of the sympathetic system was addressed by combined administration of propranolol and phentolamine. Centrally applied ANF did not modify blood pressure but diminished bile flow and bile acid output. It also reduced sodium and potassium secretion but did not modify protein or phospholipid excretion. Neither bilateral truncal vagotomy nor atropine administration abolished ANF response. Furthermore, combined administration of adrenergic antagonists did not alter ANF inhibitory effect on bile flow. In conclusion, centrally applied ANF reduced bile acid dependent flow not through a vagal or adrenergic pathway in the rat, suggesting the involvement of a peptidergic pathway. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Bile flow; Natriuretic peptides; Bile acids; Central regulation
1. Introduction Atrial natriuretic factor ŽANF. is a hormone produced by the heart that is released in response to atrial stretch, endothelin-1 and a 1 adrenergic stimulation w1x. ANF is intimately related to the control of blood arterial pressure as well as hydro saline homeostasis w2x. Although the heart is the main source of ANF, increasing evidence indicates the existence of extra-cardiac sources such as the central nervous system, the gastrointestinal tract, liver and the salivary glands, where this peptide is likely to have a paracrine role w3–5x. Natriuretic peptides receptors, both coupled and uncoupled to guanylate cyclase, are widely distributed in many tissues and cell types. Receptors for ) Corresponding author. Tel.: q54-11-4964-8268r8280; fax: q54-114964-8268. E-mail address: [email protected] ŽL.G. Bianciotti..
the natriuretic peptides were described in the gastrointestinal tract w6x, liver w7x, pancreas w8x, and salivary glands w5x as well as in the central nervous system w9,10x. The central areas include the dorsal motor nucleus of the vagus and the nucleus of the solitary tract, which are important sites for the autonomic nervous regulation of gastrointestinal functions w9,10x. We have previously reported that systemically administered ANF alkalinizes bile and reduces spontaneous as well as evoked bile secretion in the rat. ANF diminishes bile acid dependent flow since it diminishes bile acid secretion but also the output of sodium and potassium that accompany this bile fraction w11x. Furthermore, it also increases bicarbonate output, which supports an increase in the bile acid independent flow fraction, and is partially related to this anion output. ANF slightly decreases mean arterial pressure but it does not affect portal venous pressure in the rat w11x.
0167-0115r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 1 1 5 Ž 0 1 . 0 0 3 1 0 - X
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Several peptides and neuropeptides that regulate gastrointestinal physiology also influence either gastrointestinal motility andror digestive secretions when they are centrally applied. However, little is known about the central regulation of bile secretion by peptides or neuropeptides as compared with the wide literature on the brain regulation of gastric secretion and gastrointestinal motor function. The secretion of bile is increased by centrally applied neuropeptide Y through a vagal pathway w12,13x whereas it is reduced by the intracerebroventricular Žicv. injection of bombesin w14x. These findings support the involvement of peptides or neuropeptides in the regulation of bile secretion, acting through the modulation of the autonomic nervous system. On this basis, the aim of the present work was to study the effects of central injections of ANF on bile secretion as well as the possible pathways involved. Our findings show that centrally applied ANF diminished bile acid dependent flow, although the inhibition of neither the parasympathetic nor the sympathetic activity abolished ANF response, suggesting the possible participation of a peptidergic pathway.
2. Materials and methods Male Sprague–Dawley strain ŽFacultad de Farmacia y Bioquimica, Universidad de Buenos Aires. rats of 300– 330-g body weight were used in the experiments. One week previous to the bile secretion experiments, rats under anesthesia Žchloral hydrate, 0.4 mgrkg, ip. ŽSigma, St Louis, MO. were placed in a stereotaxic instrument ŽKopf model 900 David Kopf Instruments, USA. and a steel cannula was introduced in the right ventricle of the brain. Briefly, rats were placed on a stereotaxic frame and the skin overlying the midline of the skull was incised. A small hole was drilled through the appropriate area of the skull and a 23-gauge stainless steel cannula was placed in the right lateral ventricle Ž1.3 mm posterior to the bregma, 2.0 mm lateral to the midline and 4.0 mm ventral to the skull surface. w15x. The cannula was anchored with a stainless steel screw in the skull and covered by dental acrylic. Rats were individually caged and allowed to recover from surgical procedures for a week. Water and Purina commercial chow were given ad libitum except 14 h prior to bile secretion experiments in that food was removed to avoid possible release of peptides andror hormones that may eventually influence bile secretion. Rats were anesthetized with urethane Ž1.3 mgrkg, ip., and through a midline abdominal incision, the common bile duct was exposed and cannulated with a polyethylene catheter ŽPC-10 Intramedic, USA.. Rats remained anesthetized during bile collection that was performed between 9:00 and 11:00 to avoid possible circadian changes w16x. Body temperature was kept at 37 8C with a heating pad.
Bile secretion was allowed to flow for 10 min Žbasal period. to stabilize bile flow. ANF ŽPeninsula, Belmont, CA. Ž1, 10 and 100 ngrml. or artificial cerebrospinal fluid Žcontrol group. was icv injected Ž1 ml volume. at a rate of 1 mlrmin. Bile was collected every 15 min for 60 min in pretared microcentrifuge tubes. The composition of artificial cerebrospinal fluid used was ŽmM.: NaCl, 125; CaCl 2 , 1.2; MgCl 2 , 0.9; NaHCO 3 , 25; Na 2 HPO4 , 0.5; KH 2 PO4 , 0.5; glucose, 4.3; and urea 6.5. The accuracy of icv injections was assessed at the end of each experiment by icv administration of 1 ml methylene blue ŽSigma.. Animals were killed, and through the opening of the skull, the brain was removed, and the presence of methylene blue was verified strictly in the lateral ventricle. Bile volume was determined gravimetrically assuming the specific gravity of rat bile to be 1.0. Biliary sodium and potassium were measured by the ion analyzer method ŽTechnolab, Argentina.. Bile acid concentration was assessed by the 3a-hydroxysteroid dehydrogenase assay w17x. Phospholipid and total protein concentrations were assessed by modified Bartleet w18x and Lowry w19x methods, respectively. Bile flow Žmlrminr100 g body weight. and the excretion rate of the different constituents of bile were calculated. The role of the vagal cholinergic pathway in mediating ANF effect was assessed by two different experimental approaches: bilateral truncal vagotomy and atropine sulphate administration. Cervical vagotomy was not done to avoid possible hemodynamic alterations resulting from the section of cardiac vagal branches. Bilateral truncal vagotomy was performed by the section of both branches of the vagus and vagal afferents at the level of the lower esophagus 2 h before bile collection experiments. Atropine sulfate was administered in bolus 30 min before bile collection and infused at 75 mgrkgrh during the collection of bile. Control rats were infused at the same rate with saline. The participation of the sympathetic system was assessed by the administration of phentolamine Ž a-adrenergic blocker. and propranolol, Žb-adrenergic antagonist.. A bolus of 0.5 mgrkg phentolamine was given intravenously 30 min before ANF injection and then the antagonist was infused at a constant rate of 0.2 mgrkgrh throughout the period of bile collection. A bolus of propranolol 0.5 mgrkg was administered intravenously 30 min before icv injection of ANF. The dose of ANF used in these experiments was 100 ngrml. Blood pressure was monitored throughout the collection of bile after the administration of ANF using a blood pressure transducer ŽStatham 923Db. by inserting a polyethylene cannula ŽP-50 Rivero y Cia, Argentina. into the right carotid artery. The signals were recorded on a polygraph ŽCoulbourn Inst.. with data acquisition software ŽDATAQ Inst... Three readings were registered for each rat. Results are expressed as mean arterial pressure. Statistical analysis was performed by using ANOVA and the t-test modified by Bonferroni. Results are ex-
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Fig. 1. Effect of centrally applied ANF on bile flow Žmlrminr100 g BW.. I: Control; v: 1 ngrml ANF; B: 10 ngrml; ': 100 ngrml. ) : p- 0.05, ) ) : p- 0.01 and ) ) ) : p- 0.001 vs. control. Number of cases: 5–7. BW: body weight.
pressed as the mean " S.E.M.; p F 0.05 was considered statistically significant.
3. Results In order to evaluate the central effect of ANF on bile secretion, different concentrations of ANF were centrally applied. Results showed that central administration of 1, 10 and 100 ngrml ANF significantly reduced spontaneous bile flow at 15, 30, 45 and 60 min in a dose-dependent manner ŽFig. 1.. With the aim to assess possible hemodynamic alterations as the result of icv ANF injections, we measured blood pressure. Centrally applied ANF Ž1, 10, 100 ngrml. did not modify mean arterial pressure in the rat in line with previous findings w20x ŽTable 1.. Blood pressure remains unchanged during the whole period of bile collection after icv ANF. Only values up to 15 min are shown. The involvement of a cholinergic pathway in ANF response on bile secretion was evaluated by truncal vagotomy and atropine administration. Results showed that the administration of atropine alone slightly reduced bile flow. However, muscarinic blockade did not abolish ANF effect ŽFig. 2A.. In the same way, bilateral truncal vagotomy also reduced bile flow, but did not inhibit ANF effect ŽFig. 2B.. The reduction of bile flow induced either by vagotomy or by atropine administration was similar. Furthermore, the decrease in bile flow elicited by 100 ngrml ANF plus
Fig. 2. ŽA. Effect of centrally applied 100 ngrml ANF and atropine administration on bile flow. I: Control; v: Atropine; ': ANF; B: ANFqAtropine. ) : p- 0.05, ) ) : p- 0.01 and ) ) ) : p- 0.001 vs. control. §§: p- 0.01 and §§§: p- 0.001 vs. atropine. a: p- 0.05 and aa: p- 0.01 vs. ANF. Number of cases: 5–7. ŽB. Effect of centrally applied 100 ngrml ANF on bile flow in rats with truncal vagotomy. I: Control; v: vagotomy; ': ANF; B: ANFqvagotomy. ) : p- 0.05, ) ) : p- 0.01 and ) ) ) : p- 0.001 vs. control; §§: p- 0.01 and §§§: p0.001 vs. truncal vagotomy; aa: p- 0.01 and aaa: p- 0.001 vs. ANF 100 ngrml. Number of cases: 5–7.
vagotomy or atropine was more pronounced in all studied times than the reduction induced by ANF, vagotomy or atropine administration alone.
Table 1 Time Žmin.
Control ŽACSF.
ANF Ž1 ngrml.
ANF Ž10 ngrml.
ANF Ž100 ngrml.
0 0.5 5 15
90"7 87"8 91"8 95"7
96"5 93"6 98"7 101"6
99"10 98"7 102"7 102"10
98"8 95"7 102"9 100"11
Effects of icv ANF Ž1, 10 and 100 ngrml. on mean arterial pressure ŽMAP.. ACSF: artificial cerebrospinal fluid. Number of cases: 6.
Fig. 3. Effect of adrenergic blockade and centrally applied 100 ngrml ANF on bile flow. I: Control; v: phentolamineqpropranolol; ': ANF; B: ANFqphentolamineqpropranolol. ) ) : p- 0.01 and ) ) ) : p- 0.001 vs. control and propranololqphentolamine. Number of cases: 7–8.
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The participation of a sympathetic pathway in ANF effect on bile flow was assessed by the administration of phentolamine and propranolol Ž a and b adrenergic antagonists, respectively.. The adrenergic blockade modified neither basal bile flow nor ANF inhibitory effect on bile secretion ŽFig. 3.. When the concentration of the different bile constituents was measured, results showed that ANF did not only reduce bile flow, but it also diminished the output of bile acids. ANF dose-dependently reduced the output of bile
Fig. 5. Effects of icv ANF Ž1, 10 and 100 ngrml. on sodium ŽA. and potassium ŽB. secretion. ) : p- 0.05, ) ) : p- 0.01 and ) ) ) : p- 0.001 vs. control. Number of cases: 5–7. I: Control; v: 1 ngrml ANF; B: 10 ngrml ANF; ': 100 ngrml ANF. Number of cases: 6–8.
acids at 15, 30, 45 and 60 min ŽFig. 4A.. Centrally applied ANF did not modify total proteins or phospholipid excretion rates ŽFig. 4B and C.. The secretion of sodium and potassium was also decreased by icv ANF ŽFig. 5A and B.. These results support that the reduction of bile flow occurs as the result of decreased bile acid output.
4. Discussion
Fig. 4. Effects of centrally applied ANF Ž1, 10 and 100 ngrml. on the output of bile acid ŽA., proteins ŽB. and phospholipid ŽC.. ) : p- 0.05 and ) ) : p- 0.01 vs. control. Number of cases: 5–7. I: Control; v: 1 ngrml ANF; B: 10 ngrml ANF; ': 100 ngrml ANF. Number of cases: 6–8.
This is the first report to show an effect on bile secretion induced by centrally applied ANF. This study further supports the role of peptides and neuropeptides in the central regulation of bile secretion. The liver is known to have a vast supply of autonomic nerves, which originate in the hypothalamus and enter the liver with the major vessels in the porta hepatis. Nerves innervating the liver include afferent and efferent sympathetic, parasympathetic as well as peptidergic components w21x. Besides, adrenergic and cholinergic innervation, a peptidergic innervation ŽVIP, substance P and neurotensin. has also been found in livers of several species w21,22x. Although the liver possesses many intrinsic and extrinsic
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nerve innervations, the functional role of these nerves in biliary physiology is poorly understood. The brain–liver relation has been widely studied as regards to the control of glucose metabolism, in which the hypothalamus plays an important role w23,24x. However, little is known about the existence of a central regulation of bile secretion. In the present work, centrally applied ANF diminished bile flow in a dose-dependent manner. ANF reduced the secretion of bile acids and also the output of sodium and potassium. Bile flow is conceptually divided into two fractions, bile acid dependent flow, which depends upon the canalicular excretion of bile acids w25x, and the bile acid independent flow whose origin is still controversial. Diverse reports show that bicarbonate excretion accounts for the generation of bile acid independent flow. However, at present, the excretion of glutation and its derivatives are considered to play a major role in the formation of bile acid independent flow w26x. ANF reduced total bile flow but also the output of bile acids. These results indicate that the atrial factor diminishes bile acid dependent flow. Moreover, ANF also reduced the output of sodium and potassium, which are two electrolytes mainly associated with this fraction. On the other hand, the excretion of proteins and phospholipids was not modified by icv ANF although their excretion is associated with bile acid output. We have previously reported that when ANF is systemically applied it reduces bile flow but does not affect portal venous pressure in the rat w11x. ANF-evoked reduction of bile flow results from a decrease in bile acid dependent flow, since ANF reduces bile acid output as well as the excretion of sodium and potassium. Moreover, it increases bicarbonate output as well as bile pH suggesting an increase in bile acid dependent flow. Present results show that when ANF is either centrally or systemically applied, it induces similar changes in bile secretion, which does not mean that the mechanisms underlying the observed effects are necessarily the same. Mean arterial pressure was not affected by central administration of ANF in line with previous investigations w20x. Central administration of ANF even in higher concentrations than those used in the present work does not affect mean arterial pressure in the anesthetized rat w20x. These findings exclude the possibility that centrally induced hemodynamic alterations may mediate ANF effect on bile secretion. Among the few reports about the central regulation of bile secretion by peptides or neuropeptides, Yoneda et al. w12x, and Farouk et al. w13x reported that central neuropeptide Y enhances bile secretion in the rat through a vagal pathway. These findings suggest that bile secretion can be influenced by brain neuropeptides through the modulation of the autonomic nervous system. The reduction of bile flow induced by centrally applied ANF raised the possibility that ANF could diminish cholinergic activity. Bile secretion is regulated by hormones, peptides as well as by
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the autonomic nervous system. Diverse studies have shown that vagal stimulation tends to increase bile flow whereas noradrenergic stimulation reduces bile secretion w27,28x. Previous investigations have reported central ANF effects on the digestive system mediated by a cholinergic pathway. ANF inhibits gastric motility w29x and regulates gastric secretion through a vagal pathway w30x. Present results show that the inhibition of cholinergic activity either by truncal vagotomy or atropine administration slightly reduced bile flow in a similar manner. However, neither vagotomy nor atropine abolished ANF response. Moreover, ANF effect was additive to the blockade of parasympathetic pathways suggesting that ANF and the parasympathetic nervous system operate through different mechanisms to regulate bile flow. These results indicate that the parasympathetic nervous system does not mediate ANF effect on bile secretion in the rat, and further muscarinic receptors are not involved. The reduction of bile flow induced by centrally administered ANF also suggests that the atrial peptide may increase sympathetic outflow to the liver, thus resulting in diminished bile secretion. The increase of noradrenergic transmission has been associated with a reduction in bile flow w27,28x. Stimulation of the hepatic sympathetic nerves has been shown to release both noradrenaline and galanin. This activation decreases bile flow and bile acid secretion and causes an overflow of noradrenaline into the hepatic vein. However, ANF has been reported to reduce noradrenergic neurotransmission in the central nervous system. ANF diminishes norepinephrine release w31,32x and increases the neuronal uptake and endogenous content of the amine w33,34x. Moreover, it also diminishes norepinephrine turnover and the activity of tyrosine hydroxylase, which is the key enzyme in the biosynthetic pathway of catecholamines w35x. In addition, centrally applied ANF has been reported to decrease sympathetic outflow in sinoaortic-denervated rats but not in normal rats w20x. All these data do not support that an increase in noradrenergic activity evoked by ANF is responsible for the reduction of bile secretion. In agreement with this observation, present results show that the combined administration of phentolamine and propranolol did not modify the inhibitory effect of centrally applied ANF on bile secretion. All these data exclude the possibility that an increase in noradrenergic activity mediates the reduction of bile secretion evoked by centrally applied ANF. As ANF induces the same effects on bile secretion when either centrally or peripherally applied, it is possible to assume that central ANF may increase circulating ANF by stimulating the peptide release from the atria. Nevertheless, although centrally applied ANF increases circulating levels of ANF in situations of hemodynamic overload, it does not modify plasma ANF under physiological conditions w36x. ANF circulating levels remain unchanged after ANF icv injections in normal rats. It is important to point out that ANF is mainly produced by the heart, but ANF
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transcripts are also present in the liver and the gastrointestinal tract. Extra-cardiac ANF seems to function as paracrine or autacrine factor rather than a hormone. The stimuli that promote the release of ANF from extracardiac sources, such as the gastrointestinal tract, remain unknown. The fact that neither the sympathetic nor the parasympathetic nervous system seems to be involved in the effect of centrally applied ANF on bile secretion supports the possible participation of a peptidergic regulation, mediated by ANF itself or other peptides or neuropeptides central or peripherally released. Natriuretic peptide receptors have been characterized and are present in the liver w7x. ANF binding to NPR-A and NPR-B receptor subtypes increases intracellular cGMP. This intracellular messenger has been shown to reduce bile acid dependent flow and increase bile acid independent flow in rats w37x. It is likely to assume that ANF inhibitory effect on bile secretion may be mediated by cGMP generation. ANF reduced bile flow as well as the output of bile acids and electrolytes. These findings suggest that ANF may in a direct or indirect way affect the transport system responsible for the uptake of bile acids from the sinusoid, resulting in a reduced rate of excretion of bile acid through the canalicular membrane. Bile acid uptake is preferentially mediated by a sodium-coupled transporter localized on the sinusoidal membrane of the hepatocyte that is dependent on the extracellular sodium concentration w38x. Increasing the level cAMP can enhance the activity of this transporter. ANF has been reported to affect sodium-coupled transporters as the glucose–sodium transporter localized in the intestine w39x. However, the mechanisms underlying a possible effect of centrally applied ANF on the bile acid cotransporter is unknown. In conclusion, the present results show that centrally applied ANF reduced bile acid dependent flow, which results in a reduction of total bile flow. Although bile secretion is reduced when ANF is centrally applied, neither the parasympathetic nor the sympathetic nervous system is involved. Our results suggest that ANF plays a role in the central modulation of bile secretion in the rat. Acknowledgements This work was supported by grants from the Consejo Nacional de Investigaciones Cientıficas y Tecnologicas de ´ ´ la Republica Argentina ŽCONICET. and the University of ´ Buenos Aires ŽUBACYT.. References w1x de Bold AJ, Bruneau BG, Kurosky de Bold ML. Mechanical and neuroendocrine regulation of the endocrine heart. Cardiovasc Res 1996;31:7–18. w2x Brenner BM, Ballermann BJ, Gunning ME, Zeidel ML. Diverse biological actions of atrial natriuretic peptide. Physiol Rev 1990; 70:665–99.
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w34x Vatta MS, Rodriguez-Fermepin M, Bianciotti LG, Perazzo J, Monserrat A, Fernandez BE. Atrial natriuretic factor enhances norepinephrine uptake in circumventricular organs, locus coeruleus and nucleus tractus solitarii of the rat. Neurosci Lett 1995;197:29–32. w35x Vatta MS, Rodriguez-Fermepin M, Durante G, Bianciotti LG, Fernandez BE. Atrial natriuretic factor inhibits norepinephrine biosynthesis and turnover in the rat hypothalamus. Regul Pept 1999; 85:101–7. w36x Puyo AM, Vatta MS, Donoso AS, Bianciotti LG, Fernandez BE. Central natriuretic peptides regulation of peripheral atrial natriuretic factor release. Regul Pept 2000;90:93–9. w37x Myers NC, Grune S, Jamenson HL, Sawkat-Anwer M. cGMP stimulates bile acid-independent bile formation and biliary bicarbonate excretion. Am J Physiol 1996;270:G418–24. w38x Hagenbuch B, Steiger B, Foguet M, Lubbert H, Meier PJ. Functional expression cloning and characterization of the hepatocyte Naqrbile acid cotransport system. Proc Natl Acad Sci U S A 1991; 88:10629–30. w39x Martinez-Seeber A, Vidal NA, Carchio SM, Karara AL. Inhibition of water and sodium intestinal absorption by an atrial extract. Can J Physiol Pharmacol 1986;64:244–7.
Centrally applied atrial natriuretic factor diminishes bile secretion in the rat Liliana G. Bianciotti a,) , Marcelo S. Vatta b, Cristina Vescina c , Valeria Trippodi c , Maria E. Sabbatini b, Belisario E. Fernandez a a
Catedra de Fisiopatologıa, UniÕersidad de Buenos Aires, Junın ´ ´ Facultad de Farmacia y Bioquımica, ´ ´ 956, 5 Piso, 1113 Buenos Aires, Argentina b Catedra de Fisiologıa, UniÕersidad de Buenos Aires, Buenos Aires, Argentina ´ ´ Facultad de Farmacia y Bioquımica, ´ c Catedra de Quımica Analıtica, Facultad de Farmacia y Bioquımica, UniÕersidad de Buenos Aires, Buenos Aires, Argentina ´ ´ ´ ´ Received 30 May 2001; received in revised form 1 August 2001; accepted 14 August 2001
Abstract Little is known about the role of centrally applied peptides in the regulation of bile secretion. We previously reported that the intravenous injection of atrial natriuretic factor ŽANF. reduces bile acid dependent flow without affecting portal venous pressure in the rat. In the present work, we studied the effects of centrally applied ANF on bile secretion and the possible pathways involved. Rats were cannulated in the brain lateral ventricle for the administration of 1, 10 and 100 ngrml ANF. After 1 week, the common bile duct was cannulated and bile samples were collected every 15 min for 60 min after the administration of ANF. The excretion rate of various biliary components was assessed. Bile secretion experiments were also performed after bilateral truncal vagotomy or atropine administration to evaluate the participation of a vagal pathway. In addition, the role of the sympathetic system was addressed by combined administration of propranolol and phentolamine. Centrally applied ANF did not modify blood pressure but diminished bile flow and bile acid output. It also reduced sodium and potassium secretion but did not modify protein or phospholipid excretion. Neither bilateral truncal vagotomy nor atropine administration abolished ANF response. Furthermore, combined administration of adrenergic antagonists did not alter ANF inhibitory effect on bile flow. In conclusion, centrally applied ANF reduced bile acid dependent flow not through a vagal or adrenergic pathway in the rat, suggesting the involvement of a peptidergic pathway. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Bile flow; Natriuretic peptides; Bile acids; Central regulation
1. Introduction Atrial natriuretic factor ŽANF. is a hormone produced by the heart that is released in response to atrial stretch, endothelin-1 and a 1 adrenergic stimulation w1x. ANF is intimately related to the control of blood arterial pressure as well as hydro saline homeostasis w2x. Although the heart is the main source of ANF, increasing evidence indicates the existence of extra-cardiac sources such as the central nervous system, the gastrointestinal tract, liver and the salivary glands, where this peptide is likely to have a paracrine role w3–5x. Natriuretic peptides receptors, both coupled and uncoupled to guanylate cyclase, are widely distributed in many tissues and cell types. Receptors for ) Corresponding author. Tel.: q54-11-4964-8268r8280; fax: q54-114964-8268. E-mail address: [email protected] ŽL.G. Bianciotti..
the natriuretic peptides were described in the gastrointestinal tract w6x, liver w7x, pancreas w8x, and salivary glands w5x as well as in the central nervous system w9,10x. The central areas include the dorsal motor nucleus of the vagus and the nucleus of the solitary tract, which are important sites for the autonomic nervous regulation of gastrointestinal functions w9,10x. We have previously reported that systemically administered ANF alkalinizes bile and reduces spontaneous as well as evoked bile secretion in the rat. ANF diminishes bile acid dependent flow since it diminishes bile acid secretion but also the output of sodium and potassium that accompany this bile fraction w11x. Furthermore, it also increases bicarbonate output, which supports an increase in the bile acid independent flow fraction, and is partially related to this anion output. ANF slightly decreases mean arterial pressure but it does not affect portal venous pressure in the rat w11x.
0167-0115r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 1 1 5 Ž 0 1 . 0 0 3 1 0 - X
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Several peptides and neuropeptides that regulate gastrointestinal physiology also influence either gastrointestinal motility andror digestive secretions when they are centrally applied. However, little is known about the central regulation of bile secretion by peptides or neuropeptides as compared with the wide literature on the brain regulation of gastric secretion and gastrointestinal motor function. The secretion of bile is increased by centrally applied neuropeptide Y through a vagal pathway w12,13x whereas it is reduced by the intracerebroventricular Žicv. injection of bombesin w14x. These findings support the involvement of peptides or neuropeptides in the regulation of bile secretion, acting through the modulation of the autonomic nervous system. On this basis, the aim of the present work was to study the effects of central injections of ANF on bile secretion as well as the possible pathways involved. Our findings show that centrally applied ANF diminished bile acid dependent flow, although the inhibition of neither the parasympathetic nor the sympathetic activity abolished ANF response, suggesting the possible participation of a peptidergic pathway.
2. Materials and methods Male Sprague–Dawley strain ŽFacultad de Farmacia y Bioquimica, Universidad de Buenos Aires. rats of 300– 330-g body weight were used in the experiments. One week previous to the bile secretion experiments, rats under anesthesia Žchloral hydrate, 0.4 mgrkg, ip. ŽSigma, St Louis, MO. were placed in a stereotaxic instrument ŽKopf model 900 David Kopf Instruments, USA. and a steel cannula was introduced in the right ventricle of the brain. Briefly, rats were placed on a stereotaxic frame and the skin overlying the midline of the skull was incised. A small hole was drilled through the appropriate area of the skull and a 23-gauge stainless steel cannula was placed in the right lateral ventricle Ž1.3 mm posterior to the bregma, 2.0 mm lateral to the midline and 4.0 mm ventral to the skull surface. w15x. The cannula was anchored with a stainless steel screw in the skull and covered by dental acrylic. Rats were individually caged and allowed to recover from surgical procedures for a week. Water and Purina commercial chow were given ad libitum except 14 h prior to bile secretion experiments in that food was removed to avoid possible release of peptides andror hormones that may eventually influence bile secretion. Rats were anesthetized with urethane Ž1.3 mgrkg, ip., and through a midline abdominal incision, the common bile duct was exposed and cannulated with a polyethylene catheter ŽPC-10 Intramedic, USA.. Rats remained anesthetized during bile collection that was performed between 9:00 and 11:00 to avoid possible circadian changes w16x. Body temperature was kept at 37 8C with a heating pad.
Bile secretion was allowed to flow for 10 min Žbasal period. to stabilize bile flow. ANF ŽPeninsula, Belmont, CA. Ž1, 10 and 100 ngrml. or artificial cerebrospinal fluid Žcontrol group. was icv injected Ž1 ml volume. at a rate of 1 mlrmin. Bile was collected every 15 min for 60 min in pretared microcentrifuge tubes. The composition of artificial cerebrospinal fluid used was ŽmM.: NaCl, 125; CaCl 2 , 1.2; MgCl 2 , 0.9; NaHCO 3 , 25; Na 2 HPO4 , 0.5; KH 2 PO4 , 0.5; glucose, 4.3; and urea 6.5. The accuracy of icv injections was assessed at the end of each experiment by icv administration of 1 ml methylene blue ŽSigma.. Animals were killed, and through the opening of the skull, the brain was removed, and the presence of methylene blue was verified strictly in the lateral ventricle. Bile volume was determined gravimetrically assuming the specific gravity of rat bile to be 1.0. Biliary sodium and potassium were measured by the ion analyzer method ŽTechnolab, Argentina.. Bile acid concentration was assessed by the 3a-hydroxysteroid dehydrogenase assay w17x. Phospholipid and total protein concentrations were assessed by modified Bartleet w18x and Lowry w19x methods, respectively. Bile flow Žmlrminr100 g body weight. and the excretion rate of the different constituents of bile were calculated. The role of the vagal cholinergic pathway in mediating ANF effect was assessed by two different experimental approaches: bilateral truncal vagotomy and atropine sulphate administration. Cervical vagotomy was not done to avoid possible hemodynamic alterations resulting from the section of cardiac vagal branches. Bilateral truncal vagotomy was performed by the section of both branches of the vagus and vagal afferents at the level of the lower esophagus 2 h before bile collection experiments. Atropine sulfate was administered in bolus 30 min before bile collection and infused at 75 mgrkgrh during the collection of bile. Control rats were infused at the same rate with saline. The participation of the sympathetic system was assessed by the administration of phentolamine Ž a-adrenergic blocker. and propranolol, Žb-adrenergic antagonist.. A bolus of 0.5 mgrkg phentolamine was given intravenously 30 min before ANF injection and then the antagonist was infused at a constant rate of 0.2 mgrkgrh throughout the period of bile collection. A bolus of propranolol 0.5 mgrkg was administered intravenously 30 min before icv injection of ANF. The dose of ANF used in these experiments was 100 ngrml. Blood pressure was monitored throughout the collection of bile after the administration of ANF using a blood pressure transducer ŽStatham 923Db. by inserting a polyethylene cannula ŽP-50 Rivero y Cia, Argentina. into the right carotid artery. The signals were recorded on a polygraph ŽCoulbourn Inst.. with data acquisition software ŽDATAQ Inst... Three readings were registered for each rat. Results are expressed as mean arterial pressure. Statistical analysis was performed by using ANOVA and the t-test modified by Bonferroni. Results are ex-
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Fig. 1. Effect of centrally applied ANF on bile flow Žmlrminr100 g BW.. I: Control; v: 1 ngrml ANF; B: 10 ngrml; ': 100 ngrml. ) : p- 0.05, ) ) : p- 0.01 and ) ) ) : p- 0.001 vs. control. Number of cases: 5–7. BW: body weight.
pressed as the mean " S.E.M.; p F 0.05 was considered statistically significant.
3. Results In order to evaluate the central effect of ANF on bile secretion, different concentrations of ANF were centrally applied. Results showed that central administration of 1, 10 and 100 ngrml ANF significantly reduced spontaneous bile flow at 15, 30, 45 and 60 min in a dose-dependent manner ŽFig. 1.. With the aim to assess possible hemodynamic alterations as the result of icv ANF injections, we measured blood pressure. Centrally applied ANF Ž1, 10, 100 ngrml. did not modify mean arterial pressure in the rat in line with previous findings w20x ŽTable 1.. Blood pressure remains unchanged during the whole period of bile collection after icv ANF. Only values up to 15 min are shown. The involvement of a cholinergic pathway in ANF response on bile secretion was evaluated by truncal vagotomy and atropine administration. Results showed that the administration of atropine alone slightly reduced bile flow. However, muscarinic blockade did not abolish ANF effect ŽFig. 2A.. In the same way, bilateral truncal vagotomy also reduced bile flow, but did not inhibit ANF effect ŽFig. 2B.. The reduction of bile flow induced either by vagotomy or by atropine administration was similar. Furthermore, the decrease in bile flow elicited by 100 ngrml ANF plus
Fig. 2. ŽA. Effect of centrally applied 100 ngrml ANF and atropine administration on bile flow. I: Control; v: Atropine; ': ANF; B: ANFqAtropine. ) : p- 0.05, ) ) : p- 0.01 and ) ) ) : p- 0.001 vs. control. §§: p- 0.01 and §§§: p- 0.001 vs. atropine. a: p- 0.05 and aa: p- 0.01 vs. ANF. Number of cases: 5–7. ŽB. Effect of centrally applied 100 ngrml ANF on bile flow in rats with truncal vagotomy. I: Control; v: vagotomy; ': ANF; B: ANFqvagotomy. ) : p- 0.05, ) ) : p- 0.01 and ) ) ) : p- 0.001 vs. control; §§: p- 0.01 and §§§: p0.001 vs. truncal vagotomy; aa: p- 0.01 and aaa: p- 0.001 vs. ANF 100 ngrml. Number of cases: 5–7.
vagotomy or atropine was more pronounced in all studied times than the reduction induced by ANF, vagotomy or atropine administration alone.
Table 1 Time Žmin.
Control ŽACSF.
ANF Ž1 ngrml.
ANF Ž10 ngrml.
ANF Ž100 ngrml.
0 0.5 5 15
90"7 87"8 91"8 95"7
96"5 93"6 98"7 101"6
99"10 98"7 102"7 102"10
98"8 95"7 102"9 100"11
Effects of icv ANF Ž1, 10 and 100 ngrml. on mean arterial pressure ŽMAP.. ACSF: artificial cerebrospinal fluid. Number of cases: 6.
Fig. 3. Effect of adrenergic blockade and centrally applied 100 ngrml ANF on bile flow. I: Control; v: phentolamineqpropranolol; ': ANF; B: ANFqphentolamineqpropranolol. ) ) : p- 0.01 and ) ) ) : p- 0.001 vs. control and propranololqphentolamine. Number of cases: 7–8.
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The participation of a sympathetic pathway in ANF effect on bile flow was assessed by the administration of phentolamine and propranolol Ž a and b adrenergic antagonists, respectively.. The adrenergic blockade modified neither basal bile flow nor ANF inhibitory effect on bile secretion ŽFig. 3.. When the concentration of the different bile constituents was measured, results showed that ANF did not only reduce bile flow, but it also diminished the output of bile acids. ANF dose-dependently reduced the output of bile
Fig. 5. Effects of icv ANF Ž1, 10 and 100 ngrml. on sodium ŽA. and potassium ŽB. secretion. ) : p- 0.05, ) ) : p- 0.01 and ) ) ) : p- 0.001 vs. control. Number of cases: 5–7. I: Control; v: 1 ngrml ANF; B: 10 ngrml ANF; ': 100 ngrml ANF. Number of cases: 6–8.
acids at 15, 30, 45 and 60 min ŽFig. 4A.. Centrally applied ANF did not modify total proteins or phospholipid excretion rates ŽFig. 4B and C.. The secretion of sodium and potassium was also decreased by icv ANF ŽFig. 5A and B.. These results support that the reduction of bile flow occurs as the result of decreased bile acid output.
4. Discussion
Fig. 4. Effects of centrally applied ANF Ž1, 10 and 100 ngrml. on the output of bile acid ŽA., proteins ŽB. and phospholipid ŽC.. ) : p- 0.05 and ) ) : p- 0.01 vs. control. Number of cases: 5–7. I: Control; v: 1 ngrml ANF; B: 10 ngrml ANF; ': 100 ngrml ANF. Number of cases: 6–8.
This is the first report to show an effect on bile secretion induced by centrally applied ANF. This study further supports the role of peptides and neuropeptides in the central regulation of bile secretion. The liver is known to have a vast supply of autonomic nerves, which originate in the hypothalamus and enter the liver with the major vessels in the porta hepatis. Nerves innervating the liver include afferent and efferent sympathetic, parasympathetic as well as peptidergic components w21x. Besides, adrenergic and cholinergic innervation, a peptidergic innervation ŽVIP, substance P and neurotensin. has also been found in livers of several species w21,22x. Although the liver possesses many intrinsic and extrinsic
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nerve innervations, the functional role of these nerves in biliary physiology is poorly understood. The brain–liver relation has been widely studied as regards to the control of glucose metabolism, in which the hypothalamus plays an important role w23,24x. However, little is known about the existence of a central regulation of bile secretion. In the present work, centrally applied ANF diminished bile flow in a dose-dependent manner. ANF reduced the secretion of bile acids and also the output of sodium and potassium. Bile flow is conceptually divided into two fractions, bile acid dependent flow, which depends upon the canalicular excretion of bile acids w25x, and the bile acid independent flow whose origin is still controversial. Diverse reports show that bicarbonate excretion accounts for the generation of bile acid independent flow. However, at present, the excretion of glutation and its derivatives are considered to play a major role in the formation of bile acid independent flow w26x. ANF reduced total bile flow but also the output of bile acids. These results indicate that the atrial factor diminishes bile acid dependent flow. Moreover, ANF also reduced the output of sodium and potassium, which are two electrolytes mainly associated with this fraction. On the other hand, the excretion of proteins and phospholipids was not modified by icv ANF although their excretion is associated with bile acid output. We have previously reported that when ANF is systemically applied it reduces bile flow but does not affect portal venous pressure in the rat w11x. ANF-evoked reduction of bile flow results from a decrease in bile acid dependent flow, since ANF reduces bile acid output as well as the excretion of sodium and potassium. Moreover, it increases bicarbonate output as well as bile pH suggesting an increase in bile acid dependent flow. Present results show that when ANF is either centrally or systemically applied, it induces similar changes in bile secretion, which does not mean that the mechanisms underlying the observed effects are necessarily the same. Mean arterial pressure was not affected by central administration of ANF in line with previous investigations w20x. Central administration of ANF even in higher concentrations than those used in the present work does not affect mean arterial pressure in the anesthetized rat w20x. These findings exclude the possibility that centrally induced hemodynamic alterations may mediate ANF effect on bile secretion. Among the few reports about the central regulation of bile secretion by peptides or neuropeptides, Yoneda et al. w12x, and Farouk et al. w13x reported that central neuropeptide Y enhances bile secretion in the rat through a vagal pathway. These findings suggest that bile secretion can be influenced by brain neuropeptides through the modulation of the autonomic nervous system. The reduction of bile flow induced by centrally applied ANF raised the possibility that ANF could diminish cholinergic activity. Bile secretion is regulated by hormones, peptides as well as by
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the autonomic nervous system. Diverse studies have shown that vagal stimulation tends to increase bile flow whereas noradrenergic stimulation reduces bile secretion w27,28x. Previous investigations have reported central ANF effects on the digestive system mediated by a cholinergic pathway. ANF inhibits gastric motility w29x and regulates gastric secretion through a vagal pathway w30x. Present results show that the inhibition of cholinergic activity either by truncal vagotomy or atropine administration slightly reduced bile flow in a similar manner. However, neither vagotomy nor atropine abolished ANF response. Moreover, ANF effect was additive to the blockade of parasympathetic pathways suggesting that ANF and the parasympathetic nervous system operate through different mechanisms to regulate bile flow. These results indicate that the parasympathetic nervous system does not mediate ANF effect on bile secretion in the rat, and further muscarinic receptors are not involved. The reduction of bile flow induced by centrally administered ANF also suggests that the atrial peptide may increase sympathetic outflow to the liver, thus resulting in diminished bile secretion. The increase of noradrenergic transmission has been associated with a reduction in bile flow w27,28x. Stimulation of the hepatic sympathetic nerves has been shown to release both noradrenaline and galanin. This activation decreases bile flow and bile acid secretion and causes an overflow of noradrenaline into the hepatic vein. However, ANF has been reported to reduce noradrenergic neurotransmission in the central nervous system. ANF diminishes norepinephrine release w31,32x and increases the neuronal uptake and endogenous content of the amine w33,34x. Moreover, it also diminishes norepinephrine turnover and the activity of tyrosine hydroxylase, which is the key enzyme in the biosynthetic pathway of catecholamines w35x. In addition, centrally applied ANF has been reported to decrease sympathetic outflow in sinoaortic-denervated rats but not in normal rats w20x. All these data do not support that an increase in noradrenergic activity evoked by ANF is responsible for the reduction of bile secretion. In agreement with this observation, present results show that the combined administration of phentolamine and propranolol did not modify the inhibitory effect of centrally applied ANF on bile secretion. All these data exclude the possibility that an increase in noradrenergic activity mediates the reduction of bile secretion evoked by centrally applied ANF. As ANF induces the same effects on bile secretion when either centrally or peripherally applied, it is possible to assume that central ANF may increase circulating ANF by stimulating the peptide release from the atria. Nevertheless, although centrally applied ANF increases circulating levels of ANF in situations of hemodynamic overload, it does not modify plasma ANF under physiological conditions w36x. ANF circulating levels remain unchanged after ANF icv injections in normal rats. It is important to point out that ANF is mainly produced by the heart, but ANF
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transcripts are also present in the liver and the gastrointestinal tract. Extra-cardiac ANF seems to function as paracrine or autacrine factor rather than a hormone. The stimuli that promote the release of ANF from extracardiac sources, such as the gastrointestinal tract, remain unknown. The fact that neither the sympathetic nor the parasympathetic nervous system seems to be involved in the effect of centrally applied ANF on bile secretion supports the possible participation of a peptidergic regulation, mediated by ANF itself or other peptides or neuropeptides central or peripherally released. Natriuretic peptide receptors have been characterized and are present in the liver w7x. ANF binding to NPR-A and NPR-B receptor subtypes increases intracellular cGMP. This intracellular messenger has been shown to reduce bile acid dependent flow and increase bile acid independent flow in rats w37x. It is likely to assume that ANF inhibitory effect on bile secretion may be mediated by cGMP generation. ANF reduced bile flow as well as the output of bile acids and electrolytes. These findings suggest that ANF may in a direct or indirect way affect the transport system responsible for the uptake of bile acids from the sinusoid, resulting in a reduced rate of excretion of bile acid through the canalicular membrane. Bile acid uptake is preferentially mediated by a sodium-coupled transporter localized on the sinusoidal membrane of the hepatocyte that is dependent on the extracellular sodium concentration w38x. Increasing the level cAMP can enhance the activity of this transporter. ANF has been reported to affect sodium-coupled transporters as the glucose–sodium transporter localized in the intestine w39x. However, the mechanisms underlying a possible effect of centrally applied ANF on the bile acid cotransporter is unknown. In conclusion, the present results show that centrally applied ANF reduced bile acid dependent flow, which results in a reduction of total bile flow. Although bile secretion is reduced when ANF is centrally applied, neither the parasympathetic nor the sympathetic nervous system is involved. Our results suggest that ANF plays a role in the central modulation of bile secretion in the rat. Acknowledgements This work was supported by grants from the Consejo Nacional de Investigaciones Cientıficas y Tecnologicas de ´ ´ la Republica Argentina ŽCONICET. and the University of ´ Buenos Aires ŽUBACYT.. References w1x de Bold AJ, Bruneau BG, Kurosky de Bold ML. Mechanical and neuroendocrine regulation of the endocrine heart. Cardiovasc Res 1996;31:7–18. w2x Brenner BM, Ballermann BJ, Gunning ME, Zeidel ML. Diverse biological actions of atrial natriuretic peptide. Physiol Rev 1990; 70:665–99.
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w34x Vatta MS, Rodriguez-Fermepin M, Bianciotti LG, Perazzo J, Monserrat A, Fernandez BE. Atrial natriuretic factor enhances norepinephrine uptake in circumventricular organs, locus coeruleus and nucleus tractus solitarii of the rat. Neurosci Lett 1995;197:29–32. w35x Vatta MS, Rodriguez-Fermepin M, Durante G, Bianciotti LG, Fernandez BE. Atrial natriuretic factor inhibits norepinephrine biosynthesis and turnover in the rat hypothalamus. Regul Pept 1999; 85:101–7. w36x Puyo AM, Vatta MS, Donoso AS, Bianciotti LG, Fernandez BE. Central natriuretic peptides regulation of peripheral atrial natriuretic factor release. Regul Pept 2000;90:93–9. w37x Myers NC, Grune S, Jamenson HL, Sawkat-Anwer M. cGMP stimulates bile acid-independent bile formation and biliary bicarbonate excretion. Am J Physiol 1996;270:G418–24. w38x Hagenbuch B, Steiger B, Foguet M, Lubbert H, Meier PJ. Functional expression cloning and characterization of the hepatocyte Naqrbile acid cotransport system. Proc Natl Acad Sci U S A 1991; 88:10629–30. w39x Martinez-Seeber A, Vidal NA, Carchio SM, Karara AL. Inhibition of water and sodium intestinal absorption by an atrial extract. Can J Physiol Pharmacol 1986;64:244–7.