Atrial natriuretic factor modifies bile flow and composition in the rat

Regulatory Peptides, 43 (1993) 177-184 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00

177

REGPEP 01265

Atrial natriuretic factor modifies bile flow and composition in the rat Belisario E. Fernandez, Liliana G. Bianciotti, Marcelo S. Vatta, Antonio E. Dominguez and Cristina Vescina. Catedras de Fisiopatologia and Quimica Analitica, Programa de Sistemas Vasodepresores - Consejo Nacional de Investigaciones Cientificas y Tecnicas (PR OSIVAD-CONICET), Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires (Argentina) (Received 9 June 1992; revised version received 15 October 1992; accepted 28 October 1992)

Key words: Bile flow; Bile acids excretion; Electrolyte excretion Summary The effects of atrial natriuretic factor (ANF) on bile secretion were studied in the rat. ANF was injected 'in bolus' (5.0/zg/kg) every 30 min into the jugular vein. The common bile duct was cannulated and bile samples were collected every 30 min for 120 min. Systemic blood arterial pressure was registered before and after the administration of ANF. Results showed that ANF decreased bile flow and the excretion rate of sodium, postassium, chloride, bile acids, cholesterol and proteins. On the other hand, it increased pH and the excretion of bicarbonate and calcium. As ANF slighltly reduces mean arterial pressure, its vascular effect may not be considered the major event. In additon, increased excretion of bicarbonate and calcium, and the fact that ANF vascular effect is short in time suggest that the peptide may have a non-vascular effect on the processes of bile formation and its modifications along the bile ducts. This extravascular effect may be exerted on the hepatocyte ions exchangers and/or at the ductal level on the processes of excretion and reabsorption of electrolytes and water.

Introduction Atrial natriuretic factor (ANF) first characterized by de Bold etal. in 1981 [1] is synthetized and Correspondence to: B.E. Fernandez, Catedras de Fisiopatologia and Quimica Analitica, Programa de Sistemas Vasodepresores Consejo Nacional de Investigaciones Cientificas y Tecnicas (PROSIVAD-CONICET), Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956-5to piso, 1113 Buenos Aires, Argentina.

released by mammalian atrial cardiocytes in response to atrial stretch. It has been demonstrated that ANF has hypotensive effects since it induces smooth muscle relaxation and increases diuresis and natriuresis [2,3]. ANF also inhibits aldosterone [4], vasopressin [ 5 ], prolactin [6] and corticotrophin releasing factor [7] secretion and controls sympathetic activity through modifications at the catecholamine metabolism level [8-10]. All these effects are opposite to those of angiotensin II and angiotensin III. Thus,

178

ANF behaves as a physiological antagonist of both angiotensins. It has been reported that A N F regulates water and sodium transport not only in the kidney [ 11-13 ], but also in the intestine [14,15], and in capillaries [16]. In addition, A N F is also involved in the production of the aqueous humor of the eye and the cerebrospinal fluid [7]. These findings support the hypothesis that A N F regulates water and sodium transport across biological membranes. Some authors [17,18] have demonstrated the existance of A N F receptors in exocrine pancreas, the internalization of A N F and the resulting rise of cGMP. The presence of ANF receptors in the hepatocyte [ 19] suggests that ANF may be involved in the regulation of liver functionality. The aim of the present work was to investigate whether ANF could regulate bile secretion in the rat.

BILE FLOW 6 5.5 5, 4,5" .;

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OSMOLALITY

Materials and Methods Animals and treatments

Wistar strain male rats weighing between 200 and 250 g were used in the experiments. The rats were housed in steel cages and maintained at 22-25 °C in a controlled room with 12 h light/dark cycle (light from 7:00 to 19:00 h). Animals were allowed free access to food and tap water. Rats were anaesthetized with pentobarbital (50 mg/kg i.p.). The common bile duct and the jugular vein were cannulated with polyethylene catheters (PE-10 Intramedic, USA and PC-40, PL Rivero and Cia, Argentina respectively). The catheter was introduced in the common bile duct near its bifurcation to avoid contamination with pancreatic juice [20]. Bile samples were collected in pre-weighed ice-cold tubes immediately after the administration of ANF (Rat A N F 99-126, Peninsula, Belmont, USA) (experimental rats) or saline (control rats). Rats remained anaesthetized during bile collection periods which were performed between 9:00 and 11:00 h am to avoid possible circadian variations [21]. Rectal temperature was kept at 38°C with a heating lamp.

11uE(...)

pH

Fig. 1. Effectsof ANF (5 p.g/kg)on bile flow and osmolalityand pH of bile. (): Numberof cases; *P< 0.05 comparedwith control.

179

SODIUM

Systemic blood pressure was measured using a blood pressure transducer (Statham 923 Db) by inserting a cannula (PC-50 PL Rivero and Cia, Argentina) into the right carotid artery and being recorded on a Grass Model 7D polygraph.

085" 0.8. 0.75" 0.70.65" O.e. 0.55" 0.5

Experimental procedure

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A bolus of ANF (5.0 #g/kg) was injected just prior to the begining of each bile collection period. Bile samples were collected for 120 min in periods of 30 min each. In control studies, rats were injected with saline solution.

Analytical methods

POTASSIUM 2

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CHLORIDE o.~

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In each sample, the volume of bile was determined by weighing assuming a density of 1.0 g/ml. The concentration of the organic and inorganic constituents of bile were determined as follows: sodium and potassium: flame photometry; chloride: chloride titrator; calcium: atomic absorption (Varian AA-475); bicarbonate: gas analyzer (ABL-2 Radiometer, Copenhagen); total proteins: Lowry method using bovine albumin as standard; bile acids: enzymatic method [22]; phospholipids: modified Bartleet method [ 23 ]; cholesteroh enzymatic Trinder method; phosphate: colorimetric method; bilirubin: bilirubin meter (Bilmeter, Machida Luketron). Bile osmolality was determined by freezing point depression (Osmette S) and bile pH was meassured in a gas analyzer. Bile flow was calculated as #l/min x 100 g body weight. With these values the rate of excretion of the different constituents of bile were determined.

Statistical analysis

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The statistical analysis was performed using Student's t-test. P values of 0.05 or less were considered statistically significant.

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Fig. 2. Effects of A N F (5 #g/kg) on the excretion rates of bile electrolytes (sodium, potassium and chloride). (): Number of cases; * P < 0.05 compared with control.

180

BICARBONATE

Results

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Bile flow and the excretion of bile constituents showed a progressive decrease in control as well as experimental with the time of bile collection since the enterohepatic circulation of bile salts was interrupted due to the cannulation of the common bile duct. A N F decreased bile flow and the excretion of both organic and inorganic constituents of bile, except for bicarbonate and calcium. Fig. 1 illustrates that A N F reduced bile flow and increased p H at 30, 60, 90 and 120 min, while no modifications were observed as regards bile osmolality. The excretion of bile electrolytes was modified as follows: the excretion of sodium and potassium were decreased at 30, 60, 90 and 120 min; the excretion of chloride was also reduced by A N F but only at 30 and 60 min (Fig. 2); bicarbonate excretion was increased in all experimental periods and calcium excretion was enhanced only at 90 and 120 min; phosphate excretion showed no modifications as compared with control studies (Fig. 3). The excretion rate of bile acids decreased along all experimental periods. On the other hand, cholesterol excretion was reduced at 30 rain while proteins excretion diminished only at 30 and 60 min. Neither bilirubin nor phospholipids excretions showed modifications by the administration of A N F (Figs. 4 and

5).

4.25 4,1

Intravenous administration of A N F led to a rapid and short decrease in mean arterial pressure of 10 m m H g in the first 15 s but reverted to control values within 3 rain (Table I).

3.35

Discussion

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The data from the present study provide further information on the role of A N F in fluid and electrolyte homeostasis. Present results showed that A N F Fig. 3. Effects of ANF (5 pg/kg) on the excretion rates of bile electrolytes (bicarbonate, calcium and phosphate). (): Number of cases; *P<0.05 compared with control.

181

PROTEINS

BILE ACIDS 25

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PHOSPHOLIPIDS BILIRUBIN

109.5

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CHOLESTEROL

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Fig. 5. Effects of A N F (5 #g/kg) on the excretion rate of proteins and bilirubin. ( ): Number of cases. *P < 0.05 compared with control.

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reduced bile flow as well as the excretion rate of several of bile constituents. Bile that reaches the common bile duct is not an accurate reflection of the bile secreted by the hepatocyte. Canalicular bile is then modified by reabsorption and/or secretion of

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Fig. 4. Effects of A N F (5 #g/kg) on the excretion rates of bile acids, phospholipids and cholesterol. (): Number of cases; *P<0.05 compared with control.

182

TABLE I C h a n g e s in m e a n arterial pressure ( M A P ) n = 8; * P < 0 . 0 5 c o m p a r e d with control. MAP pressure changes (mmHg) Control 15 s 30 s 1 min 3 min 5 min 10 m i n

98 + 2 - 18+2" - 1 6 + 2* - 8 _+ 2* -4_+2 +2+2 + 1+ 1

water and electrolytes by the bile ducts [24]. Therefore, according to the present results A N F effect may be exerted on the hepatocyte itself and/or at the ductal level affecting the different processes of absorption and/or secretion of electrolytes and water. In addition, A N F vascular effect may also be implicated in this process. A N F reduced total bile flow. The total bile flow accounts for the decrease observed in the excretion of sodium, potassium, chloride and organic constituents of bile although it fails to explain the increased excretion of bicarbonate and calcium. If portal blood flow is increased, bile flow and the excretion of bile main electrolytes (sodium, potassium, bicarbonate and calcium) and bile acids are also usually increased [24]. Bile acid excretion rate was reduced by ANF, which implies that bile acid dependent flow fraction is reduced. In addition, this view is supported by the fall of the excretion rates of sodium and potassium which also contribute to this fraction formation. Bile acids uptake from the sinusoidal blood is an active process [25] (sodium-bile acid cotransport mechanism) which seems to be hormonally modulated perhaps via intracellular cAMP levels [24]. It has been demonstrated that in several tissues A N F inhibits basal and hormone-stimulated adenylate cyclase activity [26]. Conversely, other studies showed the lack

of ANF-induced reductions in cell-associated cAMP levels [26). Although this point still remains unclear due to the fact that there are no studies 'in vivo' on this subject and that the literature is controversial, a possible effect of ANF on the sodium-bile acid cotransport mechanism can not be excluded. The excretion of proteins was decreased at 30 and 60 min by ANF. This result may be explained by the fact that bile flow is reduced but also because there exists a certain dependance between proteins and bile acids output [25]. With regard to phospholipids and cholesterol excretion, it is well known that bile acids appear to facilitate intracellular lipid transport to the canaliculus by means of micelles formation [271. Results showed that the intravenous administration of ANF caused a rapid decrease of mean arterial pressure which reverted to control levels within 3 min. Several authors have demonstrated that A N F reduced portal blood flow and hepatic arterial flow [28], likely due to a reduction of cardiac output since there is little support for a hypothesis that ANF causes a direct effect on the splachnic vasculature [29]. On the other hand, although A N F reduces portal and arterial hepatic flows, the liver is able to minimize these changes by autoregutatory mechanisms which protect hepatic functionality. Changes in portal blood flow result in changes in portal venous pressure, however, a distensible venous resistance leads to a much smaller change in portal venous pressure in response to altered flow. The modifications of sphincters in response to changes in portal venous pressure minimizes such variations in portal pressure [30]. Although it is likely to think that ANF vascular effect may account for the reduction of bile flow and the excretion of several of bile constituents, the AN F vascular effect was too short in time to be considered the main event. In addition, A N F did not induce a marked fall of mean arterial pressure in order to overcome hepatic autorregulation. Therefore, ANF may exert a non-vascuar effect on the process of bile secretion. This hypothesis is also supported by the

183

fact that ANF increased bicarbonate and calcium excretions. The ANF non-vascular effect may be likely exerted at two different levels. On one hand, the peptide may affect the ions exchangers located on the hepatocyte membranes such as the canalicular bicarbonate-chloride exchanger and/or the basolateral calcium-ATPase and sodium-proton exchangers. The origin of the bile acid independent portion of bile still remains unknown, but the mechanisms are thought to involve active canalicular secretion of bicarbonate and/or endogenous organic anions [27], although several studies provide no evidence for the canalicular secretion of bicarbonate in generation of bile acid independent flow [25 ]. Nevertheless, bicarbonate secretion seems to contribute to this fraction of bile. Therefore, as bicarbonate excretion was increased by ANF, the peptide may stimulate bile acid independent fraction. As this fraction of bile is small compared to bile acid dependent flow, the increase may not reflect the total bile flow, since the bile acid dependent fraction was significantly diminished. On the other hand, ANF may affect the processes of reabsorption and/or secretion of electrolytes and water at the ductal level, increasing bicarbonate and calcium excretion and sodium, potassium, chloride and water reabsorption. However, it has been demonstrated that ANF reduced the net absorption of sodium and chloride across the small intestine [ 14,15 ], although the increase of duodenal bicarbonate output mediated by ANF has been reported after blood volume expansion [31]. In addition, in the kidney, ANF increases sodium and chloride excretion [ 1]. According to the present results ANF does not appear to affect bile excretion of electrolytes as it does in the kidney and the small intestine. ANF reduced bile flow and the excretion of sodium, potassium, chloride and bile acids although it increased the excretion of calcium and bicarbonate. The mechanisms underlying such a regulation may involve an effect of ANF on the the ion exchangers located on the hepatocyte membranes and/or on the biliary ducts affecting reabsorption and secretion of water and

electrolytes. The ANF effect on vasculature is too rapidly reverted to be considered the major event affecting bile excretion. Further studies are in progress in our laboratory to determine ANF mechanisms underlying the modulation of bile secretion. Increasing evidence suggests that ANF may be a digestive hormone in view of the different biological effects which have been reported along the gastrointestinal tract, all of which are mainly related to modifications in electrolytes and water balance which in turn may affect the different digestive secretions.

Acknowledgements We would like to thank the helpful assistance of Dr. Maria G. Marina Prendes (Physiology Department) and Mr. Daniel Gonzalez (Toxicology Department). This work was supported by grants of the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) of the Republica Argentina.

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20

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31

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