Secretin stimulates bile ductular through the CAMP system
secretory
activity
ROMANA LENZEN, GIANFRANCO ALPINI, AND NICOLA TAVOLONI Division of Gastroenterology, Department of Medicine, Heinrich-Heine- University, W-4000 Dusseldorf, Federal Republic of Germany; and Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029 Lenzen, Romana, Gianfranco Alpini, and Nicola Tavoloni. Secretin stimulates bile ductular secretory activity through the CAMP system. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G527-G532,1992.-Although convincing evidence has been obtained to support a ductular origin of secretin choleresis, the precise mechanism of the choleretic effect of the hormone is poorly understood. The present studies were carried out to 1) further clarify the anatomic site at which secretin stimulates bile flow and 2) establish the signal transduction system underlying this effect. To this end, parenchymal and nonparenchymal liver cells, the latter enriched in bile duct cells, were isolated from rats with ductular cell hyperplasia, and the effect of secretin on intracellular formation of both adenosine 3’,5’-cyclic monophosphate (CAMP) and inositol phosphates (IPs) was compared with that observed with glucagon and (SG-secretin). In the pancreas, CTY r10J3 Phe22,Trp25]secretin secretin stimulates both messenger systems, while SG-secretin activates only the CAMP cascade. In isolated hepatocytes, both secretin and SG-secretin failed to increase formation of CAMP and IPs, which were instead activated by glucagon. In isolated bile duct cells, secretin induced formation of both CAMP and IPs, while SG-secretin stimulated solely the CAMP system, as in the pancreas. Glucagon did not stimulate either messenger system in this cell preparation. In vivo, both secretin and SGsecretin stimulated a bicarbonate-rich fluid in rats with bile ductular cell hyperplasia and in normal guinea pigs, which was demonstrated to originate at the distal biliary epithelium. These findings support the existing view that glucagon stimulates canalicular bile flow, while secretin increases secretory activity at the bile ductules and/or ducts. More importantly, they indicate that stimulation of ductular secretory activity by secretin is mediated by the CAMP system and does not involve the IP signal transduction pathway. adenosine glucagon
3’,5’-cyclic
monophosphate;
inositol
phosphates;
is a 27-amino acid peptide that shares striking structural similarities with glucagon, vasoactive intestinal peptide, and gastric inhibitory peptide (5). Secretin-producing cells, known as S cells, are located in the mucosa of the upper part of the small intestine (10, 37,39). Although the main biological effect of secretin is stimulation of a bicarbonate-rich pancreatic juice (13)) the hormone also increases gastric pepsin (41) and duodenal bicarbonate secretion (2 1), and inhibits gastric acid output and food-stimulated gastrin release (46). Furthermore, secretin decreases upper small intestinal motility and lower esophageal sphincter pressure (ZO), and stimulates hepatic bile flow (1, 24, 51). Finally, secretin has pharmacological effects in organs like the heart, kidney, lung, and brain (11, 12, 14, l&35,40,45), and the mRNA for the hormone and/or its plasma membrane receptor have been demonstrated in the intestine, heart, and pancreas (22, 29,30). However, the biological effect of secretin in these tissues is poorly understood.
SECRETIN
0193-1857/92
$2.00 Copyright
Although the precise mechanism by which secretin stimulates cellular function is not known, activation of adenylate cyclase has been thought to be the fundamental event underlying the biological effects of the hormone (7, 19). Recently, however, it has been demonstrated that at least in the pancreas, secretin triggers also the inositol phosphate (IP) pathway (49), a messenger system that leads to calcium release from intracellular stores (6, 16, 25). This, together with the recent findings that receptors of other hormones may be coupled to either the adenosine 3’,5’-cyclic monophosphate (CAMP) and/or the IP systems (4, 31, 50), has raised new intriguing questions as to the role of each signal transduction pathway in hormone stimulation of cellular activity. In the liver, secretin stimulates a bicarbonate-rich choleresis in most animal species, including humans (8, 9, 24, 51). Although the precise target cells of secretin stimulation have not as yet been identified, compelling evidence has accumulated to indicate that the hormone regulates fluid secretion at the bile ductules and/or ducts (1, 51). Furthermore, since secretin choleresis is associated with increased plasma and biliary levels of CAMP, it has been suggested that at least in some species the hormone exerts its effect via activation of the adenylate cyclase system (32). However, the liver is a pleotrophic and pleomorphic organ so that the increase in plasma and biliary CAMP concentrations induced by the hormone may not necessarily originate from the cells involved in fluid transport. Other studies, in fact, have suggested that secretin choleresis is not regulated by the CAMP system (26). Moreover, since in the pancreas secretin stimulates both the CAMP and the IP cascades (49), it remains to be demonstrated which of the two messenger systems is involved in secretin stimulation of fluid secretion at the biliary epithelium. Accordingly, the objective of the present studies was to clarify 1) the liver epithelial cell type (hepatocyte vs. bile duct cell) responsive to the stimulatory effect of secretin and 2) the second messenger system mediating the effect of the hormone on fluid secretion. METHODS Animals and chemicals. Male Sprague-Dawley rats (200-250 g) and male Hartley albino guinea pigs (200-300 g) were obtained from Charles River Breeding Laboratories (Wilm.ington, MA) and housed in a temperature-controlled room (22°C) with alternating 12-h light-dark cycles. The animals were fed standard laboratory food ad libitum, had free access to water, and were not fasted before use. Experiments were carried out in three groups of animals. Groups 1 and 2 consisted of normal guinea pigs and normal rats, respectively, while group 3 included rats in which the common bile duct was ligated for 14 days
0 1992 the American
Physiological
Society
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(BDL rats) to induce biliary epithelial cell hyperplasia. BDL rats were prepared as described (1). In vivo experiments consisted of measurements of bile flow and composition and were carried out in each group of animals. In vitro studies instead included determinations of CAMP and IP levels and were conducted in liver-derived cells isolated from BDL rats. Phenobarbital sodium was purchased from Abbott (North Chicago, IL), sodium glycocholate and pronase from Calbiothem (La Jolla, CA), collagenase from Boehringer Mannheim (Indianapolis, IN), minimum essential medium and Joklikmodified essential medium from GIBCO (Grand Island, NY), calf serum from Flow Laboratories (McLean, VA), the dowex resin (AG l-X8, mesh size 200-400, formate form) from BioRad (Richmond, CA), and myo-[2-3H]inositol (sp act 19 Ci/ mmol) and the radioimmunoassay kit for CAMP determination from Amersham (Arlington Heights, IL). Glucagon and vasopressin were obtained from Sigma (St. Louis, MO), and secretin from Ferring Laboratories (Suffern, NY). [Tyr10~13,Phe22, Trp25]secretin (SG-secretin) was a generous gift of Dr. R. B. Merrifield (Rockefeller University, New York City). SG-secretin is an analogue in which tyrosine is substituted for leucine at positions 10 and 13, and phenylalanine and tryptophan are substituted for leucine and glycine at positions 22 and 25, respectively (3). All other chemicals were purchased from Sigma, unless otherwise indicated. Studies of Mary physiology. Measurements of bile flow and composition were conducted in anesthetized, bile duct-cannulated animals using standard procedures (1, 31). Animal temperature was invariably kept at 37°C. Secretin and SG-secretin were infused each at 1.1 nmol kg-l. h-l in rats and 2.2 nmol . kg-l h-l in guinea pigs. Glucagon was given at 1.4 nmol kg-l min-l in both species. Each hormone was infused for 30-45 min until steady-state choleresis was achieved. Throughout the experiment, blood samples (-1 ml) were obtained from the carotid artery 10 min before and 10 min after hormone infusion was initiated, and used for glucose determination. Bile was collected every 5 min, except during the initial 60-min equilibration period in which collections were made over lo-min intervals. At the end of hormone infusion, the animal was killed with an overdose of pentobarbital sodium, and the liver was removed and its weight determined. Bile concentrations of electrolytes were determined by standard clinical chemistry methods (1). Bile acids in bile were measured by the hydroxysteroid dehydrogenase procedure. Determination of Mary tree volume. These experiments were carried out as described previously (1, 31) using [3H] taurocholate as a marker of biliary transit time. Biliary tree volume (BTV) was measured during administration of secretin, SGsecretin, glucagon, glycocholate (4 hmol min-l kg-l in guinea pigs), or taurocholate (4 pmol min-l kg-l in BDL rats). Bile was collected over 3-min intervals. Blood glucose determination. Glucose in plasma was determined with an ASTRA Automated Stat-Routine Analyzer System (Beckman Instruments, Somerset, NJ), using the Beckman glucose oxidase reagent and ASTRA system calibration standards (1, 31). Isolation and purification of hepatocytes and bile duct epithelial cells. Hepatocytes were isolated by collagenase perfusion and purified by differential centrifugation using standard procedures. Nonparenchymal liver cells were isolated from BDL rats as described previously (2). After isolation, nonparenchymal liver cells were fractioned by centrifugal centrifugation to obtain a population enriched in bile duct cells. The cells in the fraction obtained at the pump flow rate of 22-34 ml/min were then resuspended in minimal essential medium containing 10% fetal calf serum (l-4 x lo5 cells/ml) and maintained overnight in a 5% C02-air-equilibrated incubator at 37°C. The morning after, cells were harvested and used for the in vitro determinations l
l
l
l
l
l
l
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described below. This nonparenchymal liver cell population contained 50-70% of biliary epithelial cells as judged by their positivity for y-glutamyl transpeptidase and cytokeratin no. 19 (2,44). Hepatocytes in this fraction accounted for Cl%, and the majority (20-30%) of contaminating cells were mesenchymal in nature (endothelial and Kupffer cells, fibroblasts) as established by vimentin positivity (2, 44). For sake of simplicity, this fraction will be referred to as “intrahepatic bile duct epithelial cells” (IBDECs). Viability of IBDECs was 197%. CAMP determination. To determine the effect of the various hormones on CAMP formation, 0.2 x lo6 hepatocytes and 1 x lo6 IBDECs were incubated at 37°C in 2.5 ml of KrebsHenseleit buffer (pH 7.4) containing 1% bovine serum albumin and gassed with 95% 02-5% CO,. After a 15-min incubation period, IO mM 3-isobutyl-1-methylxanthine was added to the cell suspension to prevent CAMP degradation. Secretin (200 nM), SG-secretin (200 nM), or glucagon (10 nM) was then added and the incubation continued for an additional 5-min (glucagon) or 30-min (secretin and SG-secretin) period. Time of incubation and hormone concentrations was established in preliminary experiments and from previously published studies that used isolated cell systems (49, 50). One milliliter of the cell suspension was obtained both 1 min before hormone addition and at the end of the hormone incubation period and was used for CAMP determination as described previously from our laboratory (31). Determination of total pH]inositol phosphates. For determination of total IPs, 10 x lo6 hepatocytes or 15 X lo6 IBDECs (pooled from 2-3 isolation experiments) were incubated at 37°C in 2.5 ml of gassed Krebs-Henseleit buffer solution (pH 7.4) containing 10 mM glucose, 1% bovine serum albumin, and 50 &i of myo-[“Hlinositol. After a 90-min labeling period, cells were centrifuged, washed, and resuspended in the original medium free of myo-[3H]inositol. We chose a 90-min labeling period based on previous studies with isolated cells (49,50) and on preliminary experiments carried out in our laboratory that demonstrated significant radioactivity incorporation after such a period. The cell suspension was then incubated for 15 min with 10 mM LiCl to avoid IP degradation (42). Secretin (200 nM), SG-secretin (200 nM), glucagon (1 nM), or vasopressin (100 nM) was finally added and the incubation continued for either 5 min (secretin, SG-secretin, vasopressin) or 30 min (glucagon). As for CAMP determination, optimal hormone concentrations and incubation periods were established in preliminary experiments conducted in our laboratory and from previously published reports using isolated cell preparations (49, 50). One milliliter of the cell suspension was then taken both 1 min before hormone addition and at the end of the hormone incubation period. Total IPs were determined as described previously from this laboratory (31). Specific IP metabolites (e.g., IP2, IP3, and IP4) were not measured because our objective was to determine the signal transdunction system (CAMP vs. IP) involved in secretin stimulation of ductular activity. In fact, IP3 is the messenger molecule and both IP2 and IP4 are metabolites of IP3 (6, 16, 25). RESULTS
Experimental strategy. Because the normal rat is very poorly responsive to the choleretic effect of secretin (1) and a low enrichment in IBDECs is obtained when these are isolated from a normal rat liver (Z), the present studies were performed in rats with bile ductular cell hyperplasia induced by BDL. This experimental strategy not only lends itself to greatly amplify bile ductular secretory activity in vivo (I), but also permits the isolation of IBDECs at a much higher yield and purity (2, 43). To
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ascertain the physiological relevance of the findings obtained in BDL rats, selective in vivo experiments were carried out in normal guinea pigs, a species highly responsive to the choleretic effect of secretin (31). BiLiary physiology. The results from these experiments are reported in Table 1. In normal rats, glucagon produced a moderate and inconsistent increase (about +10% of basal rate) in bile flow, while secretin and SG-secretin displayed no measurable stimulatory effect. With each hormone, bicarbonate concentration in bile did not change significantly. In BDL rats, however, both secretin and SG-secretin induced a large increase in bile flow and bicarbonate biliary concentration. Conversely, glucagon choleresis was not affected by bile duct proliferation as the effect of the hormone in BDL rats was the same as that seen in normal rats. In normal guinea pigs, both secretin and SG-secretin stimulated bile flow and bicarbonate biliary concentration. As expected (3 1), glucagon also stimulated bile secretion in this species. BTV results. Measurement of BTV is an approach used before in our laboratory and proved valid to establish the ductular and canalicular origin of secretin and glucagon choleresis, respectively (1, 3 1). Accordingly, to establish whether SG-secretin, which is structurally more closely related to glucagon than the parent hormone, stimulated fluid secretion at the bile ductules and/or ducts, the BTV was measured during infusion of SG-secretin and compared with that obtained when secretin, glucagon, or bile acids were administered. As summarized in Table 2, the BTVs determined during SG-secretin choleresis in both normal guinea pigs and BDL rats were similar to those obtained in these respective animals when secretin was infused and were significantly lower than those calculated when either glucagon or taurocholate was given. This indicates that SG-secretin, like the parent hormone (1, 31), stimulates fluid secretion at the distal biliary epithelium. Blood glucose ZeueZs.To establish whether hepatocellular function was affected by SG-secretin, we measured blood glucose levels in BDL rats before and after hormone Table 1. Hormone effects on bile flow and biliary bicarbonate concentrations in normal guinea pigs and BDL rats Guinea Bile flow, pl-min-l-kg-l
Basal Secretin SG-secretin Glucagon
206.6214.3 287.3t9.3* 304.6&38.5* 302.6t35.0*
Pigs
BDL
[HCO,l, n-w/l
64.2t4.2 79.lt7.3* 78.2t5.4* 76.3t6.6*
Bile flow, pl-min-l-kg-l
252.4k36.8 343.5&4&g* 363.9t44.7* 264.7t42.3
Rats
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Table 2. Biliary tree volumes in normal guinea pigs and BDL rats during hormone and bile acid choleresis Guinea
Secretin SG-secretin Glucagon Bile acids
Pigs
BDL
16.lt3.6* 17.5t5.1* 27.0~12.2 28.1t0.5
Rats
35.9t9.1* 32.8tll.2* ND 64.7t14.2
Values are means t SD and are expressed as pi/g liver; n = 3-5 rats for both groups. Biliary tree volume (BTV) was measured to determine the biliary site (canaliculi vs. bile ductules/ducts) of choleresis associated with hormone infusion. BTV was determined also during bile acid [glycocholate in guinea pigs and taurocholate in bile duct-ligated (BDL) rats, 4 pmolmin-l . kg-l] choleresis for comparison, since the latter originates at the canalicular level. Note that in both species, the BTVs measured during secretin and SG-secretin administration are significantly lower than the respective ones determined during bile acid infusion, indicating the distal origin of choleresis induced by both hormones. On the other hand, the BTV determined during infusion of glucagon into guinea pigs is quite similar to that observed with bile acid choleresis, supporting the canalicular nature of the choleresis induced by glucagon. In BDL rats, this hormone induced a small and inconsistent increase in bile flow (see Table 1); hence the BTV could not be determined (ND). * Significantly different (P < 0.05 by Student’s paired t test) compared with respective values obtained during bile acid choleresis.
infusion. As shown in Table 3, both secretin and SGsecretin failed to modify blood glucose concentration, while glucagon induced hyperglycemia as previously reported (31). CAMP formation in isolated hepatocytes and IBDECs.
As summarized in Table 4, both secretin and SG-secretin failed to modify CAMP production in isolated hepatocytes, while glucagon induced an approximately sixfold increase in CAMP baseline values. On the other hand, glucagon did not affect CAMP levels in IBDECs, which were instead increased significantly after addition of either secretin or SG-secretin. IP formation in isolated hepatocytes and IBDECs. The results are reported in Table 5. In isolated hepatocytes, glucagon induced a modest but statistically significant increase in IP formation, as previously found in vivo (31). Both secretin and SG-secretin failed to induce formation of IPs. In isolated IBDECs, however, the opposite was observed. Glucagon and SG-secretin displayed no stimulatory effect, while secretin increased IP formation significantly. Addition of vasopressin to suspensions of hepatocytes resulted in a large increase in IP formation.
[HCO,l, n-w/l
23.4k3.8 37.4t5.2* 35.3t6.1* 24.4t4.1
Values are means t SD and were obtained from either normal guinea pigs (n = 4-6) or rats in which the common bile duct was ligated for 14 days (BDL rats, n = 4-5) to induce bile duct proliferation. Secretin and SG-secretin ([TyrlOJ”, Phe22, Trp25]secretin) were infused at 1.1 nmol kg-’ h-l in BDL rats and 2.2 nmol kg-l. h-l in guinea pigs. Glucagon was infused at 1.4 nmol kg-l. min-l in both species. Values shown here were obtained under steady-state conditions. In normal rats, secretin and SG-secretin failed to stimulate bile flow rate. Glucagon produced an inconsistent effect similar in magnitude (+5-10 &min-‘Sk g-‘) to that observed in BDL rats. * Significantly different (P < 0.05) compared with respective basal values. l
IN BILE
Table 3. Blood glucose levels during hormone infusion into BDL rats Basal
Control Secretin SG-secretin Glucagon
142tl4 136tl3 14ltl3 144tl6
Hormone
Infusion
134&12 138tl5 139tl7 197tl8*
Values are means t SD and are expressed as mg/lOO ml. Samples for blood glucose determinations were obtained from 3 to 5 bile duct-cannulated rats under both basal (before hormone infusion) and experimental (during hormone infusion) conditions. Control rats did not receive hormone administration throughout the bile collection period. * Significantly different (P < 0.05 by Student’s paired t test) compared with values determined under basal conditions.
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Table 4. CAMP formation by isolated rat hepatocytes and intrahepatic bile duct epithelial cells Hepatocytes
Basal Secretin SG-secretin Glucagon
IBDECs
12.11t2.43 9.99t3.02 12.65t2.94 73.06&9.72*
0.54t0.05 0.75t0.07* 0.72t0.07* 0.56kO.06 Values are means t SD and are expressed as pmol/106 cells. Hepatocytes and intrahepatic bile duct epithelial cells (IBDECs) were isolated from bile duct-ligated rats (n = 4) and purified as described in METHODS. * Significantly different (P < 0.05 by Student’s paired t test) compared with respective basal values.
Table 5. Formation of total IPs by isolated rat hepatocytes and intrahepatic bile duct epithelial
Basal Secretin SG-secretin Glucagon Vasopressin
10,715+375 11,498&1,168 11,080+1,574 12,783+1,096* 40,182+2,173*
cells
1,208&179 1,589+224* 1,246+191 1,278+285 1,214+257
Values are means t SD and are expressed as radioactivity (my~-[~H]inositol) incorporation into cells. Results were obtained from 4 (hepatocytes) and 5 (IBDECs) incubation experiments. In the case of IBDECs, incubation experiments involved cells isolated from 2 to 3 BDL rats. * Significantly different (P < 0.05-0.001 by paired t test) compared with respective basal values.
In isolated IBDECs, vasopressin failed to induce formation of IPs. Vasopressin is a well-known stimulator of the IP messenger system (36) and was included in the present studies as a positive control. DISCUSSION
The results of the present studies lead to two conclusions. First, they indicate that secretin stimulates ductular transport function, while glucagon produces its effects solely at the hepatocellular level. Support for a canalicular and d istal 0 rigin of glucagon and secretin choleresis, respectively, has already been obtained from in vivo animal investigations (1, 31, 51). However, the present in vitro findings that 1) glucagon stimulates both the CAMP and IP systems in isolated hepatocytes, but neither of these second messenger systems in IBDECs, whereas 2) secretin activates both of these two signal transduction pathways in IBDECs, but not in isolated hepatocytes, provide more direct support for this view. Accordingly, together with the in vivo data on bile secretion and the recent autoradiographic observation demonstrating secretin binding at the bile ductules/ducts (17)) the present experiments provide conclusive evidence that the biliary epithelium is the target hepatic structure of secretin stimulation of fluid transport. The second and more important conclusion drawn from the present studies is that secretin induction of ductular secretory activity is mediated by the CAMP system. That this second messenger is involved in secretin choleresis has been suggested previously on the basis of the finding that secretin administration in vivo is associated with increased plasma and biliary levels of CAMP (32). In this previous report (3Z), however, increased
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CAMP formation was observed in some species and not in others, and the cellular source of the augmented nucleotide was not established. Furthermore, other studies have questioned the role of CAMP in secretin stimulation of bile flow, since the hormone failed to increase biliary CAMP in bile fistula dogs (26). Finally, recent experiments have shown that, at least in the pancreas, secretin stimulates production of both CAMP and IPs (49), thus raising the possibility that the hormone may induce activation of both messenger systems at the biliary epithehum as well. The present studies shed light on these uncertainties as they demonstrate that first, secretin stimulation of CAMP does originate at the bile ductules and/or ducts and not at the parenchymal level. More importantly, they indicate that secretin stimulation of ductular function involves the CAMP and not the IP system. In fact, while secretin induced formation of both CAMP and IPs, SG-secretin stimulated only the CAMP system and induced ductular secretion in vivo in a fashion similar to that observed with the parent hormone. Our in vitro results are similar to those obtained in pancreatic acini (49), thus supporting the conclusion that, as in the pancreas (49) and liver parenchyma (31, 50), two signal transduction pathways underlie hormonal regulation of cellular function in IBDECs. The significance of secretin-induced IP formation in IBDECs is not clear at the present time, also because the role of this second messenger system in cell function is not completely understood. Hydrolysis of membranebound phosphoinositides results in the formation of IPs and diacylglycerol, the former being associated with calcium mobilization from cytoplasmic stores (6, 16), the latter with activation of protein kinase C (6, 34). Intracellular calcium and protein kinase C have been suggested to regulate a variety of functions (6,16), including transport activity in hepatocytes (15, 33). Thus, although our results support a role of CAMP in secretin-induced choleresis by IBDECs, we cannot rule out that secretin induction of IPs and, presumably, of diacylglycerol formation may also play a regulatory role in IBDEC transport function. Also, it must be pointed out that a relationship between the CAMP and IP system has been demonstrated in a number of tissues (28, 38, 47, 48); hence an interaction between these two messenger systems in IBDECs is not inconceivable. Although our studies provide qualitative evidence for a role of CAMP in secretin stimulation of ductular activity, the hormone induced a modest response in IBDECs with respect to both CAMP and IP formation. In fact, secretin and SG-secretin increased CAMP levels by ~50%, and the parent hormone augmented IP formation only by -30%. This differs from the response reported in isolated pancreatic acini in which concentrations of secretin similar to those used here induced an -275% increase in basal levels of IPs and an -80% augment in CAMP content, and SG-secretin produced an even greater effect (about +90%) on CAMP formation (49). The CAMP stimulation observed in the present experiments is also more modest than that recently reported by Kato et al. (27), who found a secretin-induced increase of up to 218% in preparations of isolated IBDECs. Although the precise
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cause for these quantitative differences remains to be established, one possible explanation is that only a 5070% enrichment in IBDECs was obtained in the present studies, while the pancreatic preparation included presumably 100% acini (49). In fact, if our data are corrected for such an enrichment factor, a fractional response virtually the same as that obtained in the pancreas can be estimated. This extrapolation is justified, since the cells contaminating the IBDEC fraction were primarily mesenchymal @O-30% endothelial and Kupffer cells), and we have found that fractions enriched in these cell types are not responsive to the effect of secretin (unpublished observations). As to greater responsiveness of IBDECs observed by Kato et al. (27), the preliminary nature of this report does not allow a meaningful comparison to our study. Presumably, the cell fraction used by these investigators also involved an IBDEC enrichment (23) greater than that achieved in our experiments. Furthermore, in the study by Kato et al. (27), IBDECS were isolated from normal rats, whereas bile duct-ligated animals were used in the present studies. It is possible that hyperplastic IBDECs are less responsive to the stimulatory effect of secretin on CAMP formation. As to IP formation, little can also be said about the low responsiveness to secretin stimulation observed here, relative to that reported for pancreatic acini (49). It is possible that the modest increase detected in our experiments reflects either a low affinity of the hormone for this receptor system in IBDECs or the somewhat harsh conditions that the cells were subjected to during either the isolation (e.g., pronase digestion) and/or fractionation (centrifugal elutriation) procedures. It is also possible that, as mentioned for CAMP, the use of hyperplastic IBDECs prevented their full responsiveness to secretin stimulation. Alternatively, it can be postulated that the IP system does not play a major role in regulating ductular cell transport function, a possibility that should not be discarded, since it is consistent with the SG-secretin finding demonstrating stimulation of fluid transport in vivo without apparent involvement of IP formation. Because the present experiments were conducted in BDL rats, the question whether our findings are of physiological relevance warrants consideration. In an attempt to circumvent this problem, preliminary experiments were carried out with IBDECs isolated from normal rat livers; however, the low enrichment and poor yield obtained from these preparations prevented a clear appreciation of the hormone effect on both IP and CAMP formation. Furthermore, since both secretin and SGsecretin produced a minimal, if any, effect on bile secretion in normal rats in vivo, we judged it essential to use an experimental model in which the in vitro data could be related to findings obtained in vivo. As a result, the use of BDL rats was not only justified but imperative. Clearly, since the BDL rat model involves chronic extrahepatic biliary obstruction in order to induce proliferation of IBDECs, normal hepatic function may not fully be preserved under these conditions; thus the physiological relevance of our data may be questioned. With respect to the nature of the proliferating IBDECs in BDL rats, however, strong evidence has been presented before to support
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both their origin from preexisting bile ductules and/or ducts (2, 44) and their retention of structural and functional integrity (2,43,44). Furthermore, we have demonstrated here that SG-secretin stimulates ductular bile flow in normal guinea pigs in a fashion similar to that observed with the parent hormone, and both peptides failed to influence hepatocellular activity, as judged by the lack of their effect on plasma glucose levels. Accordingly, although some caution is obviously justified before interpreting our in vitro findings as conclusive, it seems highly unlikely that technical artifacts underlie the present results; hence reservations on their physiological relevance are unwarranted. In conclusion, the present studies have demonstrated that, while glucagon induces formation of CAMP and IPs in isolated hepatocytes and not in IBDECs, secretin stimulates both messenger systems in IBDECs but not in isolated preparations of parenchymal liver cells. Because SG-secretin, an analogue that increases ductular fluid secretion in vivo like the parent hormone, activated solely the CAMP system in IBDECs, these findings are construed to suggest that secretin stimulation of ductular transport function is mediated by the CAMP system and does not involve the IP signal transduction pathway. ’ This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42346 and by Deutsche Forschungsgemeinschaft Grant LE 606/l-l (R. Lenzen). Address for reprint requests: R Lenzen, Dept. of Medicine, Div. of Gastroenterology, Heinrich-Heine Univ. of Dusseldorf, Moorenstrasse 5, W-4000 Dusseldorf, Federal Republic of Germany. Received
28 October
1991; accepted
in final
form
15 May
1992.
REFERENCES G., R. Lenzi, L. Sarkozi, and N. Tavoloni. Biliary 1. Alpini, physiology in rats with ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules. J. Clin. Invest. 81: 569-578, 1988. 2. Alpini, G., R. Lenzi, W. 2. Zhai, M. H. Liu, P. A. Slott, F. Paronetto, and N. Tavoloni. Isolation of a nonparenchymal liver cell fraction enriched in cells with biliary epithelial phenotypes. Gastroenterology 97: 1248-1260, 1989. 3. Andrew, D., and R. B. Merrifield. Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs. Eur. J. Biochem. 164: 585-590, 1987. 4. Athari, A., and K. Jungermann. Direct activation by prostaglandin Fza but not thromboxane AZ of glycogenolysis via an increase in inositol 1,4,5-trisphosphate in rat hepatocytes. Biochem. Biophys. Res. Commun. 163: 1235-1242, 1989. 5. Bataille, D., P. Ferychet, and G. Rosselin. Interactions of glucagon, gut glucagon, VIP, and secretin with liver and fat cell plasma membranes. EndocrinoZogy 95: 713-720, 1974. 6. Berridge, M. J., and R. F. Irvine. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature Lond. 312: 315-321, 1984. B. M., M. J. Collen, H. Adachi, R. T. Jensen, 7. Bissonnette, and J. D. Gardner. Receptors for vasoactive intestinal peptide and secretin on rat pancreatic acini. Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9): G710-G717, 1984. J. L., and J. R. Bloomer. Canalicular bile secretion in 8. Boyer, man. Studies utilizing the biliary clearance of [14C]mannitol. J. Clin. Invest. 54: 773-781, 1974. 9. Buanes, T., T. Grotmol, T. Landsverk, and M. G. Raeder. Secretin empties bile duct cell cytoplasm of vesicles when it initiates ductular HCO; secretion in the pig. Gastroenterology 95: 417-424, 1988. G., C. Capella, E. Solcia, G. Vassallo, and P. 10. Bussolati, Vezzadini. Ultrastructural and immunofluorescent investigations on the secretin cell in the dog intestinal mucosa. Histochemie
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26: 218-227, 1971. 11. Carstairs, J. R., and P. J. Barnes. Visualization of vasoactive intestinal peptide receptors in human and guinea pig lung. J. Pharmacol. Exp. Ther. 239: 249-255, 1986. 12. Charlton, C. G., R. Quirion, G. E. Handelmann, R. L. Miller, R. T. Jensen, M. S. Finkel, and T. L. O’Donohue. Secretin receptors in the rat kidney: adenylate cyclase activation and renal effects. Peptides 7: 865-871, 1986. 13. Christodoulopoulos, J. B., W. H. Jacobs, and A. P. Klotz. Action of secretin on pancreatic secretion. Am. J. Physiol. 201: 1020-1024, 1961. 14. Christophe, J., M. Waelbroeck, P. Chatelain, and P. Robberecht. Heart receptors for VIP, PHI, and secretin are able to activate adenylate cyclase and to mediate inotropic and chronotropic effects. Species variations and physiopathology. Peptides 5: 341-353, 1984. J., N. D. Smith, E. Gordon, and J. L. Boyer. 15. Corasanti, Protein kinase C agonists inhibit bile secretion independently of effects on the microcirculation in the isolated perfused rat liver. HepatoZogy 10: 8-13, 1989. 16. Exton, J. H. Role of phosphoinositides in the regulation of liver function. Hepatology 8: 152-166, 1988. 17. Farouk, M., D. C. McVey, S. R. Vigna, G. D. Branum, and W. C. Meyers. Localization and characterization of secretin binding sites in rat intrahepatic bile ducts (Abstract). Hepatology 12: 917, 1990. 18. Fremeau, R. T., L. Y. Korman, and T. W. Moody. Secretin stimulates CAMP formation in the rat brain. J. Neurochem. 46: 1947-1955, 1986. 19. Gardner, J., T. Conlon, and T. Adams. Cyclic AMP in pancreatic acinar cells: effects of gastrointestinal hormones. Gastroenterology 70: 29-35, 1976. 20. Hubel, K. Secretin: a long progress note. Gastroenterology 62: 318-341, 1972. 21. Isenberg, J., B. Wallin, C. Johannsson, B. Smedfors, V. Mutt, K. Takemoto, and S. Emas. Secretin, VIP, and PHI stimulate rat proximal duodenal surface epithelial bicarbonate secretion in vivo. Regul. Pept. 8: 315-320, 1984. 22. Ishihara, T., S. Nakamura, Y. Kaziro, T. Takahashi, K. Takahashi, and S. Nagata. Molecular cloning and expression of a cDNA encoding the secretin receptor. EMBO J. 10: 16351641, 1991. 23. Ishii, M., B. Vroman, and N. F. LaRusso. Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver. Gastroenterology 97: 1236-1247, 1989. 24. Jones, R., R. Geist, and A. Hall. The choleretic effects of glucagon and secretin in the dog. Gastroenterology 60: 64-68, 1971. 25. Joseph, S. K., and J. R. Williamson. Inositol polyphosphates and intracellular calcium release. Arch. Biochem. Biophys. 273: l-15, 1989. 26. Kaminski, D. L., M. J. Ruwart, and Y. G. Deshpande. The role of cyclic AMP in canine secretin-stimulated bile flow. J. Surg. Res. 27: 57-61, 1979. 27. Kato, A., G. Gores, S. Bronk, and N. F. LaRusso. Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism (Abstract). CZin. Res. 39: 30lA, 1991. 28. Kelleher, D. J., J. E. Pessin, A. E. Ruoho, and G. L. Johnson. Phorbol ester induces desensitization of adenylate cyclase and phosphorylation of the ,8-adrenergic receptor in turkey erythrocytes. Proc. NatZ. Acad. Sci. USA 81: 4316-4320, 1984. 29. Kopin, A. S., M. B. Wheeler, and A. B. Leiter. Secretin: structure of the precursor and tissue distribution of the mRNA. Proc. NatZ. Acad. Sci. USA 87: 2299-2303, 1990. 30. Kopin, A. S., M. B. Wheeler, J. Nishitani, E. W. McBride, T. M. Chang, W. Y. Chey, and A. B. Leiter. The secretin gene: evolutionary history, alternative spicing, and developmental regulation. Proc. Natl. Acad. Sci. USA 88: 5335-5339, 1991. 31. Lenzen, R., V. J. Hruby, and N. Tavoloni. Mechanism of glucagon choleresis in guinea pigs. Am. J. Physiol. 259 (Gastrointest. Liver Physiol. 22): G736-G744, 1990.
32.
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36
37
38
39
40
41
42
43.
44.
45.
46. 47.
48.
49.
50.
51.
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Levine, R. A., and R. C. Hall. Cyclic AMP in secretin choleresis. Evidence for a regulatory role in man and baboons but not in dogs. Gastroenterology 70: 537-544, 1976. Nathanson, M. H., A. Gautam, R. Bruck, C. M. Isales, and J. L. Boyer. Effects of Ca2+ agonists on cytosolic Ca2+ in isolated hepatocytes and on bile secretion in the isolated perfused rat liver. Hepatology 15: 107-116, 1992. Nishizuka, Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature Lond. 308: 693-698, 1984. O’Donohue, T. L., C. G. Charlton, R. L. Miller, G. Boden, and D. M. Jacobowitz. Identification, characterization, and distribution of secretin immunoreactivity in rat and pig brain. Proc. Natl. Acad. Sci. USA 78: 5221-5224, 1981. Palmer, S., P. T. Hawkins, R. H. Mitchell, and C. J. Kirk. The labeling of polyphosphoinositides with [32P]Pi and the accumulation of inositol phosphates in vasopressin-stimulated hepatocytes. Biochem. J. 238: 491-499, 1986. Paquette, T. L., D. F. Schulman, D. H. Alpers, and B. M. Jaffe. Postnatal development of intestinal secretin in rats and guinea pigs. Am. J. Physiol. 243 (Gastrointest. Liver Physiol. 6): G5lLG517, 1982. Pittner, R. A., and J. N. Fain. Exposure of cultured hepatocytes to cyclic AMP enhances the vasopressin-mediated stimulation of inositol phosphate production. Biochem. J. 257: 455-460, 1989.. Polak, J., I. Colling, S. Bloom, and A. Pearse. Immunofluorescent localization of secretin and enteroglucagon in human intestinal mucosa. Stand. J. Gastroenterol. 6: 739-744, 1971. Roth, B. L., M. C. Beinfeld, and A. C. Howlett. Secretin receptors on neuroblastoma cell membranes: characterization of 12”1-labeled secretin binding and association with adenylate cyclase. J. Neurochem. 42: 1145-1152, 1984. Sanders, M., D. Amirian, A. Ayalon, and A. Soll. Regulation of pepsinogen release from canine chief cells in primary monolayer culture. Am. J. Physiol. 245 (Gastrointest. Liver Physiol. 8): G641G646, 1983. Sherman, W., B. Gish, M. Honchar, and L. Munsell. Effect of lithium on phosphoinositide metabolism in vivo. FASEB J. 45: 2639-2646, 1986. Sirica, A. E., C. A. Sattler, and H. P. Cihla. Characterization of a primary bile ductular cell culture from livers of rats during extrahepatic cholestasis. Am. J. PathoZ. 120: 67-78, 1985. Slott, P. A., M. H. Liu, and N. Tavoloni. Origin, pattern, and mechanism of bile duct proliferation following biliary obstruction in the rat. Gastroenterology 99: 466-477, 1990. Solcia, E., L. Usellini, R. Buffa, G. Rindi, L. Villani, A. Aguzzi, and E. Silini. Endocrine cells producing regulatory peptides. Experientia Base& Suppl. 56: 220-246, 1989. Straus, E., A. Greenstein, and R. Yalow. Effect of secretin on release of heterogeneous forms of gastrin. Gut 16: 999-1005, 1975. Sugden, D., J. Vanecek, D. C. Klein, T. P. Thomas, and W. B. Anderson. Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes. Nature Lond. 314: 359-361, 1985. Taylor, M. V., J. C. Metcalfe, T. R. Hesketh, G. A. Smith, and J. P. Moore. Mitogens increase phosphorylation of phosphoinositides in thymocytes. Nature Lond. 312: 462-465, 1984. Trimble, E. R., R. Bruzzone, T. J. Binden, C. J. Meehan, D. Andrew, and R. B. Merrifield. Secretin stimulates cyclic AMP and inositol trisphosphate production in rat pancreatic acinar tissue by two fully independent mechanisms. Proc. NatZ. Acad. Sci. USA 84: 3146-3150, 1987. Wakelam, M., G. Murphy, V. Hruby, and M. Houslay. Activation of two signal transduction systems in hepatocytes by glucagon. Nature Lond. 323: 68-71, 1986. Wheeler, H. O., and P. L. Mancusi-Ungaro. Role of bile ducts during secretin choleresis in dogs. Am. J. Physiol. 210: 1153-1159, 1966.
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secretory
activity
ROMANA LENZEN, GIANFRANCO ALPINI, AND NICOLA TAVOLONI Division of Gastroenterology, Department of Medicine, Heinrich-Heine- University, W-4000 Dusseldorf, Federal Republic of Germany; and Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029 Lenzen, Romana, Gianfranco Alpini, and Nicola Tavoloni. Secretin stimulates bile ductular secretory activity through the CAMP system. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G527-G532,1992.-Although convincing evidence has been obtained to support a ductular origin of secretin choleresis, the precise mechanism of the choleretic effect of the hormone is poorly understood. The present studies were carried out to 1) further clarify the anatomic site at which secretin stimulates bile flow and 2) establish the signal transduction system underlying this effect. To this end, parenchymal and nonparenchymal liver cells, the latter enriched in bile duct cells, were isolated from rats with ductular cell hyperplasia, and the effect of secretin on intracellular formation of both adenosine 3’,5’-cyclic monophosphate (CAMP) and inositol phosphates (IPs) was compared with that observed with glucagon and (SG-secretin). In the pancreas, CTY r10J3 Phe22,Trp25]secretin secretin stimulates both messenger systems, while SG-secretin activates only the CAMP cascade. In isolated hepatocytes, both secretin and SG-secretin failed to increase formation of CAMP and IPs, which were instead activated by glucagon. In isolated bile duct cells, secretin induced formation of both CAMP and IPs, while SG-secretin stimulated solely the CAMP system, as in the pancreas. Glucagon did not stimulate either messenger system in this cell preparation. In vivo, both secretin and SGsecretin stimulated a bicarbonate-rich fluid in rats with bile ductular cell hyperplasia and in normal guinea pigs, which was demonstrated to originate at the distal biliary epithelium. These findings support the existing view that glucagon stimulates canalicular bile flow, while secretin increases secretory activity at the bile ductules and/or ducts. More importantly, they indicate that stimulation of ductular secretory activity by secretin is mediated by the CAMP system and does not involve the IP signal transduction pathway. adenosine glucagon
3’,5’-cyclic
monophosphate;
inositol
phosphates;
is a 27-amino acid peptide that shares striking structural similarities with glucagon, vasoactive intestinal peptide, and gastric inhibitory peptide (5). Secretin-producing cells, known as S cells, are located in the mucosa of the upper part of the small intestine (10, 37,39). Although the main biological effect of secretin is stimulation of a bicarbonate-rich pancreatic juice (13)) the hormone also increases gastric pepsin (41) and duodenal bicarbonate secretion (2 1), and inhibits gastric acid output and food-stimulated gastrin release (46). Furthermore, secretin decreases upper small intestinal motility and lower esophageal sphincter pressure (ZO), and stimulates hepatic bile flow (1, 24, 51). Finally, secretin has pharmacological effects in organs like the heart, kidney, lung, and brain (11, 12, 14, l&35,40,45), and the mRNA for the hormone and/or its plasma membrane receptor have been demonstrated in the intestine, heart, and pancreas (22, 29,30). However, the biological effect of secretin in these tissues is poorly understood.
SECRETIN
0193-1857/92
$2.00 Copyright
Although the precise mechanism by which secretin stimulates cellular function is not known, activation of adenylate cyclase has been thought to be the fundamental event underlying the biological effects of the hormone (7, 19). Recently, however, it has been demonstrated that at least in the pancreas, secretin triggers also the inositol phosphate (IP) pathway (49), a messenger system that leads to calcium release from intracellular stores (6, 16, 25). This, together with the recent findings that receptors of other hormones may be coupled to either the adenosine 3’,5’-cyclic monophosphate (CAMP) and/or the IP systems (4, 31, 50), has raised new intriguing questions as to the role of each signal transduction pathway in hormone stimulation of cellular activity. In the liver, secretin stimulates a bicarbonate-rich choleresis in most animal species, including humans (8, 9, 24, 51). Although the precise target cells of secretin stimulation have not as yet been identified, compelling evidence has accumulated to indicate that the hormone regulates fluid secretion at the bile ductules and/or ducts (1, 51). Furthermore, since secretin choleresis is associated with increased plasma and biliary levels of CAMP, it has been suggested that at least in some species the hormone exerts its effect via activation of the adenylate cyclase system (32). However, the liver is a pleotrophic and pleomorphic organ so that the increase in plasma and biliary CAMP concentrations induced by the hormone may not necessarily originate from the cells involved in fluid transport. Other studies, in fact, have suggested that secretin choleresis is not regulated by the CAMP system (26). Moreover, since in the pancreas secretin stimulates both the CAMP and the IP cascades (49), it remains to be demonstrated which of the two messenger systems is involved in secretin stimulation of fluid secretion at the biliary epithelium. Accordingly, the objective of the present studies was to clarify 1) the liver epithelial cell type (hepatocyte vs. bile duct cell) responsive to the stimulatory effect of secretin and 2) the second messenger system mediating the effect of the hormone on fluid secretion. METHODS Animals and chemicals. Male Sprague-Dawley rats (200-250 g) and male Hartley albino guinea pigs (200-300 g) were obtained from Charles River Breeding Laboratories (Wilm.ington, MA) and housed in a temperature-controlled room (22°C) with alternating 12-h light-dark cycles. The animals were fed standard laboratory food ad libitum, had free access to water, and were not fasted before use. Experiments were carried out in three groups of animals. Groups 1 and 2 consisted of normal guinea pigs and normal rats, respectively, while group 3 included rats in which the common bile duct was ligated for 14 days
0 1992 the American
Physiological
Society
G527
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G528
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(BDL rats) to induce biliary epithelial cell hyperplasia. BDL rats were prepared as described (1). In vivo experiments consisted of measurements of bile flow and composition and were carried out in each group of animals. In vitro studies instead included determinations of CAMP and IP levels and were conducted in liver-derived cells isolated from BDL rats. Phenobarbital sodium was purchased from Abbott (North Chicago, IL), sodium glycocholate and pronase from Calbiothem (La Jolla, CA), collagenase from Boehringer Mannheim (Indianapolis, IN), minimum essential medium and Joklikmodified essential medium from GIBCO (Grand Island, NY), calf serum from Flow Laboratories (McLean, VA), the dowex resin (AG l-X8, mesh size 200-400, formate form) from BioRad (Richmond, CA), and myo-[2-3H]inositol (sp act 19 Ci/ mmol) and the radioimmunoassay kit for CAMP determination from Amersham (Arlington Heights, IL). Glucagon and vasopressin were obtained from Sigma (St. Louis, MO), and secretin from Ferring Laboratories (Suffern, NY). [Tyr10~13,Phe22, Trp25]secretin (SG-secretin) was a generous gift of Dr. R. B. Merrifield (Rockefeller University, New York City). SG-secretin is an analogue in which tyrosine is substituted for leucine at positions 10 and 13, and phenylalanine and tryptophan are substituted for leucine and glycine at positions 22 and 25, respectively (3). All other chemicals were purchased from Sigma, unless otherwise indicated. Studies of Mary physiology. Measurements of bile flow and composition were conducted in anesthetized, bile duct-cannulated animals using standard procedures (1, 31). Animal temperature was invariably kept at 37°C. Secretin and SG-secretin were infused each at 1.1 nmol kg-l. h-l in rats and 2.2 nmol . kg-l h-l in guinea pigs. Glucagon was given at 1.4 nmol kg-l min-l in both species. Each hormone was infused for 30-45 min until steady-state choleresis was achieved. Throughout the experiment, blood samples (-1 ml) were obtained from the carotid artery 10 min before and 10 min after hormone infusion was initiated, and used for glucose determination. Bile was collected every 5 min, except during the initial 60-min equilibration period in which collections were made over lo-min intervals. At the end of hormone infusion, the animal was killed with an overdose of pentobarbital sodium, and the liver was removed and its weight determined. Bile concentrations of electrolytes were determined by standard clinical chemistry methods (1). Bile acids in bile were measured by the hydroxysteroid dehydrogenase procedure. Determination of Mary tree volume. These experiments were carried out as described previously (1, 31) using [3H] taurocholate as a marker of biliary transit time. Biliary tree volume (BTV) was measured during administration of secretin, SGsecretin, glucagon, glycocholate (4 hmol min-l kg-l in guinea pigs), or taurocholate (4 pmol min-l kg-l in BDL rats). Bile was collected over 3-min intervals. Blood glucose determination. Glucose in plasma was determined with an ASTRA Automated Stat-Routine Analyzer System (Beckman Instruments, Somerset, NJ), using the Beckman glucose oxidase reagent and ASTRA system calibration standards (1, 31). Isolation and purification of hepatocytes and bile duct epithelial cells. Hepatocytes were isolated by collagenase perfusion and purified by differential centrifugation using standard procedures. Nonparenchymal liver cells were isolated from BDL rats as described previously (2). After isolation, nonparenchymal liver cells were fractioned by centrifugal centrifugation to obtain a population enriched in bile duct cells. The cells in the fraction obtained at the pump flow rate of 22-34 ml/min were then resuspended in minimal essential medium containing 10% fetal calf serum (l-4 x lo5 cells/ml) and maintained overnight in a 5% C02-air-equilibrated incubator at 37°C. The morning after, cells were harvested and used for the in vitro determinations l
l
l
l
l
l
l
IN BILE
DUCT
CELLS
described below. This nonparenchymal liver cell population contained 50-70% of biliary epithelial cells as judged by their positivity for y-glutamyl transpeptidase and cytokeratin no. 19 (2,44). Hepatocytes in this fraction accounted for Cl%, and the majority (20-30%) of contaminating cells were mesenchymal in nature (endothelial and Kupffer cells, fibroblasts) as established by vimentin positivity (2, 44). For sake of simplicity, this fraction will be referred to as “intrahepatic bile duct epithelial cells” (IBDECs). Viability of IBDECs was 197%. CAMP determination. To determine the effect of the various hormones on CAMP formation, 0.2 x lo6 hepatocytes and 1 x lo6 IBDECs were incubated at 37°C in 2.5 ml of KrebsHenseleit buffer (pH 7.4) containing 1% bovine serum albumin and gassed with 95% 02-5% CO,. After a 15-min incubation period, IO mM 3-isobutyl-1-methylxanthine was added to the cell suspension to prevent CAMP degradation. Secretin (200 nM), SG-secretin (200 nM), or glucagon (10 nM) was then added and the incubation continued for an additional 5-min (glucagon) or 30-min (secretin and SG-secretin) period. Time of incubation and hormone concentrations was established in preliminary experiments and from previously published studies that used isolated cell systems (49, 50). One milliliter of the cell suspension was obtained both 1 min before hormone addition and at the end of the hormone incubation period and was used for CAMP determination as described previously from our laboratory (31). Determination of total pH]inositol phosphates. For determination of total IPs, 10 x lo6 hepatocytes or 15 X lo6 IBDECs (pooled from 2-3 isolation experiments) were incubated at 37°C in 2.5 ml of gassed Krebs-Henseleit buffer solution (pH 7.4) containing 10 mM glucose, 1% bovine serum albumin, and 50 &i of myo-[“Hlinositol. After a 90-min labeling period, cells were centrifuged, washed, and resuspended in the original medium free of myo-[3H]inositol. We chose a 90-min labeling period based on previous studies with isolated cells (49,50) and on preliminary experiments carried out in our laboratory that demonstrated significant radioactivity incorporation after such a period. The cell suspension was then incubated for 15 min with 10 mM LiCl to avoid IP degradation (42). Secretin (200 nM), SG-secretin (200 nM), glucagon (1 nM), or vasopressin (100 nM) was finally added and the incubation continued for either 5 min (secretin, SG-secretin, vasopressin) or 30 min (glucagon). As for CAMP determination, optimal hormone concentrations and incubation periods were established in preliminary experiments conducted in our laboratory and from previously published reports using isolated cell preparations (49, 50). One milliliter of the cell suspension was then taken both 1 min before hormone addition and at the end of the hormone incubation period. Total IPs were determined as described previously from this laboratory (31). Specific IP metabolites (e.g., IP2, IP3, and IP4) were not measured because our objective was to determine the signal transdunction system (CAMP vs. IP) involved in secretin stimulation of ductular activity. In fact, IP3 is the messenger molecule and both IP2 and IP4 are metabolites of IP3 (6, 16, 25). RESULTS
Experimental strategy. Because the normal rat is very poorly responsive to the choleretic effect of secretin (1) and a low enrichment in IBDECs is obtained when these are isolated from a normal rat liver (Z), the present studies were performed in rats with bile ductular cell hyperplasia induced by BDL. This experimental strategy not only lends itself to greatly amplify bile ductular secretory activity in vivo (I), but also permits the isolation of IBDECs at a much higher yield and purity (2, 43). To
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SIGNAL
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ascertain the physiological relevance of the findings obtained in BDL rats, selective in vivo experiments were carried out in normal guinea pigs, a species highly responsive to the choleretic effect of secretin (31). BiLiary physiology. The results from these experiments are reported in Table 1. In normal rats, glucagon produced a moderate and inconsistent increase (about +10% of basal rate) in bile flow, while secretin and SG-secretin displayed no measurable stimulatory effect. With each hormone, bicarbonate concentration in bile did not change significantly. In BDL rats, however, both secretin and SG-secretin induced a large increase in bile flow and bicarbonate biliary concentration. Conversely, glucagon choleresis was not affected by bile duct proliferation as the effect of the hormone in BDL rats was the same as that seen in normal rats. In normal guinea pigs, both secretin and SG-secretin stimulated bile flow and bicarbonate biliary concentration. As expected (3 1), glucagon also stimulated bile secretion in this species. BTV results. Measurement of BTV is an approach used before in our laboratory and proved valid to establish the ductular and canalicular origin of secretin and glucagon choleresis, respectively (1, 3 1). Accordingly, to establish whether SG-secretin, which is structurally more closely related to glucagon than the parent hormone, stimulated fluid secretion at the bile ductules and/or ducts, the BTV was measured during infusion of SG-secretin and compared with that obtained when secretin, glucagon, or bile acids were administered. As summarized in Table 2, the BTVs determined during SG-secretin choleresis in both normal guinea pigs and BDL rats were similar to those obtained in these respective animals when secretin was infused and were significantly lower than those calculated when either glucagon or taurocholate was given. This indicates that SG-secretin, like the parent hormone (1, 31), stimulates fluid secretion at the distal biliary epithelium. Blood glucose ZeueZs.To establish whether hepatocellular function was affected by SG-secretin, we measured blood glucose levels in BDL rats before and after hormone Table 1. Hormone effects on bile flow and biliary bicarbonate concentrations in normal guinea pigs and BDL rats Guinea Bile flow, pl-min-l-kg-l
Basal Secretin SG-secretin Glucagon
206.6214.3 287.3t9.3* 304.6&38.5* 302.6t35.0*
Pigs
BDL
[HCO,l, n-w/l
64.2t4.2 79.lt7.3* 78.2t5.4* 76.3t6.6*
Bile flow, pl-min-l-kg-l
252.4k36.8 343.5&4&g* 363.9t44.7* 264.7t42.3
Rats
DUCT
G529
CELLS
Table 2. Biliary tree volumes in normal guinea pigs and BDL rats during hormone and bile acid choleresis Guinea
Secretin SG-secretin Glucagon Bile acids
Pigs
BDL
16.lt3.6* 17.5t5.1* 27.0~12.2 28.1t0.5
Rats
35.9t9.1* 32.8tll.2* ND 64.7t14.2
Values are means t SD and are expressed as pi/g liver; n = 3-5 rats for both groups. Biliary tree volume (BTV) was measured to determine the biliary site (canaliculi vs. bile ductules/ducts) of choleresis associated with hormone infusion. BTV was determined also during bile acid [glycocholate in guinea pigs and taurocholate in bile duct-ligated (BDL) rats, 4 pmolmin-l . kg-l] choleresis for comparison, since the latter originates at the canalicular level. Note that in both species, the BTVs measured during secretin and SG-secretin administration are significantly lower than the respective ones determined during bile acid infusion, indicating the distal origin of choleresis induced by both hormones. On the other hand, the BTV determined during infusion of glucagon into guinea pigs is quite similar to that observed with bile acid choleresis, supporting the canalicular nature of the choleresis induced by glucagon. In BDL rats, this hormone induced a small and inconsistent increase in bile flow (see Table 1); hence the BTV could not be determined (ND). * Significantly different (P < 0.05 by Student’s paired t test) compared with respective values obtained during bile acid choleresis.
infusion. As shown in Table 3, both secretin and SGsecretin failed to modify blood glucose concentration, while glucagon induced hyperglycemia as previously reported (31). CAMP formation in isolated hepatocytes and IBDECs.
As summarized in Table 4, both secretin and SG-secretin failed to modify CAMP production in isolated hepatocytes, while glucagon induced an approximately sixfold increase in CAMP baseline values. On the other hand, glucagon did not affect CAMP levels in IBDECs, which were instead increased significantly after addition of either secretin or SG-secretin. IP formation in isolated hepatocytes and IBDECs. The results are reported in Table 5. In isolated hepatocytes, glucagon induced a modest but statistically significant increase in IP formation, as previously found in vivo (31). Both secretin and SG-secretin failed to induce formation of IPs. In isolated IBDECs, however, the opposite was observed. Glucagon and SG-secretin displayed no stimulatory effect, while secretin increased IP formation significantly. Addition of vasopressin to suspensions of hepatocytes resulted in a large increase in IP formation.
[HCO,l, n-w/l
23.4k3.8 37.4t5.2* 35.3t6.1* 24.4t4.1
Values are means t SD and were obtained from either normal guinea pigs (n = 4-6) or rats in which the common bile duct was ligated for 14 days (BDL rats, n = 4-5) to induce bile duct proliferation. Secretin and SG-secretin ([TyrlOJ”, Phe22, Trp25]secretin) were infused at 1.1 nmol kg-’ h-l in BDL rats and 2.2 nmol kg-l. h-l in guinea pigs. Glucagon was infused at 1.4 nmol kg-l. min-l in both species. Values shown here were obtained under steady-state conditions. In normal rats, secretin and SG-secretin failed to stimulate bile flow rate. Glucagon produced an inconsistent effect similar in magnitude (+5-10 &min-‘Sk g-‘) to that observed in BDL rats. * Significantly different (P < 0.05) compared with respective basal values. l
IN BILE
Table 3. Blood glucose levels during hormone infusion into BDL rats Basal
Control Secretin SG-secretin Glucagon
142tl4 136tl3 14ltl3 144tl6
Hormone
Infusion
134&12 138tl5 139tl7 197tl8*
Values are means t SD and are expressed as mg/lOO ml. Samples for blood glucose determinations were obtained from 3 to 5 bile duct-cannulated rats under both basal (before hormone infusion) and experimental (during hormone infusion) conditions. Control rats did not receive hormone administration throughout the bile collection period. * Significantly different (P < 0.05 by Student’s paired t test) compared with values determined under basal conditions.
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Table 4. CAMP formation by isolated rat hepatocytes and intrahepatic bile duct epithelial cells Hepatocytes
Basal Secretin SG-secretin Glucagon
IBDECs
12.11t2.43 9.99t3.02 12.65t2.94 73.06&9.72*
0.54t0.05 0.75t0.07* 0.72t0.07* 0.56kO.06 Values are means t SD and are expressed as pmol/106 cells. Hepatocytes and intrahepatic bile duct epithelial cells (IBDECs) were isolated from bile duct-ligated rats (n = 4) and purified as described in METHODS. * Significantly different (P < 0.05 by Student’s paired t test) compared with respective basal values.
Table 5. Formation of total IPs by isolated rat hepatocytes and intrahepatic bile duct epithelial
Basal Secretin SG-secretin Glucagon Vasopressin
10,715+375 11,498&1,168 11,080+1,574 12,783+1,096* 40,182+2,173*
cells
1,208&179 1,589+224* 1,246+191 1,278+285 1,214+257
Values are means t SD and are expressed as radioactivity (my~-[~H]inositol) incorporation into cells. Results were obtained from 4 (hepatocytes) and 5 (IBDECs) incubation experiments. In the case of IBDECs, incubation experiments involved cells isolated from 2 to 3 BDL rats. * Significantly different (P < 0.05-0.001 by paired t test) compared with respective basal values.
In isolated IBDECs, vasopressin failed to induce formation of IPs. Vasopressin is a well-known stimulator of the IP messenger system (36) and was included in the present studies as a positive control. DISCUSSION
The results of the present studies lead to two conclusions. First, they indicate that secretin stimulates ductular transport function, while glucagon produces its effects solely at the hepatocellular level. Support for a canalicular and d istal 0 rigin of glucagon and secretin choleresis, respectively, has already been obtained from in vivo animal investigations (1, 31, 51). However, the present in vitro findings that 1) glucagon stimulates both the CAMP and IP systems in isolated hepatocytes, but neither of these second messenger systems in IBDECs, whereas 2) secretin activates both of these two signal transduction pathways in IBDECs, but not in isolated hepatocytes, provide more direct support for this view. Accordingly, together with the in vivo data on bile secretion and the recent autoradiographic observation demonstrating secretin binding at the bile ductules/ducts (17)) the present experiments provide conclusive evidence that the biliary epithelium is the target hepatic structure of secretin stimulation of fluid transport. The second and more important conclusion drawn from the present studies is that secretin induction of ductular secretory activity is mediated by the CAMP system. That this second messenger is involved in secretin choleresis has been suggested previously on the basis of the finding that secretin administration in vivo is associated with increased plasma and biliary levels of CAMP (32). In this previous report (3Z), however, increased
IN BILE
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CELLS
CAMP formation was observed in some species and not in others, and the cellular source of the augmented nucleotide was not established. Furthermore, other studies have questioned the role of CAMP in secretin stimulation of bile flow, since the hormone failed to increase biliary CAMP in bile fistula dogs (26). Finally, recent experiments have shown that, at least in the pancreas, secretin stimulates production of both CAMP and IPs (49), thus raising the possibility that the hormone may induce activation of both messenger systems at the biliary epithehum as well. The present studies shed light on these uncertainties as they demonstrate that first, secretin stimulation of CAMP does originate at the bile ductules and/or ducts and not at the parenchymal level. More importantly, they indicate that secretin stimulation of ductular function involves the CAMP and not the IP system. In fact, while secretin induced formation of both CAMP and IPs, SG-secretin stimulated only the CAMP system and induced ductular secretion in vivo in a fashion similar to that observed with the parent hormone. Our in vitro results are similar to those obtained in pancreatic acini (49), thus supporting the conclusion that, as in the pancreas (49) and liver parenchyma (31, 50), two signal transduction pathways underlie hormonal regulation of cellular function in IBDECs. The significance of secretin-induced IP formation in IBDECs is not clear at the present time, also because the role of this second messenger system in cell function is not completely understood. Hydrolysis of membranebound phosphoinositides results in the formation of IPs and diacylglycerol, the former being associated with calcium mobilization from cytoplasmic stores (6, 16), the latter with activation of protein kinase C (6, 34). Intracellular calcium and protein kinase C have been suggested to regulate a variety of functions (6,16), including transport activity in hepatocytes (15, 33). Thus, although our results support a role of CAMP in secretin-induced choleresis by IBDECs, we cannot rule out that secretin induction of IPs and, presumably, of diacylglycerol formation may also play a regulatory role in IBDEC transport function. Also, it must be pointed out that a relationship between the CAMP and IP system has been demonstrated in a number of tissues (28, 38, 47, 48); hence an interaction between these two messenger systems in IBDECs is not inconceivable. Although our studies provide qualitative evidence for a role of CAMP in secretin stimulation of ductular activity, the hormone induced a modest response in IBDECs with respect to both CAMP and IP formation. In fact, secretin and SG-secretin increased CAMP levels by ~50%, and the parent hormone augmented IP formation only by -30%. This differs from the response reported in isolated pancreatic acini in which concentrations of secretin similar to those used here induced an -275% increase in basal levels of IPs and an -80% augment in CAMP content, and SG-secretin produced an even greater effect (about +90%) on CAMP formation (49). The CAMP stimulation observed in the present experiments is also more modest than that recently reported by Kato et al. (27), who found a secretin-induced increase of up to 218% in preparations of isolated IBDECs. Although the precise
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cause for these quantitative differences remains to be established, one possible explanation is that only a 5070% enrichment in IBDECs was obtained in the present studies, while the pancreatic preparation included presumably 100% acini (49). In fact, if our data are corrected for such an enrichment factor, a fractional response virtually the same as that obtained in the pancreas can be estimated. This extrapolation is justified, since the cells contaminating the IBDEC fraction were primarily mesenchymal @O-30% endothelial and Kupffer cells), and we have found that fractions enriched in these cell types are not responsive to the effect of secretin (unpublished observations). As to greater responsiveness of IBDECs observed by Kato et al. (27), the preliminary nature of this report does not allow a meaningful comparison to our study. Presumably, the cell fraction used by these investigators also involved an IBDEC enrichment (23) greater than that achieved in our experiments. Furthermore, in the study by Kato et al. (27), IBDECS were isolated from normal rats, whereas bile duct-ligated animals were used in the present studies. It is possible that hyperplastic IBDECs are less responsive to the stimulatory effect of secretin on CAMP formation. As to IP formation, little can also be said about the low responsiveness to secretin stimulation observed here, relative to that reported for pancreatic acini (49). It is possible that the modest increase detected in our experiments reflects either a low affinity of the hormone for this receptor system in IBDECs or the somewhat harsh conditions that the cells were subjected to during either the isolation (e.g., pronase digestion) and/or fractionation (centrifugal elutriation) procedures. It is also possible that, as mentioned for CAMP, the use of hyperplastic IBDECs prevented their full responsiveness to secretin stimulation. Alternatively, it can be postulated that the IP system does not play a major role in regulating ductular cell transport function, a possibility that should not be discarded, since it is consistent with the SG-secretin finding demonstrating stimulation of fluid transport in vivo without apparent involvement of IP formation. Because the present experiments were conducted in BDL rats, the question whether our findings are of physiological relevance warrants consideration. In an attempt to circumvent this problem, preliminary experiments were carried out with IBDECs isolated from normal rat livers; however, the low enrichment and poor yield obtained from these preparations prevented a clear appreciation of the hormone effect on both IP and CAMP formation. Furthermore, since both secretin and SGsecretin produced a minimal, if any, effect on bile secretion in normal rats in vivo, we judged it essential to use an experimental model in which the in vitro data could be related to findings obtained in vivo. As a result, the use of BDL rats was not only justified but imperative. Clearly, since the BDL rat model involves chronic extrahepatic biliary obstruction in order to induce proliferation of IBDECs, normal hepatic function may not fully be preserved under these conditions; thus the physiological relevance of our data may be questioned. With respect to the nature of the proliferating IBDECs in BDL rats, however, strong evidence has been presented before to support
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DUCT
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CELLS
both their origin from preexisting bile ductules and/or ducts (2, 44) and their retention of structural and functional integrity (2,43,44). Furthermore, we have demonstrated here that SG-secretin stimulates ductular bile flow in normal guinea pigs in a fashion similar to that observed with the parent hormone, and both peptides failed to influence hepatocellular activity, as judged by the lack of their effect on plasma glucose levels. Accordingly, although some caution is obviously justified before interpreting our in vitro findings as conclusive, it seems highly unlikely that technical artifacts underlie the present results; hence reservations on their physiological relevance are unwarranted. In conclusion, the present studies have demonstrated that, while glucagon induces formation of CAMP and IPs in isolated hepatocytes and not in IBDECs, secretin stimulates both messenger systems in IBDECs but not in isolated preparations of parenchymal liver cells. Because SG-secretin, an analogue that increases ductular fluid secretion in vivo like the parent hormone, activated solely the CAMP system in IBDECs, these findings are construed to suggest that secretin stimulation of ductular transport function is mediated by the CAMP system and does not involve the IP signal transduction pathway. ’ This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42346 and by Deutsche Forschungsgemeinschaft Grant LE 606/l-l (R. Lenzen). Address for reprint requests: R Lenzen, Dept. of Medicine, Div. of Gastroenterology, Heinrich-Heine Univ. of Dusseldorf, Moorenstrasse 5, W-4000 Dusseldorf, Federal Republic of Germany. Received
28 October
1991; accepted
in final
form
15 May
1992.
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