Effect of Prolactin on Sugar and Amino Acid Transport by the Rat Jejunum JAMES R. MAINOYA' Department of Zoology and Cancer Research Laboratory, University of California, Berkeley, California 94720
ABSTRACT After two daily ovine prolactin injections in rats, significant increases in jejunal absorption of glucose, glycine and proline, as well as of fluid and NaC1, occurred. Although ouabain caused dramatic reductions in fluid, sodium, chloride and glucose transport, prolactin had an effect even in the presence of ouabain. Mucosal hyperosmolality significantly decreased fluid, sodium and chloride absorption but had no significant effect on glucose transport. Addition of prolactin in vitro had no effect on intestinal absorption. Prolactin-induced increases in transport of fluid, NaCl and organic nutrients by the mammalian jejunum may play a n important role in nutritional and osmoregulatory a d a p tations.
Prolactin has several physiological actions in mammals (Bern and Nicoll, '68; Nicoll and Bern, '72; Horrobin, '73) including the stimulation of intestinal absorption of fluid and electrolytes (Ramsey and Bern, '72; Mainoya et al., '74). Although there are numerous studies on the effects of hormones on intestinal absorption of sugars and amino acids, only a few authors have considered the role of the pituitary gland in their transport (Levin, '69; Gelb and Gerson, '69). Consequently, the actions of prolactin on intestinal absorption of sugar and amino acids have never been reported. Moreover, there is some question as to whether the action of prolactin on jejunal transport primarily subserves an osmoregulatory or a nutritional role in the animal. The purpose of the present study was to determine the effects of prolactin on jejunal transport of D-glucose, L-glycine and L-proline, in comparison with its already known effects on fluid and NaCl transport. MATERIAL A N D METHODS
Male Sprague-Dawley rats (Simonsen Labs, Gilroy, Calif.) weighing 250-300 g were used in this study. The animals were given a White diet (Simonsen Labs.) and water ad libitum. Intestinal transport of fluid, NaCI, Dglucose, L-glycine and L-proline was studied in vitro using the everted sacs of the midjejunal segment (111) as described preJ. ExP. Z ~ O L 192; ., 149-154.
viously (Ramsey and Bern, '72; Mainoya et al., '74). The everted jejunal segment was weighed empty using a Mettler H balance, filled with 1.0 ml Krebs-Henseleit bicarbonate Ringer (pH 7.4) containing 28 mM glucose (serosal fluid), reweighed and incubated in 20 ml of the same solution (mucosal fluid) for one hour, or for 30 minutes in some experiments, at 3 7 ° C in equilibrium with a gas phase of 95% 02/5% Con. The procedure for the determination of fluid, sodium and chloride transport was described previously (Mainoya et al., '74). Glucose concentrations were determined by the glucose oxidase method as described by Raabo and Terkildsen ('60) and readings were made with a Klett-Summerson Photoelectric Colorimeter. The osmolality of solutions was measured by the freezing point depression method (Advanced Instruments 0smometer). Glycine and proline transport was investigated at a concentration of 5 mM, so that the initial amount of unlabelled amino acid in the mucosal solution was 100 pmole and in the serosal solution 5 pmole. The specific activity of 2-JH-L-glycine added to the mucosal solution was 5.2 pCi/mM and that of 3H-L-prolinewas 5.4 pCi/mM. The labelled amino acids were obtained from Schwarzmann (Orangeburg, N. Y.) and unlabelled amino acids from Calbio1 Present address: Department of Zoology. University of Dar-es-Salaam, Dar-es-salaam, Tanzania.
149
150
JAMES R MAINOYA
dium containing 500 ng/ml oPRL. The final incubation was conducted for one hour using the same concentration of prolactin as in the preincubation fluid.
chem (Los Angeles). Radioactivity was measured in aliquots of samples with a Liquid Scintillation Counter (Packard); the scintillation fluid contained 4 g BBOT [2, 5-bis2 - (5 - tert-butyl-benzoxazolyl) - thiophene] and 80 g naphthalene in 600 ml toluene plus 400 ml 1-methoxyethanol (Packard). Criteria for glucose, glycine and proline transport were increases in their quantities in the serosal fluid, expressed as pmole/g initial wet weight/hr or 30 minutes, and the concentration gradients (serosaumucosal) developed by the end of the incubation. The values are expressed as mean t standard error and the statistical significance determined using the unpaired t-test. Hormonal treatment. Ovine prolactin (oPRL: N.I.H. PS-10; 25.6 I.U./mg) was prepared for injection as described before (Ramsey and Bern, '72); controls were injected with saline (vehicle). Injections were given subcutaneously in the midafternoon, 48 and 24 hours before the animal was killed. Incubation with prolactin. Jejunal sacs (segment 111) from untreated rats were first preincubated for 30 minutes in a me-
RESULTS
The effects of prolactin on intestinal fluid, NaCl and glucose transport are shown in tables 1-4. Mucosal fluid, sodium and chloride transfer is significantly increased by prolactin injections (table 1). In the same preparations glucose absorption is also enhanced by prolactin: both serosal transfer and the concentration gradient (serosaVmucosa1) were significantly higher in sacs from prolactin-injected rats than in sacs from the control group. When ouabain was added to the serosal fluid at a concentration of 4 X 1 0 - 4 M , glucose, fluid, sodium and chloride transport were significantly decreased in both groups (table 2 ) . The data also indicate that inhibition of sodium transport was not complete. However, there was a significant positive correlation between glucose and sodium transport (r = 0.92, p < 0.001) as well as between fluid and sodium transport (r = 0.96, p < 0.001).
TABLE 1
meets of
o v i n e p r o l a c t i n ( o P R L ) o n j e j u n a l t r a n s p o r t of f l u i d , NaCl a n d g l u c o s e Mucosal fluid transport (mug wet wt/h)
Treatment (number of an iin als)
Mucosal CItransport (yWg wet wtih)
Mucosal Na+ transport (gEq/g wet wtlh)
~
Control 1.0mgoPRL
(7) (9)
0.88 f 0.06 1.49t0.063
128.7 f 10.7 233.3-12.93
33.1 f 5.8 66.8f7.92
Glucose conc. gradient (serosall mucosal)
Mucosal glucose transport (yMoUg wet wt/h)
*
~
34.8 4.0 67.725.83
~~
1.46 f 0.09 1.87f0.101
10.05 > p > 0.01, 0.01 > p > 0.001. 3 p < 0.001.as compared with control values
2
TABLE 2
Effect of prolactin ( o P R L ) on j e j u n a l t r a n s p o r t o f f l u i d , N a C l a n d glucose in the p r e s e n c e of 4 X 10-4 M o u a b a i n Treatment (number of animals)
Addition to serosal solution
Control Control
(5)
1.0 mg oPRL 1.0 mg oPRL
(5) (5)
3
None 4x10-4~ ouabain None 4X M ouabain
Mucosal Na+ transport igEq/g wet wt/h)
Mucosal CItransport (FEqlg wet w 4 h )
Mucosal glucose transport iFMollg w e t wUh)
0.74 t 0.02
104.8 f 14.1
43.0 t 8.2
19.1 f 2 . 3
0.20 t 0.08 3 1.08 t 0.08
30.5 2 15.6 2 167.0f 15.7
0.1 6.2 2 74.2 f 6.6
1.3t 3.7 2 43.9 f 5.3
69.6 k 5.2
10.6 f 5.0 :1
15.8 24.0
0.43 t 0.04
> p > 0.01. 0.01 > p =. 0.001. p < 0 001, as compared with normal Ringer values.
10.05 2
(5)
Mucosal fluid transport (mug wet wt/h)
3
3
2
151
PROLACTIN AND INTESTINAL NUTRIENT ABSORPTION
Increased mucosal osmolality has been glucose transport are shown in table 4. In shown to decrease intestinal absorption of contrast to the results obtained with in fluid and solutes (Dinda et al., '73). Table vivo administration of prolactin, its addi3 presents results of an experiment car- tion in vitro has no apparent effect on ried out to assess the enhancement of je- fluid, sodium, chloride and glucose absorpjunal absorption by prolactin when the tion in the quantity employed. osmolality of the mucosal solution was Tables 5 and 6 show the effects of proraised from 292 to 402 mOsmlkg by the lactin on glycine and proline transport, addition of 110 mM mannitol. Mucosal respectively. Glycine and proline transport hyperosmolality caused a significant de- was significantly enhanced in prolactincrease in fluid and sodium transport in injected rats. The concentration gradient both groups. Under these conditions chlo- (serosal concentration/mucosal concentraride absorption was converted to secretion. tion > 1) is indicative of active transIt is noteworthy, however, that the reduc- port and is significantly increased for protion in glucose transport was not signifi- line (p < 0.01). The transport of amino cant. The increase in the serosal osmolality acids also correlated significantly with sowas highly significant in both groups (p < dium transport: (r = 0.79, p < 0.01, for 0.001). glycine and r = 0.76, p < 0.01, for proThe effects of adding prolactin in vitro line). Similarly, in these experiments as in on intestinal fluid, sodium, chloride and the previous ones, fluid and NaCl transTABLE 3
w f e c t of prolactin (oPRL) on jejunal transport offluid, NaCl and glucose f r o m a hyperosmotic mucosal medium Treat men t (number of animals)
Addition to mucosal solution
Control Control
(5) (5)
1.0 mg oPRL 1.0 mg oPRL
(5) (5)
None 110mM mannitol None 110mM mannitol
Mucosal fluid transport (mug wet wtlh)
Mucosal Na+ transport (pEq/g wet wVh)
Mucosal C1transport (pEqlg wet wtlh)
Mucosal glucose transport (pMollg wet wtlh)
Osmolality of serosal fluid (mOsmoUkg)
0.73 2 0.06
103.0 2 7.4
42.7 k 3.9
21.9 k 2 . 2
285.9k0.5
0.04 2 0.04 3 1.10 t 0.03
33.6 t 9.1 3 - 11.62 6.4 3 153.8 2 6.5 63.3 k 7.0
12.0 k 4.9 46.3 2 4.6
331.1 k 2 . 3 3 289.2 k 3.0
32.4 k 8.4
337.6 k 3.3 3
0.18 & 0.01
3
54.3 t 5.1
3
- 8.0k2.4
3
10.05 > p > 0.01. 2 0.01 > p > 0.001. 3 p < 0.001. as compared with normal Ringer values. TABLE 4
meet of
prolactin (oPRL) added in vitro onjejunal transport offluid, NaCl and glucose
Treatment (number of animals)
Mucosal fluid transport (mug wet wwh)
Mucosal Na+ transport (pEq/g wet wffh)
Mucosal C1transport (pEq/g wet wtlh)
Mucosal glucose transport (fiMol/g wet wtlh)
Control (4) 500 nglml oPRL (5)
0.91 & 0.08 0.94 k 0.05
142.8 +- 10.4 156.0 t 7.6
5 2 . 9 2 3.7 65.1 ? 5.7
29.1 2 3.7 27.6 2 2.6
TABLE 5
mfect of prolactin ( o P R L ) on jejunal transport offluid, N a C l and glycine Treat men t (number of animals)
Control (4) 1 . 0 m g o P R L (6)
Mucosal fluid transport (mug wet wt/h)
0.91 2 0 . 0 3 1.47&0.09*
Mucosal Na+ transport (pEslg wet wt/h)
144.4k11.8 222.7k12.31
10.01 > p > 0.001. p < 0.001,as compared with control values.
2
Mucosal C1transport (pEs/g wet wtlh)
28.1 2 2 . 8 57.724.91
Mucosal glycine transport ( pMol/g wet wuh)
12.4 % 0.9 18.5k0.62
Glycine conc gradient (serosall mucosal)
1.6tO.l 1.9kO.l
152
JAMES R. MAINOYA TABLE 6
Effect of p r o l a c t z n ( o P R L ) on jejunal t r a n s p o r t offluid, N a C l a n d proline Mucosal fluid transport (mug wet wtih)
Treatment (number of animals)
Control 1.0 mg oPRL 10.05 20.01
p
p > 0.01. > p > 0.001. 0.001. as compared with control values
port is significantly higher in prolactininjected rats than in the control group. DISCUSSION
Hormones have been shown to influence intestinal transport of nutrients (Levin, '69; Gelb and Gerson, '69; Mahmood et al., '70). The present data strongly indicate that prolactin administration produced significant increases in glucose, glycine and proline transport, as well as in fluid and NaCl absorption. The stimulatory effects of prolactin on intestinal fluid and ions have been reported previously (Ramsey and Bern, '72; Mainoya et al., '74; Mainoya, '75). Although glucose absorption by the small intestine of hypophysectomized rats was decreased when measured in vivo (Althausen, '49; Righetti and Levitan, '70), Fabry et al. ('59) showed that growth hormone had no effect on glucose absorption in the normal rat but increased absorption in the adrenalectomized rat. Finkelstein and Schachter ('62) reported that growth hormone depressed galactose absorption in hypophysectomized rats but was without effect on proline absorption. Increased intestinal transport of methionine following ACTH administration has been claimed (Mahmood et al., '70). These workers also reported that cortisol and deoxycorticosterone had no significant influence on intestinal methionine transport. Important increases in prolactin secretion have been demonstrated in the rat during pregnancy and lactation (Meites et al., '72). Pregnancy results in increased absorption of glucose (Larralde and Fernandez-Otero, '68). Penzes and Simon ('68) reported significant increases in DL-methionine absorption in lactating rats but no significant changes in pregnancy. In a previous study, prolactin, estrogen, preg-
nancy and lactation were shown to enhance intestinal absorption of fluid and NaC1, whereas ovariectomy and progesterone treatments had no significant effect on absorption (Mainoya, '74). These findings are consistent with the reported elevated prolactin secretion following estrogen, but not progesterone, administration during early and late pregnancy; and throughout suckled lactation (Meites et al., '72). The dependence of fluid transport in the mammalian jejunum on sodium transport and on the presence of glucose is well known (Gilman and Koelle, '60; Schultz et al., '74; Kimmich, '73). Analysis of the present data showed a statistically significant positive correlation between fluid and sodium transported across the jejunal mucosa. Neutral amino acids are transported across the intestinal mucosa by a n active transport process (Wilson and Wiseman, '54). The action of prolactin on absorption of glucose and amino acids was examined because of their importance in nutrition and because their active transport is sodium-dependent (Schultz and Curran, '70; Reiser and Christiansen, '72). The present results are also in agreement with those of Bihler and Crane ('62) and those of Wheeler et al. ('65) who showed a correlation between glucose and sodium transport, and between amino acids and sodium transport, respectively. Furthermore, ouabain, a n inhibitor of sodium transport (cf. Glynn, '64), inhibits intestinal glucose absorption (Schultz and Zalusky, '64; Schultz and Curran, '70; Kimmic h, '73). Despite claims by Forster and Matthaus ('73) that active transport of glucose in the small intestine is not sodium-dependent, the prevailing view is that active transport of sugar is dependent on active
PROLACTIN AND INTESTINAL NUTRIENT ABSORPTION
sodium transport (Kimmich, '73). The residual transport of sodium and glucose observed in the presence of ouabain could be due to inaccessibility of ouabain to the sodium pump in the intact tissue (Kimmich, '73). The fluid transported from the mucosal to the serosal side of in vitro preparations of rat jejunum was found to be isotonic whether the mucosal fluid was isotonic, hypotonic or hypertonic (Lee, '68). A similar isosmotic transport of fluid by the rabbit gall bladder has also been reported (Diamond, '64). In the present study an attempt was made to elucidate the effects of prolactin on fluid, NaCl and glucose transfer from hypertonic mucosal fluids. An elevation of osmolality of the mucosal solution produced significant changes not only in mucosal fluid and NaCl transfer but also in serosal fluid osmolality. Glucose transport was also decreased but the differences were not statistically significant. These observations are essentially in accord with those of Dinda et al. ('73). Parsons and Wingate ('61) also found that elevating the mucosal osmolality by using sodium chloride had little effect on glucose transport in the rat small intestine. The present results suggest that the primary effect of prolactin on jejunal absorption is on active transport of solutes rather than on increase in water permeability. In view of the fact that the intestinal mucosal is impermeable to mannitol (Lindermann and Solomon, '62), the increased serosal osmolality must be due to a higher concentration of sodium and glucose in the absorbate (Dinda et al., '73). The absence of stimulation of intestinal transport when prolactin is added in vitro shows that the time required to induce increases in transport processes is considerably longer than one and one-half hours, Unfortunately, it is difficult to keep the intestine in good condition in vitro for more than a few hours. The present observations are also consistent with those of Ramsey and Bern ('72) who showed that prolactin added in vitro had no effect on intestinal fluid absorption. Doneen and Bern ('74) found that prolactin had effects on fish urinary bladder in vitro, but only after two days of culture in a medium containing prolactin. The report by Snart and Dalton ('73) that prolactin added in vitro
153
to toad bladder stimulated transport in less than one hour is difficult to reconcile with other data and calls for further reevaluation. The present study has provided results that are consistent with previous suggestions regarding the effects of prolactin in water and electrolyte accumulation in mammals. Although the role of the gut in osmoregulation is not so dramatically apparent as that of the kidney, it provides sites for the initial processes involved in the accumulation of water and salts (Bentley, '71). This study provides the first evidence that prolactin enhances glucose and amino acid transport by the small intestine. The latter effects of prolactin are of potential nutritional significance in view of the fact that the bulk of the transport of sugars and amino acids normally takes place in the small intestine. It is suggested that increased prolactin secretion could partly explain the increased intestinal transport of sugar and amino acids during pregnancy and lactation (Larralde and Fernandez-Otero, '68; Penzes and Simon, '68). It is postulated that the action of prolactin on intestinal transport may primarily subserve a nutritional role rather than an osmoregulatory one. ACKNOWLEDGMENTS
Aided by NIH grant CA-05388 and a Rockefeller Foundation Fellowship. I am indebted to Professor Howard A. Bern for his valuable advice and useful suggestions at every phase of this study and for reading the manuscript. LITERATURE CITED Althausen, T. L. 1949 Hormonal and vitamin factors in intestinal absorption. Gastroenterology, 12: 467480. Bentley, P. J. 1971 Endocrines and Osmoregulation, A Comparative Account of the Regulation of Water and Salt in Vertebrates. SpringerVerlag, Berlin. Bern, H. A,, and C. S . Nicoll 1968 The comparative endocrinology of prolactin. Recent Progr. Horm. Res., 24: 681-720. Bihler, I., and R. K. Crane 1962 On the mechanism of intestinal absorption of sugars. Biochim. biophys. Acta, 59: 78-93. Diamond, J. M. 1964 The mechanism of isotonic water transport. J. gen. Physiol., 48: 1 5 4 2 . Dinda, P. K., M. Beck and I. T. Beck 1973 On the mechanism of isosmotic transport across the small intestine. The composition of the absorb-
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JAMES R. MAINOYA
ate transported from a mucosal solution made hypertonic by the addition of mannitol. Can. J. Physiol. Pharmacol., 51: 1311-138. Doneen, B. A., and H. A. Bern 1974 In uitro effects of prolactin and cortisol on water permeability of the urinary bladder of the teleost Gillichthys mirabilis. J. Exp. Zool., 187: 173-179. Fabry, P., V. Kujalova and B. Mosinger 1959 Influence of somatotropic hormone on small intestine resorption of glucose. Endokrinologie, 38: 152-158. Finkelstein, J. D., and D. Schacter 1962 Active transport of calcium by the intestine: effects of hypophysectomy and growth hormone. Am. J. Physiol., 203: 873-880. Forster, H., and M. Matthaus 1973 Some comments on the couplinrr between intestinal absorption of glucose-and-of sodium. FEBS Letters, 31 : 75-80. Gelb, A. M., and C. D. Gerson 1969 Influence of the endocrine glands on small intestine absorption. Am. J. Clin. Nutr., 2 2 : 305-310. Gilman, A,, and E. S . Koelle 1960 Substrate requirements for ion transport by the rat intestine studied in vitro. Am. J. Physiol., 199: 10251029. Glynn,,I. M. 1964 The action of cardiac glycosides on ion movements. Pharmacol. Rev., 16: 381-407. Horrobin, D.F. 1973 Prolactin: Physiology and Clinical Significance. MTP, St. Lmnardgate, Lancaster. Kimmich, G. A. 1973 Coupling between Na+ and sugar transport in smali intestine. Biochim. biophy. Acta, 300: 31-78. Larralde, J., and P. Fernandez-Otero 1968 The effect of pregnancy on the in uitro intestinal absorption of glucose and glycine. Revta. esp. Fisiol., 24: 49-50. Larralde, J., P. Fernandez-Otero and M. Gonzalez 1966 increased active transport of glucose through the intestine during pregnancy. Nature (London), 209: 1356-1357. Lee, J . S. 1968 Isosmotic absorption of fluid from rat jejunum in vitro. Gastroenterology, 5 4 : 365-374. Levin, R. J . 1969 The effects of hormones on the absorptive, metabolic and digestive functions of the small intestine. J. Endocr., 45: 315348. Lindermann, B., and A. K. Solomon 1962 Permeability of the luminal surface of intestinal mucosal cells. J. gen. Physiol., 45: 8 0 1 4 1 0 . Mahmood, A , , S. D. Varma and D. S . Wagle 1970 Effect of hormonal treatments in rats on the intestinal transport of methionine in uitro. Indian J. Exp. Biol., 8: 330-331. Mainoya, J. R. 1975 Further studies on the action of prolactin on fluid and ion absorption by the rat jejunum. Endocrinology, 96: 11901196. 1974 Influence of prolactin, estrus cycle, pregnancy and lactation on intestinal fluid
and ion transport by the rat jejunum. Am. Zool.. 14: 1244 (abstract 15). Mainoya, J. R., H. A. Bern and J . W. Regan 1974 Influence of ovine prolactin on transport of fluid and sodium chloride by the mammalian intestine and gall bladder. J. Endocr., 63: 311317. Meites, J., K. H. Lu, W. Wuttke, C. W. Welsch, H. Nagasawa and S . K . Quadri 1972 Recent studies on the function and control of prolactin secretion in rats. Recent Prog. Horm. Res., 28: 471-524. Nicoll, C . S . , and H. A. Bern 1972 On the action of prolactin among the vertebrates: is there a common denominator? In: Lactogenic Hormones. G. E. W. Wolstenholme and J . Knight, eds. Churchill and Livingston, London, 1972. p. 299. Parsons, D. S . , and D. L. Wingate 1961 The effect of osmotic gradients on fluid transfer across rat intestine in witro. Biochim. biophys. Acta., 46.; 170-183. Penzes, L., and C . T. Simon 1968 Intestinal absorption and turnover of DL-methionine during reproduction in the rat. Jap. J. Physiol., 18: 288-296. Raabo, E., and T. C. Terkildsen 1960 On the enzymatic determination of blood glucose. Scand. J. Clin. Lab. Invest., 1 2 ; 402-407. Ramsey, D. H., and H. A . Bern 1972 Stimulation by ovine prolactin of fluid transfer in everted sacs of rat small intestine. J. Endocr., 53: 453459. Reiser, S . , and P. A. Christiansen 1972 Relative effectiveness of extracellular sodium in supporting leucine uptake by isolated intestinal epithelial cells. Proc. SOC.exp. Biol. Med., 140: 362366. Righetti, A , , and R. Levitan 1970 Small bowel absorption in hypophysectomized rats. Am. J. digest. Dis., 15: 218-225. Schultz, S . G.. and P. F. Curran 1970 COUDICW~ transport of sodium and organic solutes. Physiol. Rev., 50: 637-718. Schultz, S. G., R. A. Frizzell and H. N . Nellans 1974 Ion transport by mammalian small intestine. Ann. Rev. Physiol., 36: 51-93. Schultz, S. G., and R. Zalusky 1964 Ion transport in the isolated rabbit ileum. 11. The interaction between active sodium and active sugar transport. J. gen. Physioi., 4 7 : 1043-1059. Snart, R. S . , and T. Dalton 1973 Response of toad bladder to prolactin. Comp. Biochem. Physiol., 4 5 A : 307-313. Wheeler, K. P., Y. Inui, P. E. HoLlenberg, E. Eavenson and H. N Christensen 1965 Relation of amino acid transport to sodium ion concentration. Biochim. biophys. Acta., 109: 620-622. Wilson, T. H., and G. Wiseman 1954 The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J. Physiol., 123: 116-125. ~
~
~~
.
ABSTRACT After two daily ovine prolactin injections in rats, significant increases in jejunal absorption of glucose, glycine and proline, as well as of fluid and NaC1, occurred. Although ouabain caused dramatic reductions in fluid, sodium, chloride and glucose transport, prolactin had an effect even in the presence of ouabain. Mucosal hyperosmolality significantly decreased fluid, sodium and chloride absorption but had no significant effect on glucose transport. Addition of prolactin in vitro had no effect on intestinal absorption. Prolactin-induced increases in transport of fluid, NaCl and organic nutrients by the mammalian jejunum may play a n important role in nutritional and osmoregulatory a d a p tations.
Prolactin has several physiological actions in mammals (Bern and Nicoll, '68; Nicoll and Bern, '72; Horrobin, '73) including the stimulation of intestinal absorption of fluid and electrolytes (Ramsey and Bern, '72; Mainoya et al., '74). Although there are numerous studies on the effects of hormones on intestinal absorption of sugars and amino acids, only a few authors have considered the role of the pituitary gland in their transport (Levin, '69; Gelb and Gerson, '69). Consequently, the actions of prolactin on intestinal absorption of sugar and amino acids have never been reported. Moreover, there is some question as to whether the action of prolactin on jejunal transport primarily subserves an osmoregulatory or a nutritional role in the animal. The purpose of the present study was to determine the effects of prolactin on jejunal transport of D-glucose, L-glycine and L-proline, in comparison with its already known effects on fluid and NaCl transport. MATERIAL A N D METHODS
Male Sprague-Dawley rats (Simonsen Labs, Gilroy, Calif.) weighing 250-300 g were used in this study. The animals were given a White diet (Simonsen Labs.) and water ad libitum. Intestinal transport of fluid, NaCI, Dglucose, L-glycine and L-proline was studied in vitro using the everted sacs of the midjejunal segment (111) as described preJ. ExP. Z ~ O L 192; ., 149-154.
viously (Ramsey and Bern, '72; Mainoya et al., '74). The everted jejunal segment was weighed empty using a Mettler H balance, filled with 1.0 ml Krebs-Henseleit bicarbonate Ringer (pH 7.4) containing 28 mM glucose (serosal fluid), reweighed and incubated in 20 ml of the same solution (mucosal fluid) for one hour, or for 30 minutes in some experiments, at 3 7 ° C in equilibrium with a gas phase of 95% 02/5% Con. The procedure for the determination of fluid, sodium and chloride transport was described previously (Mainoya et al., '74). Glucose concentrations were determined by the glucose oxidase method as described by Raabo and Terkildsen ('60) and readings were made with a Klett-Summerson Photoelectric Colorimeter. The osmolality of solutions was measured by the freezing point depression method (Advanced Instruments 0smometer). Glycine and proline transport was investigated at a concentration of 5 mM, so that the initial amount of unlabelled amino acid in the mucosal solution was 100 pmole and in the serosal solution 5 pmole. The specific activity of 2-JH-L-glycine added to the mucosal solution was 5.2 pCi/mM and that of 3H-L-prolinewas 5.4 pCi/mM. The labelled amino acids were obtained from Schwarzmann (Orangeburg, N. Y.) and unlabelled amino acids from Calbio1 Present address: Department of Zoology. University of Dar-es-Salaam, Dar-es-salaam, Tanzania.
149
150
JAMES R MAINOYA
dium containing 500 ng/ml oPRL. The final incubation was conducted for one hour using the same concentration of prolactin as in the preincubation fluid.
chem (Los Angeles). Radioactivity was measured in aliquots of samples with a Liquid Scintillation Counter (Packard); the scintillation fluid contained 4 g BBOT [2, 5-bis2 - (5 - tert-butyl-benzoxazolyl) - thiophene] and 80 g naphthalene in 600 ml toluene plus 400 ml 1-methoxyethanol (Packard). Criteria for glucose, glycine and proline transport were increases in their quantities in the serosal fluid, expressed as pmole/g initial wet weight/hr or 30 minutes, and the concentration gradients (serosaumucosal) developed by the end of the incubation. The values are expressed as mean t standard error and the statistical significance determined using the unpaired t-test. Hormonal treatment. Ovine prolactin (oPRL: N.I.H. PS-10; 25.6 I.U./mg) was prepared for injection as described before (Ramsey and Bern, '72); controls were injected with saline (vehicle). Injections were given subcutaneously in the midafternoon, 48 and 24 hours before the animal was killed. Incubation with prolactin. Jejunal sacs (segment 111) from untreated rats were first preincubated for 30 minutes in a me-
RESULTS
The effects of prolactin on intestinal fluid, NaCl and glucose transport are shown in tables 1-4. Mucosal fluid, sodium and chloride transfer is significantly increased by prolactin injections (table 1). In the same preparations glucose absorption is also enhanced by prolactin: both serosal transfer and the concentration gradient (serosaVmucosa1) were significantly higher in sacs from prolactin-injected rats than in sacs from the control group. When ouabain was added to the serosal fluid at a concentration of 4 X 1 0 - 4 M , glucose, fluid, sodium and chloride transport were significantly decreased in both groups (table 2 ) . The data also indicate that inhibition of sodium transport was not complete. However, there was a significant positive correlation between glucose and sodium transport (r = 0.92, p < 0.001) as well as between fluid and sodium transport (r = 0.96, p < 0.001).
TABLE 1
meets of
o v i n e p r o l a c t i n ( o P R L ) o n j e j u n a l t r a n s p o r t of f l u i d , NaCl a n d g l u c o s e Mucosal fluid transport (mug wet wt/h)
Treatment (number of an iin als)
Mucosal CItransport (yWg wet wtih)
Mucosal Na+ transport (gEq/g wet wtlh)
~
Control 1.0mgoPRL
(7) (9)
0.88 f 0.06 1.49t0.063
128.7 f 10.7 233.3-12.93
33.1 f 5.8 66.8f7.92
Glucose conc. gradient (serosall mucosal)
Mucosal glucose transport (yMoUg wet wt/h)
*
~
34.8 4.0 67.725.83
~~
1.46 f 0.09 1.87f0.101
10.05 > p > 0.01, 0.01 > p > 0.001. 3 p < 0.001.as compared with control values
2
TABLE 2
Effect of prolactin ( o P R L ) on j e j u n a l t r a n s p o r t o f f l u i d , N a C l a n d glucose in the p r e s e n c e of 4 X 10-4 M o u a b a i n Treatment (number of animals)
Addition to serosal solution
Control Control
(5)
1.0 mg oPRL 1.0 mg oPRL
(5) (5)
3
None 4x10-4~ ouabain None 4X M ouabain
Mucosal Na+ transport igEq/g wet wt/h)
Mucosal CItransport (FEqlg wet w 4 h )
Mucosal glucose transport iFMollg w e t wUh)
0.74 t 0.02
104.8 f 14.1
43.0 t 8.2
19.1 f 2 . 3
0.20 t 0.08 3 1.08 t 0.08
30.5 2 15.6 2 167.0f 15.7
0.1 6.2 2 74.2 f 6.6
1.3t 3.7 2 43.9 f 5.3
69.6 k 5.2
10.6 f 5.0 :1
15.8 24.0
0.43 t 0.04
> p > 0.01. 0.01 > p =. 0.001. p < 0 001, as compared with normal Ringer values.
10.05 2
(5)
Mucosal fluid transport (mug wet wt/h)
3
3
2
151
PROLACTIN AND INTESTINAL NUTRIENT ABSORPTION
Increased mucosal osmolality has been glucose transport are shown in table 4. In shown to decrease intestinal absorption of contrast to the results obtained with in fluid and solutes (Dinda et al., '73). Table vivo administration of prolactin, its addi3 presents results of an experiment car- tion in vitro has no apparent effect on ried out to assess the enhancement of je- fluid, sodium, chloride and glucose absorpjunal absorption by prolactin when the tion in the quantity employed. osmolality of the mucosal solution was Tables 5 and 6 show the effects of proraised from 292 to 402 mOsmlkg by the lactin on glycine and proline transport, addition of 110 mM mannitol. Mucosal respectively. Glycine and proline transport hyperosmolality caused a significant de- was significantly enhanced in prolactincrease in fluid and sodium transport in injected rats. The concentration gradient both groups. Under these conditions chlo- (serosal concentration/mucosal concentraride absorption was converted to secretion. tion > 1) is indicative of active transIt is noteworthy, however, that the reduc- port and is significantly increased for protion in glucose transport was not signifi- line (p < 0.01). The transport of amino cant. The increase in the serosal osmolality acids also correlated significantly with sowas highly significant in both groups (p < dium transport: (r = 0.79, p < 0.01, for 0.001). glycine and r = 0.76, p < 0.01, for proThe effects of adding prolactin in vitro line). Similarly, in these experiments as in on intestinal fluid, sodium, chloride and the previous ones, fluid and NaCl transTABLE 3
w f e c t of prolactin (oPRL) on jejunal transport offluid, NaCl and glucose f r o m a hyperosmotic mucosal medium Treat men t (number of animals)
Addition to mucosal solution
Control Control
(5) (5)
1.0 mg oPRL 1.0 mg oPRL
(5) (5)
None 110mM mannitol None 110mM mannitol
Mucosal fluid transport (mug wet wtlh)
Mucosal Na+ transport (pEq/g wet wVh)
Mucosal C1transport (pEqlg wet wtlh)
Mucosal glucose transport (pMollg wet wtlh)
Osmolality of serosal fluid (mOsmoUkg)
0.73 2 0.06
103.0 2 7.4
42.7 k 3.9
21.9 k 2 . 2
285.9k0.5
0.04 2 0.04 3 1.10 t 0.03
33.6 t 9.1 3 - 11.62 6.4 3 153.8 2 6.5 63.3 k 7.0
12.0 k 4.9 46.3 2 4.6
331.1 k 2 . 3 3 289.2 k 3.0
32.4 k 8.4
337.6 k 3.3 3
0.18 & 0.01
3
54.3 t 5.1
3
- 8.0k2.4
3
10.05 > p > 0.01. 2 0.01 > p > 0.001. 3 p < 0.001. as compared with normal Ringer values. TABLE 4
meet of
prolactin (oPRL) added in vitro onjejunal transport offluid, NaCl and glucose
Treatment (number of animals)
Mucosal fluid transport (mug wet wwh)
Mucosal Na+ transport (pEq/g wet wffh)
Mucosal C1transport (pEq/g wet wtlh)
Mucosal glucose transport (fiMol/g wet wtlh)
Control (4) 500 nglml oPRL (5)
0.91 & 0.08 0.94 k 0.05
142.8 +- 10.4 156.0 t 7.6
5 2 . 9 2 3.7 65.1 ? 5.7
29.1 2 3.7 27.6 2 2.6
TABLE 5
mfect of prolactin ( o P R L ) on jejunal transport offluid, N a C l and glycine Treat men t (number of animals)
Control (4) 1 . 0 m g o P R L (6)
Mucosal fluid transport (mug wet wt/h)
0.91 2 0 . 0 3 1.47&0.09*
Mucosal Na+ transport (pEslg wet wt/h)
144.4k11.8 222.7k12.31
10.01 > p > 0.001. p < 0.001,as compared with control values.
2
Mucosal C1transport (pEs/g wet wtlh)
28.1 2 2 . 8 57.724.91
Mucosal glycine transport ( pMol/g wet wuh)
12.4 % 0.9 18.5k0.62
Glycine conc gradient (serosall mucosal)
1.6tO.l 1.9kO.l
152
JAMES R. MAINOYA TABLE 6
Effect of p r o l a c t z n ( o P R L ) on jejunal t r a n s p o r t offluid, N a C l a n d proline Mucosal fluid transport (mug wet wtih)
Treatment (number of animals)
Control 1.0 mg oPRL 10.05 20.01
p
p > 0.01. > p > 0.001. 0.001. as compared with control values
port is significantly higher in prolactininjected rats than in the control group. DISCUSSION
Hormones have been shown to influence intestinal transport of nutrients (Levin, '69; Gelb and Gerson, '69; Mahmood et al., '70). The present data strongly indicate that prolactin administration produced significant increases in glucose, glycine and proline transport, as well as in fluid and NaCl absorption. The stimulatory effects of prolactin on intestinal fluid and ions have been reported previously (Ramsey and Bern, '72; Mainoya et al., '74; Mainoya, '75). Although glucose absorption by the small intestine of hypophysectomized rats was decreased when measured in vivo (Althausen, '49; Righetti and Levitan, '70), Fabry et al. ('59) showed that growth hormone had no effect on glucose absorption in the normal rat but increased absorption in the adrenalectomized rat. Finkelstein and Schachter ('62) reported that growth hormone depressed galactose absorption in hypophysectomized rats but was without effect on proline absorption. Increased intestinal transport of methionine following ACTH administration has been claimed (Mahmood et al., '70). These workers also reported that cortisol and deoxycorticosterone had no significant influence on intestinal methionine transport. Important increases in prolactin secretion have been demonstrated in the rat during pregnancy and lactation (Meites et al., '72). Pregnancy results in increased absorption of glucose (Larralde and Fernandez-Otero, '68). Penzes and Simon ('68) reported significant increases in DL-methionine absorption in lactating rats but no significant changes in pregnancy. In a previous study, prolactin, estrogen, preg-
nancy and lactation were shown to enhance intestinal absorption of fluid and NaC1, whereas ovariectomy and progesterone treatments had no significant effect on absorption (Mainoya, '74). These findings are consistent with the reported elevated prolactin secretion following estrogen, but not progesterone, administration during early and late pregnancy; and throughout suckled lactation (Meites et al., '72). The dependence of fluid transport in the mammalian jejunum on sodium transport and on the presence of glucose is well known (Gilman and Koelle, '60; Schultz et al., '74; Kimmich, '73). Analysis of the present data showed a statistically significant positive correlation between fluid and sodium transported across the jejunal mucosa. Neutral amino acids are transported across the intestinal mucosa by a n active transport process (Wilson and Wiseman, '54). The action of prolactin on absorption of glucose and amino acids was examined because of their importance in nutrition and because their active transport is sodium-dependent (Schultz and Curran, '70; Reiser and Christiansen, '72). The present results are also in agreement with those of Bihler and Crane ('62) and those of Wheeler et al. ('65) who showed a correlation between glucose and sodium transport, and between amino acids and sodium transport, respectively. Furthermore, ouabain, a n inhibitor of sodium transport (cf. Glynn, '64), inhibits intestinal glucose absorption (Schultz and Zalusky, '64; Schultz and Curran, '70; Kimmic h, '73). Despite claims by Forster and Matthaus ('73) that active transport of glucose in the small intestine is not sodium-dependent, the prevailing view is that active transport of sugar is dependent on active
PROLACTIN AND INTESTINAL NUTRIENT ABSORPTION
sodium transport (Kimmich, '73). The residual transport of sodium and glucose observed in the presence of ouabain could be due to inaccessibility of ouabain to the sodium pump in the intact tissue (Kimmich, '73). The fluid transported from the mucosal to the serosal side of in vitro preparations of rat jejunum was found to be isotonic whether the mucosal fluid was isotonic, hypotonic or hypertonic (Lee, '68). A similar isosmotic transport of fluid by the rabbit gall bladder has also been reported (Diamond, '64). In the present study an attempt was made to elucidate the effects of prolactin on fluid, NaCl and glucose transfer from hypertonic mucosal fluids. An elevation of osmolality of the mucosal solution produced significant changes not only in mucosal fluid and NaCl transfer but also in serosal fluid osmolality. Glucose transport was also decreased but the differences were not statistically significant. These observations are essentially in accord with those of Dinda et al. ('73). Parsons and Wingate ('61) also found that elevating the mucosal osmolality by using sodium chloride had little effect on glucose transport in the rat small intestine. The present results suggest that the primary effect of prolactin on jejunal absorption is on active transport of solutes rather than on increase in water permeability. In view of the fact that the intestinal mucosal is impermeable to mannitol (Lindermann and Solomon, '62), the increased serosal osmolality must be due to a higher concentration of sodium and glucose in the absorbate (Dinda et al., '73). The absence of stimulation of intestinal transport when prolactin is added in vitro shows that the time required to induce increases in transport processes is considerably longer than one and one-half hours, Unfortunately, it is difficult to keep the intestine in good condition in vitro for more than a few hours. The present observations are also consistent with those of Ramsey and Bern ('72) who showed that prolactin added in vitro had no effect on intestinal fluid absorption. Doneen and Bern ('74) found that prolactin had effects on fish urinary bladder in vitro, but only after two days of culture in a medium containing prolactin. The report by Snart and Dalton ('73) that prolactin added in vitro
153
to toad bladder stimulated transport in less than one hour is difficult to reconcile with other data and calls for further reevaluation. The present study has provided results that are consistent with previous suggestions regarding the effects of prolactin in water and electrolyte accumulation in mammals. Although the role of the gut in osmoregulation is not so dramatically apparent as that of the kidney, it provides sites for the initial processes involved in the accumulation of water and salts (Bentley, '71). This study provides the first evidence that prolactin enhances glucose and amino acid transport by the small intestine. The latter effects of prolactin are of potential nutritional significance in view of the fact that the bulk of the transport of sugars and amino acids normally takes place in the small intestine. It is suggested that increased prolactin secretion could partly explain the increased intestinal transport of sugar and amino acids during pregnancy and lactation (Larralde and Fernandez-Otero, '68; Penzes and Simon, '68). It is postulated that the action of prolactin on intestinal transport may primarily subserve a nutritional role rather than an osmoregulatory one. ACKNOWLEDGMENTS
Aided by NIH grant CA-05388 and a Rockefeller Foundation Fellowship. I am indebted to Professor Howard A. Bern for his valuable advice and useful suggestions at every phase of this study and for reading the manuscript. LITERATURE CITED Althausen, T. L. 1949 Hormonal and vitamin factors in intestinal absorption. Gastroenterology, 12: 467480. Bentley, P. J. 1971 Endocrines and Osmoregulation, A Comparative Account of the Regulation of Water and Salt in Vertebrates. SpringerVerlag, Berlin. Bern, H. A,, and C. S . Nicoll 1968 The comparative endocrinology of prolactin. Recent Progr. Horm. Res., 24: 681-720. Bihler, I., and R. K. Crane 1962 On the mechanism of intestinal absorption of sugars. Biochim. biophys. Acta, 59: 78-93. Diamond, J. M. 1964 The mechanism of isotonic water transport. J. gen. Physiol., 48: 1 5 4 2 . Dinda, P. K., M. Beck and I. T. Beck 1973 On the mechanism of isosmotic transport across the small intestine. The composition of the absorb-
154
JAMES R. MAINOYA
ate transported from a mucosal solution made hypertonic by the addition of mannitol. Can. J. Physiol. Pharmacol., 51: 1311-138. Doneen, B. A., and H. A. Bern 1974 In uitro effects of prolactin and cortisol on water permeability of the urinary bladder of the teleost Gillichthys mirabilis. J. Exp. Zool., 187: 173-179. Fabry, P., V. Kujalova and B. Mosinger 1959 Influence of somatotropic hormone on small intestine resorption of glucose. Endokrinologie, 38: 152-158. Finkelstein, J. D., and D. Schacter 1962 Active transport of calcium by the intestine: effects of hypophysectomy and growth hormone. Am. J. Physiol., 203: 873-880. Forster, H., and M. Matthaus 1973 Some comments on the couplinrr between intestinal absorption of glucose-and-of sodium. FEBS Letters, 31 : 75-80. Gelb, A. M., and C. D. Gerson 1969 Influence of the endocrine glands on small intestine absorption. Am. J. Clin. Nutr., 2 2 : 305-310. Gilman, A,, and E. S . Koelle 1960 Substrate requirements for ion transport by the rat intestine studied in vitro. Am. J. Physiol., 199: 10251029. Glynn,,I. M. 1964 The action of cardiac glycosides on ion movements. Pharmacol. Rev., 16: 381-407. Horrobin, D.F. 1973 Prolactin: Physiology and Clinical Significance. MTP, St. Lmnardgate, Lancaster. Kimmich, G. A. 1973 Coupling between Na+ and sugar transport in smali intestine. Biochim. biophy. Acta, 300: 31-78. Larralde, J., and P. Fernandez-Otero 1968 The effect of pregnancy on the in uitro intestinal absorption of glucose and glycine. Revta. esp. Fisiol., 24: 49-50. Larralde, J., P. Fernandez-Otero and M. Gonzalez 1966 increased active transport of glucose through the intestine during pregnancy. Nature (London), 209: 1356-1357. Lee, J . S. 1968 Isosmotic absorption of fluid from rat jejunum in vitro. Gastroenterology, 5 4 : 365-374. Levin, R. J . 1969 The effects of hormones on the absorptive, metabolic and digestive functions of the small intestine. J. Endocr., 45: 315348. Lindermann, B., and A. K. Solomon 1962 Permeability of the luminal surface of intestinal mucosal cells. J. gen. Physiol., 45: 8 0 1 4 1 0 . Mahmood, A , , S. D. Varma and D. S . Wagle 1970 Effect of hormonal treatments in rats on the intestinal transport of methionine in uitro. Indian J. Exp. Biol., 8: 330-331. Mainoya, J. R. 1975 Further studies on the action of prolactin on fluid and ion absorption by the rat jejunum. Endocrinology, 96: 11901196. 1974 Influence of prolactin, estrus cycle, pregnancy and lactation on intestinal fluid
and ion transport by the rat jejunum. Am. Zool.. 14: 1244 (abstract 15). Mainoya, J. R., H. A. Bern and J . W. Regan 1974 Influence of ovine prolactin on transport of fluid and sodium chloride by the mammalian intestine and gall bladder. J. Endocr., 63: 311317. Meites, J., K. H. Lu, W. Wuttke, C. W. Welsch, H. Nagasawa and S . K . Quadri 1972 Recent studies on the function and control of prolactin secretion in rats. Recent Prog. Horm. Res., 28: 471-524. Nicoll, C . S . , and H. A. Bern 1972 On the action of prolactin among the vertebrates: is there a common denominator? In: Lactogenic Hormones. G. E. W. Wolstenholme and J . Knight, eds. Churchill and Livingston, London, 1972. p. 299. Parsons, D. S . , and D. L. Wingate 1961 The effect of osmotic gradients on fluid transfer across rat intestine in witro. Biochim. biophys. Acta., 46.; 170-183. Penzes, L., and C . T. Simon 1968 Intestinal absorption and turnover of DL-methionine during reproduction in the rat. Jap. J. Physiol., 18: 288-296. Raabo, E., and T. C. Terkildsen 1960 On the enzymatic determination of blood glucose. Scand. J. Clin. Lab. Invest., 1 2 ; 402-407. Ramsey, D. H., and H. A . Bern 1972 Stimulation by ovine prolactin of fluid transfer in everted sacs of rat small intestine. J. Endocr., 53: 453459. Reiser, S . , and P. A. Christiansen 1972 Relative effectiveness of extracellular sodium in supporting leucine uptake by isolated intestinal epithelial cells. Proc. SOC.exp. Biol. Med., 140: 362366. Righetti, A , , and R. Levitan 1970 Small bowel absorption in hypophysectomized rats. Am. J. digest. Dis., 15: 218-225. Schultz, S . G.. and P. F. Curran 1970 COUDICW~ transport of sodium and organic solutes. Physiol. Rev., 50: 637-718. Schultz, S. G., R. A. Frizzell and H. N . Nellans 1974 Ion transport by mammalian small intestine. Ann. Rev. Physiol., 36: 51-93. Schultz, S. G., and R. Zalusky 1964 Ion transport in the isolated rabbit ileum. 11. The interaction between active sodium and active sugar transport. J. gen. Physioi., 4 7 : 1043-1059. Snart, R. S . , and T. Dalton 1973 Response of toad bladder to prolactin. Comp. Biochem. Physiol., 4 5 A : 307-313. Wheeler, K. P., Y. Inui, P. E. HoLlenberg, E. Eavenson and H. N Christensen 1965 Relation of amino acid transport to sodium ion concentration. Biochim. biophys. Acta., 109: 620-622. Wilson, T. H., and G. Wiseman 1954 The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J. Physiol., 123: 116-125. ~
~
~~
.
