Effects of Bovine Growth Hormone, Human Placental Lactogen and Ovine Prolactin on Intestinal Fluid and Ion Transport in the Rat JAMES R. MAINOYA Department of Zoology and Cancer Research Laboratory, University of California, California 94720
potassium transport in the jejunum; in sodium, chloride, potassium and calcium transport in the ileum. Growth hormone also significantly increased fluid, sodium and chloride transport in the colon. Treatment of hypophysectomized rats with human placental lactogen enhanced fluid and ion transport in the jejunum; however, it failed to restore normal potassium transport in the ileum and colon at the 1 mg daily dose level. Growth hormone and human placental lactogen appear to affect jejunal water and electrolyte transport in the same manner as occurs with prolactin, possibly by influencing active ion transport. (Endocrinology 96: 1165, 1975)
ABSTRACT. The influence of bovine growth hormone and human placenta] lactogen on intestinal absorption was compared with that of ovine prolactin. Administration of each of these hormones in vivo daily for 2 days, resulted in increased fluid and electrolyte transport by the rat intestine, as measured in vitro. Hypophysectomy causes a fall in fluid and ion absorption in the rat jejunum but these changes are prevented by growth hormone treatment. Bovine growth hormone and ovine prolactin produce essentially similar effects in intact rats: significant increases in fluid, sodium and calcium transport in the duodenum; in fluid, sodium and
E
FFECTS of growth hormone on r,enal water, sodium and potassium retention have long been claimed (1,2,3), and prolactin and growth hormone are reported to have comparable effects on renal water, sodium and potassium retention (4,5). The role of growth hormone in intestinal absorption of fluid and ions has been examined only in a limited fashion (6), and that of placental prolactins apparently not at all. Recent experiments have demonstrated that administration of ovine prolactin to rats caused significant increases in intestinal fluid and ion transport (7,8,9). Moreover, growth hormone and prolactin are the only hypophysial hormones capable of stimulating active calcium transport in the rat duodenum in vivo (6). Available evidence indicates that large quantities of placental lactogen are secreted during the second half of human gestation (10), while plasma levels of pituitary prolactin increase gradually throughout pregnancy to term (11). In the rat, however, pituitary prolactin levels are Received June 24, 1974.
Berkeley,
highest during early and late pregnancy and low during mid-pregnancy (12,13), whereas placental lactogen is highest in mid-pregnancy (14). Presumably, under these conditions placental lactogen could play a significant role in the regulation of maternal water and mineral metabolism. In man, at least, there are indications that hPL like hGH increases urinary calcium excretion and sodium retention (15). Intestinal absorption of fluid and ions in the rat is significantly higher during pregnancy and lactation (16). The present experiments were designed to investigate the actions of bovine growth hormone and human placental lactogen on intestinal absorption of fluid and ions in the adult male rat, and to compare the effects of growth hormone with those of ovine prolactin. Materials and Methods Male adult Sprague-Dawley rats (Simonsen Labs, Gilroy, Calif) were used and were not fasted prior to experimentation. Normal rats weighed 250-300 g and hypophysectomized rats 200-220 g. They were maintained on white diet (Simonsen Labs, Gilroy Calif.) and water
1165
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
1166
Endo • 1975 Vol 96 • No 5
MAINOYA
given ad libitum. Hypophysectomized rats were given 5% glucose solution in place of drinking water because of decreased caloric intake and were used 10 days after the operation. Animals with pituitary remnants at the time of the experiment were discarded. From nembutal-anesthetized animals, the small intestine was exposed and rinsed out in situ with 0.9% NaCl. Twelve-cm long everted intestinal sacs were prepared as described by Wilson and Wiseman (17). In experiment 1, sacs were prepared from segments III and IV of the jejunum (cf. 7). In experiment 2, sacs were made from segments III and IV and one sac was prepared from the entire colon. In experiment 3, a 6-cm long sac was prepared from the proximal duodenum, in addition to two 12-cm sacs; one from the jejunum (segment III) and another from the distal ileum. In experiment 4, sacs were prepared from the jejunum (segment III), from the distal ileum and from the entire colon. In general, hypophysectomized rats had thinner intestines than intact rats. Each sac was weighed empty using a Mettler H balance. One end was ligated, and aerated Ringer solution (18) containing 28 mM glucose was introduced into the everted sac by means of a blunt-ended needle with attached syringe; this end was then ligated and the sac reweighed. Each sac was then placed in 20 ml of Ringer contained in a 125-ml Erlenmeyer flask. The flasks were placed in a metabolic incubator shaken at 80 oscillations/min, and the contents of each flask were aerated continuously with 95% O2/5% CO2 at 37 C for 1 h. Following incubation each sac was reweighed to determine mucosal transfer, and both mucosal and serosal fluids recovered for ion determination. Sodium, potassium, calcium and magnesium were measured using a PerkinElmer Atomic Absorption Spectrophotometer (Model 290 B) and chloride concentrations were determined electrometrically using the BuchlerCotlove Chloridometer.
Hormones. 1.0 mg bovine growth hormone (bGH: NIH BS-8; 0.8 USP U/mg containing 1.0 IU PRL activity/mg); ovine prolactin (oPRL: NIH PS-10; 25.6 IU/mg) and purified human placental lactogen (hPL; human chorionic somatomammotropin: provided by Professor C. H. Li) were prepared for injection by first dissolving 10 mg in 1 ml of 0.002M NaOH and then diluting to 20 ml with 0.9% NaCl. In experiments where 0.1 and/or 0.25 mg of bGH or hPL were used, 1.0 or 2.5 mg were first dissolved in 1 ml of 0.002M NaOH and then diluted with 0.9% NaCl to the required concentration. Injections were given subcutaneously in mid-afternoon 48 and 24 h before the animal was used.
Results
Bovine growth hormone stimulates intestinal absorption (Tables 1 and 2). As shown in Table 1, hypophysectomy decreased sodium, potassium and magnesium absorption significantly; fluid, calcium and chloride transport was also reduced, but the variations in responses were large. In other experiments (9), the reduction of fluid and sodium transport by hypophysectomy was consistently significant; 0.25 mg or 1.0 mg bGH significantly increased absorption to well above the normal control level. Moreover, 1.0 mg growth hormone stimulated absorption to the same extent in hypophysectomized and in intact rats. In Table 2, jejunal and colonic fluid, sodium, potassium, chloride, calcium and magnesium absorption was measured following 0.1 and 1.0 mg bGH treatment for 2 days; both doses significantly stimulated fluid and sodium transport in the jejunum (P < 0.01). In the colon 1.0 mg bGH significantly increased fluid, sodium and chloride transport (P < 0.05) but only slightly enhanced potassium, calcium and Expression of results. Mucosal transport is magnesium transport (F > 0.05). defined as loss of fluid or ion from the mucosal The results presented in Table 3 show to the serosal compartment during incubation. that growth hormone and prolactin are This is expressed as ml fluid or /ueq ion/g initial equally effective in stimulating fluid, wet weight/h in a 6-cm or 12-cm sac. All values sodium and calcium transport in the are expressed as mean ± standard error and duodenum. In addition, growth hormone statistical significance determined according to significantly increased chloride transport in the unpaired Student's t test.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
1167
GH AND hPL AND INTESTINAL ABSORPTION TABLE 1. Effect of hypophysectomy and bGH on mucosal fluid and ion transport by the rat jejunum Intestinal sac III
IV
Fluid transport (ml/g wet wt/h)
Na+ transport (/xeq/g wet wt/h)
control (7)
0.97 ± 0.10
131.5 ± 6.2*
47.5 ± 10.9
Hypophysectomized (HX) (8)
0.67 ± 0.13
94.7 + 11.0
49.7 ± 5.7
HX + 0.25 mgbGH(5)
1.03 ± 0.07
161.6 ± 10.7**
63.5 ± 5.3
HX + 1.0 mgbGH(8)
1.79 ± 0.14***
235.0 ± 17.0***
99.2 ± 4.0***
1.0 mg bGH (10)
1.57 ± 0.13***
230.3 ± 14.5***
84.8 ± 4.6***
Treatment (number)
Cl" transport (/xeq/g wet wt/h)
K+ transport (/xeq/g wet wt/h)
Ca++ transport (/xeq/g wet wt/h)
Mg++ transport (/xeq/g wet wt/h)
2.84 ± 0.36**
0.95 + 0.18
0.58 ± 0.32**
0.64 + 0.19
-0.78 + 0.15
3.78 ± 0.68* **
1.22 ± 0.15
-0.13 ±0.17*
4.39 ± 0.30***
1.03 + 0.13
-0.39 ± 0.65
0.51 +. 0.20*** 0.59 ± 0.19***
control (7)
1.09 ± 0.11
136.9 ± 9.4
44.5 + 6.2
1.96 + 0.66*
0.78 ±0.17
HX(8)
0.73 ±0.14
106.3 ± 18.5
32.5 + 6.2
0.12 + 0.42
0.54 ± 0.17
HX + 0.25 mgbCH(5)
1.16 ±0.10*
186.5 ± 16.9*
52.7 ± 9.6
HX + 1.0 mgbCH(8)
1.79 ± 0.17***
235.9 + 14.8***
87.5 ± 5.3***
3.89 ± 0.81**
1.12 ±0.14*
0.05 ±0.15***
1.0 mg bGH (10)
1.84 + 0.08***
225.4 ± 7.5***
82.0 + 7.0***
3.18 ± 0.49***
1.10 ±0.14*
0.58 ± 0.12***
-0.94 a: 0.09
' P < 0.05, ** P < 0.01, *** P < 0.001 compared with hypophysectomized animals.
the duodenum (P < 0.01). In the jejunum, growth hormone and prolactin significantly increased fluid, sodium (P < 0.01) and potassium transport (P < 0.01). In the distal ileum prolactin significantly increased fluid, sodium, chloride, potassium and calcium transport whereas growth hormone increased sodium, chloride, potassium and calcium transport. In all instances, both hormones show the same general effects; however, in some cases the differences
from the control are not significant at P < 0.05. The effects of human placental lactogen on intestinal fluid and ion transport in hypophysectomized rats are shown in Table 4 for the jejunum (segment III), distal ileum and entire colon. In the jejunum, hPL at all three dosage levels increased fluid, sodium and chloride absorption. 1.0 mg hPL increased potassium and calcium absorption rates without af-
TABLE 2. Effect of bGH on mucosal fluid and ion transport by the rat jejunum and colon Intestinal sac III
IV
Colon
1
Treatment (number)
Fluid transport (ml/g wet wt/h)
Na+ transport (/ieq/g wet wt/h)
Cl" transport (/xeq/g wet wt/h)
K+ transport (jieq/g wet wt/h)
Ca++ transport (/i.eq/g wet wt/h)
Mg++ transport (/xeq/g wet wt/h)
control (6) 0.1 mg bGH (6) 1.0 mg bGH (7)
0.74 ± 0.13 1.22 + 0.07** 1.55 ± 0.08***
89.6 ± 9.6 152.8 ± 17.4** 158.9 + 10.6*'*
15.2 + 7.6 19.0 ± 4.4 34.5 ± 11.6
1.37 ± 0.51
1.63 ± 0.13
0.74 + 0.28
2.63 + 0.52
1.97 ±0.29
0.52 ± 0.21
control (6) 0.1 mg bGH (6) 1.0 mg bGH (7)
0.86 ± 0.14 1.24 + 0.06* 1.62 ± 0.10***
97.6 ± 11.8 138.8 ± 9.8* 169.4 ± 16.5**
13.6 ± 11.0 19.9 + 7.7 36.9 + 14.1
1.66 ± 0.55
1.88 + 0.21
0.55 + 0.32
2.83 ± 0.64
2.18 ±0.26
0.96 ± 0.27
control (6) 0.1 mg bGH (6) 1.0 mg bGH (7)
1.14 + 0.08 1.31 + 0.08 1.40 + 0.08*
226.2 + 14.7 249.2 + 15.8 293.4 ± 22.9*
222.2 ± 18.2 251.5 + 9.8 278.1 ± 16.4*
11.35 ± 0.88
3.72 ± 0.19
2.94 + 0.18
12.81 + 1.62
4.58 ± 0.34
3.21 + 0.34
P < 0.05, ** P < 0.01, *** P < 0.001 compared with control animals.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
1168
Endo • 1975 Vol 96 • No 5
MAINOYA
TABLE 3. Effect of oPRL and bGH on mucosal fluid and ion transport by the rat small intestine ci-
Treatment (number)
Fluid transport (ml/g wet wt/h)
Na + transport (jteq/g wet wt/h)
transport (/*ecj / g wet v/t/h)
control (8) 1.0 mg oPRL (8) 1.0 ing bCH (9)
0.33 ±0.07 0.64 ± 0.05** 0.65 ± 0.06**
55.4 ± 9.5 103.2 ± 8.9** 91.1 ± 9.7*
28.5 ± 6.0 43.1 ± 3.7 51.8 ± 3.6**
Mid-Jejunum
control (8) 1.0 mg oPRL (8) 1.0 mg bCH (9)
0.81 ±0.12 1.37 + 0.12** 1.33 + 0.11**
106.8 + 13.8 193.6 + 17.4** 184.4 + 16.1**
30.6 ± 4.6 46.4 + 7.5 46.3 + 5.1*
Distal Ileum
control (8) 1.0 mg oPRL (8) 1.0 mg bGH (9)
0.78 + 0.14 1.31 ±0.11** 1.11 ±0.10
147.6 ± 19.7 228.0 + 17.7** 210.9 ± 15.7*
105.2 + 17.5 174.3 ± 13.6** 154.8 ± 10.6*
Intestinal region Proximal Duodenum
Ca ++ transport (/xeq/g wet wt/h)
Mg ++ transport (^eq/g wet wt/h)
1.40 ± 0.50 1.44 ± 0.52 2.12 ± 0.53
3.71 ± 0.69 7.04 ± 0.60** 7.19 + 0.60**
0.91 ± 0 . 1 7 0.96 ± 0 . 1 2 1.17 ± 0 . 1 1
0.12 ± 0.24 2.53 ± 0.44*** 2.69 + 0.58**
1.05 + 0.16 1.56 ± 0.13* 1.42 ± 0.13
0.31 ± 0.08 0.41 ±0.11 0.42 ± 0.12
0.12 ± 0.38 1.16 ± 0.22* 1.50 ± 0.49*
0.84 ± 0.22 1.78 + 0.09** 1.63 ± 0.21*
0.92 ± 0.24 1.49 + 0.15 1.15 ±0.12
K* transport
(fieq/g wet wt/h)
* P < 0.05, ** P < 0.01, *** P < 0.001 compared with control animals.
fecting magnesium absorption. In the terminal ileum hPL restored fluid, sodium, chloride, calcium and magnesium transport but was without effect on potassium ab-
sorption. Hypophysectomy produced a dramatic reduction in fluid and ion absorption in the colon; hPL significantly increased colonic fluid, sodium, chloride and
TABLE 4. Effect of hypophysectomy and hPL on fluid and ion transport by the rat gut
Intestinal region Mid-Jejunum
Distal Ileum
Entire Colon
Treatment (number)
Na+ transport
Fluid transport (ml/g wet wt/h)
(jteq/g wet wt/h)
Cl" transport (jieq/g wet wt/h)
control (9)
0.92 ± 0.09
121.9 ± 12.9
56.9 ± 10.2*
HX(7)
0.68 ± 0.14
95.9 ± 19.8
23.5 ± 7.2
HX + O.lmg hPL (5)
1.21 ± 0.14*
167.6 ± 25.8*
40.8+ 9.3
HX + 0.25 mg hPL (6)
1.57 ± 0.18**
219.4 + 18.0***
59.9+ 9.1**
HX + 1.0 mg hPL (8)
1.73 ±0.08***
253.4 ± 17.6***
74.5+ 9.9**
control (9)
0.98 ± 0.09*
180.2 ± 9.8**
137.6 ± 7.0**
HX(7)
0.60 + 0.14
110.9 ±21.6
HX + 0.1 mg hPL (5)
1.00 ±0.12
156.4 + 10.9
116.6 ±
HX + 0.25 mghPL(6)
1.02 ± 0.16
192.4 ± 33.4
157.2 + 20.8*
79.6 ± 16.8
K+ transport (/xeq/g wet wt/h)
Ca++ transport (neq/g wet wt/h)
Mg++ transport (/teq/g wet wt/h)
0.96 + 0.26
0.94 ± 0.21
0.46 + 0.51
-0.26 ± 0.27
2.63 ± 0.56**
0.89 ± 0.20
0.19 ± 0.27
1.67 ± 0.72*
1.13 ± 0.31
1.48 ±0.22***
0.66 + 0.39
0.21 ± 0.19
2.15 ± 0.54* -0.14 + 0.46
-2.38 ± 1.26
5.1
HX + 1.0 mg hPL (8)
1.09 ±0.13*
216.0 ± 26.9*
157.4 + 21.2*
control (9)
1.15 ± 0.13***
201.7 + 21.8**
172.7 ± 18.5**
HX(7)
0.48 + 0.03
116.0 ± 9.3
100.2+
HX + 0.1 mg hPL (5)
0.64 ± 0.08*
163.2 ± 20.9*
127.7 ± 16.0
HX + 0.25 mg hPL (6)
0.75 ± 0.05***
169.0 ± 11.9**
152.7+
HX + 1.0 mg hPL (8)
0.76 ± 0.04***
185.3 ± 8.7***
152.1 ± 7.4***
9.3
1.31 + 0.24
1.09 ± 0.13**
2.06 ±0.17*
2.25 ± 0.20***
-0.11 ± 0.97
1.24 ± 0.24
1.23 ± 0.09
-1.44 + 0.74
1.80 ±0.17
1.72 ± 0.12**
-3.45 + 0.55 5.64 + 0.60***
8.7**
P < 0.05, * P < 0.01, •*• P < 0.001 compared with hypophysectomized (HX) animals.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
GH AND hPL AND INTESTINAL ABSORPTION
magnesium transport over the hypophysectomized levels. Discussion Actions of growth hormone and prolactin on renal water and electrolyte handling have been reported. Both hormones effectively caused water, sodium and potassium retention (3,4,5,19). In man, sodium chloride retention often accompanies the administration of human growth hormone (20,21,22); in addition, higher doses produced fluid retention (23). In general, the present results indicate that all three hormones significantly stimulate fluid, sodium and chloride transport. Hypophysectomy decreased fluid and electrolyte absorption but the several parts of the gut were not uniformly affected. The decrement in absorption was not always significant in jejunal segments but was clearly so in the ileum and colon. Hypophysectomy, however, significantly decreased potassium transport in the three regions of the gut. On the other hand, calcium transport was significantly decreased only in the colon. Although it has been previously claimed that growth hormone was more effective than prolactin in stimulating active calcium transport in the rat duodenum (6), both hormones stimulated calcium absorption in the duodenum to the same extent in our experiment. At similar dosages the three hormones produced comparable stimulation of fluid and ion transport. Possible sensitization of the gut by hypophysectomy to hormonal injections could explain why some dosages of growth hormone and human placental lactogen resulted in greater fluid and electrolyte transport compared with that seen in intact control rats. The differences in transport rates observed among some experiments are difficult to explain. Although changes in the diet have been shown to affect intestinal absorption (7), there were no dietary changes in the present study. The mechanism(s) by which the hormones stimulate intestinal fluid and ion
1169
transport is unclear. These protein hormones show similar structural properties and biological actions (10,24). Though growth hormone is mitogenic on intestinal crypts (25), this action would not adequately explain the present results. It is possible that stimulation of sodium transport could explain the failure of prolactin to augment fluid absorption in absence of sodium or in the presence of ouabain in the serosal fluid (9) and decreased amino acidtransporting properties of growth hormone in the absence of sodium (26). The observations that growth hormone increased renal water, sodium and potassium retention in adrenalectomized rats (2,3) and in hypophysectomized and in adrenalectomized-hypophysectomized rats (3), and that prolactin stimulated intestinal fluid absorption in adrenalectomizednephrectomized rats (7) suggest that the actions of these hormones are not mediated through the adrenal gland. It is not certain whether the similar effects of these hormones on intestinal transport is due to stimulation of similar or different aspects of cell metabolism essential for ion-transport processes. The stimulatory action of these three different protein hormones on intestinal transport of water and salts further suggests that pituitary and placental lactogenic and growth hormones may play physiological roles of significance in water and electrolyte metabolism. Acknowledgments Aided by NIH Grant CA-05388 and a Rockefeller Foundation Fellowship. I wish to thank Professor Howard A. Bern for his valuable advice and for useful criticisms during the preparation of the manuscript. oPRL and bGH generously provided by NIAMDD.
References 1. Whitney, J. E., L. L. Bennett, and C. H. Li, Proc Soc Exp Biol Med 79: 584, 1952. 2. Stein, J. D. Jr., L. L. Bennett, A. A. Batts, and C. H. Li, Am] Physiol 171: 587, 1952. 3. Croxatto, H., E. Labarca, G. Swaneck, and P. Garfias, AmJ Physiol 211: 588, 1966. 4. Lockett, M. F.J Physiol 181: 192, 1965.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
1170
MAINOYA
-, and B. Nail,/ Physiol 180: 147, 1965. 5. 6. Finkelstein, J. D., and D. Schachter, Am J Physiol 203: 873, 1962. 7. Ramsey, D. H., and H. A. Bern,./ Endocrinol 53: 453, 1972. 8. Mainoya, J. R., H. A. Bern, and J. W. Regan, J Endocrinol 63: 311, 1974. 9. , Endocrinology 96: 1158, 1975. 10. Grumbach, M. M., S. L. Kaplan, J. J. Sciarra, and I. M. Burr, Ann NY Acad Sci 148: 501, 1968. 11. Hwang, P., H. Guyda, and H. Friesen, Proc Natl Acad Sci (USA) 68: 1902, 1971. 12. Meites, J., K. H. Lu, W. Wuttke, C. W. Welsch, H. Nagasawa, and S. K. Quadri, Recent Progr Horm Res 28: 417, 1972. 13. Simpson, A. A., M. H. W. Simpson, Y. N. Sinha, and G. H. Schmidt,/ Endocrinol 58: 675, 1973. 14. Linkie, D. M., and G. D. Niswender, Biol Reprod 8: 48, 1973. 15. McGarry, E. E., and J. C. Beck, In Wolstenholme, G. E. W., and J. Knight (eds.), Lactogenic Hormones, Churchill and Livingston, London, 1972, p. 299.
Endo • 1975 Vol 96 • No 5
16. Mainoya, J. R., Am Zool 14: 1244, 1974. 17. Wilson, T. H., and G. W. Wiseman J Physiol 123: 116, 1954. 18. Krebs, H. A., and K. Henseleit, Hoppe-Seyler's Z Physiol Chem 210: 33, 1932. 19. Horrobin, D. F., P. G. Burstyn, I. J. Loyd, N. Durkin, A. Lipton, and K. L. Muiruri, Lancet ii: 352, 1971. 20. Bergenstal, D. M., and M. B. Lipsett, / Clin Endocrinol Metab 20: 1427, 1960. 21. Gershberg, H . J Clin Endocrinol Metab 20: 1107, 1960. 22. Biglieri, E. G., C. O. Watlington, and P. H. ForshamJ Clin Endocrinol Metab 21: 361, 1961. 23. Hutchings, J. J., R. F. Escamilla, W. C. Deamer, and C. H. Li, / Clin Endocrinol Metab 19: 759, 1959. 24. Li, C. H., In Wolstenholme, G. E. W., and J. Knight (eds.), Lactogenic Hormones, Churchill and Livingston, London, 1972, p. 7. 25. Leblond, C. P., and R. Carriere, Endocrinology 56: 261, 1955. 26. Kostyo, J. L., Endocrinology 75: 113, 1964.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
4
potassium transport in the jejunum; in sodium, chloride, potassium and calcium transport in the ileum. Growth hormone also significantly increased fluid, sodium and chloride transport in the colon. Treatment of hypophysectomized rats with human placental lactogen enhanced fluid and ion transport in the jejunum; however, it failed to restore normal potassium transport in the ileum and colon at the 1 mg daily dose level. Growth hormone and human placental lactogen appear to affect jejunal water and electrolyte transport in the same manner as occurs with prolactin, possibly by influencing active ion transport. (Endocrinology 96: 1165, 1975)
ABSTRACT. The influence of bovine growth hormone and human placenta] lactogen on intestinal absorption was compared with that of ovine prolactin. Administration of each of these hormones in vivo daily for 2 days, resulted in increased fluid and electrolyte transport by the rat intestine, as measured in vitro. Hypophysectomy causes a fall in fluid and ion absorption in the rat jejunum but these changes are prevented by growth hormone treatment. Bovine growth hormone and ovine prolactin produce essentially similar effects in intact rats: significant increases in fluid, sodium and calcium transport in the duodenum; in fluid, sodium and
E
FFECTS of growth hormone on r,enal water, sodium and potassium retention have long been claimed (1,2,3), and prolactin and growth hormone are reported to have comparable effects on renal water, sodium and potassium retention (4,5). The role of growth hormone in intestinal absorption of fluid and ions has been examined only in a limited fashion (6), and that of placental prolactins apparently not at all. Recent experiments have demonstrated that administration of ovine prolactin to rats caused significant increases in intestinal fluid and ion transport (7,8,9). Moreover, growth hormone and prolactin are the only hypophysial hormones capable of stimulating active calcium transport in the rat duodenum in vivo (6). Available evidence indicates that large quantities of placental lactogen are secreted during the second half of human gestation (10), while plasma levels of pituitary prolactin increase gradually throughout pregnancy to term (11). In the rat, however, pituitary prolactin levels are Received June 24, 1974.
Berkeley,
highest during early and late pregnancy and low during mid-pregnancy (12,13), whereas placental lactogen is highest in mid-pregnancy (14). Presumably, under these conditions placental lactogen could play a significant role in the regulation of maternal water and mineral metabolism. In man, at least, there are indications that hPL like hGH increases urinary calcium excretion and sodium retention (15). Intestinal absorption of fluid and ions in the rat is significantly higher during pregnancy and lactation (16). The present experiments were designed to investigate the actions of bovine growth hormone and human placental lactogen on intestinal absorption of fluid and ions in the adult male rat, and to compare the effects of growth hormone with those of ovine prolactin. Materials and Methods Male adult Sprague-Dawley rats (Simonsen Labs, Gilroy, Calif) were used and were not fasted prior to experimentation. Normal rats weighed 250-300 g and hypophysectomized rats 200-220 g. They were maintained on white diet (Simonsen Labs, Gilroy Calif.) and water
1165
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
1166
Endo • 1975 Vol 96 • No 5
MAINOYA
given ad libitum. Hypophysectomized rats were given 5% glucose solution in place of drinking water because of decreased caloric intake and were used 10 days after the operation. Animals with pituitary remnants at the time of the experiment were discarded. From nembutal-anesthetized animals, the small intestine was exposed and rinsed out in situ with 0.9% NaCl. Twelve-cm long everted intestinal sacs were prepared as described by Wilson and Wiseman (17). In experiment 1, sacs were prepared from segments III and IV of the jejunum (cf. 7). In experiment 2, sacs were made from segments III and IV and one sac was prepared from the entire colon. In experiment 3, a 6-cm long sac was prepared from the proximal duodenum, in addition to two 12-cm sacs; one from the jejunum (segment III) and another from the distal ileum. In experiment 4, sacs were prepared from the jejunum (segment III), from the distal ileum and from the entire colon. In general, hypophysectomized rats had thinner intestines than intact rats. Each sac was weighed empty using a Mettler H balance. One end was ligated, and aerated Ringer solution (18) containing 28 mM glucose was introduced into the everted sac by means of a blunt-ended needle with attached syringe; this end was then ligated and the sac reweighed. Each sac was then placed in 20 ml of Ringer contained in a 125-ml Erlenmeyer flask. The flasks were placed in a metabolic incubator shaken at 80 oscillations/min, and the contents of each flask were aerated continuously with 95% O2/5% CO2 at 37 C for 1 h. Following incubation each sac was reweighed to determine mucosal transfer, and both mucosal and serosal fluids recovered for ion determination. Sodium, potassium, calcium and magnesium were measured using a PerkinElmer Atomic Absorption Spectrophotometer (Model 290 B) and chloride concentrations were determined electrometrically using the BuchlerCotlove Chloridometer.
Hormones. 1.0 mg bovine growth hormone (bGH: NIH BS-8; 0.8 USP U/mg containing 1.0 IU PRL activity/mg); ovine prolactin (oPRL: NIH PS-10; 25.6 IU/mg) and purified human placental lactogen (hPL; human chorionic somatomammotropin: provided by Professor C. H. Li) were prepared for injection by first dissolving 10 mg in 1 ml of 0.002M NaOH and then diluting to 20 ml with 0.9% NaCl. In experiments where 0.1 and/or 0.25 mg of bGH or hPL were used, 1.0 or 2.5 mg were first dissolved in 1 ml of 0.002M NaOH and then diluted with 0.9% NaCl to the required concentration. Injections were given subcutaneously in mid-afternoon 48 and 24 h before the animal was used.
Results
Bovine growth hormone stimulates intestinal absorption (Tables 1 and 2). As shown in Table 1, hypophysectomy decreased sodium, potassium and magnesium absorption significantly; fluid, calcium and chloride transport was also reduced, but the variations in responses were large. In other experiments (9), the reduction of fluid and sodium transport by hypophysectomy was consistently significant; 0.25 mg or 1.0 mg bGH significantly increased absorption to well above the normal control level. Moreover, 1.0 mg growth hormone stimulated absorption to the same extent in hypophysectomized and in intact rats. In Table 2, jejunal and colonic fluid, sodium, potassium, chloride, calcium and magnesium absorption was measured following 0.1 and 1.0 mg bGH treatment for 2 days; both doses significantly stimulated fluid and sodium transport in the jejunum (P < 0.01). In the colon 1.0 mg bGH significantly increased fluid, sodium and chloride transport (P < 0.05) but only slightly enhanced potassium, calcium and Expression of results. Mucosal transport is magnesium transport (F > 0.05). defined as loss of fluid or ion from the mucosal The results presented in Table 3 show to the serosal compartment during incubation. that growth hormone and prolactin are This is expressed as ml fluid or /ueq ion/g initial equally effective in stimulating fluid, wet weight/h in a 6-cm or 12-cm sac. All values sodium and calcium transport in the are expressed as mean ± standard error and duodenum. In addition, growth hormone statistical significance determined according to significantly increased chloride transport in the unpaired Student's t test.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
1167
GH AND hPL AND INTESTINAL ABSORPTION TABLE 1. Effect of hypophysectomy and bGH on mucosal fluid and ion transport by the rat jejunum Intestinal sac III
IV
Fluid transport (ml/g wet wt/h)
Na+ transport (/xeq/g wet wt/h)
control (7)
0.97 ± 0.10
131.5 ± 6.2*
47.5 ± 10.9
Hypophysectomized (HX) (8)
0.67 ± 0.13
94.7 + 11.0
49.7 ± 5.7
HX + 0.25 mgbGH(5)
1.03 ± 0.07
161.6 ± 10.7**
63.5 ± 5.3
HX + 1.0 mgbGH(8)
1.79 ± 0.14***
235.0 ± 17.0***
99.2 ± 4.0***
1.0 mg bGH (10)
1.57 ± 0.13***
230.3 ± 14.5***
84.8 ± 4.6***
Treatment (number)
Cl" transport (/xeq/g wet wt/h)
K+ transport (/xeq/g wet wt/h)
Ca++ transport (/xeq/g wet wt/h)
Mg++ transport (/xeq/g wet wt/h)
2.84 ± 0.36**
0.95 + 0.18
0.58 ± 0.32**
0.64 + 0.19
-0.78 + 0.15
3.78 ± 0.68* **
1.22 ± 0.15
-0.13 ±0.17*
4.39 ± 0.30***
1.03 + 0.13
-0.39 ± 0.65
0.51 +. 0.20*** 0.59 ± 0.19***
control (7)
1.09 ± 0.11
136.9 ± 9.4
44.5 + 6.2
1.96 + 0.66*
0.78 ±0.17
HX(8)
0.73 ±0.14
106.3 ± 18.5
32.5 + 6.2
0.12 + 0.42
0.54 ± 0.17
HX + 0.25 mgbCH(5)
1.16 ±0.10*
186.5 ± 16.9*
52.7 ± 9.6
HX + 1.0 mgbCH(8)
1.79 ± 0.17***
235.9 + 14.8***
87.5 ± 5.3***
3.89 ± 0.81**
1.12 ±0.14*
0.05 ±0.15***
1.0 mg bGH (10)
1.84 + 0.08***
225.4 ± 7.5***
82.0 + 7.0***
3.18 ± 0.49***
1.10 ±0.14*
0.58 ± 0.12***
-0.94 a: 0.09
' P < 0.05, ** P < 0.01, *** P < 0.001 compared with hypophysectomized animals.
the duodenum (P < 0.01). In the jejunum, growth hormone and prolactin significantly increased fluid, sodium (P < 0.01) and potassium transport (P < 0.01). In the distal ileum prolactin significantly increased fluid, sodium, chloride, potassium and calcium transport whereas growth hormone increased sodium, chloride, potassium and calcium transport. In all instances, both hormones show the same general effects; however, in some cases the differences
from the control are not significant at P < 0.05. The effects of human placental lactogen on intestinal fluid and ion transport in hypophysectomized rats are shown in Table 4 for the jejunum (segment III), distal ileum and entire colon. In the jejunum, hPL at all three dosage levels increased fluid, sodium and chloride absorption. 1.0 mg hPL increased potassium and calcium absorption rates without af-
TABLE 2. Effect of bGH on mucosal fluid and ion transport by the rat jejunum and colon Intestinal sac III
IV
Colon
1
Treatment (number)
Fluid transport (ml/g wet wt/h)
Na+ transport (/ieq/g wet wt/h)
Cl" transport (/xeq/g wet wt/h)
K+ transport (jieq/g wet wt/h)
Ca++ transport (/i.eq/g wet wt/h)
Mg++ transport (/xeq/g wet wt/h)
control (6) 0.1 mg bGH (6) 1.0 mg bGH (7)
0.74 ± 0.13 1.22 + 0.07** 1.55 ± 0.08***
89.6 ± 9.6 152.8 ± 17.4** 158.9 + 10.6*'*
15.2 + 7.6 19.0 ± 4.4 34.5 ± 11.6
1.37 ± 0.51
1.63 ± 0.13
0.74 + 0.28
2.63 + 0.52
1.97 ±0.29
0.52 ± 0.21
control (6) 0.1 mg bGH (6) 1.0 mg bGH (7)
0.86 ± 0.14 1.24 + 0.06* 1.62 ± 0.10***
97.6 ± 11.8 138.8 ± 9.8* 169.4 ± 16.5**
13.6 ± 11.0 19.9 + 7.7 36.9 + 14.1
1.66 ± 0.55
1.88 + 0.21
0.55 + 0.32
2.83 ± 0.64
2.18 ±0.26
0.96 ± 0.27
control (6) 0.1 mg bGH (6) 1.0 mg bGH (7)
1.14 + 0.08 1.31 + 0.08 1.40 + 0.08*
226.2 + 14.7 249.2 + 15.8 293.4 ± 22.9*
222.2 ± 18.2 251.5 + 9.8 278.1 ± 16.4*
11.35 ± 0.88
3.72 ± 0.19
2.94 + 0.18
12.81 + 1.62
4.58 ± 0.34
3.21 + 0.34
P < 0.05, ** P < 0.01, *** P < 0.001 compared with control animals.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
1168
Endo • 1975 Vol 96 • No 5
MAINOYA
TABLE 3. Effect of oPRL and bGH on mucosal fluid and ion transport by the rat small intestine ci-
Treatment (number)
Fluid transport (ml/g wet wt/h)
Na + transport (jteq/g wet wt/h)
transport (/*ecj / g wet v/t/h)
control (8) 1.0 mg oPRL (8) 1.0 ing bCH (9)
0.33 ±0.07 0.64 ± 0.05** 0.65 ± 0.06**
55.4 ± 9.5 103.2 ± 8.9** 91.1 ± 9.7*
28.5 ± 6.0 43.1 ± 3.7 51.8 ± 3.6**
Mid-Jejunum
control (8) 1.0 mg oPRL (8) 1.0 mg bCH (9)
0.81 ±0.12 1.37 + 0.12** 1.33 + 0.11**
106.8 + 13.8 193.6 + 17.4** 184.4 + 16.1**
30.6 ± 4.6 46.4 + 7.5 46.3 + 5.1*
Distal Ileum
control (8) 1.0 mg oPRL (8) 1.0 mg bGH (9)
0.78 + 0.14 1.31 ±0.11** 1.11 ±0.10
147.6 ± 19.7 228.0 + 17.7** 210.9 ± 15.7*
105.2 + 17.5 174.3 ± 13.6** 154.8 ± 10.6*
Intestinal region Proximal Duodenum
Ca ++ transport (/xeq/g wet wt/h)
Mg ++ transport (^eq/g wet wt/h)
1.40 ± 0.50 1.44 ± 0.52 2.12 ± 0.53
3.71 ± 0.69 7.04 ± 0.60** 7.19 + 0.60**
0.91 ± 0 . 1 7 0.96 ± 0 . 1 2 1.17 ± 0 . 1 1
0.12 ± 0.24 2.53 ± 0.44*** 2.69 + 0.58**
1.05 + 0.16 1.56 ± 0.13* 1.42 ± 0.13
0.31 ± 0.08 0.41 ±0.11 0.42 ± 0.12
0.12 ± 0.38 1.16 ± 0.22* 1.50 ± 0.49*
0.84 ± 0.22 1.78 + 0.09** 1.63 ± 0.21*
0.92 ± 0.24 1.49 + 0.15 1.15 ±0.12
K* transport
(fieq/g wet wt/h)
* P < 0.05, ** P < 0.01, *** P < 0.001 compared with control animals.
fecting magnesium absorption. In the terminal ileum hPL restored fluid, sodium, chloride, calcium and magnesium transport but was without effect on potassium ab-
sorption. Hypophysectomy produced a dramatic reduction in fluid and ion absorption in the colon; hPL significantly increased colonic fluid, sodium, chloride and
TABLE 4. Effect of hypophysectomy and hPL on fluid and ion transport by the rat gut
Intestinal region Mid-Jejunum
Distal Ileum
Entire Colon
Treatment (number)
Na+ transport
Fluid transport (ml/g wet wt/h)
(jteq/g wet wt/h)
Cl" transport (jieq/g wet wt/h)
control (9)
0.92 ± 0.09
121.9 ± 12.9
56.9 ± 10.2*
HX(7)
0.68 ± 0.14
95.9 ± 19.8
23.5 ± 7.2
HX + O.lmg hPL (5)
1.21 ± 0.14*
167.6 ± 25.8*
40.8+ 9.3
HX + 0.25 mg hPL (6)
1.57 ± 0.18**
219.4 + 18.0***
59.9+ 9.1**
HX + 1.0 mg hPL (8)
1.73 ±0.08***
253.4 ± 17.6***
74.5+ 9.9**
control (9)
0.98 ± 0.09*
180.2 ± 9.8**
137.6 ± 7.0**
HX(7)
0.60 + 0.14
110.9 ±21.6
HX + 0.1 mg hPL (5)
1.00 ±0.12
156.4 + 10.9
116.6 ±
HX + 0.25 mghPL(6)
1.02 ± 0.16
192.4 ± 33.4
157.2 + 20.8*
79.6 ± 16.8
K+ transport (/xeq/g wet wt/h)
Ca++ transport (neq/g wet wt/h)
Mg++ transport (/teq/g wet wt/h)
0.96 + 0.26
0.94 ± 0.21
0.46 + 0.51
-0.26 ± 0.27
2.63 ± 0.56**
0.89 ± 0.20
0.19 ± 0.27
1.67 ± 0.72*
1.13 ± 0.31
1.48 ±0.22***
0.66 + 0.39
0.21 ± 0.19
2.15 ± 0.54* -0.14 + 0.46
-2.38 ± 1.26
5.1
HX + 1.0 mg hPL (8)
1.09 ±0.13*
216.0 ± 26.9*
157.4 + 21.2*
control (9)
1.15 ± 0.13***
201.7 + 21.8**
172.7 ± 18.5**
HX(7)
0.48 + 0.03
116.0 ± 9.3
100.2+
HX + 0.1 mg hPL (5)
0.64 ± 0.08*
163.2 ± 20.9*
127.7 ± 16.0
HX + 0.25 mg hPL (6)
0.75 ± 0.05***
169.0 ± 11.9**
152.7+
HX + 1.0 mg hPL (8)
0.76 ± 0.04***
185.3 ± 8.7***
152.1 ± 7.4***
9.3
1.31 + 0.24
1.09 ± 0.13**
2.06 ±0.17*
2.25 ± 0.20***
-0.11 ± 0.97
1.24 ± 0.24
1.23 ± 0.09
-1.44 + 0.74
1.80 ±0.17
1.72 ± 0.12**
-3.45 + 0.55 5.64 + 0.60***
8.7**
P < 0.05, * P < 0.01, •*• P < 0.001 compared with hypophysectomized (HX) animals.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
GH AND hPL AND INTESTINAL ABSORPTION
magnesium transport over the hypophysectomized levels. Discussion Actions of growth hormone and prolactin on renal water and electrolyte handling have been reported. Both hormones effectively caused water, sodium and potassium retention (3,4,5,19). In man, sodium chloride retention often accompanies the administration of human growth hormone (20,21,22); in addition, higher doses produced fluid retention (23). In general, the present results indicate that all three hormones significantly stimulate fluid, sodium and chloride transport. Hypophysectomy decreased fluid and electrolyte absorption but the several parts of the gut were not uniformly affected. The decrement in absorption was not always significant in jejunal segments but was clearly so in the ileum and colon. Hypophysectomy, however, significantly decreased potassium transport in the three regions of the gut. On the other hand, calcium transport was significantly decreased only in the colon. Although it has been previously claimed that growth hormone was more effective than prolactin in stimulating active calcium transport in the rat duodenum (6), both hormones stimulated calcium absorption in the duodenum to the same extent in our experiment. At similar dosages the three hormones produced comparable stimulation of fluid and ion transport. Possible sensitization of the gut by hypophysectomy to hormonal injections could explain why some dosages of growth hormone and human placental lactogen resulted in greater fluid and electrolyte transport compared with that seen in intact control rats. The differences in transport rates observed among some experiments are difficult to explain. Although changes in the diet have been shown to affect intestinal absorption (7), there were no dietary changes in the present study. The mechanism(s) by which the hormones stimulate intestinal fluid and ion
1169
transport is unclear. These protein hormones show similar structural properties and biological actions (10,24). Though growth hormone is mitogenic on intestinal crypts (25), this action would not adequately explain the present results. It is possible that stimulation of sodium transport could explain the failure of prolactin to augment fluid absorption in absence of sodium or in the presence of ouabain in the serosal fluid (9) and decreased amino acidtransporting properties of growth hormone in the absence of sodium (26). The observations that growth hormone increased renal water, sodium and potassium retention in adrenalectomized rats (2,3) and in hypophysectomized and in adrenalectomized-hypophysectomized rats (3), and that prolactin stimulated intestinal fluid absorption in adrenalectomizednephrectomized rats (7) suggest that the actions of these hormones are not mediated through the adrenal gland. It is not certain whether the similar effects of these hormones on intestinal transport is due to stimulation of similar or different aspects of cell metabolism essential for ion-transport processes. The stimulatory action of these three different protein hormones on intestinal transport of water and salts further suggests that pituitary and placental lactogenic and growth hormones may play physiological roles of significance in water and electrolyte metabolism. Acknowledgments Aided by NIH Grant CA-05388 and a Rockefeller Foundation Fellowship. I wish to thank Professor Howard A. Bern for his valuable advice and for useful criticisms during the preparation of the manuscript. oPRL and bGH generously provided by NIAMDD.
References 1. Whitney, J. E., L. L. Bennett, and C. H. Li, Proc Soc Exp Biol Med 79: 584, 1952. 2. Stein, J. D. Jr., L. L. Bennett, A. A. Batts, and C. H. Li, Am] Physiol 171: 587, 1952. 3. Croxatto, H., E. Labarca, G. Swaneck, and P. Garfias, AmJ Physiol 211: 588, 1966. 4. Lockett, M. F.J Physiol 181: 192, 1965.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
1170
MAINOYA
-, and B. Nail,/ Physiol 180: 147, 1965. 5. 6. Finkelstein, J. D., and D. Schachter, Am J Physiol 203: 873, 1962. 7. Ramsey, D. H., and H. A. Bern,./ Endocrinol 53: 453, 1972. 8. Mainoya, J. R., H. A. Bern, and J. W. Regan, J Endocrinol 63: 311, 1974. 9. , Endocrinology 96: 1158, 1975. 10. Grumbach, M. M., S. L. Kaplan, J. J. Sciarra, and I. M. Burr, Ann NY Acad Sci 148: 501, 1968. 11. Hwang, P., H. Guyda, and H. Friesen, Proc Natl Acad Sci (USA) 68: 1902, 1971. 12. Meites, J., K. H. Lu, W. Wuttke, C. W. Welsch, H. Nagasawa, and S. K. Quadri, Recent Progr Horm Res 28: 417, 1972. 13. Simpson, A. A., M. H. W. Simpson, Y. N. Sinha, and G. H. Schmidt,/ Endocrinol 58: 675, 1973. 14. Linkie, D. M., and G. D. Niswender, Biol Reprod 8: 48, 1973. 15. McGarry, E. E., and J. C. Beck, In Wolstenholme, G. E. W., and J. Knight (eds.), Lactogenic Hormones, Churchill and Livingston, London, 1972, p. 299.
Endo • 1975 Vol 96 • No 5
16. Mainoya, J. R., Am Zool 14: 1244, 1974. 17. Wilson, T. H., and G. W. Wiseman J Physiol 123: 116, 1954. 18. Krebs, H. A., and K. Henseleit, Hoppe-Seyler's Z Physiol Chem 210: 33, 1932. 19. Horrobin, D. F., P. G. Burstyn, I. J. Loyd, N. Durkin, A. Lipton, and K. L. Muiruri, Lancet ii: 352, 1971. 20. Bergenstal, D. M., and M. B. Lipsett, / Clin Endocrinol Metab 20: 1427, 1960. 21. Gershberg, H . J Clin Endocrinol Metab 20: 1107, 1960. 22. Biglieri, E. G., C. O. Watlington, and P. H. ForshamJ Clin Endocrinol Metab 21: 361, 1961. 23. Hutchings, J. J., R. F. Escamilla, W. C. Deamer, and C. H. Li, / Clin Endocrinol Metab 19: 759, 1959. 24. Li, C. H., In Wolstenholme, G. E. W., and J. Knight (eds.), Lactogenic Hormones, Churchill and Livingston, London, 1972, p. 7. 25. Leblond, C. P., and R. Carriere, Endocrinology 56: 261, 1955. 26. Kostyo, J. L., Endocrinology 75: 113, 1964.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 16:41 For personal use only. No other uses without permission. . All rights reserved.
4
