Effects of Calcitonin and Substance P on the Transport of Ca, Na, and Cl Across Rat Ileum in Vitro

G ASTROE N TEROLOGY 73:89-94, 1977 Copyright © 1977 by the American Gastroenterological Association

Vol. 73, No. 1 Printed in U .S.A.

EFFECTS OF CALCITONIN AND SUBSTANCE P ON THE TRANSPORT OF Ca, Na, AND Cl ACROSS RAT ILEUM IN VITRO MARLIN

w.

WALLING, THOMAS

A.

BRASITUS,

AND DANIEL

v.

KIMBERG

Department of Medicine, Harvard Medical S chool and the Gastrointestinal Unit of the Department of Medicine, Beth Israel Hospital , B oston , Massachusetts

Both salmon calcitonin (SCT) and substance P decreased ileal Na absorption, changed Cl transport from net absorption to net secretion and elevated the short circuit current when added in vitro at concentrations of 10 p,g per ml to solutions bathing the serosal surface of rat ileum which had been stripped of its serosal muscle coat. The effects of substance P were of greater magnitude but shorter duration than SCT. Both peptides also increased the bidirectional fluxes of Ca but did not alter net Ca movement. The changes in Na and Cl fluxes and short circuit current are identifical to those which occur when cellular levels of cyclic AMP increased. However, incubation of ileal mucosa with SCT or substance P did not cause a detectable change in cellular levels of cyclic AMP or cyclic GMP. Both the mechanism of action and the possible physiological functions of SCT and substance P in the regulation of electrolyte transport require further investigation. The results with SCT appear to confirm prior suggestions that calcitonin may act directly to produce secretory diarrhea under pathophysiological conditions. Recent reports have provided evidence that intravenous infusion of calcitonin (CT) can cause fluid and electrolyte secretion by the small intestine of man and rabbits. 1- 3 It has also been shown that increased levels of intestinal mucosal 3' ,5' -cyclic adenosine monophosphate (cAMP) can cause small intestinal fluid and electrolyte secretion. 4 • :; Whether the small intestinal effect of CT is mediated by cAMP and whether it is attributable to a direct interaction of CT with the mucosal cell remains unknown. Of interest in this regard are the prior studies from this laboratory in which high concentrations of CT failed to exert a direct influence on adenylate cyclase activity in crude membrane preparations from mucosal cells6 or on cAMP levels in rabbit ileal mucosa incubated in vitro. 7 Because the previous studies concerning the effects of CT on intestinal fluid and electrolyte transport were conducted in intact organisms, 1- 3 the possibility that the CT effect may be indiReceived September 7, 1976. Accepted January 30, 1977. Address r equests for r eprints to: Dr. Daniel V. Kimberg, Department of Medicine , College of Physicians and Surgeons of Columbia University , 630 West 168th Street , New York, New York 10032. This work was supported by Grants AM-13696 and AM-05114 from the National Institute of Arthritis, Metabolism and Digestive Diseases. Dr. K imberg was the recipient of Research Career Development Award AM-19377. Dr. Walling is the recipient of a Clinical Investigator Award from the Veterans Administration . Dr. Walling's present address is: Veterans Administration Wadsworth Hospital Center , Los Angeles, California 90073 . Dr. Brasitus' present address is: Internal Medicine, USAF Regional Hospital, Maxwell AFB , Montgomery, Alabama 36112. The authors thank Richard R. Feinberg and E . Timothy O'Brien for their expert technical assistance.

rect and mediated by the release of another hormone remains to be explored. Thus for example, CT could conceivably cause the release of another hormone, such as vasoactive intestinal peptide (VIP) , which has been found in small intestinal mucosa8 • ~~ and which acts as a potent stimulator of mucosal adenylate cyclase activity and intestinal electrolyte secretion. 7 In preliminary experiments in which we examined the effects of a number of hormones and peptides on small intestinal mucosa we found that substance P caused a striking increase in short circuit current (SCC) , suggesting that it too might alter intestinal electrolyte transport. This undecapeptide has been isolated from neural tissue and intestine, 10 • 11 and it has been shown to stimulate intestinal contraction and salivary secretion. 11-1 3 The role, if any, of substance P in modulating small intestinal secretion has not been previously assessed. The present experiments were undertaken to determine whether or not CT and substance P are capable of exerting direct effects on rat ileum in vitro. In these studies mucosal cAMP and 3' ,5' -cyclic guanosine monophosphate (cGMP) levels were examined along with the transport of Ca, N a, and Cl.

Materials and Methods Male Holtzman rats (Holtzman Company, Madison, Wis.), weighing more than 400 g were raised on a normal laboratory chow diet and used for these studies. In experiments concerned with the effects of CT, the animals were fed a diet altered to contain 0.02% Ca 14 for 18 hr before study in order to diminish the likelihood of significant. endogenous CT secretion. Synthetic salmon calcitonin (SCT), 4790 Medical Research Council (MRC) U per mg, was generously supplied by Dr. J. W. Bastian, Armour Pharmaceutical Company, Kankakee, 89

90

WALLING ET AL.

Illinois. The SCT was dissolved in 0.1 N acetic acid containing 0.1% bovine serum albumin (BSAJ and stored as a 1 mg per ml solution at -soc. Synthetic substance P was provided by Dr. Susan E. Leeman, Department of Physiology and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts. Substance P was dissolved in 0.9% NaCl and stored as a 1 mg per ml solution at -S°C. Prostaglandin E, (PGE,), which was used in some of the experiments, was generously provided by Dr. John Pike of the Upjohn Company, Kalamazoo, Michigan. The transmural fluxes of Ca, Na, and Cl were measured isotopically across rat ileum which had been stripped of the serosal musculature in a manner identical to that previously described by Walling and Kimberg for rat duodenum.'·'· '" A Krebs-Ringer-bicarbonate buffer"' containing 11 mM n-glucose and 0.1% BSA (fraction V, fatty acid-poor, Sigma Chemical Company, St. Louis, Mo.) was used. The BSA had been heated at S6°C for 1 hr to eliminate proteolytic activity. The presence of BSA in the chambers necessitated the use of a silicone defoamer (antifoam A spray, Dow-Corning Corporation, Midland, Mich.). Nulling of the intestinal potential difference and conductance measurements were made as previously described.'·' The SCT flux experiments were conducted in one group of animals, and the control and substance P studies in a second group, and this probably accounts for the differences in base line flux values. Initial experiments with rat ileum indicated that an SCC response was demonstrable when 48 MRC U per ml (10 J.tg per ml) of SCT were added, whereas neither S nor SOO mMRC U per ml caused a detectable change. Because substance P effected a large SCC response at 10 j.tg per ml (-8 X 10 -~ M), this concentration was used in all experiments. cAMP and cGMP levels were measured in rat ileal mucosa stripped of its muscularis and incubated as previously described from this laboratory. 7 The extraction and separation of nucleotides was performed by a modification of the method of Murad et al. 17 as described in an earlier publication, ' 8 and measurements were made using the Gilman method for cAMP"' and the radioimmunoassay technique of Steiner et al.2°· 2 1 for cGMP. Protein was measured by the method of Lowry et al. 22 using BSA as a standard. Plasma Ca determinations for the bioassay of SCT were done by atomic absorption spectroscopy (Perkin-Elmer model 290 B, Perkin-Elmer Corporation, Norwalk, Conn.).

Results Ileal ion fluxes. Base line fluxes of Ca, Na, and Cl were measured at 15-min intervals during an initial 75 min (period I, table 1), and then either at 10-min intervals for SCT-solvent or SCT, or at 5-min intervals for substance P during the 20 to 30 min subsequent to the addition ofO.l ml of solvent, SCT, or substance P (period II, table 1). In the control experiments there was active secretion ofCa (P < 0.005), and active absorption ofNa (P < 0.005), and of Cl (P < 0.001) . In these control experiments there were no significant flux changes between periods I and II (table 1). The addition of 10 JJ-g per ml of SCT to the serosal compartment of the 13 pairs of tissue studied caused an increase in sec which was always apparent within 2 min, was usually maximal by 5 mm, and persisted above base line values for 30 min in 62% of the preparations, for 20 min in 31%, and for 15 min in the remaining 7% of the tissues. The maximal increment m SCC ranged from 7 to 51%, with a 15 to 20% increase in 50% of

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CALCITONIN, SUBSTANCE P , AND INTESTINAL TRANSPORT

the tissues. A second addition of SCT elicited only a small, transient increase in SCC. Only the seven pairs of tissue in which sec was elevated for 30 min (fig. 1) were used in the flux determinations in table 1, and only those flux values in these tissues that were apparently at the new steady state are reported. The SCC values in table 1 are the average for the 30-min period during which there was a response. As shown in table 1, SCT increased both Ca JMs and JsM but failed to influence the net secretion of Ca. On the other hand, this peptide did cause a decrease in Na JMs and JNet as well as a change in Cl J Net from absorption to secretion. This latter effect resulted both from a decrease in Cl JMs and an increase in Cl J sM· Although the residual ion flux was apprently decreased, this estimate may be subject to considerable inaccuracy because of the transient nature of the sec responses. The addition of substance P to the serosal compartment at a final concentration of 10 /Lg per ml caused a profound and precipitous rise in sec that was maximal within 1 min (152% increase), then fell to 50% by 5 min and to 9% by 15 min, and was generally gone by 20 min (fig. 2). A second addition of substance P at this point produced only a slight rise in SCC. The SCC values reported in table 1 are the average of the first 10 min after additon of substance P . The effects of substance P on ion fluxes were determined by using the mean of the first four 5-min periods after addition of the peptide. As was the case with SCT, substance P increased both unidirectional Ca fluxes but failed to affect the net Ca secretion . The effects of substance P on Na and Cl fluxes were also similar to those induced by SCT, but were of much greater magnitude (table 1). There is uncertainty about the equilibration of the isotopic tracers to a new steady state during the 15 to 20-min interval of sec perturbation. However, we found that the unidirectional and net fluxes of Na and Cl returned to values that were not different from those observed in the control period over the 30 min after the cessation of the sec response. Because of the rapid changes in SCC after substance P addition, residual ion flux was not estimated. Although there was a significant increase in tissue conductance in both treatment groups, this was also ob-

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served in the control groups after 30 min; this phenomenon may, at least in part, be attributable to the increased duration of in vitro incubation of the tissues (table 1). Bioassay of SCT. One possible explanation for the transient nature of the SCC response to SCT is that the hormone may be inactivated in the transport apparatus by binding to glass, oxidation, or proteolysis. To test this possibility, the hypocalcemic activity of SCT was assayed in normal 90- to 100-g Holtzman rats by the method of Cooper et aP 3 The animals received either 1 ml of buffer used in the transport studies, with 10 /Lg of stock SCT added just before administration, or 1 ml of the serosal medium after completion of the transport experiments. Pretreatment plasma Ca was 2.34 mM ± 0.03 (n = 12). The freshly prepared SCT reduced plasma Ca to 1.69 mM ± 0.02, whereas the material from the flux experiments produced a fall to 1.87 mM ± 0.07. Although there was a 28% decrease in hypocalcemic activity, most of the hormone was clearly still active at the end of the in vitro incubations. Variation between species . Contrary to the esults reported by Kisloff and Moore,2 Gray and his co-workers24 failed to-obtain a SCT effect on rabbit ileum during intravenous infusion of the hormone. Furthermore, the recent report by Gray et al.2 4 also indicated that high u 20 concentrations of SCT (10 to 144 MRC U per ml) added u to Ussing type chambers containing rabbit ileum pro~ duced only a 25% increase in sec that was apparent for w only 4 min. In preliminary studies we too had observed <( a smaller and more transient response to SCT with w 0::: 10 u rabbit than with rat ileum. To assess the species differ~ ence further, the effects of substance P were compared Iz in the rat and rabbit. When 10 /Lg per ml of substance P w u was added to the serosal surface of rabbit ileum stripped 0::: w of both muscle coats, the mean maximal sec increase 0 10 20 30 was 11% (range 6 to 16%, n = 4), whereas the increase in TIME (MIN) rat ileum was 152% (range 123 to 191%, n = 6) (fig. 2). Effects of SCT and substance P on ileal mucosal Fra . 1. Effects of salmon calcitonin on rat ileal short-circuit current (SCC) in 7 of 13 pairs of tissues examined in which the response cyclic nucleotide levels . The results of experiments in which SCT or substance P were incubated with rat ileal persisted for 30 min. (f)

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WALLING ET AL . TABLE

Substance

Vol . 73 , No.1

2. Effects of salmon calcitonin (SCT), substance P, and PGE 1 on ileal mucosal cyclic nucleotide levels • Cyclic AMP Cyclic GMP 2 min

5 min

2 min

5 min

pmoles /mg protein

SCT-control SCT (10 ILg/mll Substance P-control Substance P (10 !Lgiml) PGE,-control PGE, (5 X 10-" M)

11. 7 ± 0.7 (6) 11. 7 ± 7. 6 ± 9. 3 ± 8. 7 ± 17.0 ±

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(6) (6) (4) (4)

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± 0.04 (6) ± 0.03 (6) ± 0.04 (5 ) ± 0.05 (5)

0.0 7 ± 0.01 (5) 0.0 6 ± 0.03 (5)

" Values are means ± 1 SEM for experiments performed as described in Methods. Numbers of experiments are indicated in parentheses. Incubations with SCT (or control) contained 0.1 % heat-inactivated BSA . b P < 0.02. No other significant differences between control and test substance-conta ining incubations at either 2 or 5 min (P > 0.05).

mucosa in order to assess the potential effects of these agents on cAMP and cGMP levels are presented in table 2. PGE 1 was included as a "positive" control, for it is known to increase adenylate cyclase activity and cAMP levels in rodent small intestine. 6 • 25 Although PGE 1doubled mucosal cAMP levels after 2 min (P < 0.02), neither SCT nor substance P affected the levels of this nucleotide during the 2 to 5-min period when sec changes were found to be maximal (see above). Furthermore, there was no change in the cGMP content of ileal mucosa after the same hormonal treatment. Discussion The results of the present studies conducted with adult rat ileum in vitro indicate that pharmacological concentrations of SCT and substance P cause inhibition of active Na absorption, stimulation of active Cl secretion, and presumably a reversal in the direction of net water movement from mucosa to serosa to serosa to mucosa (table 1). Thus, SCT has effects on rat small intestine in vitro which are quite similar to those observed during intravenous administration of the hormone in both manl,3 and the rabbit. 2 In view of the present demonstration of a direct effect of SCT on intestinal ion transport in vitro, it seems quite unlikely that the previous reports of in vivo effects of this hormone on these parameters can be attributed to SCT-stimulated release of another intestinal secretagogue. Furthermore, the failure of SCT to cause a measurable effect on rabbit ileum in a prior study24 may be explicable on the basis of differences in the sensitivity of intestine from various species to this hormone. In contrast to the previously reported in vivo studies, r- 3 the present in vitro experiments were performed with the transmural PD nulled by a SCC. The absence of an electrical gradient which could affect the diffusional movement of ions may account for the lack of an observed change in Na J sM in the present data. In two in vivo studies with SCT there was a marked increase in the plasma to lumen movement of Na, but in neither were changes in PD measured. 1· 2 In man, Gray et aP observed a decrease in lumen to plasma flux in ileum and jejunum, but no change in plasma to lumen flux, findings in accord with the present data. Because residual ion flux (JR) acrOSS intestine [JR = sec in ion equivalents - (L net Na absorption - net Cl absorp-

tion)] has been shown to result primarily from HC03 movement,2r; the apparent decrease in JR after SCT addition (table 1) is suggestive of a decrease in HC03 secretion. In the in vivo studies 1-3 it has been reported that there is either a CT-induced decrease in HC0 3 absorption or an increase in HC0 3 secretion. The effects of SCT and of substance P on rat ileum noted in the present study, and of SCT on human 1· 3 and rabbit2 small intestine in previous in vivo studies, are identical to those observed when cAMP levels are increased by cholera enterotoxin,.j-{j· 2"· 27· ~ certain prostaglandins,6· 25 • 2 &-30 vasoactive intestinal polypeptide, 7 theophylline, 4· ·' · 27· 2 ~· 31 and the direct addition of dibutyryl cAMP. 4· :;, 31 In spite he similarity of the effects of SCT and substance P to those of other agents which influence the intestine by virtue of an interaction with the adenylate cyclase system, we have thus far been unable to document such an interaction in the case of the two peptides in question. Thus, in previous studies calcitonin failed to stimulate adenylate cyclase activity in a crude preparation of mucosal cell membranes prepared from rat intestine, 6 and this peptide also failed to influence cAMP levels in rabbit ileal mucosa incubated in vitro.7 In the present study neither SCT nor substance P had a demonstrable influence on either cAMP or cGMP levels in rat ileum. These results are consistent with the possibility that cAMP may not play an obligatory role in modulating the small intestinal secretion of N a, Cl, and water, and that certain agents may cause an apparently identical intestinal response by another mechanism. A similar phenomenon has also recently been described with serotonin. 32 Alternatively, one must consider the possiblity that the methods in this study may not have detected small changes in the cAMP concentration in a compartment of the cellular pool responsible for modulating absorption and secretion. With currently available techniques a phenomenon such as this cannot be excluded. Patients with medullary carcinoma of the thyroid have very high levels of CT33· 34 and, in many instances, of circulating prostaglandins. 3"· 3G Furthermore, many of these patients have severe watery diarrhea. 3"· 36 It is quite possible that prostaglandins contribute to this secretory diarrhea by a cAMP-dependent mechanism. G, 2 " · 2&-30 Based on the results of the in vivo human studies by Gray et al., 1' 3 as well as the present experi2

July 1977

CALCITONIN, SUBSTANCE P , AND INTESTINAL TRANSPORT

ments, it seems likely that the CT excess in these patients may also contribute to the secretory diarrhea, perhaps by a mechanism which is independent of the cAMP system. Another aspect of the present study that is worthy of mention is the failure of high concentrations of SCT to influence theCa J Net· These results are consonant with those obtained by Gray et al. 1 and with results in several species of animals 37-39 where SCT infusion in vivo failed to influence small intestinal Ca absorption. Employing an isolated vascularly perfused rat small intentinal preparation, Olson and co-workers, 40 on the other hand, found that an acute infusion of a low dose of procine CT (10 mMRC U) into the arterial perfusate or a chronic infusion of 1. 25 mMRC U per hr produced a drop in the lumen to blood flux of calcium. However, an acute infusion of a large dose (500 mMRC U) produced a substantial increase in lumen to blood calcium flux. The increase in Ca JMs in the present study (table 1) is consistent with the response observed by Olson et al. to the high dose ofCT. 4° Furthermore, the similar increase inCa J sM which was noted in the present study (table 1), and which accounted for the lack of a change in net absorption, may explain the apparent difference between the results of Olson et al. 40 and the work of others previously cited.1· 37- 39 The mechanism underlying the increase in both Ca JMs and Ca J sM observed in this study is unclear. It could result from a bidirectional increase in passive permeability, stimulation of both active absorption and active secretion, or a combination of these processes which would increase JMs and J sM without affecting J Net· Substance P is an interesting undecapeptide that was isolated initially from equine brain and small intestine, 10 • 11 and more recently from bovine hypothalamus.1 3· 41 This peptide has been shown to cause transient hypotension upon intravenous injection into anesthetized rabbits, 10· 11 and it is also capable of contracting guinea pig ileum and rat duodenum, 10· 11 as well as stimulating salivation in the rat. 11 · 13 Substance P can also cause the release of histamine from mast cells, 42 and when infused into the renal arteries of anethetized dogs it has a potent natriuretic effect. 43 This peptide has been localized in certain primary sensory neurons, and a transmitter or modulator role in the nervous system has been proposed.4 4-4 7 In addition to the protean effects of substance P already described, the present studies have documented that this peptide can also influence the intestinal transport ofNa and Cl, and presumably of water. The elucidation of the physiological role, if any, of substance P and of SCT in modulating intestinal ion transport, and the mechanism underlying the effects of the high concentrations of these substances noted in the present study, should provide interesting avenues for future investigation. REFERENCES 1. Gray TK, Bieberdorf FA, Fordtran JS: Thyrocalcitonin and the jejunal absorption of calcium, water, and electrolytes in normal subjects. J Clin Invest 52:3084-3088, 1973

93

2. Kisloff B, Moore EW: Effect of thyrocalcitonin on rabbit jejunal and ileal H,O and electrolyte transport (abstr). Gastroenterology 68:960, 1975. 3. Gray TK , Brannan P, Juan D, et al: Ion transport changes during calcitonin-induced intestinal secretion in man. Gastroenterology 71:392-398 , 1976 4. Field M: Intestinal secretion: effect of cyclic AMP and its role in cholera. N Engl J Med 284:1137-1144, 1971 5. Field M: Intestinal secretion. Gastroenterology 66:1063-1084, 1974 6. Kimberg DV , Field M, Johnson J, et al: Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins. J Clin Invest 50:1218-1230, 1971 7. Schwartz CJ, Kimberg DV , Sheerin HE, et al: Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J Clin Invest 54:536-544 , 1974 8. Said SI , Mutt V: Polypeptide with broad biological activity: isolation from small intestine. Science 169:1217-1218, 1970 9. Said SI, Mutt V: Isolation from porcine-intestinal wall of a vasoactive octocosapeptide related to secretin and to glucagon. Eur J Biochem 28:199-204, 1972 10. von Euler US, Gaddum JH: An unidentified depressor substance in certain tissue extracts. J Physiol (Lond) 72:74-87, 1931 11. Chang MM , Leeman SE : Isolation of a sialagogic peptide from bovine hypothalamic tissue and its characterization as substance P. J Bioi Chern 245:4784-4790, 1970 12. Horton EW: Human urinary kinin excretion. Br J Pharmacol 14:125-132, 1959 13. Leeman SE, Hammerschlag R: Stimulation of salivary secretion by a factor extracted from hypothalamic tissue. Endocrinology 81:803-810, 1967 14. Walling MW, Kimberg DV: Calcium absorption or secretion by rat ileum in vitro: effects of dietary calcium intake . Am J Physiol 226:1124-1129, 1974 15. Walling MW, Kimberg DV: Active secretion of calcium by adult rat ileum and jejunum in vitro. Am J Physiol225:415-422 , 1973 16. Walling MW, Kimberg DV: Effects of 1 a,25-dihydroxyvitamin D3 and Solanum glaucophyllum on intestinal calcium and phosphate transport and on plasma Ca, Mg and P levels in the rat. Endocrinology 97:1567-1576, 1975 17. Murad F, Manganiello V, Vaughan M: A simple sensitive protein-binding assay for guanosine 3',5'-monophosphate. Proc Natl Acad Sci USA 68:736-737, 1971 18. Wojcik JD, Grand RJ, Kimberg DV: Amylase secretion by rabbit parotid gland: role of cyclic AMP and cyclic GMP. Biochim Biophys Acta 411:250-262, 1975 19. Gilman AG: A protein binding assay for adenosine 3' ,5' -cyclic monophosphate. Proc Nat! Acad Sci USA 67:305-312, 1970 20. Steiner AL, Parker CW, Kipnis DM: Radioimmunoassay for cyclic nucleotides. I. Preparation of antibodies and iodinated cyclic nucleotides. J Bioi Chern 247:1106-1113, 1972 21. Steiner AL, Pagliara AS , Chase LR, et a!: Radioimmunoassay for cyclic nucleotides. II. Adenosine 3' ,5' -monophosphate and guanosine 3' ,5' -monophosphate in mammalian tissues and body fluids . J Bioi Chern 247:1114-1120, 1972 22. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Bioi Chern 193:265-275, 1951 23. Cooper CW, Hirsch PF, Toverud SU, et al: An improved method for the biological assay of thyrocalcitonin. Endocrinology 81:610616 , 1967 24. Gray TK, Juan D, Powell DW: Salmon calcitonin and water and electrolyte transport in rabbit ileum. Proc Soc Exp Biol Med 150:151-154, 1975 25. Kimberg DV, Field M, Gershon E, eta!: Effects of prostaglandins and cholera enterotoxin on intestinal mucosal cyclic AMP

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26 . 27.

28.

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33. 34.

35. 36.

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