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The neurohumoral secretagogues carbachol, substance P and neurotensin increase Ca++ influx and calcium content in rabbit ileum
Life Sciences, Vol. 31, pp. 1929-1937 Printed in the U.S.A.
Pergamon Press
THE NEUROHUMORAL SECRETAGOGUES CARBACHOL, SUBSTANCE P AND NEUROTENSIN INCREASE Ca ++ INFLUX AND CALCIUM CONTENT IN RABBIT ILEUM Mark Donowitz, Ronald Fogel, Laurie Battisti and Nancy Asarkof Departments of Medicine and Physiology; Tufts University School of Medicine and New England Medical Center; Boston, MA 02111 (Received in final form July 22, 1982) Summary Several neurohumoral substances, which cause active electrolyte secretion or inhibit absorption in rabbit ileum but do not affect the adenylate cyclase-cAMP system, were shown to increase Ca++ influx across the serosal surface of rabbit ileum and also to increase total ileal calcium content. These neurohumoral substances include carbachol, substance P and neurotensin. It is possible that many neurohumoral substances which cause similar changes in ileal electrolyte transport act by increasing the basolateral membrane permeability to Ca++. In spite of detailed descriptions of the nature and location of intestinal electrolyte pumps and transport proteins, little is known about what normally regulates intestinal electrolyte absorption and secretion at the intracellular level. Intracellular regulators of intestinal electrolyte secretory processes are the best defined. To date, increases in adenylate cyclase activity or cAMP content and probably guanylate cyclase activity and cGMP content have been found in intestine which is actively secreting electrolytes (1-3). However, the number of agents which both cause active intestinal electrolyte secretion and alter either cAMP or cGMP content in intestinal mucosa is quite small (4-5). Recently, evidence has been accumulating which suggests that intracellular *Ca++ is also involved in the regulation of active intestinal Na and C1 transport. The Ca ++ ionophore A23187 qualitatively duplicated the effects of increased cAMP content in altering rabbit ileal and colonic, and flounder intestinal electrolyte transport (6-8). Conversely, lowering intracellular Ca++ in rabbit ileum by exposure to 1-verapamil or bathing solutions from which the Ca++ was omitted stimulated active Na and CI absorption (9). In addition, it appears that the intracellular Ca ++ binding protein calmodulin may be involved in regulation of active intestinal electrolyte secretion. This was suggested by the observation that the phenothiazine trifluoperazine, which is known to bind to and inhibit intracellular Ca++-calmodulin complexes in non-intestinal tissue, partially inhibited the effect of cAMP on ileal electrolyte transport as well as the transport effect of the Ca ++ ionophore A23187 (i0,ii).
*In this manuscript, Ca ++ , while calcium intracellular stores.
Ca + + refers to free cytosolic r--efers to total cell calcium
0024-3205/82/181929-09503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
Ca ++ or
or ionized calcium in
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That neurohumoral substances may alter intestinal electrolyte transport by a mechanism involving intracellular Ca++ has also been suggested recently. Serotonin has been shown to inhibit the neutral NaCI uptake process in rabbit ileum, by a mechanism dependent on the presence of Ca ++ in the solution bathing the serosal surface. The effect is associated with an increase in 4bCa++ influx across the basolateral membrane and in total ileal calcium content (12,13). The effects of serotonin on electrolyte transport could be inhibited by the presence of verapamil, a "Ca ++ channel blocker" (13). In contrast, dopamine stimulated active Na and CI absorption in rabbit ileum, decreased 45Ca++ influx across the basolateral membrane and decreased calcium content (14). Thus, Ca++ might serve as an intraeellular regulator of both active intestinal absorption and secretion, and agents which alter active intestinal electrolyte transport might do so by altering intracellular calcium. The purpose of this study was to determine whether any of several neurohumoral substances which alter active intestinal electrolyte transport by as yet undefined means might act through Ca ++ . This was studied by looking for changes in 45Ca++ influx across the basolateral membrane and intracellular calcium content as determined both by atomic absorption spectrometry and by 45Ca++ uptake at close to isotopic equilibrium. The substances tested include carbachol, substance P, neurotensin, cholecystokinin and bombesin. These substances were chosen because they all affect active intestinal electrolyte transport (5,6,15-18), do not alter the adenylate cyclase-cAMP system in rabbit ileum and also because most have been shown by Gardner and co-workers to increase enzyme secretion from i s ~ a t e d guinea pig pancreatic acinar cells by a mechanism which involves Ca (19,20). These hormones had diverse effects on intestinal electrolyte transport. Carbachol~ substance P and neurotensin caused small intestinal electrolyte secretion (15,16,21). Less well studied are cholecystokinin and bombesin. Crude cholecystokinin stimulated active C1 absorption (17). Detailed evaluation of the effects on intestinal transport of bombesin have not been reported. However, it did increase rabbit ileal short-circuit current, which represents a change in net ion movement (18). Serotonin was also studied to compare several techniques for measuring ileal calcium content. Materials and Methods Male New Zealand albino rabbits weighing 2-2 1/2 kg were maintained on a standard rabbit chow diet with free access to water. The distal ileum was removed from anesthetized animals and epithelial sheets weighing 50-100 mg were prepared which consisted of mucosa, lamina propria, muscularis mucosa, and submucosa (13,14). Calcium content was determined by atomic absorption spectrometry and estimated by 45Ca ++ uptake after exposure to the test substances by techniques we have previously described (13,14). In brief, the epithelial sheets were maintained at 37°C in a shaking water bath, gassed with 95% 03-5% CO 2. Tissue was exposed for 60 min to Ringer's-HCO 3 containing 4~Ca ++ (New England Nuclear, Boston, MA) and [3H]polyethylene glycol, molecular weight 900 (New England Nuclear), as an extracellular space marker. Then the ileal mucosa was transferred to flasks containing 45Ca ~-~, [3H]polyethylene glycol of the same specific activities in Ringer's-HCO 3 alone, or Ringer's-HCOR containing the test substance. We ++ uptake appeared constant between 30 and previously reported that 45 Ca._ 105 min after addition of 45Ca +~ (13); calcium content was measured during this period. Following exposure to 45Ca++, tissue was weighed
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and dissolved in i ml Protosol (New England Nuclear); then 45Ca ++ and 3H were determined by liouid scintillation spectrometry using the method of external standards. ~ Uptake values of 45Ca++ were calculated by dividing intracellular 45Ca++ by the external fluid specific activity, and the 45Ca++ content was expressed in nmoles per mg wet weight. Calcium uptake was also measured by atomic absorption spectrometry (14). Ileal mucosa identically treated to that described above was removed in triplicate 25 min after exposure to the neurohumoral substance or Ringer's-HC03, blotted lightly, weighed, homogenized with a Polytron in 2 ml LaCI 3 (0.3mM in 5N HCI) (J.T. Baker Chemical Co., Phillipsburg, NJ). After centrifugation in a Beckman microfuge, the supernatant was diluted 1:5 (v/v) with LaCI 3 and calcium content determined using standards of ultrapure CaCI 2 (Alfa Products, Danvers, MA) with atomic absorption spectroscope model #551 (IL Instrumentation Laboratory, Inc., Lexington, MA). Calcium in the extracellular fluid as determined above was subtracted from total calcium content to give intracellular calcium. Calcium content was expressed in nmoles per mg wet weight. The rate of 45Ca ++ influx from the serosal bathing solution into the ileal epithelial sheet was determined using an influx chamber as previously described (14,22). Tissue was not short-circuited during these studies. Tissue was mounted in this chamber mucosal surface down, gassed with 95% 02-5% C02, and maintained at 37°C. The exposed surface area was 0.33cm 2, and circulation on the mucosal surface by a gas lift system was augmented with a stirring bar. Bathing solutions consisted of Ringer's-HCO 3 with lOmM glucose on the serosal and lOmM mannitol on the mucosal surface. After 25 min of exposure to Ringer's-HCO3, the serosal bathing solution was changed to one containing Ringer's-HCO3, lOmM glucose plus 0.03 uCi/ml 45Ca++ and 1 uCi/ml [JH]PEG. In addition, some of the tissue was exposed to neurohumoral substances on the serosal surface. 1, 1.5, 2 and 3 min later, the serosal bathing solution was rapidly removed, the tissue flushed with cold isotonic mannitol, punched out. blotted and weighed. The tissue was dissolved in Protosol, and 45Ca++ and 3H determined in both tissue and serosal bathing solutions. 45Ca++ influx was expressed in nmoles per mg wet weight per min. The rate of 45Ca ++ influx was determined for each experiment by performing linear regression analyses of data obtained at the four times studied. In preliminary experiments, it was determined that 45Ca++ influx in untreated control tissue was linear between 30 sec and 3 min after exposure to 45Ca++. In addition, up to 5 min after addition of 45Ca++ to the serosal surface, no 45Ca 4-+ was detectable in the mucosal bathing solution. Thus, o v e r this period of time, Ca++ influx and 45Ca++ influx are equivalent; and will be referred to as Ca++ influx. 45Ca ++ efflux was measured into a Ca++-free solution as previously described (13) or calculated as previously discussed (23). Epithelial sheets of approximately i00 mg wet weight were incubated for 60 mln in Ringer's-HCO 3 containing 45Ca +q', 50 uCi/ml, in a shaking water bath at 37°C, gassed with 95% 02-5% CO 2. Following this labeling period, the ~issue was rinsed three times in iced Ringer's-HCO 3 and efflux of 5Ca++ measured from each piece of tissue over nine lO--m~n periods by placing each tissue separately into an oxygenated flask in a shaking water bath at 37°C containing 2 ml of solutions not containing 4bCa ++ and consisting of Ringer's-HCO 3 with Ca ++ omitted (EGTA not added). After each i0 min period, each piece of tissue was transferred to another similar flask. For the first 3 lO-mln periods, all tissue was in Ca++-free Ringer's-HCO3, and for the subsequent six 10-min periods, tissue was
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exposed to Ca++-free Ringer's-HCO 3 or to similar solutions which also contained neurohumoral substances. After measuring the efflux rate for 90 min, each piece of tissue was blotted lightly, weighed, placed into I ml Protosol, and incubated overnight at 55°C to dissolve the tissue, and 45Ca++ determined by liquid scintillation spectrometry. The radioactivity was also determined in tissues handled identically up to the initial eff1~x measurement but in which the Ca++ efflux rate was not measured. 45Ca++ in the external bathing fluid was also determined. The 45Ca++ efflux rate was expressed in nmoles per mg wet weight per minute. In addition, the Ca ++ efflux rate was calculated from the influx rate and the net change in ileal calcium content as previously described. Ca++ efflux rate equals the Ca++ influx rate minus the change in ileal calcium content (23). Sources of the neurohumoral substances utilized included: serotonin creatinine sulfate and carbachol (Sigma Chemical Co., St. Louis, MO); substance P (Beckman Instruments, Inc., Palo Alto, CA); neurotensln and bombesin (Bachem, Inc., Torrence, CA). The octapeptide of cholecystokinin was a gift from Dr. M. Ondettl, Squibb Institute of Medical Research, Princeton, NJ. l-verapamil, the stereolsomer with specific "Ca ++ channel" blocking properties, was supplied by Mr. A. Graham, Knoll Pharmaceutical Co., Whippany, NJ. Results As expected normal ileal mucosal cells were still loading with ~5Ca q-+ after 95 min but by this time had already taken up approximately 60% of the total calcium content determined by atomic absorption spectrometry (Tables I,II). Ileal calcium content determined by atomic absorption spectrometry (Table I), was significantly increased by carbachol, substance P, neurotensln and serotonin, but not by bombesln (SxlO-6M and 10-SM) or cholecystokinin octapeptide (10-6M - Io-SM) compared to simultaneously studied control tissue exposed only to standard Ringer's-HCO 3 • The percent of total calcium which was extracellular was not affected by the neurohumoral substances, and for control tissue was 10.4 ~ 0.8% (N=20). The same four neurohumoral substances (carbachol, substance P, neurotensin, serotonin) also increased ileal calcium content estimated by more than 60 min of exposure to 45Ca++. These changes were detectable within 5 minutes of exposure to the test substances (Table II). As in the case of total calcium content, bombesln and cholecystokinin did not alter ileal calcium content measured by this technique. The effect of all neurohumoral substances studied on ileal 45Ca ~- content was constant 5-35 minutes after their addition. For all neurohumoral substances as well as control tissue, calcium content estimated with 45Ca ++ was 55-65% of that determined by atomic absorption spectrometry (compare Tables I and II). Similar to results with atomic absorption spectrometry, the percent of total calcium which was extracellular was not affected by the neurohumoral substances. For control tissue this was 15.4 + 0.8% (N=20); the increased extracellular contribution compared to that det~rmlned by atomic absorption spectrometry reflects the smaller estimate of calcium content. 45Ca ++ influx linear between 30
across seconds
the and
basolateral membrane in rabbit 3 minutes in untreated control
ileum tissue
was and
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after exposure to the neurohumoral substances described in Table III. Initially to characterize this influx technique, the effect on 45Ca++ influx of l-verapamil (10-4M) was determined, l-verapamil is a stereoisomer of verapamil; while verapamil is thought to have both "Na and Ca++ channel-blocking" properties, the i-form is thought to possess only "Ca++ channel blocking" properties (24). Compared to untreated control tissue, l-verapamil caused a significant decrease in the rate of 45Ca++ influx (0.049 + 0.006 nmoles/mg wet weight/minute vs. 0.034 + 0.007, in untreated control and l-verapamil exposed tissue, respective-ly, N=6, P <0.025). As demonstrated in Table III, carbachol, substance P and neurotensin all significantly increased 45Ca++ influx across the basolateral membrane of rabbit ileum. Similar data have previously been reported for serotonin (13). TABLE I Changes in lleal Intracellular Calcium Content, as Determined by Atomic Absorption Spectrometry,
Induced by
Intestinal Secretagogues*
Neurohumoral Calcium Content Substance (nmoles/mg wet weight) Control 1.17 + 0.12 Serotonin 1.40 ~ 0.17 (2.6 x 10-4M) -Carbachol 1.60 + 0.ii (10-SM) -Substance P 1.32 + 0.07 (10-5M) -Neurotensin 1.52 + 0.12 (10-5M) -Bombesln 1.02 + 0.13 (5 x 10-6M) -Cholecystokinln I.ii + 0.16 (5 x 10-6M) --
N 20 8
Percentage Chanse +15 + 8%
P <0.05
7
+35 + 13% <0.02
7
+18 + 4%
<0.01
8
+14 + 5%
<0.01
ii
0 + 9%
NS
7
- i + 7%
NS
*Calcium content of ileum under untreated control conditions (Ringer's-HCO 3) and after exposure to test substances. Approximately 50-100 mg wet weight epithelial sheets of rabbit ileum were studied. All tissue was exposed to Ringer's-HCO 3 in vitro for 60 mln; then neurohumoral substances were added and the tissue was exposed for an additional 25 min. The calcium in the extracellular fluid, as described in Table II was subtracted from the total calcium content to give Intracellular calcium. Calcium content was expressed in nmoles/mg wet weight. Data presented represents the mean + SEM. N represents the number of animals studied. P values refer to comparisons of control tissue and simultaneously studied tissue exposed to the neurohumoral substances (paired t test). The rate of 45Ca + + efflux measured from untreated control and after exposure to carbachol (10-5M) was determined. As described in Table III, carbachol did not significantly affect 45Ca++ efflux measured into medium from which Ca + + was omitted. In addition, changes in Ca ++ efflux
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were estimated from Ca ++ influx and changes in calcium content (23). The calculated Ca + + efflux was slightly increased compared to untreated control tissue by exposure to carbachol, substance P and neurotensin; estimated changes in the calculated Ca++ efflux rate were carbachol (+12%), substance P (+24%) and neurotensin (+16%). Discussion Several of the neurohumoral substances studied (substance P, neurotensin, serotonin and carbachol, the cholinergic agonist) had similar effects on active ileal electrolyte transport (5,12,15,16,18). Except for neurotensin, which causes secretion in vivo (21), and which has not been studied in detail on active electrolyte transport, these neurohumoral TABLE Changes in lleal Intracellular
Content,
II as Estimated
to 45Ca~-+, Induced by Intestinal
Neurohumoral Substance
45Ca + + Content (nmoles/mg wet weight)
Control Serotonin (2.6 x IO-4M) Carbachol (10-5M) Substance P (IO-5M) Neurotensin (IO-5M) Bombesin (5 x 10-6M ~j Cholecystoklnin (5 x IO-6M
0.70 + 0.04 0.82 ~ 0.08 -0.84 + 0.07 -
-
0.73 + -0.82 + -0.71 + -0.71 + --
Change From Simultaneously Studied Control ..... 0.15 + 0.06 -O.11 + 0.03
Secretagogues*
N 20 6 8
_
0.03 0.06 0.04 0.09
0.06 + _ 0.05 + _ 0.00 + _ 0.02 + --
by Prolonged Exposure
Percentage Change
P
........ +23 + 7% < 0 . 0 2 -+i6 + 2% < 0 . 0 0 5 _
0.03
6
0.02
7
0.04
7
0.01
9
+i0 + _ + 8 + _ + i + _ + 4 + --
3%
<0.O5
3%
<0.025
4%
NS
3%
NS
*In vitro 45Ca4+ uptake into ileum 65-95 min after 45Ca + + addition and 5-35 min after exposure to test substances. 45Ca ++ uptake was determined in the presence of [3H]Polyethylene glycol as an extracellular space marker. Starting 60 mln after addition of 45Ca++, untreated control tissue (exposed to Ringer's-HCO3) and tissue exposed to several test substances were studied simultaneously. 45Ca + + uptake was ~alculated as previously described by subtracting the 4bCa++ in the extracellular fluid from the total 45Ca++ to give the intracellular 45Ca++ and dividing the intracellular 45Ca + + by the media specific activity (14,22). 45Ca++ uptake was expressed in nmoles/mg wet weight. Data presented represents the mean 45Ca++ uptake 65-95 min after 45Ca + + exposure + SEM. N represents the number of animals studied. P valu~ refer to comparisons of untreated control tissue and simultaneously studied tissue exposed to individual neurohumoral substances (paired t test).
0
TABLE III Z 0
Changes in lleal 45Ca ++ Influx and Efflux Induced by Intestinal Secretagogues
Calcium Content* (nmoles/mg wet wt/min) control carhaehol (10-5M)
..... 0.016
(7)
45Ca++ Influx (nmoles/mg wet wt/min)
Calculated Ca + + Efflux** (nmoles/mg wet wt/mln
Measured 45Ca++ Efflux (nmoles/mg wet wt/min)
0.042 + 0.006
(i0)
0.042
0.040 + 0.010
(9)
0.063 + 0.006
(8)
0.047
0.036 + 0.003
(9)
p < 0.05
NS
t.D
Z f0 C 0
B
substance P (10-5M)
0.008
(7)
0.060 + 0.007
(7)
0.052
(9)
0.049
o
p < 0.05 neurotensin
0.008
(8)
0.057 + 0.009
(10-5M) p <0.05
f~ m
*Calculated as (calcium content after 25 min of exposure to neurohumoral substance - untreated control tissue from same anlmal)/25 mln; ** calculated as A calcium content = Ca ~-F influx - Ca + + efflux according to (23). Assumptions made for thls calculation include i) in studies of the neurohumoral substances described, changes in calcium content were similar after 5 and 25 minutes of exposure. Thus calcium content and 45Ca++ influx were assumed to have occurred over similar times and 2) 4 5 C a + + ~ 4 0 C a + + exchange is considered negligible, p compared to untreated control tissue (paired t test). Numbers in parentheses are number of animals studied.
b-J m F-~
Lo Ln
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Neurohumoral Substances and Ileal Calcium
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substances have been demonstrated either to inhibit active Na and CI absorption or cause active C1 secretion in ileum. Their effects on active ileal transport were similar to the effects of the calcium ionophore A23187 which is assumed to increase cytoplasmic free Ca ++ . Serotonin, carbachol. substance P and neurotensin all appear to affect rabbit ileal Ca +# similarly. The changes in Ca ++ handling by the intestine are most compatible with the primary effect being a change in the basolateral membrane permeability to Ca ++ , with changes in calcium content caused secondarily to the change in influx as the calcium stores rapidly take up most of the increased intracellular Ca++. While calcium efflux appeared slightly increased by these neurohumoral substances (based on our previous measurement of efflux of 45Ca++ caused by serotonin and the calculated changes caused by carbachol, substance P and neurotensin, even though the measured effect of carbachol was not to significantly alter 45Ca++ efflux), if these effects on efflux were primary, it would be expected that a decrease in calcium content would have occurred. Oppositely, an increase in ileal calcium content was detected experimentally. Consequently, we propose that Ca ++ mobilization from stores does not represent the primary event. Involvement of Ca ++ in the serotonin effect on ileal electrolyte transport is established (13), but the current study represents the first demonstration that serotonin increases total ileal calcium content. The involvement of Ca++ in this effect of serotonin is not surprising since in non-mammilian tissue, several actions of serotonin have been demonstrated to be Ca++ - dependent and associated with stimulation of Ca ++ uptake (25,26). For instance, serotonin-induced salivary gland secretion in the blowfly (Calliphora erythrocephala) is Ca++-dependent and associated with an increase in 45Ca++ uptake (26). In addition, in A p ~ s i a , serotonin elicited an inward current carried exclusively by Ca in a caudal quarter of the abdominal ganglion and in certain cells of the buccal ganglia
(27). Carbachol causes active electrolyte secretion in rabbit ileum (6,15). Many of the actions of carbachol are Ca++-dependent, and the carbachol-induced increase in rabbit ileal short-circuit current has previously been shown to be Ca++-dependent (6). Involvement of Ca ++ in the effects of substance P has only recently been suggested (19,20,28). Substance P inhibits NaCI absorption and causes electrogenic C1 secretion in the rat ileum (16). Less well studied in rabbit ileum, substance P caused a short-lived increase in short-circuit current which was dependent on the presence of Ca++ in the solution bathing the serosal surface (18). Other evidence of involvement of Ca++ in the actions of substance P include the substance P-induced increase in 45Ca++ uptake into and efflux from isolated cells of the rat parotid gland (28). In addition, in isolated guinea pig pancreatic acinar cells, substance P competes for surface receptors with physalemin, a polypeptide which increases Ca++ efflux from the same cells (19,20). Ca++ dependence of the effects of neurotensin has only recently been suggeste~ since the neurotensin-induced increase in rabbit ileal short-circuit current was dependent on Ca++ in the solution bathing the serosal surface (18). These findings suggest that agents which either inhibit active rabbit ileal electrolyte absorption (serotonin, substance P), or cause secretion (carbachol, substance P), can do so by a mechanism which is dependent on external Ca + + and which is associated with an increase in ileal basolateral membrane Ca++ influx and intracellular calcium content. Since
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serotonin, carbachol, substance P and neurotensin all previously have been shown not to alter intestinal adenylate cyclase activity or cAMP content, (4 and M. Donowitz, unpublished observations) these results suggest that calcium is a common intracellular mediator of neurohumoral substance-induced changes in active electrolyte transport, independent of cAMP. References l. 2. 3.
4. 5.
M.FIELD, Mechanisms of Intestinal Secretion, ed. H.J. Binder, pp 83-91, Alan R. Liss, Inc., New York (1979). M. FIELD, L.H. GRAF, ., W.J. LAIRD, and P.L. SMITH, Proc. Natl. Acad. Sci. USA 275 2800-2804 (1978). W.G. MARNANE, Y.-H. TAI, E.C. BOEDEKER, R.A. DECKER, A.N. CHARNEY, and M. DONOWITZ, Gastroenterology 81 90-100 (1980). H.J. BINDER, G.F. LEMP and J.D. GARDNER, Am. J. Physiol. 238 GI90-GI96 (1980). T.A. BRASITUS, M. FIELD and D.V. KIMBERG, Am. J. Physiol. 231 275-282
(1976). 6. 7. 8. 9. i0. ii. 12.
13. 14. 15. 16.
J.E. BOLTON and M. FIELD, J. Membr. Biol. 35 159-173 (1977). R.A. FRIZZELL, J. Membr. Biol. 35 159-173 (1977). M. DONOWITZ, L. BATTISTI, J. MADARA, J. TRIER, S. CUSOLITO, S. CARLSON, and M. FIELD, Bull. Mt. Desert. Is. Biol. Lab. 2 1 22-26 (1981). M. DONOWITZ and N. ASARKOF, Am. J. Physiol. in press (1982). P.L. SMITH and M. FIELD, Gastroenterology 78 1545-1553 (1980). A. ILUNDAIN and R.J. NAFTALIN, Nature 279 446-448 (1979). M. DONOWTIZ, Y.-H. TAI and N. ASARKOF, Am. J. Physiol. 239 G463-G472 (1980). M. DONOWTIZ, N. ASARKOF and G, PIKE, J . C l i n . I n v e s t . 66 341-352 ( 1 9 8 0 ) . M. DONOWITZ, S. CUSOLITO, L. BATTISTI, R. FOGEL and G.W.G. SHARP, J. Clin. Invest. 69 1008-1016 (1982). E.J. TAPPER, D.W. POWELL and S.M. MORRIS, Am. J. Physiol. 235 E402-E409 (1978). M.S. WALLING, T.A. BRASITUS and D.V. KIMBERG, Gastroenterology 73 89-94
(1977). 17. D. FROMM and W. SILEN, Current Topics in Sur$ical Research V.I, eds. G.D. Zuidema and D.B. Skinner, pp 249-261, Academic Press, New York (1969). 18. R.J. MILLER, J.F. KACHAR, M. FIELD and J. RIVIER, Hormonal Resulation of Epithelia Transport of lons and Water, eds. W.N. Scott, D.B.P. Goodman, pp 571-593, N.Y. Acad. Sci., New York (1981). 19. R.T. JENSEN and J.D. GARDNER, Proc. Natl. Sci. USA 76 5679-5683 (1979). 20. J.D. GARDNER, Ann. Rev. Physiol. 41 55-66 (1979). 21. P. REASBECK, G. BARBEZAT, A. SHALKES and D. FLETCHER, Gastroenterology 8 2 1156(a) (1982). 22. H.N. NELLANS and S.G. SCHULTZ, J. Gen. Physiol. 68 441-463 (1976). 23. C.B. WOLLHEIM and G.W.G. SHARP, Physiol. Rev. 61 914-973 (1981). 24. R. BAYER, R. HENNEKER, R. KAUFMANN and R. MANNHOLD, Arch. Pharm. 290 81-97 (1975). 25. M.J. BERRIDGE, J. Exp. Biol. 53 171-186 (1970). 26. W.T. PRINCE, M.J. BERRIDGE and H. RASMUSSEN, Proc. Natl. Acad. Sci. USA 69 552-557 (1972). 27. T.C. PELLMAR and D.O. CARPENTER, Nature 277 483-484 (1979). 28. F.R. BUTCHER, Biochim. Biophys. Acta 630 254-260 (1980).
Pergamon Press
THE NEUROHUMORAL SECRETAGOGUES CARBACHOL, SUBSTANCE P AND NEUROTENSIN INCREASE Ca ++ INFLUX AND CALCIUM CONTENT IN RABBIT ILEUM Mark Donowitz, Ronald Fogel, Laurie Battisti and Nancy Asarkof Departments of Medicine and Physiology; Tufts University School of Medicine and New England Medical Center; Boston, MA 02111 (Received in final form July 22, 1982) Summary Several neurohumoral substances, which cause active electrolyte secretion or inhibit absorption in rabbit ileum but do not affect the adenylate cyclase-cAMP system, were shown to increase Ca++ influx across the serosal surface of rabbit ileum and also to increase total ileal calcium content. These neurohumoral substances include carbachol, substance P and neurotensin. It is possible that many neurohumoral substances which cause similar changes in ileal electrolyte transport act by increasing the basolateral membrane permeability to Ca++. In spite of detailed descriptions of the nature and location of intestinal electrolyte pumps and transport proteins, little is known about what normally regulates intestinal electrolyte absorption and secretion at the intracellular level. Intracellular regulators of intestinal electrolyte secretory processes are the best defined. To date, increases in adenylate cyclase activity or cAMP content and probably guanylate cyclase activity and cGMP content have been found in intestine which is actively secreting electrolytes (1-3). However, the number of agents which both cause active intestinal electrolyte secretion and alter either cAMP or cGMP content in intestinal mucosa is quite small (4-5). Recently, evidence has been accumulating which suggests that intracellular *Ca++ is also involved in the regulation of active intestinal Na and C1 transport. The Ca ++ ionophore A23187 qualitatively duplicated the effects of increased cAMP content in altering rabbit ileal and colonic, and flounder intestinal electrolyte transport (6-8). Conversely, lowering intracellular Ca++ in rabbit ileum by exposure to 1-verapamil or bathing solutions from which the Ca++ was omitted stimulated active Na and CI absorption (9). In addition, it appears that the intracellular Ca ++ binding protein calmodulin may be involved in regulation of active intestinal electrolyte secretion. This was suggested by the observation that the phenothiazine trifluoperazine, which is known to bind to and inhibit intracellular Ca++-calmodulin complexes in non-intestinal tissue, partially inhibited the effect of cAMP on ileal electrolyte transport as well as the transport effect of the Ca ++ ionophore A23187 (i0,ii).
*In this manuscript, Ca ++ , while calcium intracellular stores.
Ca + + refers to free cytosolic r--efers to total cell calcium
0024-3205/82/181929-09503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
Ca ++ or
or ionized calcium in
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Vol. 31, No. 18, 1982
That neurohumoral substances may alter intestinal electrolyte transport by a mechanism involving intracellular Ca++ has also been suggested recently. Serotonin has been shown to inhibit the neutral NaCI uptake process in rabbit ileum, by a mechanism dependent on the presence of Ca ++ in the solution bathing the serosal surface. The effect is associated with an increase in 4bCa++ influx across the basolateral membrane and in total ileal calcium content (12,13). The effects of serotonin on electrolyte transport could be inhibited by the presence of verapamil, a "Ca ++ channel blocker" (13). In contrast, dopamine stimulated active Na and CI absorption in rabbit ileum, decreased 45Ca++ influx across the basolateral membrane and decreased calcium content (14). Thus, Ca++ might serve as an intraeellular regulator of both active intestinal absorption and secretion, and agents which alter active intestinal electrolyte transport might do so by altering intracellular calcium. The purpose of this study was to determine whether any of several neurohumoral substances which alter active intestinal electrolyte transport by as yet undefined means might act through Ca ++ . This was studied by looking for changes in 45Ca++ influx across the basolateral membrane and intracellular calcium content as determined both by atomic absorption spectrometry and by 45Ca++ uptake at close to isotopic equilibrium. The substances tested include carbachol, substance P, neurotensin, cholecystokinin and bombesin. These substances were chosen because they all affect active intestinal electrolyte transport (5,6,15-18), do not alter the adenylate cyclase-cAMP system in rabbit ileum and also because most have been shown by Gardner and co-workers to increase enzyme secretion from i s ~ a t e d guinea pig pancreatic acinar cells by a mechanism which involves Ca (19,20). These hormones had diverse effects on intestinal electrolyte transport. Carbachol~ substance P and neurotensin caused small intestinal electrolyte secretion (15,16,21). Less well studied are cholecystokinin and bombesin. Crude cholecystokinin stimulated active C1 absorption (17). Detailed evaluation of the effects on intestinal transport of bombesin have not been reported. However, it did increase rabbit ileal short-circuit current, which represents a change in net ion movement (18). Serotonin was also studied to compare several techniques for measuring ileal calcium content. Materials and Methods Male New Zealand albino rabbits weighing 2-2 1/2 kg were maintained on a standard rabbit chow diet with free access to water. The distal ileum was removed from anesthetized animals and epithelial sheets weighing 50-100 mg were prepared which consisted of mucosa, lamina propria, muscularis mucosa, and submucosa (13,14). Calcium content was determined by atomic absorption spectrometry and estimated by 45Ca ++ uptake after exposure to the test substances by techniques we have previously described (13,14). In brief, the epithelial sheets were maintained at 37°C in a shaking water bath, gassed with 95% 03-5% CO 2. Tissue was exposed for 60 min to Ringer's-HCO 3 containing 4~Ca ++ (New England Nuclear, Boston, MA) and [3H]polyethylene glycol, molecular weight 900 (New England Nuclear), as an extracellular space marker. Then the ileal mucosa was transferred to flasks containing 45Ca ~-~, [3H]polyethylene glycol of the same specific activities in Ringer's-HCO 3 alone, or Ringer's-HCOR containing the test substance. We ++ uptake appeared constant between 30 and previously reported that 45 Ca._ 105 min after addition of 45Ca +~ (13); calcium content was measured during this period. Following exposure to 45Ca++, tissue was weighed
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Neurohumoral
Substances and Ileal Calcium
1931
and dissolved in i ml Protosol (New England Nuclear); then 45Ca ++ and 3H were determined by liouid scintillation spectrometry using the method of external standards. ~ Uptake values of 45Ca++ were calculated by dividing intracellular 45Ca++ by the external fluid specific activity, and the 45Ca++ content was expressed in nmoles per mg wet weight. Calcium uptake was also measured by atomic absorption spectrometry (14). Ileal mucosa identically treated to that described above was removed in triplicate 25 min after exposure to the neurohumoral substance or Ringer's-HC03, blotted lightly, weighed, homogenized with a Polytron in 2 ml LaCI 3 (0.3mM in 5N HCI) (J.T. Baker Chemical Co., Phillipsburg, NJ). After centrifugation in a Beckman microfuge, the supernatant was diluted 1:5 (v/v) with LaCI 3 and calcium content determined using standards of ultrapure CaCI 2 (Alfa Products, Danvers, MA) with atomic absorption spectroscope model #551 (IL Instrumentation Laboratory, Inc., Lexington, MA). Calcium in the extracellular fluid as determined above was subtracted from total calcium content to give intracellular calcium. Calcium content was expressed in nmoles per mg wet weight. The rate of 45Ca ++ influx from the serosal bathing solution into the ileal epithelial sheet was determined using an influx chamber as previously described (14,22). Tissue was not short-circuited during these studies. Tissue was mounted in this chamber mucosal surface down, gassed with 95% 02-5% C02, and maintained at 37°C. The exposed surface area was 0.33cm 2, and circulation on the mucosal surface by a gas lift system was augmented with a stirring bar. Bathing solutions consisted of Ringer's-HCO 3 with lOmM glucose on the serosal and lOmM mannitol on the mucosal surface. After 25 min of exposure to Ringer's-HCO3, the serosal bathing solution was changed to one containing Ringer's-HCO3, lOmM glucose plus 0.03 uCi/ml 45Ca++ and 1 uCi/ml [JH]PEG. In addition, some of the tissue was exposed to neurohumoral substances on the serosal surface. 1, 1.5, 2 and 3 min later, the serosal bathing solution was rapidly removed, the tissue flushed with cold isotonic mannitol, punched out. blotted and weighed. The tissue was dissolved in Protosol, and 45Ca++ and 3H determined in both tissue and serosal bathing solutions. 45Ca++ influx was expressed in nmoles per mg wet weight per min. The rate of 45Ca ++ influx was determined for each experiment by performing linear regression analyses of data obtained at the four times studied. In preliminary experiments, it was determined that 45Ca++ influx in untreated control tissue was linear between 30 sec and 3 min after exposure to 45Ca++. In addition, up to 5 min after addition of 45Ca++ to the serosal surface, no 45Ca 4-+ was detectable in the mucosal bathing solution. Thus, o v e r this period of time, Ca++ influx and 45Ca++ influx are equivalent; and will be referred to as Ca++ influx. 45Ca ++ efflux was measured into a Ca++-free solution as previously described (13) or calculated as previously discussed (23). Epithelial sheets of approximately i00 mg wet weight were incubated for 60 mln in Ringer's-HCO 3 containing 45Ca +q', 50 uCi/ml, in a shaking water bath at 37°C, gassed with 95% 02-5% CO 2. Following this labeling period, the ~issue was rinsed three times in iced Ringer's-HCO 3 and efflux of 5Ca++ measured from each piece of tissue over nine lO--m~n periods by placing each tissue separately into an oxygenated flask in a shaking water bath at 37°C containing 2 ml of solutions not containing 4bCa ++ and consisting of Ringer's-HCO 3 with Ca ++ omitted (EGTA not added). After each i0 min period, each piece of tissue was transferred to another similar flask. For the first 3 lO-mln periods, all tissue was in Ca++-free Ringer's-HCO3, and for the subsequent six 10-min periods, tissue was
1932
Neurohumoral
Substances and Ileal Calcium
Vol. 31, No. 18, 1982
exposed to Ca++-free Ringer's-HCO 3 or to similar solutions which also contained neurohumoral substances. After measuring the efflux rate for 90 min, each piece of tissue was blotted lightly, weighed, placed into I ml Protosol, and incubated overnight at 55°C to dissolve the tissue, and 45Ca++ determined by liquid scintillation spectrometry. The radioactivity was also determined in tissues handled identically up to the initial eff1~x measurement but in which the Ca++ efflux rate was not measured. 45Ca++ in the external bathing fluid was also determined. The 45Ca++ efflux rate was expressed in nmoles per mg wet weight per minute. In addition, the Ca ++ efflux rate was calculated from the influx rate and the net change in ileal calcium content as previously described. Ca++ efflux rate equals the Ca++ influx rate minus the change in ileal calcium content (23). Sources of the neurohumoral substances utilized included: serotonin creatinine sulfate and carbachol (Sigma Chemical Co., St. Louis, MO); substance P (Beckman Instruments, Inc., Palo Alto, CA); neurotensln and bombesin (Bachem, Inc., Torrence, CA). The octapeptide of cholecystokinin was a gift from Dr. M. Ondettl, Squibb Institute of Medical Research, Princeton, NJ. l-verapamil, the stereolsomer with specific "Ca ++ channel" blocking properties, was supplied by Mr. A. Graham, Knoll Pharmaceutical Co., Whippany, NJ. Results As expected normal ileal mucosal cells were still loading with ~5Ca q-+ after 95 min but by this time had already taken up approximately 60% of the total calcium content determined by atomic absorption spectrometry (Tables I,II). Ileal calcium content determined by atomic absorption spectrometry (Table I), was significantly increased by carbachol, substance P, neurotensln and serotonin, but not by bombesln (SxlO-6M and 10-SM) or cholecystokinin octapeptide (10-6M - Io-SM) compared to simultaneously studied control tissue exposed only to standard Ringer's-HCO 3 • The percent of total calcium which was extracellular was not affected by the neurohumoral substances, and for control tissue was 10.4 ~ 0.8% (N=20). The same four neurohumoral substances (carbachol, substance P, neurotensin, serotonin) also increased ileal calcium content estimated by more than 60 min of exposure to 45Ca++. These changes were detectable within 5 minutes of exposure to the test substances (Table II). As in the case of total calcium content, bombesln and cholecystokinin did not alter ileal calcium content measured by this technique. The effect of all neurohumoral substances studied on ileal 45Ca ~- content was constant 5-35 minutes after their addition. For all neurohumoral substances as well as control tissue, calcium content estimated with 45Ca ++ was 55-65% of that determined by atomic absorption spectrometry (compare Tables I and II). Similar to results with atomic absorption spectrometry, the percent of total calcium which was extracellular was not affected by the neurohumoral substances. For control tissue this was 15.4 + 0.8% (N=20); the increased extracellular contribution compared to that det~rmlned by atomic absorption spectrometry reflects the smaller estimate of calcium content. 45Ca ++ influx linear between 30
across seconds
the and
basolateral membrane in rabbit 3 minutes in untreated control
ileum tissue
was and
Vol. 31, No. 18, 1982
Neurohumoral Substances and Ileal Calcium
1933
after exposure to the neurohumoral substances described in Table III. Initially to characterize this influx technique, the effect on 45Ca++ influx of l-verapamil (10-4M) was determined, l-verapamil is a stereoisomer of verapamil; while verapamil is thought to have both "Na and Ca++ channel-blocking" properties, the i-form is thought to possess only "Ca++ channel blocking" properties (24). Compared to untreated control tissue, l-verapamil caused a significant decrease in the rate of 45Ca++ influx (0.049 + 0.006 nmoles/mg wet weight/minute vs. 0.034 + 0.007, in untreated control and l-verapamil exposed tissue, respective-ly, N=6, P <0.025). As demonstrated in Table III, carbachol, substance P and neurotensin all significantly increased 45Ca++ influx across the basolateral membrane of rabbit ileum. Similar data have previously been reported for serotonin (13). TABLE I Changes in lleal Intracellular Calcium Content, as Determined by Atomic Absorption Spectrometry,
Induced by
Intestinal Secretagogues*
Neurohumoral Calcium Content Substance (nmoles/mg wet weight) Control 1.17 + 0.12 Serotonin 1.40 ~ 0.17 (2.6 x 10-4M) -Carbachol 1.60 + 0.ii (10-SM) -Substance P 1.32 + 0.07 (10-5M) -Neurotensin 1.52 + 0.12 (10-5M) -Bombesln 1.02 + 0.13 (5 x 10-6M) -Cholecystokinln I.ii + 0.16 (5 x 10-6M) --
N 20 8
Percentage Chanse +15 + 8%
P <0.05
7
+35 + 13% <0.02
7
+18 + 4%
<0.01
8
+14 + 5%
<0.01
ii
0 + 9%
NS
7
- i + 7%
NS
*Calcium content of ileum under untreated control conditions (Ringer's-HCO 3) and after exposure to test substances. Approximately 50-100 mg wet weight epithelial sheets of rabbit ileum were studied. All tissue was exposed to Ringer's-HCO 3 in vitro for 60 mln; then neurohumoral substances were added and the tissue was exposed for an additional 25 min. The calcium in the extracellular fluid, as described in Table II was subtracted from the total calcium content to give Intracellular calcium. Calcium content was expressed in nmoles/mg wet weight. Data presented represents the mean + SEM. N represents the number of animals studied. P values refer to comparisons of control tissue and simultaneously studied tissue exposed to the neurohumoral substances (paired t test). The rate of 45Ca + + efflux measured from untreated control and after exposure to carbachol (10-5M) was determined. As described in Table III, carbachol did not significantly affect 45Ca++ efflux measured into medium from which Ca + + was omitted. In addition, changes in Ca ++ efflux
1934
Neurohumoral
Substances
and lleal Calcium
Vol.
31, No. 18, 1982
were estimated from Ca ++ influx and changes in calcium content (23). The calculated Ca + + efflux was slightly increased compared to untreated control tissue by exposure to carbachol, substance P and neurotensin; estimated changes in the calculated Ca++ efflux rate were carbachol (+12%), substance P (+24%) and neurotensin (+16%). Discussion Several of the neurohumoral substances studied (substance P, neurotensin, serotonin and carbachol, the cholinergic agonist) had similar effects on active ileal electrolyte transport (5,12,15,16,18). Except for neurotensin, which causes secretion in vivo (21), and which has not been studied in detail on active electrolyte transport, these neurohumoral TABLE Changes in lleal Intracellular
Content,
II as Estimated
to 45Ca~-+, Induced by Intestinal
Neurohumoral Substance
45Ca + + Content (nmoles/mg wet weight)
Control Serotonin (2.6 x IO-4M) Carbachol (10-5M) Substance P (IO-5M) Neurotensin (IO-5M) Bombesin (5 x 10-6M ~j Cholecystoklnin (5 x IO-6M
0.70 + 0.04 0.82 ~ 0.08 -0.84 + 0.07 -
-
0.73 + -0.82 + -0.71 + -0.71 + --
Change From Simultaneously Studied Control ..... 0.15 + 0.06 -O.11 + 0.03
Secretagogues*
N 20 6 8
_
0.03 0.06 0.04 0.09
0.06 + _ 0.05 + _ 0.00 + _ 0.02 + --
by Prolonged Exposure
Percentage Change
P
........ +23 + 7% < 0 . 0 2 -+i6 + 2% < 0 . 0 0 5 _
0.03
6
0.02
7
0.04
7
0.01
9
+i0 + _ + 8 + _ + i + _ + 4 + --
3%
<0.O5
3%
<0.025
4%
NS
3%
NS
*In vitro 45Ca4+ uptake into ileum 65-95 min after 45Ca + + addition and 5-35 min after exposure to test substances. 45Ca ++ uptake was determined in the presence of [3H]Polyethylene glycol as an extracellular space marker. Starting 60 mln after addition of 45Ca++, untreated control tissue (exposed to Ringer's-HCO3) and tissue exposed to several test substances were studied simultaneously. 45Ca + + uptake was ~alculated as previously described by subtracting the 4bCa++ in the extracellular fluid from the total 45Ca++ to give the intracellular 45Ca++ and dividing the intracellular 45Ca + + by the media specific activity (14,22). 45Ca++ uptake was expressed in nmoles/mg wet weight. Data presented represents the mean 45Ca++ uptake 65-95 min after 45Ca + + exposure + SEM. N represents the number of animals studied. P valu~ refer to comparisons of untreated control tissue and simultaneously studied tissue exposed to individual neurohumoral substances (paired t test).
0
TABLE III Z 0
Changes in lleal 45Ca ++ Influx and Efflux Induced by Intestinal Secretagogues
Calcium Content* (nmoles/mg wet wt/min) control carhaehol (10-5M)
..... 0.016
(7)
45Ca++ Influx (nmoles/mg wet wt/min)
Calculated Ca + + Efflux** (nmoles/mg wet wt/mln
Measured 45Ca++ Efflux (nmoles/mg wet wt/min)
0.042 + 0.006
(i0)
0.042
0.040 + 0.010
(9)
0.063 + 0.006
(8)
0.047
0.036 + 0.003
(9)
p < 0.05
NS
t.D
Z f0 C 0
B
substance P (10-5M)
0.008
(7)
0.060 + 0.007
(7)
0.052
(9)
0.049
o
p < 0.05 neurotensin
0.008
(8)
0.057 + 0.009
(10-5M) p <0.05
f~ m
*Calculated as (calcium content after 25 min of exposure to neurohumoral substance - untreated control tissue from same anlmal)/25 mln; ** calculated as A calcium content = Ca ~-F influx - Ca + + efflux according to (23). Assumptions made for thls calculation include i) in studies of the neurohumoral substances described, changes in calcium content were similar after 5 and 25 minutes of exposure. Thus calcium content and 45Ca++ influx were assumed to have occurred over similar times and 2) 4 5 C a + + ~ 4 0 C a + + exchange is considered negligible, p compared to untreated control tissue (paired t test). Numbers in parentheses are number of animals studied.
b-J m F-~
Lo Ln
1936
Neurohumoral Substances and Ileal Calcium
Vol. 31, No. 18, 1982
substances have been demonstrated either to inhibit active Na and CI absorption or cause active C1 secretion in ileum. Their effects on active ileal transport were similar to the effects of the calcium ionophore A23187 which is assumed to increase cytoplasmic free Ca ++ . Serotonin, carbachol. substance P and neurotensin all appear to affect rabbit ileal Ca +# similarly. The changes in Ca ++ handling by the intestine are most compatible with the primary effect being a change in the basolateral membrane permeability to Ca ++ , with changes in calcium content caused secondarily to the change in influx as the calcium stores rapidly take up most of the increased intracellular Ca++. While calcium efflux appeared slightly increased by these neurohumoral substances (based on our previous measurement of efflux of 45Ca++ caused by serotonin and the calculated changes caused by carbachol, substance P and neurotensin, even though the measured effect of carbachol was not to significantly alter 45Ca++ efflux), if these effects on efflux were primary, it would be expected that a decrease in calcium content would have occurred. Oppositely, an increase in ileal calcium content was detected experimentally. Consequently, we propose that Ca ++ mobilization from stores does not represent the primary event. Involvement of Ca ++ in the serotonin effect on ileal electrolyte transport is established (13), but the current study represents the first demonstration that serotonin increases total ileal calcium content. The involvement of Ca++ in this effect of serotonin is not surprising since in non-mammilian tissue, several actions of serotonin have been demonstrated to be Ca++ - dependent and associated with stimulation of Ca ++ uptake (25,26). For instance, serotonin-induced salivary gland secretion in the blowfly (Calliphora erythrocephala) is Ca++-dependent and associated with an increase in 45Ca++ uptake (26). In addition, in A p ~ s i a , serotonin elicited an inward current carried exclusively by Ca in a caudal quarter of the abdominal ganglion and in certain cells of the buccal ganglia
(27). Carbachol causes active electrolyte secretion in rabbit ileum (6,15). Many of the actions of carbachol are Ca++-dependent, and the carbachol-induced increase in rabbit ileal short-circuit current has previously been shown to be Ca++-dependent (6). Involvement of Ca ++ in the effects of substance P has only recently been suggested (19,20,28). Substance P inhibits NaCI absorption and causes electrogenic C1 secretion in the rat ileum (16). Less well studied in rabbit ileum, substance P caused a short-lived increase in short-circuit current which was dependent on the presence of Ca++ in the solution bathing the serosal surface (18). Other evidence of involvement of Ca++ in the actions of substance P include the substance P-induced increase in 45Ca++ uptake into and efflux from isolated cells of the rat parotid gland (28). In addition, in isolated guinea pig pancreatic acinar cells, substance P competes for surface receptors with physalemin, a polypeptide which increases Ca++ efflux from the same cells (19,20). Ca++ dependence of the effects of neurotensin has only recently been suggeste~ since the neurotensin-induced increase in rabbit ileal short-circuit current was dependent on Ca++ in the solution bathing the serosal surface (18). These findings suggest that agents which either inhibit active rabbit ileal electrolyte absorption (serotonin, substance P), or cause secretion (carbachol, substance P), can do so by a mechanism which is dependent on external Ca + + and which is associated with an increase in ileal basolateral membrane Ca++ influx and intracellular calcium content. Since
Vol. 31, No. 18, 1982
Neurohumoral
Substances
and Ileal Calcium
1937
serotonin, carbachol, substance P and neurotensin all previously have been shown not to alter intestinal adenylate cyclase activity or cAMP content, (4 and M. Donowitz, unpublished observations) these results suggest that calcium is a common intracellular mediator of neurohumoral substance-induced changes in active electrolyte transport, independent of cAMP. References l. 2. 3.
4. 5.
M.FIELD, Mechanisms of Intestinal Secretion, ed. H.J. Binder, pp 83-91, Alan R. Liss, Inc., New York (1979). M. FIELD, L.H. GRAF, ., W.J. LAIRD, and P.L. SMITH, Proc. Natl. Acad. Sci. USA 275 2800-2804 (1978). W.G. MARNANE, Y.-H. TAI, E.C. BOEDEKER, R.A. DECKER, A.N. CHARNEY, and M. DONOWITZ, Gastroenterology 81 90-100 (1980). H.J. BINDER, G.F. LEMP and J.D. GARDNER, Am. J. Physiol. 238 GI90-GI96 (1980). T.A. BRASITUS, M. FIELD and D.V. KIMBERG, Am. J. Physiol. 231 275-282
(1976). 6. 7. 8. 9. i0. ii. 12.
13. 14. 15. 16.
J.E. BOLTON and M. FIELD, J. Membr. Biol. 35 159-173 (1977). R.A. FRIZZELL, J. Membr. Biol. 35 159-173 (1977). M. DONOWITZ, L. BATTISTI, J. MADARA, J. TRIER, S. CUSOLITO, S. CARLSON, and M. FIELD, Bull. Mt. Desert. Is. Biol. Lab. 2 1 22-26 (1981). M. DONOWITZ and N. ASARKOF, Am. J. Physiol. in press (1982). P.L. SMITH and M. FIELD, Gastroenterology 78 1545-1553 (1980). A. ILUNDAIN and R.J. NAFTALIN, Nature 279 446-448 (1979). M. DONOWTIZ, Y.-H. TAI and N. ASARKOF, Am. J. Physiol. 239 G463-G472 (1980). M. DONOWTIZ, N. ASARKOF and G, PIKE, J . C l i n . I n v e s t . 66 341-352 ( 1 9 8 0 ) . M. DONOWITZ, S. CUSOLITO, L. BATTISTI, R. FOGEL and G.W.G. SHARP, J. Clin. Invest. 69 1008-1016 (1982). E.J. TAPPER, D.W. POWELL and S.M. MORRIS, Am. J. Physiol. 235 E402-E409 (1978). M.S. WALLING, T.A. BRASITUS and D.V. KIMBERG, Gastroenterology 73 89-94
(1977). 17. D. FROMM and W. SILEN, Current Topics in Sur$ical Research V.I, eds. G.D. Zuidema and D.B. Skinner, pp 249-261, Academic Press, New York (1969). 18. R.J. MILLER, J.F. KACHAR, M. FIELD and J. RIVIER, Hormonal Resulation of Epithelia Transport of lons and Water, eds. W.N. Scott, D.B.P. Goodman, pp 571-593, N.Y. Acad. Sci., New York (1981). 19. R.T. JENSEN and J.D. GARDNER, Proc. Natl. Sci. USA 76 5679-5683 (1979). 20. J.D. GARDNER, Ann. Rev. Physiol. 41 55-66 (1979). 21. P. REASBECK, G. BARBEZAT, A. SHALKES and D. FLETCHER, Gastroenterology 8 2 1156(a) (1982). 22. H.N. NELLANS and S.G. SCHULTZ, J. Gen. Physiol. 68 441-463 (1976). 23. C.B. WOLLHEIM and G.W.G. SHARP, Physiol. Rev. 61 914-973 (1981). 24. R. BAYER, R. HENNEKER, R. KAUFMANN and R. MANNHOLD, Arch. Pharm. 290 81-97 (1975). 25. M.J. BERRIDGE, J. Exp. Biol. 53 171-186 (1970). 26. W.T. PRINCE, M.J. BERRIDGE and H. RASMUSSEN, Proc. Natl. Acad. Sci. USA 69 552-557 (1972). 27. T.C. PELLMAR and D.O. CARPENTER, Nature 277 483-484 (1979). 28. F.R. BUTCHER, Biochim. Biophys. Acta 630 254-260 (1980).