Effects of galanin on short circuit current and electrolyte transport in rabbit ileum

Peptides, Vol. 15, No. 8, pp. 1431-1436, 1994 Copyright© 1994ElsevierScienceLtd Printed in the USA.All rightsreserved 0196-9781/94 $6.00 + .00

Pergamon 0196-9781(94)00124-3

Effects of Galanin on Short Circuit Current and Electrolyte Transport in Rabbit Ileum F A D I A R. H O M A I D A N , * t ~ S H A O H U A T A N G , * M A R K D O N O W I T Z { A N D G E O F F R E Y W. G. S H A R P *

*Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, N Y 14853, i-Division of Gastroenterolgy, Winthrop-University Hospital and Stony Brook School of Medicine, Mineola, N Y 11501 and ¢Departments of Medicine and Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Received 21 M a r c h 1994 HOMAIDAN, F. R., S. H. TANG, M. DONOWITZ AND G. W. G. SHARP. Effects ofgalanin on short circuit current and electrolytetransportin rabbitileum PEPTIDES 15(8) 1431-1436, 1994.--Galanin decreased short circuit current (1~)and increased active Na+ and CI- absorption in rabbit ileum. In the absence of calcium, the galanin-induced decrease in 1~ was inhibited by approximately 60%. Tetrodotoxin significantlyreduced the effect of galanin on 1~, and tetrodotoxin and EGTA totally blocked the effect, indicating that the nonneuronal mediator of the effect is Ca2+ dependent. Galanin binding to basolateral membranes prepared from ileal epithelial cells was specific and of high affinity. These results suggest the involvement of this peptide in the regulation of intestinal epithelial cell function. Galanin

NaCI absorption

Electrolytetransport

THE peptide galanin, originally isolated from the upper small intestine of pigs (21), is distributed throughout the body in several mammalian species. It has been localized to the central nervous system and to neurones innervating the respiratory tract, pancreas, and gastrointestinal tract of human, rat, and pig ( 15,16). Consistent with its widespread distribution, galanin exhibits a wide variety of biological effects (16). Among the effects already known are its ability to contract smooth muscle (7,21), inhibit the release of several hormones (8,14), inhibit insulin release (1,6,18), inhibit short circuit current increases due to transmural electrical stimulation in pig intestine (3), and induce contraction in pig, guinea pig, rat, and rabbit ileal circular muscle (2). The variety of actions emphasizes the potential importance of the peptide in cell function and the need to understand the mechanisms by which it acts. In the gut, galanin is present in the neurones of the myenteric, submucosai, and mucosal plexuses. Galanin immunoreactivity is present in all layers of the gut wall and all levels of the gastrointestinal tract (15). To date, only a few reports about the role of this peptide in the regulation of intestinal electrolyte transport have been published (3,11,13). In this article, the effects ofgalanin on short circuit current (Ix) and Na + and CI transport in rabbit ileum were studied using the Ussing chamber voltage clamp technique together with isotope flux studies. Additionally, binding studies using [~25I]galanin were performed on a crude

preparation ofbasolateral membranes from ileal villus epithelial cells. METHOD Male New Zealand albino rabbits weighing 2-3 kg were maintained on a standard rabbit chow diet with free access to water. The animals were killed by an overdose of sodium pentobarbital. The distal ileum was removed, and epithelial sheets devoid of serosa and muscularis propria were prepared for transport studies as previously described (5).

In Vitro Electrolyte Transport Ileal mucosa was mounted as a flat sheet between two Lucitemodified Ussing chambers having an aperture of 1.13 cm 2, and was oxygenated and maintained at 37°C. Transmural potential difference (PD), short circuit current (I~) (which is equivalent to the sum of all electrogenic ion transport processes occurring simultaneously), conductance (G), and Na ÷ and C1- fluxes were determined. The Ix was continously recorded using a chart recorder. An automatic voltage clamp, corrected for fluid resistance between the PD sensing bridges, provided continuous short circuiting of the tissue. Mucosal-to-serosai and serosal-to-mucosal fluxes of Na + and C1- were measured 20-80 min after the addition of isotopes using 22Na+ and 36C1- on tissue matched to

t Requests for reprints should be addressed to Fadia R. Homaidan at her current address: G.I. Research Laboratories, 222 Station Plaza North, Suite 511 Winthrop-University Hospital, Mineola, NY 11501.

1431

1432

HOMAIDAN ET AL.

using a glass-Teflon homogenizer (20 strokes) in ice-cold TrisHCI (5 mM) buffer containing 50 #g/ml bacitracin and 1 mM PMSF, pH 7.4. The homogenate was centrifuged at 2500 × g for 10 rain at 4°C to remove cell debris and brush border membranes, and the resulting supernatant was centrifuged at 100,000 × g for 60 min at 4°C. The final pellet was resuspended in a small volume of HEPES buffer (20 mM, pH 7.0),and stored at - 8 0 ° C until use.

~'- -IO E

< =3_ ~

-15

-20

[1251]Galanin Binding Assay .......

i .I

........

,

........

1

[Galaninl

r 10

........

,

100

........

i

1000

nM

FIG. 1. Concentration-dependent response of the effect of galanin on rabbit ileal short circuit current (n = 4). Galanin was added to the serosal surface of the tissue, and the data, based on the maximum decrease in short circuit current, are shown for each concentration tested. A separate piece of stripped ileum was used for each determination•

differ in conductance by not more than 25%. The time periods were measured between 20-40 min, prior to the addition of galanin, 40-50 min, immediately after galanin addition, and subsequently, 50-80 min. In reporting the ion flux experiments, a negative sign indicates net secretion and a positive sign net absorption. Unless specified, the bathing solution consisted of Ringer'sHCO3 composed of (in mM): NaCI, 115; NaHCO3, 25; CaC12, 1.2; MgCI2, 1.2; K2HPO4, 2.4; and KH2PO4, 0.4; pH 7.4, after gassing with 95% 02/5% CO2. Glucose (10 mM) and mannitol (10 mM) were added to the serosal and mucosal bathing fluids, respectively, at the time of mounting the tissue. In ion substitution studies, Na ÷ ion was substituted with choline and CIwith gluconate.

Equilibrium binding assays were performed at 4°C in 20 mM HEPES buffer containing 5 m M MgCI2, 2% bovine serum albumin (BSA), and the protease inhibitors aprotinin (15 #g/ml) and bacitracin (1 mg/ml). Villus cell membranes (300 #g/ml) were incubated on ice with increasing concentrations of [125I]galanin (0.01-10 nM) in the absence (total binding) and presence (nonspecific binding) of 10 ~tM unlabeled galanin. Assays were allowed to equilibrate for 45 min and were then terminated by the addition of 150 #1 ice-cold incubation buffer to an aliquot of the incubation mixture (200 ~tl). The membranes and bound ligand were separated by centrifugation (20,000 × g for 10 min), and were rapidly washed twice with 20 m M HEPES buffer containing 10% (w/v) sucrose• Radioactivity retained in the pellet was measured using gamma spectrometry. Specific binding was calculated by the difference between total and nonspecific binding•

10

E

2b

2a

0

< -10

-20

Cell Isolation and Membrane Preparation Intestinal villus cells were isolated by the method of Sundaram et al. (20). A segment of distal ileum was removed and washed with 154 m M NaCI, everted, and filled with the same solution and incubated in citrate buffer (pregassed with 95% 02 and 5% COz for 30 min) composed of (in mM): NaCI, 96; Na citrate, 27; KCI, 1.5; KH2PO4, 8; and Na2HPO4, 5.6; containing 5000 units penicillin, 5 mg/l streptomycin, 10 mg/1 gentamycin, and 0.5 mM dithiothreitol (DTT), pH 7.2, at 37°C. After 10 min the citrate solution was discarded and the intestine was incubated in a phosphate buffer (pregassed with 95% 02 and 5% CO2 for 30 min) composed of (in mM): NaCI, 115; NaHCO3, 25; K2HPO4, 2.4; KH2PO4, 0.4; /3-hydroxy butyrate, 0.5; L-glutamine, 0.5; and EDTA, 0.3; containing 5000 units penicillin, 5 mg/l streptomycin, 10 mg/l gentamycin, and 0.5 m M dithiothreitol (DTT), pH 7.2, for 6 min at 37°C with shaking. This solution was collected, and 1 mMCaCI2 and 1 mMphenylmethyl sulfonic acid (PMSF) were added, This step was repeated at least three times. The isolated cells, which were pure epithelial cells as judged by histological studies, were collected from the phosphate buffer by centrifugation at 500 × g for 5 min (room temperature) and were resuspended in the same buffer. Cells with the highest alkaline phosphatase activity, measured according to the method of Lansing et al. (12), and the lowest thymidine kinase activity, measured according to the method of Sundaram et al. (20), were pooled together and labeled as villus cells. These were usually fractions 2 and 3. Cells were then homogenized

•

-30

,

.

10

,

.

20

,

.

30

,

.

,

•

50

40

Time 10"

2c

•

,

, 10

60

•

,

•

20

(min)

ga]anin

O"

E

.10

"

~

-20

'

~.30

-40 •

-50

•

i

,

0

,

•

10 Time

, 20

•

, 30

(min)

FIG. 2. (a) The results of a single representative experiment are shown to illustrate the transient effect of 100 nM galanin (added at the arrows) on I~. A second addition ofgalanin gave similar effects on Ix as the first addition, showing that the transient effect of galanin is not due to desensitization of the galanin receptors. (b) The effect of galanin (100 nM) on 1~ when added every2 min. The sustainedeffectunder these conditions suggests that the transient effect followinga singleaddition ofgalanin is due to breakdown of the peptide. (c) The effect of galanin (100 nM) on 1~ alone (O) or in the presence (O) of the endopeptidase inhibitor phosphoramidon (10 #M). The tissue was incubated with the inhibitor for 15 min prior to the addition of galanin. The figure is a representative tracing of a set of six experiments. Similar results were obtained using leupeptin, bacitracin, aprotinin, or a combination of these inhibtors.

EFFECT OF GALANIN ON ELECTROLYTE TRANSPORT

1433

TABLE 1 EFFECTS OF GALAN1N ON 1,~ AND ILEAL Na AND CI TRANSPORT IN RABBIT ILEUM n

1~

Js, m~ s

JNa s --* m

JNa net

Ja m --~ s

Jo s~ m

Jca net

-0.7-+0.8

5.9-+0.1

6.7-+ 1.1

-0.8-+0.9

4.9 -+ 1.0

12.9 -+ 1.6

9.0 -+ 1.5

3.9 -+ 0.4

5.6 _+ 0.8 <0.01

7.0 -+ 1.5 <0.02

2.3 _+ 0.6 <0.05

4.7 -+ 1.0 <0.02

-1.2 -+ 1.6

6.9 -+ 0.6

6.7 -+ 0.8

0.2 -+ 1.2

-0.5 -+ 0.9 NS

1.0 -+ 0.7 NS

0.0 -+ 0.7 NS

1.0 -+ 0.5 NS

Period A Control (t = 20-40 rain)

4

2.3 -+ 0.4

7.6+0.6

8.3-+ 1.1

Period B Galanin (100 nM) (t = 40-50 min)

1.1 -+ 0.4

13.0 -+ 1.1

8.1 -+ 1.8

Period B - A p

A

-1.2 _ 0.4 <0.01

5.4 -+ 0.8 <0.01

-0.2 _+ 0.8 NS

Period C Galanin (t = 50-80 min)

4

2,6 -+ 0.5

9.1 -+ 0.4

10.3 -+ 1.9

Period C - A p*

A

--0.3 -+ 0.2 NS

1.5 -+ 0.3 NS

2.0 -+ 0.8 NS

Values are means _+ SE; n, number of animals studied. In the experiments, isotopes were added at time 0. Period A flux measurements were taken between 20 and 40 min after adding isotope. Galanin was added at 40 min. Period B flux measurements were taken between 40 and 50 min, and Period C between 50 and 80 min. p, differences between Period A and Period B (paired t test); p*, differences between Period A and Period C (paired t-test). Units are vEq. cm -2 h -~ for fluxes and I~.

Materials" Verapamil a n d b u m e t a n i d e were from Sigma Chemical (St. Louis, MO); tetrodotoxin ( T T X ) was from C a l b i o c h e m Corp. (La Jolla, CA); n i t r e n d i p i n e (BAY E 5009) was kindly supplied by Miles Institute for Preclinical Pharmacology (New Haven, CT); [125I]galanin a n d galanin (porcine) were from Peninsula Laboratories, Inc. (Belmont, CA); 22Na a n d 36C1 were from New England Nuclear (Boston, MA). Statistical analyses were performed with Student's t-tests for paired a n d u n p a i r e d data; half-maximal a n d m a x i m a l effects of galanin on transport were d e t e r m i n e d by the m e t h o d o f W o o l f H a n e s (17) by plotting the c o n c e n t r a t i o n / c h a n g e in I~ vs. concentration. In these calculations, the half-maximal concentration a n d the m a x i m a l effect for each e x p e r i m e n t were determined, a n d the results are presented as the m e a n s o f these values _+ SE. RESULTS

Effects of Galanin on Isc and Na + and CI- Movement G a l a n i n , w h e n added to the ileal serosal surface, caused a c o n c e n t r a t i o n - d e p e n d e n t decrease in I~ (Fig. 1). Different concentrations o f g a l a n i n were added to different c h a m b e r s a n d the change in I~ was determined. Each tissue was exposed to only one c o n c e n t r a t i o n o f galanin. The effect o f galanin o n I~ had a n ECso o f 6 _+ 1 nh//, a n d a m a x i m u m decrease in I~ o f 18 ± 4 #A/cruZ. This effect was transient, was m a x i m a l in 2 - 5 min, a n d returned to baseline in 15 rain [Fig. 2(a)]. T h a t the transient nature o f the effect was due to b r e a k d o w n of galanin was tested in three different ways:

1. A second addition o f g a l a n i n to a tissue t h a t had completed a transient response to galanin resulted in a further response that was equivalent in m a g n i t u d e a n d time course to the first response [Fig. 2(a)]. This m e a n s also that an alternative possibility, desensitization of the receptors for galanin, is not the cause o f the transient response. 2. Repetitive additions of 100 n M g a l a n i n every 2 m i n resulted in a sustained response [Fig. 2(b)]. 3. W h e n the serosal bathing solution from one tissue, after 20 m i n with galanin, was added to a fresh u n t r e a t e d tissue, n o effect was observed o n the latter, indicating that the galanin had already b r o k e n d o w n (data not shown). In other experiments, bacitracin (10 #M), leupeptin (0.2 ~g/mi to 2 mg/ml), a p r o t i n i n (250 U / m l ) , a n d p h o s p h o r a m i d o n ( 1 0 30 ttM), added to the tissue 15 m i n prior to the addition of galanin, failed to prolong or increase the effects o f galanin [an example is s h o w n in Fig. 2(c)]. G a l a n i n h a d n o effect on I~ w h e n added to the mucosal surface (data not shown). T h e effects o f galanin o n N a ÷ a n d C1- fluxes are shown in Table 1. G a l a n i n decreased P D from 2.9 + 0.6 m V before the addition of galanin to 0.5 -+ 0.1 at the t i m e of the m a x i m u m galanin response (p < 0.01) a n d increased G from 21.7 +_ 3.6 m S / c m 2 before the addition o f g a l a n i n to 29.0 +_ 6.6 at the time of the m a x i m u m galanin response (p < 0.01). G a l a n i n increased net N a + (AjNg) a n d net CI- fluxes (z2xa~t) due to a significant increase in mucosal-to-serosal ( m - s ) fluxes. These changes in N a + a n d CI- fluxes were m e a s u r e d over l0 m i n (because of the transient nature of the galanin response o n I~) a n d probably reflect greater changes occurring over a shorter period (i.e., perhaps for only 2 - 5 min). Significant effects were detected in the

1434

H O M A I D A N ET AL.

TABLE 2

Effects of Galanin on Is, in the Presence of TTX

EFFECTS OF GALANIN, NITRENDIPINE, VERAPAMIL, AND GALANIN AFTER NITRENDIPINE OR VERAPAMIL ON SHORT CIRCUIT CURRENT Maximal Decrease in 1~ (uh/cm 2)

I. Galanin (100 nM) Nitrendipine (10 uM) Galanin after nitrendipine II. Galanin (100 nM) Verapamil (10 uM) Galanin after verapamil p+ p++

23.9 _+ 4.8 21.1 _+ 2.7 10.0 + 3.0 15.0 _+ 1.9 28.7 _+ 6.5 7.9 _+ 2.8 <0.05 <0.005

p

<0.01 n = 4 <0.005 <0.05 <0.01 n = 4 <0.01 <0.05

Values are means + SE. Stripped ileal preparations from all animals were studied under paired conditions. Galanin, nitrendipine, and verapamil were added to the serosal bathing solution, then galanin was added approximately 10 rain after nitrendipine or verapamil, p, the probability that responses are different from zero. p+, differences between galanin's effects in the absence or presence of nitrendipine or verapamil, p++, the probability that the sum of the individual effects of galanin and nitrendipine (or verapamil) are different than the effects of galanin and nitrendipine (or verapamil) when added together.

period 0 - 1 0 min after galanin and no significant effect during a flux period measured 10-20 min after galanin (data included in the 50-80-min period). In addition to the large increase in the (m-s) CI- flux, there was also a smaller increase in serosalto-mucosal (s-m) CI- flux.

Effects of Galanin on Isc in the Presence of Nitrendipine, Verapamil, and EGTA In insulin-secreting cells, galanin has been shown to hyperpolarize the cell membrane, which leads to closure of the voltagesensitive Ca 2+ channels (4) and also to directly close Ca 2+ channels (10). To determine if this might be occurring in enterocytes, or whether galanin has additional effects beyond changes in Ca 2+ channels, the effect ofgalanin on I~ was studied in the presence of nitrendipine, verapamil, or EGTA. Nitrendipine, a dihydropyridine, and verapamil, a phenylalkylamine, are Ca 2+ channel antagonists that act on two types of Ca z+ channels in ileal epithelial cells (9). Both drugs decreased I~, and these decreases have been associated previously with increases in NaC1 absorption (9). Galanin (100 nM), added after maximally effective concentrations of nitrendipine (10 gM) or verapamil (10 gM) (Table 2), caused additional decreases in I~ of 42% and 52% of the effect of galanin alone. The sum of the individual effects of galanin and nitrendipine, or galanin and verapamil, was greater than the effect of galanin plus nitrendipine, or galanin plus verapamil, when added together, indicating that these agents have actions that are overlapping, but that galanin has an additional action or actions other than closure of voltage-sensitive Ca 2+ channels. Removal of Ca 2+ from the bathing solution by addition of 2.5 m M E G T A to give a [Ca2+]i of approximately 50 n M (calculated by a computer program by R. Steinhardt, University of California, Berkely, CA) caused a decrease in L¢, measured after 10 min of the addition of EGTA, of 30 + 6/.tA/cm 2 (n = 4). Galanin, in normal Ringer solution ([Ca 2+] = 1.2 mM), decreased Ix by 20 + 3 u A / c m 2 (n = 4), but had an additional effect on Isc when added 10 min after E G T A (paired experiments), which was equivalent to 36% of the effect of galanin alone (decreased I~ by 7.1 _+ 1.6 ~tA/cm 2, n = 4).

To determine whether the effects of galanin were exerted through enteric nerves, galanin was added after T T X . The addition of T T X caused a decrease in Isc of 35 _+ 11 # A / c m 2 that was maximal in 10 min. Galanin (100 nM) added 10-15 min after T T X caused a further significant decrease in 1~ (Table 3) that was equivalent to 33% of its effect alone. Attempts to measure Na + and CI fluxes in the presence of galanin and T T X were not successful due to the small transient effect of galanin. However, Na + and C1- ion substitution studies were performed. Both ion substitutions caused complete abolition of the I~ response to galanin (the effect of galanin after T T X in Na+-free buffer = - 0 . 2 + 0.2 ~ A / c m 2, n = 5, and in C1--free buffer = 1.2 + 0.6 # A / c m 2, n = 6), indicating that only neutral NaCl is affected by galanin.

Effects of Galanin on Is,. in the Presence of TTX and EGTA In the presence of T T X the effect of galanin is partially blocked. Therefore, as might be expected, there appear to be both neuronal and nonneuronal components in galanin's effect on I~. In the presence of EGTA, the effect of galanin is also partially blocked, so there are Ca2+-dependent and Ca2+-inde pendent components in the action. To determine the nature of the nonneuronal portion of the action of galanin (that occurs in the presence of T T X ) and the Ca2+-independent action (that occurs in the presence of EGTA), the effect ofgalanin was studied in the simultaneous presence of T T X and EGTA. As expected, T T X caused a decrease in I~. E G T A after T T X caused a further decrease in I~ of 19.9 + 6.7 # A / c m 2 (n = 4). Galanin (100 nM) added after both T T X (1 uM) and E G T A (2.5 mM) had no effect (0.7 _+ 0.7 # A / c m 2, n = 4). These data suggest that galanin has at least two distinct actions on I~, neuronal and nonneuronal, and that the latter may be mediated by Ca 2+ entry into the epithelial cells.

[J2~l]Galanin Binding to Villus Cell Basolateral Membranes Equilibrium binding of [' 251]galanin was saturable and of high affinity, and was best fit by a single-site model. Shown in Fig. 3 are the results of a representative experiment of [~25I]galanin binding to a crude preparation of ileal basolateral membranes with a Scatchard/Rosenthal plot in the inset. From nonlinear regression analysis, the Kd was 0.43 + 0.07 n M a n d the maximum number of binding sites Bm~ was 28.0 + 1.7 fmol/mg membrane protein (n = 3).

TABLE 3 EFFECTS OF TTX, GALANIN, AND GALANIN AFTER TTX ON SHORT CIRCUIT CURRENT

Maximal Decrease in 1~ (#A/cm2) Galanin (100 nM) TTX (1 #M) Galanin after TTX p+

15.9 +_ 3.3 35.4 + 10.9 5.3 _+ 1.2 <0.05

<0.0l <0.05 <0.01

Values are means _+ SE. Stripped ileal preparations from five animals were studied under paired conditions. Galanin was added l0 rain after TTX. All drugs were added to the serosal surface, p, the probability that responses are different from zero. p÷, differences between galanin's effects in the absence or presence of TTX.

EFFECT OF GALANIN ON ELECTROLYTE TRANSPORT 311

e~

20 40

30

2o

= 10

1o

o

.

,

.

,

.

.

.

.

B (pM)

I 0

2

I 4

I 6

I 8

I I0

I 12

Galanin (nM)

FIG. 3. Specificbinding of [~251]galaninas a function of free ligand concentration. Points are mean of triplicate determinations from a single experiment from a series of three similar experiments. Inset: Scatchard analysis of the data presented in the figure. The data were best fit with a one-site model.

DISCUSSION Consistent with its widespread distribution, galanin exhibits a wide variety of biological effects (16). Galanin appears to have presynaptic actions as well as postsynaptic ones affecting neurotransmitter and hormone release. For example, it causes inhibition of electrically or neurochemically stimulated acetylcholine release from myenteric plexus neurons (23) and causes a decrease in bombesin-stimulated gastric acid secretion, presumably by inhibition of acetylcholine release from myenteric nerve terminals (19). It also inhibits insulin release in vivo, as well as in perfused pancreas and in insulin-secreting cells and cell lines (1,8,14,18). In the endocrine pancreas it inhibits the release of insulin and somatostatin and stimulates the release of glucagon (6). In the gastrointestinal tract, galanin-containing neurones have been found in both myenteric and submucosal plexuses of the stomach and the small and large intestine. Galanin inhibits gut motility (23); this inhibition is indirect and probably mediated by the inhibition of release of excitatory neurotransmitters such as acetylcholine and substance P (7). Besides potent and important actions on gastrointestinal motility and pancreatic function, galanin has been shown to cause the inhibition of basal and stimulated gastric acid secretion (22), and decrease I~ in porcine jejunum (3) and guinea pig colon (13), but in contrast, it increased I~ and decreased Na ÷ and C1- absorption in rat colon (11) with little effect on rat jejunum. We have now studied the effects of galanin on the small intestine of the rabbit. Two major effects were noted. Galanin

1435

stimulated Na ÷ and C1- absorption, as documented by increased m - s and net fluxes of Na ÷ and CI-, and decreased I~, an association that has been observed previously with other stimulators of this NaCI transport system. The effects on absorption and on I~ are obviously the net result of a complex of factors. I~ itself is a measure of the sum total of all active ion movements across the intestine. Additionally, the effects of galanin on I~ were found to be differentially affected by TTX, by Ca2+-channel blockers, and by the presence or absence of Ca 2÷ in the medium. Notably, in the rabbit intestine, TTX blocks 70% of the effect of galanin on I~, indicative of a major neuronal component or components to its action. The mechanisms underlying this neuronally mediated effect remain to be understood. They may be anything from increased neural release of stimulators of NaC1 absorption (e.g., norepinephrine or dopamine) to inhibition of release of inhibitors of NaC1 absorption (e.g., acetylcholine). The remaining 30% of the effect of galanin on I~, which is insensitive to TTX, is totally dependent on the presence of extracellular Ca 2+. This Ca2+-dependent, TTX-insensitive portion of the effect of galanin could be exerted by a direct action of galanin on the ileal epithelial transporting cells. Such a possibility is supported by the finding of high-affinity binding sites on a membrane preparation derived from rabbit ileal epithelial cells. Additional evidence comes from our detection of an effect of galanin on the electrical activity of an isolated enterocyte under whole-cell patch clamp conditions (work in progress; data not shown). It is possible, however, despite these data and the presence of the galanin binding sites, that the TTX-insensitive effect of galanin is also exerted indirectly, for example, by the release of mediators from submucosal inflammatory cells. Nevertheless, the results obtained with the Ca 2÷ channel blockers and Ca2+-free solutions, and the clear dependence on extracellular Ca 2÷, lead to the possibility that galanin is acting, as it does in other cell types (4,10), to reduce the intracellular Ca 2+ concentration by hyperpolarizing the epithelial cells and reducing Ca 2+ entry into the cell via voltage-dependent Ca 2+ channels (VDCC). In the pancreatic B-cell, galanin reduces [Ca2+]i by an action to activate K + channels, which, by repolarization, inhibits the VDCC (1,4,18), but also by an inhibitory effect that is targeted directly at the L-type Ca 2+ channels (10). In conclusion, galanin decreases I~ and increases NaCI absorption in the rabbit ileum. Most of the effect is mediated by TTX-sensitive neuronal mechanisms that are presumably due to the release or inhibition of release of neurotransmitters. Evidence is presented for the presence ofgalanin receptors on the ileal epithelial cells, a finding that suggests an action of galanin directly on the cells. As Ca 2+ channel blockade mimics and partially overlaps the effect of galanin, a possible mechanism of action of galanin is by repolarization of the cells and reducing [Ca2+]i, effects that are seen in other cell types. ACKNOWLEDGEMENTS The work was supported by National Institutes of Health Grants ROI DK 31667 and DK 26523.

REFERENCES

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