Endothelin: a potent stimulator of intestinal ion secretion in vitro

RegulatoryPeptides,36 (! 991) 1-19

1

© 1991 Elsevier Science Publishers B.V. All fights reserved 0167-0115/91/$03.50

REGPEP 01084

Endothelin: a potent stimulator of intestinal ion secretion in vitro M.A. Brown and P.L. Smith Department of Drug Delivery, SmithKline Beecham, King of Prussia, PA (U.S.A.) (Received 10 January 1991; revised version received 12 June 1991; accepted 14 June 1991)

Key words: Endothelin; Secretion; Intestine; Prostaglandin; Leukotriene; Ion transport

Summary Effects of endothelin (ET) on electrical properties and Na ÷ and C1- fluxes in stripped rabbit ileal mucosa were investigated in vitro in Ussing chambers. Results demonstrate that serosal addition of ET-1, ET-2, ET-3 or the precursor 38 amino acid 'big endothelin' produce dose-dependent increases in short-circuit current (1so) with maximal effects at approx. 100 nM, 100 nM, 10 nM and 100 nM, respectively and half-maximal effects at 1.4 nM, 5 riM, 1.4 nM and 20 nM, respectively. Mucosal addition of ET-3 failed to elicit a response. Changes in I,c elicited by ET-3 are accompanied by decreases in net fluxes of both Na ÷ and CI-. The cyclooxygenase inhibitors, indomethacin and piroxicam, inhibited the increase in I,¢ produced by ET-3 and indomethacin also abolished the changes in Na + and CI- fluxes produced by ET-3. However, no changes in the release of PGE2, thromboxane B2 or 6-keto-prostaglandin F1~ could be detected up to 20 min after the addition of ET-3. Preincubation of tissues with neuronal agonists or antagonists, antihistamines or an LTD4/LTE 4 receptor antagonist, SKF 104353, failed to alter the response to ET-3. Furthermore, removal of s e r o s a l C a 2 ÷ alSO failed to inhibit the change in I,~ produced by ET-3. These results indicate that endothelin is a potent intestinal secretagogue which does not appear to elicit its response through stimulation of PGE 2, thromboxane A 2 or prostacyclin.

Correspondence: P.L. Smith, Drug Delivery, L-I 11, SmithKline Beecham, PO Box 1539, King of Prussia, PA 19406-0939. U.S.A.

Introduction

In 1988, Yanagisawa and coworkers [ 1] reported on the isolation of a 21 amino acid vasoconstrictor peptide from the culture supernatant of porcine aortic endothelial cells. This peptide, endothelin-1 (ET-1), has been shown to produce a variety of pharmacological effects in addition to vasoconstriction [1] including contraction of airway, intestinal and uterine smooth muscle [2-5], stimulation of atrial natriuretic peptide secretion from cultured rat atrial myocytes [6], inhibition ofnorepinephrine release from rat mesenteric arteries in response to nerve stimulation [7], inhibition of ADP-induced platelet aggregation [8] and stimulation of N a + / K + - A T P a s e activity and (Na+,K+,C1 - )-cotransport in cultured vascular smooth muscle cells [9]. Recently, Takahashi and coworkers [ 10] demonstrated the presence of endothelinlike immunoreactivity in all regions of the rat gastrointestinal tract. In addition, this study identified specific binding sites in the rat gastrointestinal tract for [ ~25I]endothelin-1 and demonstrated that both ET-1 and ET-3 contract isolated strips of rat stomach and colon and guinea-pig ileum [10]. Several mechanisms have been shown to be involved in the actions of ET-1. These include stimulation of phospholipase C [11-14], activation of protein kinase C via stimulation of phosphytidylinositol turnover [ 12,15,16] or inhibition of a protein kinase C inhibitor [ 17], stimulation of arachidonic acid metabolism [18,19] and interaction with the dihydropyridine-sensitive Ca 2 + channels [ 16,20]. In endothelial cells, ET-1 is produced by proteolytic cleavage from a precursor peptide of 203 amino acids via an intermediate 'big endothelin' (38 amino acids for human and 39 amino acids for porcine) [ 1,21,22]. In humans, three distinct endothelinrelated genes have been shown to encode for a family of endothelins including ET-1 and two others [Trp 6, Leu 7 ]endothelin, ET-2 and [Thr 2, Phe 4, Thr ~, Tyr 6, Lys 7, Tyrt4] endothelin, ET-3 [23]. In de-endothelialized porcine coronary artery strips, ET-1 is the most potent peptide although ET-2 produces the greatest contractile response [23]. In this study, we have investigated the effects of endothelins on the electrical properties of intestinal mucosa placed in Ussing chambers. Results from this study indicate that endothelins have profound effects On the electrical properties of both the small and large intestine which are consistent with stimulation of an electrogenic CI- secretory process.

Materials and Methods

Tissue preparation Preparation of rabbit intestinal mucosa was carded out as previously described [24 ]. Briefly, distal ileum or distal colon, obtained from New Zealand White rabbits (2-3 kg) maintained on standard chow and water ad libitum, were rinsed with ice-cold HCO3- Ringer solution and placed in oxygenated ice-cold HCO3- Ringer containing the following (in mM): 144 Na + , 5 K +, 1.25 Ca 2+, 1.25 Mg 2+ , 125 CI-, 25 H C O 3 , 0.3 H2PO~-, and 1.6 HPO 2-. This solution has a pH of 7.4-7.5 when~gas~ed with 5 ~o CO2 in 02 and 37 o C. Tissues were opened along their mesenteric border and stripped

of underlying muscle by blunt dissection. Segments were mounted as flat sheets in standard Ussing chambers (1.13 cm z exposed surface area). Tissues were bathed on both surfaces with 10 ml of HCO3- Ringer, which was circulated by gas lift (5~ CO 2 in 02) and maintained at 37 °C by water-jacked reservoirs. The mucosal and serosal bathing solutions contained 10 mM mannitol and 10 mM glucose, respectively. Measurement of electrical parameters and ion fluxes

Two to six segments of ileum or colon from each animal were studied simultaneously. Transepithelial electric potential difference (PD) with reference to the mucosal bathing solution and short-circuit current (Isc) were measured with automatic voltage clamps (Custom Control, Houston, TX), which automatically compensate for the potential drop due to the fluid between the PD bridge tips and the tissue surfaces. Transepithelial conductance (Gt) was calculated from the change in current produced by briefly (< 5 s) clamping the PD to 5 mV. After being mounted in Ussing chambers, all tissues were allowed to equilibrate for a period of 60 rain. At 30 min, tissues were rinsed and the baths replaced with fresh HCO3- Ringer containing 10 mM glucose (serosal) and 10 mM mannitol (mucosal). In studies using indomethacin, indomethacin was added to the mucosal and serosal bathing solutions when tissues were first mounted and then again immediately after the 30 rain wash. Unidirectional fluxes (J) of Na and C1 from mucosa(m)-to-serosa(s) and from s-to-m were measured in paired tissues (resistance differing by < 2 5 ~ ) under I~c conditions with tracer quantifies of 22NaC1 and Na36C1 (New England Nuclear, Boston, MA). Isotopes were added after the 60 min equilibration period. Fluxes were calculated from samples taken at 15 min intervals starting 45 min after addition of isotope. Period I (first 15-rain interval) served as a control for two tissue pairs with the third pair being treated throughout with indomethacin (1 #M in both the mucosal and serosal bathing solutions). During Period II, (second 15-min interval), one control tissue pair and the indomethacin tissue pair were treated with serosal ET-3 (10 nM), while the remaining pair continued as controls. During Period III (15-min interval which began 45 min after the start of Period I), all tissues were treated with serosal prostaglandin E 1 (PGE~, 10/aM). In studies designed to investigate effects of Ca 2 + removal from the serosal bathing solution, Ca2+-free Ringer was prepared by removing all Ca 2+ salts and adding 0.24 mM EGTA. Measurement of immunoreactive thromboxane B e, PGE 2 and 6-keto-PGF2~, release

Four segments of stripped rabbit ileal mucosa mounted in Ussing chambers as described above and short-circuited throughout the experiment were equilibrated for 60 rain. One pair of these tissues was treated with indomethacin (1 #M) in the serosai and mucosai bathing solutions throughout the experiment. After 60 min the mucosai and serosal bathing solutions were replaced with fresh HCO 3 Ringer solution (containing indomethacin where appropriate). Tissues were allowed to re-equilibrate for an additional 15 min after which 1 ml samples were obtained from the serosal bathing solutions and replaced with 1 ml of fresh HCO 3 Ringer solution. 15 min later, a second

1 ml sample was removed from the serosal bath and replaced in all tissues. After removal of the second sample, ET-3 was added to the serosal bathing solutions of appropriate control and indomethacin-treated tissues. At 1.5, 3, 10 and 20 min, 1 ml serosal samples were removed and replaced from all tissues. All samples were immediately placed on ice and subsequently frozen until assay. Samples were assayed directly (without extraction) in duplicate for thromboxane B2, prostaglandin E2 (PGE2) and 6-keto-prostaglandin F ~ (PGF~) employing radioimmunoassay kits (New England Nuclear, Boston, MA).

Statistical analysis Results are shown as means + S.E.M. Unless otherwise stated, n values indicate the number of animals studied. Grouped data were analyzed using Student's t-test for paired variates. The lower limit for statistical significance was set at P < 0.05. Drugs and peptides Endothelin (ET)-1 (human, porcine), ET-2 (human), ET-3 (rat) and the 38 amino acid endothelin (big-endo, human) were obtained from Peninsula Laboratories (Belmont, CA). Leukotriene C 4 (LTC4) , LTD4, 2(S)-hydroxy-3(R)-[(2-carboxyethyl)thio]3-[2-(8-phenyloctyl)phenyl]propanoic acid (SKF 104353) and mepyramine were synthesized by SmithKline Beecham (King of Prussia, PA). Ultraviolet spectroscopy was used to determine the molar concentrations and purity of LTC4 and LTD 4. Indomethacin, atropine, piroxicam, histamine, carbachol, tetrodotoxin, EGTA, PGE~, substance P and N-formylmethionylleucylphenylalanine (FMLP) were obtained from Sigma (St. Louis, MO). Endothelins were prepared in 0.1 To bovine serum albumin (Fraction V, Sigma, St. Louis, MO). Indomethacin, piroxicam and FMLP were dissolved in dimethyl sulphoxide (DMSO) and PGE~ was dissolved in ethanol. The final concentration of DMSO or ethanol did not exceed 0.1 ~o by volume. Appropriate vehicle controls were studied in parallel tissues.

Results

Concentration dependence and time-coursesfor the effects of different endothelins on electrical properties of intestinal mucosa Fig. 1 depicts the concentration-dependence for the effects of endothelins from several species as well as the precursor molecule 'big endothelin' on the 1so across rabbit ileal mucosa. The concentration-dependence for these endothelins reveals that maximal effects for big endothelin (big endo), ET-1 and ET-2 are observed at approx. 100 nM while the maximal effect for ET-3 occurs at approx. 10 nM. Half-maximal effects for ET- 1 and ET-3 are seen at 1.4 nM while the half-maximal effects for ET-2 and big endo are seen at 5 and 20 nM, respectively. Fig. 2A illustrates the time-course for Isc and G t in ileal mucosa to which bovine serum albumin (BSA, 0.1% solvent control) was added to the serosal bathing solution. In this tissue, Is¢ and Gt remain fairly constant over the 20 min period following BSA addition. Serosal addition of PGE~ (10 #M) 20 min after addition of BSA elicits an immediate increase in I,~ and G t which is followed by a

S°0 -

A [] • O

ET-1 ET-2 ET-3

/.." (~

Big Endo 0"

6.0-

A

=L v

4.0-

g

2.0-

[Endothelin], M

Fig. l. Concentration-response curves for ET- 1, ET-2, ET-3 and big endo. Each point is the mean + S.E.M. for five animals in which all conditions were examined. One concentration of the appropriate agonist was added to each tissue and the maximal change in Isc recorded.

decrease in both Isc and G t o v e r the next 20 min. As shown in Fig. 2B, ET-3 (100 nM) elicits an immediate increase in Isc which is maximal at approx. 2 min after serosal addition and then returns toward baseline over the next 20 rain. Addition of ET-3 also appears to elicit a decrease in G t w h i c h slowly returns toward baseline over the next 20 min. Time-courses for ET-1 and ET-2 are similar to that seen with ET-3 (data not shown). Serosal addition of a second dose of ET-3 20 rain after the initial administration fails to elicit a second response. However, serosal addition of PGE~ 40 min after the initial ET-3 addition elicits a normal response (e.g., increase in I,~ and decrease in Gt). Mucosal addition of ET-3 (100 nM) did not produce any change in I,~ compared to parallel controls (A/~c(control - ET-3) = - 0 . 1 + 0.5 #Eq/h per cm 2, n = 5) and also did not inhibit the increase in I ~ elicited by serosal addition of ET-3 (10 nM) (A/s¢(control - mucosal ET-3 treated) -- 0.3 + 1.2 #Eq/h per cm:, n = 5). Fig. 3A illustrates that the time-course of effects of big endo is similar to that seen with ET-3 although the duration of the effects on 1,c and Gt appear to be prolonged. From Fig. 3B it can be seen that ET-3 elicits changes in I,¢ in rabbit colon which are similar to those seen in rabbit ileum (compare Fig. 3B with Fig. 2B). In rabbit distal

8.0-

A.

- 40

6.0-

- 30

4.0-

- 20

2.0-

-10

~,

if" 0

t~ v g

I

I

I

I

I

8.0- B.

0

-40

6.0-

g

- 30 .

,'°

Z]

,....

4.0-

- 20

-10

2.0~r~, PGE1 ET-3 I

0

20

I

I

I

40

60

80

Minutes

Fig. 2. Representative time-courses for the effects of serosal PGE], 10 #M (A) and serosal ET-3, 10 nM (B) on the short-circuit current (Is¢) and transepithelial conductance (GO across rabbit ileal mucosa.

colon, ET-3 produces changes in Gt which parallel the changes in Isc (Fig. 3B). These effects of ET-3 do not, however, alter the changes in Iso and G t produced by PGE].

Role of leukotrienes in ET-3 stimulation of lsc Qualitatively, the response to endothelins is similar to the response seen with the peptidoleukotrienes [25,26]. To determine whether the effects of ET-3 are due to release of peptidoleukotrienes, the interaction between ET-3 and LTC 4 were investigated (Fig. 4A). From the first three bars in Fig. 4A, it can be seen that pretreatment of rabbit ileum with LTC 4 (10 #M) increased Isc by 7.88 + 0.86 #Eq/h per cm z and as reported previously [26], a second administration of LTC 4 did not elicit a response indicating that the tissue was refractory to repeated stimulation. However, the observation that the tissue could still respond to PGE~ indicates that the tissue is not refractory to all secretory stimuli. The second group of tissues (middle three bars of Fig. 4A) were fh'st treated with LTC 4 and then stimulated with ET-3. The response to ET-3 was reduced compared to a non-LTC4 pretreated tissue (P < 0.05, n = 5) but was not abolished indicating that a portion of the ET-3 response can be inhibited by pretreatment with LTC 4. Further support for an interaction between LTC 4 and ET-3 is provided by the

8.0- A. t,~ ~

6.0-

~. E

-

~

~

- 40.0

4.0-

'~',

2.0-

•~,""" PGEI

,Lk

- 30.0 -2o.o

BigEndo i

o

50.0

i

I

i

o

~, '~

-1.04

_

~

g

"

10.0

~---L~

'" 0.04.0-

~ /k

[ ~

[ 9.0

••••i

[5o

~

~PGE1

2.0 -

~ET-3

7.0

0

0

I

20

I

I

40 60 Minutes

I

80

?'0

Fig. 3. Representative time-courses for the effects of serosal big endothelin (big endo), 100 nM and PGE~, 10/aM on Is¢ and Gt across rabbit ileal mucosa (A) and serosal ET-3, 10 nM and PGE~, 10 pM on l~c and Gt across rabbit distal colonic mucosa (B).

finding that pretreatment of the tissue with ET-3 reduced the responsiveness of the tissue to a subsequent stimulation by LTC 4 (P < 0.05, last group of three bars in Fig. 4A). However, as indicated in Fig. 4A, stimulation of tissues with LTC 4 and ET-3 did not alter the subsequent response to P G E t. Involvement of the peptidoleukotrienes LTD 4 and LTE 4 in the ET-3 response was examined from studies with the LTD4/LTE 4 receptor antagonist, SKF 104353, which completely abolished the response to LTD 4 without altering the response to ET-3 (Fig. 4B). However, an interaction between the response to ET-3 and LTD4 is indicated by the crossover studies which demonstrate that pretreatment of the tissue with ET-3 reduces the response to LTD 4 although pretreatment of the tissue with LTD 4 had no effect on the response to a subsequent addition of ET-3. Further studies will be required to clearly define these interactions.

Effects of cyclooxygenase inhibitors on Isc responses to ET-3 Prior studies have shown that a variety of secretory stimuli elicit their responses by cyclooxygenase dependent pathways [26-34]. To determine whether the transient

A.

m

ET-3 (10 nM) LTC4 (10pM) PGE1 (10pM)

8.0

6.0 uJ ::L

g 4.0

2.0

r-"l Control SK&F 104353 (0.1~M) k-~ ET-3 (10 nM) B B LTD4 (0.3pM) PGE1 (10pM)

B.

8.0

~

6.0 \\ \\

W v

o

4.0

2.0-

@

-2.0 Fig. 4. Maximal changes in 1,c elicited by serosal additions of ET-3, LTC 4 and PGE I as indicated (A) or SKF 104353, ET-3, LTD4 and PGEI as indicated (B) to ileal mueosa. Each group of three (A) or four (B) bars is the means +_ I S.E.M. for five animals in which all conditions were examined in separate tissues from each animal.

increases in Isc elicited by ET-3 result from the release of metabolites of the cyclooxygenase pathway, the effects of two inhibitors of this enzyme, indomethacin and piroxicam, on the response to ET-3 were determined (Fig. 5). When added to the mucosal and serosal bathing solutions, both indomethacin and piroxicam produce dose-dependent inhibition of the increase in lsc elicited by ET-3 (Fig. 5). The maximal

8.0-

• Indomethacin A Piroxlcam

A IN

O" ILl

olt......... 4.0-

1 2.0-

0

i /,x 0

t 10 .7

I

I

10''6

1~ $

!

10 -4

[Inhibitor], M

Fig. 5. Concentration-dependent effects of indomethacin or piroxicam on the increase in Isc elicited by serosal ET-3 (10 nM). Results are means + 1 S.E.M. for five animals at each point.

effect of indomethacin is produced at a concentration of 1 #M while a half-maximal effect is seen at 0.14 #M. The maximal effect of piroxicam is seen at 30 #M and a half-maximal effect occurs at 4.2 #M. Although these results suggest that the response to ET-3 is mediated by release of an arachidonic acid metabolite, measurement of release of immunoreactive PGE 2, thromboxane B2, or 6-keto-PGFl~ into the serosal bathing solution failed to demonstrate any significant change in the release of these products by ET-3 although indomethacin (1 #M) did reduce levels of these prostanoids (Figs. 6-8). Using identical techniques, we were previously able to demonstrate that the divalent cation ionophore, A23187, elicits an increase in release of immunoreactive PGE2, thromboxane B2, and 6-keto-PGF1 ~into the serosal bathing solution [34]. Although it is not possible to determine the arachidonate metabolite responsible for the changes in electrolyte transport elicited by ET-3 from these results, it is clear that the response to ET-3 is distinct from that of other secretory stimuli such as the divalent cation ionophore, A23187, bradykinin or FMLP [28-30,34].

10 4'

3"

E

o c

2"

D,.

-20

-10

0

10

20

3'0

Minutes

Fig. 6. Changes in the release of immunoreactive PGEz in control ( O ) or indomethacin (1 #M)-treated tissues (I-1) in the absence (open circles or squares) or presence (filled circles or squares) of serosal endothelin (10 nM added at time zero). Results are means + 1 S.E.M. for five animals at each point.

Effects of ET-3 on unidirectional and net fluxes of Na + and CI- and electrical properties of rabbit ileum in the absence or presence of indomethacin The ionic basis for the change in Isc elicited by ET-3 was determined from measurement of unidirectional fluxes of Na + and C1 - (Table I). In control tissues (Group A), unidirectional and net fluxes of Na + and C1- and electrical properties were constant during the initial two flux periods. Immediately after the second flux period, P G E 1 was added to the serosal bathing solution of these tissues. As previously reported, P G E 1 produced a significant decrease in the m-to-s C1- flux and a significant increase in the s-to-m C1 - flux resulting in a change in net CI- flux from absorption to secretion. This change in net C1- transport was accompanied by a significant increase in Isc. In a second group of tissues, ET-3 was added immediately after the first flux period (Group B). In addition to the increase in I ~ elicited by ET-3, a significant decrease in the net fluxes of both Na + and CI- was also observed. Subsequent addition of PGE1 to these tissues did not further alter the unidirectional or net fluxes of Na + or C1although Isc was increased. Since indomethacin abolished the increase in Isc elicited by ET-3 (Fig. 5), changes in the fluxes o f N a + and CI- produced by ET-3 were determined in tissues pretreated with indomethacin (Group C). Indomethacin completely abolished the changes in Na ÷ and CI- fluxes elicited by ET-3 but did not alter the changes in these fluxes or the electrical changes produced by PGE1.

11 4"

E

o

& x

i-

o

3'o Minutes

Fig. 7. Changes in the release of immunoreactive TxB 2 in control (O) or indomethaein (1 #M)-treated tissues (Fl) in the absence (open circles or squares) or presence (filled circles or squares) of serosal endothelin (10 nM added at time zero). Results are means + 1 S.E.M. for five animals at each point. Effect o f Ca 2 + removal or inhibitors on the changes in Isc elicited by ET-3 A variety of secretory stimuli have been shown to be dependent on the presence of Ca / + in bathing solution to elicit a response [26,28,33,35]. Since endothelin has been shown to produce its effects in some tissues via an interaction with the dihydropyridinesensitive Ca 2 + channels [ 16,20], ET-3 responses in the absence of serosal Ca 2 + were determined (Fig. 9A). Removal of Ca 2 + from the serosal bathing solution had no effect on the increase in Is¢ produced by ET-3 (10 nM). However, the response to serosal F M L P (30 riM), a secretory stimuli which produces a change in Is¢ which is similar both in terms of time-course and magnitude of response to ET-3 [28], was inhibited by greater than 80 ~o by removal of Ca 2 ÷ from the serosal bathing solution. These results clearly demonstrate that ET-3 unlike F M L P does not depend on extracellular Ca z + for its response. Fig. 9A also provides evidence that ET-3 and F M L P do not act via a common post receptor mechanism since preincubation of the tissue with F M L P does not alter the subsequent response to ET-3 (or vice versa) although as reported previously, pretreatment with F M L P would completely inhibit a second challenge to F M L P [281. Role o f enteric nervous system or mast cells in the intestinal response to ET-3 To determine whether the effects of ET-3 could be due to the release of neurotransmitters from subepithelial nerves or to histamine release from mast cells, the effects

12

TABLE I Effects of ET-3 in the absence or presence of i n d o m e t h a c i n on unidirectional a n d net fluxes of s o d i u m and chloride and electrical properties across r a b b i t ileum a Period b

Sodium

Chloride

Jms

Jsm

,)met

Jms

Jsm

IA control

16.5 ± 1.4

17.9 ± 2.4

- 1.4 + 2.0

8.2 + 1.3

7.0 ± 0.6

IIA control IIIA+PGEI c

14.2 + 1.3 13.7±1.3

13.5 + 1.0 14.3±0.6

0.7 +_ 1.6 -0.6±1.4

8.2 _+ 1.1 6.8±1.2"

5.8 _+ 0.8 8.4_+0.6*

IB control IIB+ET-3 d IIIB + PGE~ c

14.9+_1.6 14.2±1.6 15.0±1.8

15.1+1.8 16.7±1.7 17.3±1.8

-0.2±1.1 -2.5±1.2" -2.4_+1.7

8.0±1.2 8.2+1.8 7.2_+1.3

6.8±0.5 9.4±1.1 9.4_+0.7

IC indo c IIC + ET-3 IIIC+PGEI c

18.3 + 1.1 18.6 ± 0.4 17.7±0.6

14.5 ± 1.1 16.1 + 2.1 18.0±2.2

3.8 + 0.4 2.5 + 1.9 -0.3±2.2

7.5 ± 1.2 8.4 ± 1.1 6.4±1.0"

5.8 ± 0.7 7.0 ± 1.4 9.9±0.9*

Jnet

/so

Gt

1.2 _+ 1.4

2.2 ± 0.2

31.6 ± 2.7

2.4 ± 0.6 -1.7_+1.2"

2.2 ± 0.2 5.9_+0.3*

32.2 ± 2.7 30.8±3.6

1.2±0.9 -1.2±1.3" -2.1_+0.9

2.1+0.2 3.7±0.5* 5.9±0.4*

31.6±3.0 31.4_+3.0 31.0±3.6

1.6 _+0.6 1.4 ± 0.6 -3.5±0.9*

1.6 + 0.6 1.6 +_ 0.6 6.1±0.4"

32.5 ± 1.9 33.9 ± 2.3 32.4±1.8

a Results are m e a n s _+ S.E.M. for five animals (four animals with i n d o m e t h a c i n ) in w h i c h all conditions were e x a m i n e d in each animal. b P e r i o d I began 90-105 min after m o u n t i n g tissues in vitro. Periods II a n d I I I b e g a n 15 and 30 min after P e r i o d I. P G E 1 ( 1 0 / ~ M ) w a s a d d e d to the serosal b a t h i n g solution at the o u t s e t of P e r i o d III. d ET-3 (10 n M ) was a d d e d to the serosal bathing solution at the o u t s e t of P e r i o d II. e I n d o m e t h a c i n (indo, 1 # M ) was p r e s e n t in the serosal a n d m u c o s a l b a t h i n g solutions t h r o u g h o u t the experiment. * P < 0.05 c o m p a r e d to the preceeding Period.

of pretreatment of tissues with the muscarinic antagonist, atropine (10/~M), the excitable sodium channel blocker, tetrodotoxin (0.1#M), the putative intestinal neurotransmitter, substance P (0.1/~M) or the Hi-receptor antagonist, mepyramine (10 #M) were examined. None of these maneuvers significantly altered the response to ET-3 although atropine was effective in blocking the response of the tissue to the muscarinic agonist, carbachol (100/~M) and mepyramine was effective in blocking the response of the tissue to histamine (100/~M) (Figs. 9B and 10). Although pretreatment of tissues with carbachol did not significantly alter the response to ET-3, pretreatment of tissues with ET-3 did reduce the response to subsequently added carbachol (P < 0.05) (Fig. 10B). The mechanism involved in this interaction is not known and will require further investigation.

Discussion

The vasoactive peptide, endothelin, has been shown to be a potent secretagogue which acts selectively from the serosal but not the mucosal surface of rabbit ileal and colonic mucosa. The time-course for effects of endothelin are similar to those seen with a number of other secretagogues including peptidoleukotrienes [25,26,33], bradykinin

13

4"

J E

o

s

ors "S'

s /

s

M.

(,,'n 3

t......t ..........t

v

20

-10

0

1'0

2'0

3'0

Minutes Fig. 8. Changes in the release of immunoreactive 6-keto-PGF~ in control (C)) or indomethacin (1 #M)treated tissues (f-]) in the absence (open circles or squares) or presence (filled circles or squares) of serosal endothelin (10 nM added at time zero). Results are means + 1 S.E.M. for five animals at each point.

[29,31], thromboxanes [32], histamine [36], F M L P [28] and carbachol [37]. The increase in Isc in rabbit ileal mucosa elicited by ET-3 is accompanied by parallel decreases in net fluxes o f N a + and C1 -. Furthermore, in rabbit distal colon, the parallel increases in Isc and G t s e e n with ET-3 are identical to changes in electrical parameters seen with a variety of colonic secretagogues which have repeatedly been demonstrated to result from stimulation of electrogenic CI- secretion [38,39]. The concentration dependence of the endothelin response reveals half-maximal effects at 1.4 nM for ET-3 which is similar to the half-maximal concentration of 0.4 nM required to produce constriction in porcine right coronary artery strips [ 1] and approx. 1 nM required to stimulate the contractile response and increases in Ca 2 + influx in isolated rabbit aortic rings and cultured vascular smooth muscle cells [ 14]. In rabbit ileum, the concentration dependence for ET-1 and ET-2 are similar to that seen with ET-3 with half-maximal effects seen at 1.4 nM and 5 nM, respectively. The concentration dependence for big endothelin is shifted to the right in rabbit ileum with a half-maximal effect seen at 20 nM. Whether this effect is due to an interaction with a specific receptor or to metabolism of big endothelin to an active endothelin by the mucosa cannot be determined from our experiments. The mechanisms by which endothelin exerts its effects include changes in intracellular Ca 2+ [14], increases in inositol metabolism to inositol bis- and tris-phosphates [13,14,40,41 ], production of diacylglycerol from arachidonic acid [11 ], and release of

14

A. 10.0 -

E

8.06.0-

,~

4.0-

~"

~ ET-3 (10 nM) B B FMLP (30 nM) ~ PGE 1 (10pM)

~ 1

Ill

2.0_

Serosal Ca2+-free B. 10.0-

Control

[~;

Control Mepyramine (10pM) ET-3 (10 nM) BIB Histamine (100t11M) PGE 1 (101xM)

8.06.0=L 4.0-

2.0-

Fig. 9. Effects of Ca 2 ÷ removal from the serosal bathing solution on the increase in Is¢ elicited by serosal ET-3, F M L P or PGE 1 (A) or pretreatment with mepyramine on the increase in Is¢ elicited by serosal ET-3, histamine or PGE l (B). Results are means + 1 S.E.M. for tissues from five animals.

prostanoids [3,7,18,42]. The involvement of arachidonic acid metabolism and extracellular Ca 2 + in response to endothelin in intestinal mucosa was therefore examined. Stimulation of Isc by ET-3 is blocked by preincubation of tissues with the cyclooxygenase inhibitors indomethacin and piroxicam suggesting that arachidonate metabolites may be involved in the response to endothelin. However, measurement of release of immunoreactive P G E 2, TxB 2 or 6-keto-PGF~ into the serosal bathing solution did not reveal any increase in production of these prostanoids. Thus, the inhibitory effects of indomethacin and piroxicam may indicate that other prostanoids are responsible for the increase in Isc elicited by endothelin or that these agents alter

15 A,

~ ' ~ ET-3 (10 nM) m m Tetrodotoxln (0.111M) IBm Sublllllnce P (0.11xM)

10.0 -

PGE 1 (10ldVI)

8.0-

6.0-

4.0-

2.0-

0 -1.0 -

B.

10.0-

8.0-

Q

6.0-

'~

4.0-

r--1 Control m Atropllr~ (1 01JM) ET-3 (10 riM) m l CarbachoI (100plY0 PGE 1 (10pM)

2.0-

Fig. 10. Effects of pretreatment with tetrodotoxin or substance P on the increase in I~¢ elicited by serosal ET-3 or PGE~ (A) or pretreatment with atropine on the increase in lsc produced by serosal ET-3, carbaehol or PGE t (B). Results are means + 1 S.E.M. for tissues from five animals.

endothelin interaction with its receptor in the intestinal mucosa. Support for the former hypothesis is provided by prior studies showing that a variety of prostanoids can stimulate Isc in rabbit ileal mucosa [43]. The latter hypothesis is based on the fmding that cyclooxygenase inhibitors can alter the binding of FMLP to human granulocytes [44]. Further studies will be required to distinguish between these possibilities. Prior studies have shown that the peptidoleukotrienes, LTC4, LTD 4 and LTE 4 are potent secretory stimuli and that the secretory response elicited by these mediators can be inhibited by indomethacin in vitro [25,26,33]. To examine whether the response to endothelin could be mediated by stimulation of production or release of peptidoleukotrienes, the effects of pretreatment of tissues with peptidoleukotrienes or with a specific LTD4/LTE4 receptor antagonist, SKF 104353 [25] were examined. Previously, it was shown that pretreatment of tissues with the peptidoleukotrienes produced a sensitization to subsequent peptidoleukotriene stimulation [33]. Thus, if endothefin elicits its secretory response through an increase in production or release of peptidoleukotrienes, pretreatment of tissues with peptidoleukotrienes should abolish the

16 response to endothelin. Although prestimulation of ileal mucosa with LTC 4 reduced the response to endothelin, it did not abolish the response. Thus, it may be that endothelin elicits a portion of its secretory response through leukotriene production or release. An alternate explanation for the observed partial inhibition is that the leukotrienes and endothelin may share a common mediator in the pathway by which they stimulate secretion. In endothelial cells, responses to endothelin are dependent on the presence of extracellular Ca 2 + and it has been proposed that endothelin may alter membrane Ca 2 + permeability [ 1,20]. However, in the intestine, removal of Ca 2 + from the serosal bathing solution did not alter the response to endothelin. Thus, endothelin does not appear to alter intestinal ion transport via a change in the influx of Ca 2 ÷ from the serosal bathing solution. This finding is identical to the effects observed with interleukin 1 [34] but contrast with the results observed with a variety of other intestinal secretagogues, including histamine [36], bradykinin [31], F M L P [28], carbachol [37], leukotrienes [33], phorbol esters [27], substance P [35], neurotensin [35] and the divalent cation ionophore, A23187 [45], which demonstrate a marked dependence on the presence of Ca 2 + in the serosal bathing solution. To examine the role of neurotransmitters and/or mast cells in the response of the intestinal mucosa to endothelin, tissues were pretreated with the excitable N a + channel blocker, tetrodotoxin, the muscarinic receptor antagonist, atropine or the H l-histamine receptor antagonist, mepyramine. Results from these studies suggest that release of neurotransmitters or histamine are not involved in the response of the intestine to endothelin. In summary, these results demonstrate that endothelin can stimulate secretion in both the small and large intestine. It has recently been shown that endothelin can stimulate C1- secretion in the trachea [46] as well. These results together with the findings of Takahashi and coworkers [ 10] provide evidence that endothelin can play a critical role in regulation of intestinal salt and water transport.

Acknowledgement Portions of this work have been presented previously in abstract form [47].

References 1 Yanagisawa,M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K. and Masaki, T., A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature, 332 (1988) 411-415. 2 Uchida, Y., Ninomiya,H., Saotome, M., Nomura, A., Ohtsuka, M., Yanagisawa,M., Goto, K., Masaki, T. and Hasegawa, S., Endothelin, a novel vasoconstrictor peptide, as potent bronchoconstrictor, Eur. J. Pharmacol., 154 (1988) 227-228. 3 De Nucci, G., Thomas, R., D'Orleans-Juste, P., Antunes, E., Walder, C., Warner, T.D. and Vane, J.R., Pressor effectsof circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostaeyclin and endothelium-derived relaxing factor, Proc. Natl. Sci. USA, 85 (1988) 9797-9800.

17 4 Kozuka, M., Ito, T., Hirose, S., Takahashi, K. and Hagiwara, H., Endothelin induces two types of contractions of rat uterus: phasic contractions by way of voltage-dependent calcium channels and developing contractions through a second type of calcium channels, Biochem. Biophys. Res. Commun., 159 (1989) 317-323. 5 Lagente, V., Chabrier, P.E., Mencia-Huerta, J.-M. and Braquet, P., Pharmacological modulation of the bronchopulmonary action of the vasoactive peptide, endothelin, Biochem. Biophys. Res. Commun., 158 (1989) 625-632. 6 Fukuda, Y., Hirata, Y., Yoshimi, H., Kojima, T., Kobayashi, Y., Yanagisawa, M. and Masaki, T., Endothelin is a potent secretagogue for atrial natriuretic peptide in cultured rat atrial myocytes, Biochem. Biophys. Res. Commun., 155 (1988) 167-172. 7 Tabuchi, Y., Nakumaru, M., Rakugi, H., Nagano, M., Mikami, H. and Ogihara, T., Endothelin inhibits presynaptic adrenergic neurotransmission in rat mesenteric artery, Biochem. Biophys. Res. Common., 161 (1989) 803-808. 8 Thiemermann, C., Lidbury, P. S., Thomas, G. R. and Vane, J. R., Endothelin-1 releases prostacyclin and inhibits ex vivo platelet aggregation in the anesthetized rabbit, J. Cardiovasc. Pharmacol., 13 (1989) S138-S141. 9 Garay, R., Rosati, C., Braquet, M., Chabrier, P.-E. and Braquet, P., Stimulation of the Na+/K + pump and the (Na* ,K + ,CI- )-cotransport system by endothelin-1 in cultured vascular smooth muscle cells: involvement of cyclo-oxygenase products, J. Cardiovasc. Pharmacol., 13 (1989) $213-$215. 10 Takahashi, K., Jones, P.M., Kanse, S.M., Lam, H.-C., Spokes, R.A., Ghatei, M.A. and Bloom, S.R., Endothelin in the gastrointestinal tract: presence of endothelinlike immunoreactivity, endothelin-1 messenger RNA, endothelin receptors and pharmacological effect, Gastroenterology, 99 (1990) 1660-1667. 11 Resink, T.J., Scott-Burden, T. and Buhler, F.R., Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells, Biochem. Biophys. Res. Commun., 158 (1989) 279-286. 12 Takuwa, N., Takuwa, Y., Yanagisawa, M. Yamashita, K. and Masaki, T., A novel vasoactive peptide endothelin stimulates mitogenesis through inositol lipid turnover in Swiss 3T3 fibroblasts, J. Biol. Chem., 264 (1989) 7856-7861. 13 Kasuya, Y., Takuwa, Y., Yanagisawa, M., Kimura, S., Goto, K. and Masaki, T., Endothefin-1 induces vasoconstriction through two functionallydistinct pathways in porcine coronary artery: contribution of phosphoinositide turnover, Biochem. Biophys. Res. Commun., 161 (1989) 1049-1055. 14 Marsden, P.A., Danthuluri, N. R., Brenner, B.M., Ballermann, B.J. and Brock, T.A., Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization, Biochem. Biophys. Res. Commun., 158 (1989) 86-93. 15 Muldoon, L., Rodland, K.D., Forsythe, M.L. and Magun, B.E., Stimulation of phosphatidylinositol hydrolysis, diacylglycerol release, and gene expression in response to endothelin, a potent new agonist for fibroblasts and smooth muscle cells, J. Biol. Chem., 264 (1989) 8529-8536. 16 Van Renterghem, C., Vigne, P., Barhanin, J., Schmid-Alliana, A., Frelin, C. and Lazdunski, M., Molecular mechanism of action of the vasoconstrictor peptide endothelin, Biochem. Biophys. Res. Commun., 157 (1988) 977-985. 17 Sugiura, M., Inagami, T., Hare, G.M.T. and Johns, J.A., Endothelin action: inhibition by a protein kinase C inhibitor and involvement ofphosphoinositols, Biochem. Biophys. Res. Commun., 158 (1989) 170-176.

18 Rakugi, H., Nakamaru, M.,Tabuchi, Y., Nagano, M., Mikami, H. and Ogihara, T., Endothelin stimulates the release of prostacyclin from rat mesenteric arteries, Biochem. Biophys. Res. Commun., 160 (1989) 924-928. 19 Takayasu, M., Kondo, K. and Terao, S., Endothelin-induced mobilization of Ca 2+ and the possible involvement of platelet activating factor and thromboxane A2, Biochem. Biophys. Res. Commun., 160 (1989) 751-757. 20 Goto, K., Kasuya, Y., Matsuki, N., Takuwa, Y., Kurihara, H., Ishikawa, T., Kimura, S., Yanagisawa, M. and Masaki, T., Endothefin activates the dihydropyridone-sensitive voltage-dependentCa 2 + channel in vascular smooth muscle~ P~oc. Natl. Acad. Sci. USA, 86 (1989) 3915-3918. 21 Itoh, Y., Yanagisawa, M., Ohkubo, S., Kimura, C., Kosaka, T., Inoue, A., Ishida, N., Mitsui, Y., Onda, H., Fuj[n6, M. and Masaki, T., Cloning and sequence analysis of cDNA encoding the precursor of a

18

22

23

24 25

26 27 28 29 30 31 32

33

34 35 36 37 38 39 40

41 42 43 44

human endothelium-derived vasoconstrictor peptide, endothelin: identity of human and porcine endothelin, FEBS Lett. 231 (1988) 440-444. Yanagisawa, M. and Masaki, T., Endothelin, A novel endothelin-derived peptide: Pharmacological activities, regulation and possible roles in cardiovascular control, Biochem. Pharmacol., 38 (1989) 1877-1883. Inoue, A., Yanagisawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K. and Masaki, T., The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes, Proc. Natl. Acad. Sci. USA, 86 (1989) 2863-2867. Guandalini, S., Kachur, J.F., Smith, P.L., Miller, R.J. and Field, M., In vitro effects of somatostatin on ion transport in rabbit intestine, Am. J. Physiol., 238 (1980) G67-G74. Elton, E., Chiossone, D.C., McCafferty, G.P., Ryan, R. and Smith, P.L, SK&F 104353: Selective antagonism of peptidoleukotriene-induced changes in electrolyte transport by rat ileal mucosa in vitro, J. Pharmacol. Exp. Ther., 251 (1989) 484-489. Smith, P.L., Chiossone, D.C. and McCafferty, G.P., Characterization of LTC4 effects on rabbit ileal mucosa invitro, Arch. Pharmacol., 341 (1990) 94-100. Donowitz, M., Cheng, H.Y. and Sharp, G.W.G., Effect of phorbol esters on sodium and chloride transport in rat colon, Am. J. Physiol., 251 (1986) G509-G517. Finley, R.B. and Smith, P.L., Stimulation of chloride secretion by N-formyl-methionyUeucylphenylalanine (FMLP) in rabbit ileal mucosa, J. Physiol., 417 (1989) 403-419. Hojvat, S.A., Musch, M.W. and Miller, R.J., Stimulation of prostaglandin production in rabbit ileal mucosa by bradykinin, J. Pharmacol. Exp. Ther., 226 (1983) 749-755. Lawson, L.D. and Powell, D.W., Bradykinin-stimulated eicosanoid synthesis and secretion by rabbit ileal components, Am. J. Physiol., 252 (1987) G783-G790. Manning, D.C., Synder, S.H., Kachur, J.F., Miller, R.J. and Field, M., Bradykinin receptor-mediated chloride secretion in intestinal function, Nature, 299 (1982) 256-259. Smith, P.L., McCafferty, G.P., Fondacaro, J.D., Wasserman, M.A., Elton, E. and Ryan, F.M., Effects of putative thromboxane receptor agonists and antagonists on rat small intestinal ion transport, J. Pharmacol. Exp. Ther., 247 (1988) 143-149. Smith, P.L., Montzka, D.P., McCafferty, G.P., Wasserman, M.A. and Fondacaro, J.D., Effect of sulfidopeptide leukotrienes D4 and E4 on ileal ion transport in vitro in the rat and rabbit, Am. J. Physiol., 255 (1988) G175-G183. Chiossone, D. C., Simon, P. L. and Smith, P. L., Interleukin-1: effects on sodium and chloride absorption and electrical properties of rabbit ileal mucosa in vitro, Eur. J. Pharmacol., 180 (1990) 217-228. Kachur, J.F., Miller, R.J., Field, M. and Riveir, J., Neurohumoral control of ileal electrolyte transport. II. Neurotensin and substance P, J. Pharmacol. Exp. Ther., 220 (1982) 456-463. McCabe, R. D. and Smith, P. L., Effects of histamine and histamine receptor antagonists on ion transport in rabbit descending colon, Am. J. Physiol., 247 (1984) G411-G418. Tapper, E.J., Powell, D.W. and Morris, S.M., Cholinergic-adrenergic interactions on intestinal ion transport, Am. J. Physiol., 235 (1978) E404-E409. Smith, P.L. and McCabe, R.D., Mechanism and regulation of transcellular potassium transport by the colon, Am. J. Physiol., 247 (1984) G445-G456. Frizzell, R.A., Koch, M.J. and Schultz, S.G., Ion transport by rabbit colon. I. Active and passive components. J. Membr. Biol., 27 (1976) 297-316. Reynolds, E.E., Mok, L.L.S. and Kurokawa, S., Phorbol ester dissocistes endothelin-stimulated phosphoinositide hydrolysis and arachidonic acid release in vascular smooth muscle cells, Biochem. Biophys. Res. Commun., 160 (1989) 868-873. Resink, T.J., Scott-Burden, T. and Buhler, F.R., Endothelin stimulates phospholipase C in cultured vascular smooth muscle cells, Biochem. Biophys. Res. Commun., 157 (1988) 1360-1368. Rae, G.A., Trybulec, M., De Nucci, G. and Vane, J.R., Endothelin-I releases eicosanoids from rabbit isolated perfused kidney and spleen, J. Cardio. Pharmacol., 13 (1989) S89-$92. Musch, M.W., Field, M., Miller, R.J. and Stoff, J. S., Homologous desensitization to prostaglandins in rabbit ileum, Am. J. Physiol., 252 (1987) G120-G127. Van Dyke, K., Peden, D., Van Dyke, C., Jones, G., Gastronova, V. and Ma, J., Inhibition of nonsteroidal

19 antiinflammatory drugs on luminol-dependent human granulocyte chemiluminescence and [3H]FMLP binding, Inflammation, 6 (1982) 113-125. 45 BoRon, J.E. and Field, M., Ca ionophore-stimulated ion secretion in rabbit ileal mucosa: relation to actions of cyclic AMP and carbamylcholine, J. Membr. Biol., 35 (1977) 159-174. 46 Plews, P.I. and Leikauf, G. D., Endothelin stimulates chloride secretion by canine tracheal epithelium, FASEB J., 4 (1990) A352. 47 Brown, M.A. and Smith, P.L., Endothelin: a potent stimulator of intestinal secretion in vitro, FASEB J., 4 (1990) A959.