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Vasoactive intestinal polypeptide: Increased tone, enhancement of acetylcholine release, and stimulation of adenylate cyclase in intestinal smooth muscle
Life Sciences, Vol. 26, pp. 811-822
Pergamon Press
P r i n t e d in t h e U.S.A.
VASOACTIVE INTESTINAL POLYPEPTIDE: INCREASEDTONE, ENHANCEMENTOF ACETYLCHOLINE RELEASE, AND STIMULATION OF ADENYLATECYCLASE IN INTESTINAL SMOOTHMUSCLE Marlene L. Cohen and Ann Schwab Landry L i l l y Research Laboratories Eli L i l l y and Company Indianapolis, Indiana 46285 (Received in f i n a l form January 14, 1980) Summar~ Vasoactive intestinal polypeptide (VIP) was examined in vitro for effects on tone and neuronal release mechanisms in in~slgF6"~T smooth muscle since this is a site of high peptide concentration. VIP contracted the guinea Rig ileum and rabbit Jejunum in concentrations ranging from lO-~ to lO-7 M. Increased tone in the guinea pig ileum was p a r t i a l l y antagonized by the anticholinergic agent, atropine (4.3 x lO -b M) suggesting that one component of the c o n t r a c t i l e response was due to the indirect release of acetylcholine. The H1 receptor antagonist, pyrilamine, did not a l t e r the increased tone produced by VIP indicating that histamine release did not contribute to the ileal contractile response and that VIP exerted a selective effect to enhance neuronal release of acetylcholine. The a b i l i t y of VIP to modulate acetylcholine release was confirmed in f i e l d stimulated ileal preparations where VIP increased the force developed to endogenously released acetylchollne without altering the direct response to acetylcholine. In rabbit jejunum and ileal smooth muscle, VIP elevated cyclic AMP levels. However, inhibition of phosphodiesterase with papaverine did not potentiate either the VIP-induced ileal contraction or enhancement of the f i e l d stimulated response. This raises the possibility that increases in intestinal cyclic AMP may be involved more in VIP-induced alterations in ion transport or secretory phenomenon than in intestinal motility. These studies describing the a b i l i t y of VIP to modulate acetylcholine release and to increase ileal tone are consistent with the proposed role of VIP in intestinal pathologies involving excessive mucous secretion and motility. Vasoactive intestinal polyPeptide (VIP) belongs to a family of structurally related peptides that are widely distributed within the gastrointestinal and nervous systems (1). VIP stimulates several diverse biological systems such as cardiac contraction (2), pancreatic secretion (3), pituitary release of growth hormone, leuteinizing hormone (4), prolactin (5), lipolysis (6), and adenylate cyclase activity (6,7,8,9). Although l i t t l e is established regarding the physiological importance of these biological effects, the high concentrations of VIP in gastrointestinal 0024-3205/80/100811-12502.00/0 Copyright (c) 1980 Pergamon P r ess Lid
812
Effect of VIP in Intestinal Smooth Muscle
Vol. 26, No. i0, 1980
tissue coupled to a possible involvement of VIP in the watery diarrhea syndrome ( l O , l l , 1 2 ) and the defecation reflex (13) prompted the present in v i t r o study of the effects of VIP on intestinal smooth muscle m o t i l i t y . Early studies suggested that VIP did not affect longitudinal ileal smooth muscle function in v i t r o (14), but more recent preliminary reports have demonstrated a c~tr--a'~-Tle response to VIP in such tissue (15). Furthermore, a biphasic relaxant:contractile effect has been observed in canine jejunal smooth muscle (16). In addition to effects on smooth muscle function, VIP stimulated intestinal electrolyte secretion (17) and markedly stimulated intestinal adenylate cyclase (18,1g,20,21,22). Although certain stimulatory actions of VIP have been documented, the a b i l i t y of VIP to enhance acetylcholine release in intestinal smooth muscle has not been examined. In the present study, we examined the in v i t r o contractile effects of VIP on the guinea pig ileum and determined i f V l ~ P ~ t e d any stimulatory effect on acetylcholine release in this tissue. VIP was active on both parameters. In addition, we found that while VIP could elevate cyclic AMP levels in intestinal tissue, this was probably not related to its effects on neuronal acetylcholine re]ease. These studies provide further evidence for effects of VIP on intestinal tissue that are consistent with an involvement of VIP in the defecation reflex and the watery diarrhea syndrome and extend the stimulatory properties of VIP to an effect on neurotransmitter release. Methods Pharmacological Studies. Male or female New Zealand white rabbits (I-2 kg) or Hartley guinea pigs (200-300 g) (Sweetwater Farm Rabbitry, HilIsboro, Ohio), allowed free access to food, were k i l l e d by a blow to the head. Segments (2-3 om long) of rabbit jejunum or guinea pig ileum were mounted longitudinally in lO ml isolated tissue baths containing Krebs' solution of the following composition (millimolar concentrations): NaCI, 118.2; KCI, 4.6; CaCI2.2H20, 1.6; KH2P04, 1.2; MgSO4, 1.2; dextrose, lO.O and NaHCO3, 24.8. In all experiments, tissues were maintained at 37oc and aerated with 95 percent 02 and 5 percent CO2. Guinea pig ilea were mounted between two electrodes, consisting of a stainless steel rod (bottom) and a circular platinum wire (top). Square wave impulses (O.l Hz) of supramaximal voltage (40 V) and 0.7 msec duration were provided by a Grass $44 stimulator. Tissues were equilibrated for I-2 hours at 2 g of applied force. Isometric responses were recorded on Beckman Dynographs. The height of the guinea pig ileum twitch responses or spontaneous jejunal contractions were measured before and after peptide challenge. The percent response after peptide was determined by dividing the contractile height after peptide by the height before peptide administration multiplied by lO0. Statistical analysis utilized Student's t test and significant differences between mean values were assumed when p < 0.05. Biochemical Studies. Sections of rabbit jejunum or rabbit i l e a l tissue divided into mucosa or longitudinal muscle by the method of Rang (23) were cleaned and placed in Krebs' bicarbonate buffer (lO ml) (see above for composition). Tissues were maintained at 37oc, aerated with 95 percent 02 and 5 percent C02, and allowed to equilibrate for 30 min before challenge with VIP for lO minutes. In some experiments, isobutylmethylxanthine was added 5 min prior to the VIP. At the end of each experin~nt, tissues were rapidly removed, rinsed in buffer, blotted, and quick-frozen on dry ice. Tissues were weighed and stored at -70oc until assayed.
Vol. 26, No. i0, 1980
Effect of VIP in Intestinal Smooth Muscle
813
For assay of cyclic nucleotides, tissues were homogenized in ground glass homogenizers at 4oc in a total of 4.0 ml 9f 5 percent trichloroacetic acid. Trace amounts of both 3H-cAMP and JH-cGMP (4000-9200 dpm) were added to monitor loss of nucleotides during tissue processing. After centrifugation at 9go g for 20 min and ether extraction of the supernatant, cyclic AMP was isolated by passing the supernatant through anion exchange columns and eluting with 5 ml of 2 N formic acid. After suitable column washes, cyclic GMP was recovered by eluting with 7 ml of 4 N formic acid. The eluates were lyophylized and cyclic AMP or cyclic GMP resuspended in appropriate volumes of 50 mM sodium acetate, pH 6.2. Cyclic AMP and cyclic GMP were assayed by the method of Steiner et al. (24) as modified by the acetylation procedure of Harper and Brooke~'-(~'). Antiserum binding was used to separate bound from free cyclic AMP. Protein was determined in duplicate by the method of Lowry et al. (26) using bovine serum albumin standards. The source of the drugs used was as follows: vasoactive intestinal polypeptide (VIP; Peninsula Laboratories, San Carlos, CA); papaverine hydrochloride (Eli L i l l y and Company, Indianapolis, IN); 3-isobutyl-l-methylxanthine (Aldrich Chemical Co., Milwaukee, WI); acetylcholine chloride, histamine dihydrochloride, atropine sulfate, and bacitracin (Sigma Chemical Company, St. Louis, MO); pyrilamine maleate (ICN Pharmaceuticals, Irvine, CA). The source of the reagents for the cyclic AMP assay was as follows: AG I-X8 anion exchange resin (200-400 mesh, formate form) (Bio-Rad Laboratories, Richmlmd, CA); ~ll-adenosine-3',S~-cycllc phosphate, $pec. act. 23 Ci/mmol (SchwarzlMann, Orangeburg, NY); Jfl-guanosine-3',5'-cycllc phosphate, spec. act. 21Cl/uanl (Amersham/Searle, Arlington Heights, IL); bovine albumin (Armour Pharmaceutical Co., Phoenix, AZ); Aqua$ol, acetic anhydride, iethylamine, normal rabbit serum, cyclic ~ standard, cyclic ~ standard, h~I-ScAMP antigen, spec. act. 150 Ci/mol, cyclic AMP anti- serum, 1251-ScGMP antigen, spec. act. ISO Ci/mmo1., and cyclic GMP anti- serum (New England Nuclear, Boston, MS).
~
Results VIP-Induced Contraction of Intestinal Smooth Muscle. VIP increased tone in the guinea pig ileum in concentrations of 10-g to 10 -7 M (Figure I). At the higher concentrations used, the increase in tone was accompanied by initiation of phasic activity in the ileum. VIP also produced a contractile response in the rabbit jejunum, a tissue that exhibits rhythmic contract13e activity (Figure 2). In the Jejunum, low concentrations of VIP increased the height of the phasic activity, whereas higher concentrations increased tone. Since both acetylcholtne and histamine are endogenous c o n t r a c t f l e agents found in the guinea pig ileum, we investigated the p o s s i b i l i t y that VIP might be contracting the ileum by an indirect mechanism involving acetylcholine and/or histamine release. In the presence of a concentration of atropine (4.3 x 10-0 M) that abolished the twitch response to f i e l d stimulation and the response to 1 ug/ml acetylcholtne, there was a s i g n i f i c a n t reduction in the increased tone generated by VIP (Table 1). The antagonism of the VIP-tnduced tone by atropine was most apparent at low concentrations of VIP and even with this r e l a t i v e l y high concentration of atropine, a c o n t r a c t i l e response to VIP s t i l l persisted. The i n i t i a t i o n of phasic a c t i v i t y in the ileum seen with VIP was blocked after atropine. Thus, VIP-tnduced contraction of the guinea pig ileum is due in part to release of
Effect of VIP in Intestinal Smooth Muscle
814
Vol. 26, No. I0, 1980
acetyicholine. Pyrilamine (IO-6M), an Hl antagonist, in a concentration that abolished the contractile response tQ histamine (3 x 10-7 M) did not alter the contraction to VIP (lO -v to lO-/M), suggesting that histamine release is not involved in the contractile response of the guinea pig ileum to VIP (data not shown).
VIP
CONTllULCTILE FORCE
10-s
3x10- I
0.22"11.06 1.32"0.0/
104
II.llZ"0Jl?
3xi0-4
10-7
8.13"0.011
!.!!i-*11.11
(emul i.---.,4
REPflESENTATIVE oYIiOORAPH RECOROING
FIG. 1 Concentration-response of the guinea pig ileum to VIP. VIP was added to ileal preparations in the concentrations indicated at the top. Values in the middle column represent mean forces developed to VIP + S.E. for Ig ileal preparations. A representative response of one guinea pig lleum for each concentration of VIP is depicted on the bottom. VIP was added at the arrows and W indicates replacement of the Krebs' buffer with buffer containing no drug,
VIP 10-e M
VIP 1 minute
3x10-SM
VIP 10-7 M
FIG. 2 Concentration-response of the rabbit jejunum to VIP. Dynograph recording of the spontaneous contractions of the rabbit jejunum after increasing concentrations of VIP. W indicates replacement of the Krebs' buffer with buffer containing no drug. Effect of VIP on the Response of the Guinea Pig Ileum to Field Stimulation. In addition to contracting the ileum from baseline force, VIP (I0 -9 to
(6)
O*
0.27_*0.09
O*
0.41+_0.08
10 -8
O*
--
--
0.51_+0.14
O*
1.59_+0.48
0.28+0.15 *
0.87_+0.13
Contractile Force (grams)a
3 x 10 -9
--
--
0.83+_0.14
1.16_+0.14
3 x 10 -8
(VIP in M; Ach in glml)
O*
3.22+_0.92
0 . 9 9 +0.14
1.24_+0-12
10 -7
a Values are means + S.E. for the number of tissues indicated in parentheses. b After control responses to VIP and acetylcholtne were obtained, tissues were incubated with atropine for 20 minutes and responses to VIP and acetylcholtne were repeated. * Values d i f f e r s i g n i f i c a n t l y (p
After Atropine (4) b (4.3 x 10 -b H)
Acetylcholine
After Atropine (5) b (4.3 x 10 -b M)
VIP ( l )
10 -9
Concentration
Effect of Atropine on Contraction of the Guinea Pig Ileum to VIP and Acetylcholine
TABLE I
0*
5.05+1.16
10-6
~n
fD
n
X
0 Tt ~r
g
C/3
m
m Tt
o
rt
Pl P-h
0
Z 0
0
816
Effect of VIP in Intestinal Smooth Muscle
Vol. 26, No. I0, 1980
10-7 M) also increased the response to f i e l d stimulation (Figures 3 and 4). At a time Hen the response to f i e l d stimulation was increased, the contractile response to acetylcholine was unaltered by VIP (Figure 3). Thus, VIP can specifically enhance the guinea pig ileum response to f i e l d stimulation suggesting the VIP exerts a neuronal effect to increase release of acetylcholine.
CONTROLRESPONSE RESPONSETOACETYLCHOUNE TO ACETYLCHOUNE IN THE PRESENCEOF VIP (3xlO4M) A
O I nanlte
t
ACk'TYLC)'iOLIN£ 10 n41/mi
t
1
VI'P AC1ETYLCNOLIN£ 3~IO-OM 10 141/rid
FIG. 3 Effect of VIP on response of the guinea pig ileum to f i e l d stimulation and to the direct application of acetylcholine. Dynograph recording of tissue that was stimulated with square wave Impulses at 40 V, 0.1Hz and for 0.7 msec duration. Stimulation was stopped 30 seconds prior to acetylcholine challenge. A: Control response to acetylcholine (10 ng/ml); B: VIP (3 x lO-8 M) was given approximately 1 minute prior to acetylcholine (10 nglm)) challenge. W indicates replacement of the Krebs' buffer with buffer containing no drugs.
120-
~mMCN~)
8O-
4O"
VAtmAClIMEWalIIIINIIALPOLYIIqEPTIO!Imdarl
FIG. 4 Concentration-response of the field-stimulated guinea pig ileum to VIP in the absence (open circles) and presence (closed circles) of bacitracin (2 x 10-5 M). Points represent mean increases in twitch height + S.E. for the number of tissues indicated in parentheses. To examine the p o s s i b i l i t y that contraction of the guinea pig ileum caused the enhancement of f i e l d stimulated responses, we evaluated the a b i l i t y of histamine and acetylcholine, two known contractile agonists to alter the response to f i e l d stimulation. Neither agent s i g n i f i c a n t l y
Vol. 26, No. i0, 1980
Effect of VIP in I n t e s t i ~ l Smooth Muscle
817
increased the response to field stimulation in the guinea pig ileum (data not shown). Thus, enhancement of acetylcholine release by VIP is independent of its a b i l i t y to induce tone in the guinea pig ileum. Addition of bacitracin (2 x lO-5 M) to the buffer has been suggested to be useful in optimizing responses to peptides (5,27). Although we obtained good responses in the absence of bacitracin, we repeated experiments of VIP enhancement of the responses to field stimulation in the presence and absence of bacitracin (2 x I0 -~ M). The response to VIP was increased about 30 percent when bacitracin was present in the buffer (Figure 4). Role of VIP to Increase Intestinal Levels of C@clic AMP and C~clic GMP. VlP (lO -6 M) elevated both cyclic AMP (30 percent) and cyclic C~MP(24 percent) levels in the rabbit Jejunum (Table 2). In the presence of phosphodiesterase inhibition with isobutylmethylxanthine, VIP elevated cyclic nucleotide levels even further with a small increase in the percent elevation to 44 and 58 percent for cyclic AMP and cyclic GMP, respectively. VIP increased cyclic AMP in mucosal tissue from the rabbit ileum as well (Table 2). In this tissue, however, cyclic GMP levels were unaltered. Similar results were obtained in preliminary experiments with two tissues of longitudinal muscle from the rabbit ileum (data not shown). Since VIP increased the force of spontaneous contractions of the rabbit Jejunum and the height of the response to f i e l d stimulation in ileal smooth muscle, we asked i f such effects might be mediated by the elevations in cyclic nucleotide levels that occur after VIP. We reasoned that i f the iBcrease in ileal twitch height were related to activation of adenylate cyclase by VIP, then phosphodiesterase inhibition should potentiate this response to VIP. For thgse studies, we used the phosphodiesterase inhibitor, papaverine, which at lO-~ M produced a measurable 33 percent (n-g) and 90 percent (n-6) elevation in jejunal cyclic AMP and cyclic GMP levels, respectively. Because papaverine i t s e l f is a smooth muscle relaxant that produced a 14 percent (n.6) and 68 p~rcent (n.7) reduction in the response to f i e l d stimulation at 3 x lO-° M and 3 x lO-OM, respectively, a potential effect on VIP-induced responses was examined at both concentrations. VIP-induced enhancement of the response to f i e l d stimulation in the guinea pig ileum was not potentiated by either concentration of papaverine (Table 3). In fact, papaverine at 3 x lO-5 M co,~)letely inhibited the response to VIP presumably due to a nonspecific relaxant effect of papaverine. Discussion VIP exerts multiple in vitro effects on intestinal smooth muscle that might be related to its p r o p o ~ involvement in certain pathologies of the gastrointestinal tract. The present study describes a contractile effect, a presynaptic action to increase neurotransmitter release and an activation of intestinal adenylate cyclase. VlP increased tone in the guinea pig ileum and increased the rhythmic contractile force of the rabbit jejunum. These results confirm previous reports of VIP-induced contractile responses in intestinal smooth muscle (15,16). In contrast, in vitro relaxant or inhibitory effects of VIP have been reported in other ~ o ~ s c l e preparations such as the guinea pig gallbladder (28), mouse vas deferens (29), guinea pig taenia coli (30,31) and rat portal vein (unpublished observation). In the present experiments, the ileal contraction produced by VIP was p a r t i a l l y antagonized by atropine.
I0.46~0.41 13.3+1.48 19.18+2.15
VIP (lO -6 M) (9)
Isob~tylmethyixanthine (g)c (10 -4 M)
VIP (10.6 M) (g)
19.93÷1.01 19.45+0.88 29.99+1.69
VIP (10 -6 M) (6)
Isobutylmethylxanthine (6) c (10 ~ M)
VIP (10 .6 M) (6)
54.9+6.3 **
52.9+10.0"
43.8+11.3"*
31.3+4.4"
% Increase
0.384+0.11
0.428+0.10
0.181+0.19
0.171+0.04
0.457+0.05
0.292+0.03 -
0.164+0.02
0.130+0.01
Cyclic GMPa (pmoleslmcj protein)
-10.7+18.2
4.1÷10.1
58.4÷17.4"*
23.7+7.2*
% Increase
a Cyclic AMP and cyclic GMP were measured by the radtoimunoassay technique of Steiner et al. (24). Values are means + S. E. b Sections of intact- rabbit jejunum or rabbit ileal mucosa separated from the longitudinal ileal muscle by the procedure of Rang (23) were used. The nugg)er of tissues is indicated in parentheses. c Isobutylmethylxanthine was added to the incubation buffer 5 minutes prior to challenge with VIP. * Values significantly (p
13.19+1.18
Control (6)
Rabbit Ileal l~cosa: b
7.96+0.26
Centrol (9)
Rabbit Jejunum:b
Cyclic AMPa (pmoleslm¢j protein)
Effect of VIP on Cyclic AMP and Cyclic GMP Levels in Rabbit Jejunum and lleal Mucosa
TABLE 2
Oo O
t-J
Z 0
t~
0
f)
0 I'+ =r
k~
g~
rt
0
(3 rt
M t~ t~
O0
O0
m
_0.5+2.4(4)c*
5.3+_2.4(4)*
4.3+_4.0(4)*
40.6+_8.5(4)
48.2+_8.2(8)
10-8
6.3"+2.9(4)*
50.5-+11.0(4)
51.3"+!i.2(8)
3 x 10-8
Values are
-0.3-+Z./(4)c*
54.9+_13.5(4)
85.0+21.6(8)
10-7
b Papav~ine was incorporated into the Krebs' buffer containing bacitracln (2 x 10-5 M) during equilibration and for the duration of the experiment. c Negative sign indicates the percent decrease In twitch height. • Values differ significantly (p
a Tissues were stimulated with square wave impulses (O.1 Hz) at 40 V and 0.7 msec duration. means + S.E. for the number of tissues indicated in parentheses.
(3 x 10-5 M)
Papaverine b
1.34+0.08(20)*
21.7+-4.4(4)
12.1_+1.6(4)
1.85+o.08(2o)*
Papaverineb
(3 x 10 -6 M)
22.3+_4. o(e)
6.5+_4.8(8)
3 x 10 -9
2.20+0.10(40)
10 -9
VIP (Molar)
Percent Increase in Twitch Heighta
Control
Initial Twitch Height (g)a
Effect of VIP on the Field Stimulated Response of the Guinea Pig Ileum in the Presence and Absence of Papaverine
TABLE 3
~D
Oo
rJ l-a ill
o r$
#-m
re It II r~
P-4
o
fo
pl v~
0
~a
;-a 0
ON,
820
Effect of VIP in Intestinal Smooth Muscle
%'ol. 26, No. i0, 1980
Although these data are consistant with a direct interaction of VIP with the muscarinic receptor in the ileum, more l i k e l y , antagonism of the VIP-induced contraction by atropine indicates that VIP may act to release acetylcholine from neural elements in the ileum. A similar conclusion was reached with studies in the ileum using tetrodotoxin (15) and studies on intestinal absorption and blood flow with atropine (32). Since atropine did not completely abolish contractions to VIP, additional mechanisms for VIP-induced contraction must be considered. Studies with the Hl antagonist, pyrilamine, indicate that histamine release is not involved in this contractile response. VIP may be acting to contract the ileum by release of other contractile agents or by a direct mechanism in addition to the neuronally mediated cholinergic action. Further evidence for a stimulatory effect of VIP on neuronal release of acetylcholine in the ileum was derived from studies with the f i e l d stimulated ileum. VIP, in a concentration related fashion enhanced the responses to f i e l d stimulation. This effect was clearly presynaptic since responses to the direct administration of acetylcholine or histamine were unaltered. Consistent with this observation, a small (less than lO percent) potentiation of guinea pig detrusor responses to nerve stimulation has recently been reported for VIP (31). Thus, VIP joins the expanding l i s t of peptides such as the enkephalins (33), angiotensin (34,35) and somatostatin (29,36,37,38) that can modulate presynaptic neurotransmitter release mechanisms. In fact, modulatlon of transmitter release may represent the principle mechanism by which polypeptide hormones affect biological systems. In addition to these effects on smooth muscle function and neuronal release mechanisms, VIP is known to activate adenylate cyclase in broken cell preparations of intestinal origin (18,1g,20,21,22). Our studies confirm this a c t i v i t y and show that activation of adenylate cyclase by VIP occurs in intact intestinal tissue and may be accompanied by increases in cyclic GMP as occurred in rabbit jejunal tissue, although elevations in cyclic GMP may not occur in all tissues. The increases in cyclic AMP induced by VIP in intestinal smooth muscle are unlikely to be related to its effects on neurotransmitter release or ileal contractile mechanisms for the following reasons: 1) Other agents that elevate cyclic nucleotide levels do not produce similar effects; 2) I f cyclic AMP were mediating the effects of VIP, then we would anticipate an enhanced response to VIP after inhibition of phosphodiesterase. This did not occur. Certainly other explanations may be proposed for the i n a b i l i t y of these indirect experiments to reveal a positive correlation to elevations in cyclic AMP. Although further studies must be done in this regard, i t is possible that VIP-induced increases in cyclic AMP more closely relate to its effects on ion transport of intestinal secretion (18,20) than on its a b i l i t y to affect smooth muscle tone or neurotransmitter release. Nevertheless, the present studies document marked effects of VIP in intestinal smooth muscle function to promote increased muscle tone. VIP containing nerves are abundant throughout the gastrointestinal tract (39) and our studies suggest that such nerves may serve to modulate (by enhancement) neurotransmitter release in intestinal smooth muscle. This hypothesis is consistent with an involvement of VIP in the defecation reflex and intestinal pathologies associated with excessive diarrhea such as Crohn's disease (39) and the watery diarrhea syndrome (]O,]l,12).
V o l . 2 6 , No. 10, 1980
Effect
o f VIP i n l n t e s t l n a l
Smooth M u s c l e
821
References I. 2. 3. 4. 5. 6. 7. 8.
9. lO. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36.
M. G. BRYANT, S. R. BLOOM, R. H. ALBUQUERQUE, J. M. POLAK and I. MODLIN, Gut. 17 394 (1976). ~I.~ID, L. P. BOSHER, J. A. SPATH and H. A. KONTOS, Clin. Res. 20 29 (1972). S. J. KONTUREK, A. PUCHERand T. RADECKI, J. Physiol. 255 497-509 (1976). E. VIJAYAN, W. K. S/V~SON, S. I. SAID and S. M. ~CCANN,~docrinolog,v. I04 53-57 (1979). ~RUBERG, W. H. ROTSZTEJN, S. ARANCIBIA, J. BESSONand A. ENJALBERT, Eur. J. Pharm. 51 319-320 (1978). Y. SAITO, N. MA~UOKA, K. SHIRAI, M. YAMAMOTO,A. KUMAGAI and N. YANAIHARA, Endocrinol. Japon. 25 403-405 (1978). E. K. FRANDSEN, G. A. KRISHNA~ d S. I. SAID, Br. J. Pharmac. 62 367-369 (1978). M. QUIK, L. L. IVERSEN and S. R. BLOOM, Biochem. Pharmacol. 27 2209-.2213 (1978). C. BORGHI, S. NICOSIA, A. GIACHETTI and S. SAID, Life Sci. 24 65-70 (1979). S. R. BLOOM, J. M. POLAK and A. G. E. PEARSE, Lancet. 2 14-16 (1973). S. I. SAID and G. R. FALOONA, N. Engl. J. M e d . ~ 5 5 - 1 6 0 (1975). E. J. BURBIGE, R. J. MANNINGa ~ , ~ J. Gastroenterolog.v. 70 136-140 (1978). ~ FAHRENKRUG, H. GALBO, J. J, MOLSTand O. B. SCHAFFALITZKYDE MUCI(ADELL, J. Physiol. 280 405-422 (197B). P. J. PIPER, S. "I. $AID~d J. R. VANE, Nature. 225 I144-I146 (1970). S. S. 3AFFER, J. T. FARRAR, W. M. YAU a n ~ . lglX]THLOUF, ~ . 66 716 (1974). 3. KACHELHOFFER, C'~-~NDEL, J. DAUCHEL, D. HOHMATTERand J. F. GRENIER, Digestive Diseases. 21 957-962 (1976). G. O. BARBEZATand ~ I . GROSSMAN, Science. 174 422-424 (1971). C. J. SCHWARTZ, D. V. KIMBERG, H. E."~'FIL~'ITTN,T. FIELD and S. I. SAID, J. Clin. Invest. 54 536-544 (1974). ~ , ~ P. CONLON, A. G. LEVY and J. D. GARDNER, Gastroenterology. 68 667-675 (1975). D. B. WALDMAN, J. ~ GARDNER, A. M. ZFASS and G. M. MAKHLOUF, Gastroenterology. 73 518-523 (1977). B. SIMON and H. KA~ER, ~ . 74 722-725 (1978). B. AMIRANOFF, M. LABURTHE, C. DUPONT, and ~.. ROSSELIN, Biochimica et Bioph~sica Acta. 544 474-481 (1978). ___ H. P. RANG, Br. J.--ffharmaco1. 22 356-3bb (1964). A. L. STEINER, C. W. PARKERanTD. M. KIPNIS, J. Biol. Chem. 247 1106-I113 (1972). J. F. HARPERand G. BROOKER, J .C~clicL. Nucleotide Res. 1:207-218, 1975. O.H. LOWRY, N. J. ROSEBROUGH, A FARR and R. J. RANDXLL, J. Biol. Chem. 193 265-275 (1957). l.-~-~. ~RSEN, S. D. IVERSEN, F. E. BLOOM, T. VARGOand R. GUILLEMIN, Nature. 271 679-681 (1978). ?J~-l~--RYXIT-and S. RYAVE, Am. J. Physiol. 234 E44-E46 (1978). A. J. KASTIN, D. H. COY, A. V. $CH~LLY an~]--C. A. MEYERS, Pharmacol. Biochem. Behavior. g 673-676 (1978). T. COCKSand G. BURNSTOCK, Eur. J. Pharn~col. 54 251-259 (1979). A. JOHNS, Can. J. Physiol. Pharmacol. 57 106-I~ (1979). D. MAILMAN, J. Physiol. 279 121-132 (l~J'~8). J. HUGHES, Brain l~es. 88-'J~]5-308 (1975). P. J. KADOWI~'~-I~--~. ~EET and M. J. BRODY, J. Pharmacol. Exp. Ther. 176 167-173 (1971). ~ B E L L , Circ. Res. 31 348-355 (1972). R. GUILLEMIN, EndocrT~ology. 9_991653-1654 (1976).
822
37. 38. 39.
Effect of VIP in Intestinal Smooth Muscle
Vol. 26, No. I0, 1980
M. L. COHEN, E. ROSING, K. S. WILEY and I. H. SLATER, Life Sci. 23 1659-1664 (1978). J, MAGNAN, D. REGOLI, R. QUIRION, S. LEMAIRE, S. ST-PIERRE and F. RIOUX, Eur. J. Pharmacol. 55 347-354 (1979). J. M. POLAK and 5. ~ BLOOM, Adv. Exp. Med. Biol. I06 27-64 (1978).
Pergamon Press
P r i n t e d in t h e U.S.A.
VASOACTIVE INTESTINAL POLYPEPTIDE: INCREASEDTONE, ENHANCEMENTOF ACETYLCHOLINE RELEASE, AND STIMULATION OF ADENYLATECYCLASE IN INTESTINAL SMOOTHMUSCLE Marlene L. Cohen and Ann Schwab Landry L i l l y Research Laboratories Eli L i l l y and Company Indianapolis, Indiana 46285 (Received in f i n a l form January 14, 1980) Summar~ Vasoactive intestinal polypeptide (VIP) was examined in vitro for effects on tone and neuronal release mechanisms in in~slgF6"~T smooth muscle since this is a site of high peptide concentration. VIP contracted the guinea Rig ileum and rabbit Jejunum in concentrations ranging from lO-~ to lO-7 M. Increased tone in the guinea pig ileum was p a r t i a l l y antagonized by the anticholinergic agent, atropine (4.3 x lO -b M) suggesting that one component of the c o n t r a c t i l e response was due to the indirect release of acetylcholine. The H1 receptor antagonist, pyrilamine, did not a l t e r the increased tone produced by VIP indicating that histamine release did not contribute to the ileal contractile response and that VIP exerted a selective effect to enhance neuronal release of acetylcholine. The a b i l i t y of VIP to modulate acetylcholine release was confirmed in f i e l d stimulated ileal preparations where VIP increased the force developed to endogenously released acetylchollne without altering the direct response to acetylcholine. In rabbit jejunum and ileal smooth muscle, VIP elevated cyclic AMP levels. However, inhibition of phosphodiesterase with papaverine did not potentiate either the VIP-induced ileal contraction or enhancement of the f i e l d stimulated response. This raises the possibility that increases in intestinal cyclic AMP may be involved more in VIP-induced alterations in ion transport or secretory phenomenon than in intestinal motility. These studies describing the a b i l i t y of VIP to modulate acetylcholine release and to increase ileal tone are consistent with the proposed role of VIP in intestinal pathologies involving excessive mucous secretion and motility. Vasoactive intestinal polyPeptide (VIP) belongs to a family of structurally related peptides that are widely distributed within the gastrointestinal and nervous systems (1). VIP stimulates several diverse biological systems such as cardiac contraction (2), pancreatic secretion (3), pituitary release of growth hormone, leuteinizing hormone (4), prolactin (5), lipolysis (6), and adenylate cyclase activity (6,7,8,9). Although l i t t l e is established regarding the physiological importance of these biological effects, the high concentrations of VIP in gastrointestinal 0024-3205/80/100811-12502.00/0 Copyright (c) 1980 Pergamon P r ess Lid
812
Effect of VIP in Intestinal Smooth Muscle
Vol. 26, No. i0, 1980
tissue coupled to a possible involvement of VIP in the watery diarrhea syndrome ( l O , l l , 1 2 ) and the defecation reflex (13) prompted the present in v i t r o study of the effects of VIP on intestinal smooth muscle m o t i l i t y . Early studies suggested that VIP did not affect longitudinal ileal smooth muscle function in v i t r o (14), but more recent preliminary reports have demonstrated a c~tr--a'~-Tle response to VIP in such tissue (15). Furthermore, a biphasic relaxant:contractile effect has been observed in canine jejunal smooth muscle (16). In addition to effects on smooth muscle function, VIP stimulated intestinal electrolyte secretion (17) and markedly stimulated intestinal adenylate cyclase (18,1g,20,21,22). Although certain stimulatory actions of VIP have been documented, the a b i l i t y of VIP to enhance acetylcholine release in intestinal smooth muscle has not been examined. In the present study, we examined the in v i t r o contractile effects of VIP on the guinea pig ileum and determined i f V l ~ P ~ t e d any stimulatory effect on acetylcholine release in this tissue. VIP was active on both parameters. In addition, we found that while VIP could elevate cyclic AMP levels in intestinal tissue, this was probably not related to its effects on neuronal acetylcholine re]ease. These studies provide further evidence for effects of VIP on intestinal tissue that are consistent with an involvement of VIP in the defecation reflex and the watery diarrhea syndrome and extend the stimulatory properties of VIP to an effect on neurotransmitter release. Methods Pharmacological Studies. Male or female New Zealand white rabbits (I-2 kg) or Hartley guinea pigs (200-300 g) (Sweetwater Farm Rabbitry, HilIsboro, Ohio), allowed free access to food, were k i l l e d by a blow to the head. Segments (2-3 om long) of rabbit jejunum or guinea pig ileum were mounted longitudinally in lO ml isolated tissue baths containing Krebs' solution of the following composition (millimolar concentrations): NaCI, 118.2; KCI, 4.6; CaCI2.2H20, 1.6; KH2P04, 1.2; MgSO4, 1.2; dextrose, lO.O and NaHCO3, 24.8. In all experiments, tissues were maintained at 37oc and aerated with 95 percent 02 and 5 percent CO2. Guinea pig ilea were mounted between two electrodes, consisting of a stainless steel rod (bottom) and a circular platinum wire (top). Square wave impulses (O.l Hz) of supramaximal voltage (40 V) and 0.7 msec duration were provided by a Grass $44 stimulator. Tissues were equilibrated for I-2 hours at 2 g of applied force. Isometric responses were recorded on Beckman Dynographs. The height of the guinea pig ileum twitch responses or spontaneous jejunal contractions were measured before and after peptide challenge. The percent response after peptide was determined by dividing the contractile height after peptide by the height before peptide administration multiplied by lO0. Statistical analysis utilized Student's t test and significant differences between mean values were assumed when p < 0.05. Biochemical Studies. Sections of rabbit jejunum or rabbit i l e a l tissue divided into mucosa or longitudinal muscle by the method of Rang (23) were cleaned and placed in Krebs' bicarbonate buffer (lO ml) (see above for composition). Tissues were maintained at 37oc, aerated with 95 percent 02 and 5 percent C02, and allowed to equilibrate for 30 min before challenge with VIP for lO minutes. In some experiments, isobutylmethylxanthine was added 5 min prior to the VIP. At the end of each experin~nt, tissues were rapidly removed, rinsed in buffer, blotted, and quick-frozen on dry ice. Tissues were weighed and stored at -70oc until assayed.
Vol. 26, No. i0, 1980
Effect of VIP in Intestinal Smooth Muscle
813
For assay of cyclic nucleotides, tissues were homogenized in ground glass homogenizers at 4oc in a total of 4.0 ml 9f 5 percent trichloroacetic acid. Trace amounts of both 3H-cAMP and JH-cGMP (4000-9200 dpm) were added to monitor loss of nucleotides during tissue processing. After centrifugation at 9go g for 20 min and ether extraction of the supernatant, cyclic AMP was isolated by passing the supernatant through anion exchange columns and eluting with 5 ml of 2 N formic acid. After suitable column washes, cyclic GMP was recovered by eluting with 7 ml of 4 N formic acid. The eluates were lyophylized and cyclic AMP or cyclic GMP resuspended in appropriate volumes of 50 mM sodium acetate, pH 6.2. Cyclic AMP and cyclic GMP were assayed by the method of Steiner et al. (24) as modified by the acetylation procedure of Harper and Brooke~'-(~'). Antiserum binding was used to separate bound from free cyclic AMP. Protein was determined in duplicate by the method of Lowry et al. (26) using bovine serum albumin standards. The source of the drugs used was as follows: vasoactive intestinal polypeptide (VIP; Peninsula Laboratories, San Carlos, CA); papaverine hydrochloride (Eli L i l l y and Company, Indianapolis, IN); 3-isobutyl-l-methylxanthine (Aldrich Chemical Co., Milwaukee, WI); acetylcholine chloride, histamine dihydrochloride, atropine sulfate, and bacitracin (Sigma Chemical Company, St. Louis, MO); pyrilamine maleate (ICN Pharmaceuticals, Irvine, CA). The source of the reagents for the cyclic AMP assay was as follows: AG I-X8 anion exchange resin (200-400 mesh, formate form) (Bio-Rad Laboratories, Richmlmd, CA); ~ll-adenosine-3',S~-cycllc phosphate, $pec. act. 23 Ci/mmol (SchwarzlMann, Orangeburg, NY); Jfl-guanosine-3',5'-cycllc phosphate, spec. act. 21Cl/uanl (Amersham/Searle, Arlington Heights, IL); bovine albumin (Armour Pharmaceutical Co., Phoenix, AZ); Aqua$ol, acetic anhydride, iethylamine, normal rabbit serum, cyclic ~ standard, cyclic ~ standard, h~I-ScAMP antigen, spec. act. 150 Ci/mol, cyclic AMP anti- serum, 1251-ScGMP antigen, spec. act. ISO Ci/mmo1., and cyclic GMP anti- serum (New England Nuclear, Boston, MS).
~
Results VIP-Induced Contraction of Intestinal Smooth Muscle. VIP increased tone in the guinea pig ileum in concentrations of 10-g to 10 -7 M (Figure I). At the higher concentrations used, the increase in tone was accompanied by initiation of phasic activity in the ileum. VIP also produced a contractile response in the rabbit jejunum, a tissue that exhibits rhythmic contract13e activity (Figure 2). In the Jejunum, low concentrations of VIP increased the height of the phasic activity, whereas higher concentrations increased tone. Since both acetylcholtne and histamine are endogenous c o n t r a c t f l e agents found in the guinea pig ileum, we investigated the p o s s i b i l i t y that VIP might be contracting the ileum by an indirect mechanism involving acetylcholine and/or histamine release. In the presence of a concentration of atropine (4.3 x 10-0 M) that abolished the twitch response to f i e l d stimulation and the response to 1 ug/ml acetylcholtne, there was a s i g n i f i c a n t reduction in the increased tone generated by VIP (Table 1). The antagonism of the VIP-tnduced tone by atropine was most apparent at low concentrations of VIP and even with this r e l a t i v e l y high concentration of atropine, a c o n t r a c t i l e response to VIP s t i l l persisted. The i n i t i a t i o n of phasic a c t i v i t y in the ileum seen with VIP was blocked after atropine. Thus, VIP-tnduced contraction of the guinea pig ileum is due in part to release of
Effect of VIP in Intestinal Smooth Muscle
814
Vol. 26, No. I0, 1980
acetyicholine. Pyrilamine (IO-6M), an Hl antagonist, in a concentration that abolished the contractile response tQ histamine (3 x 10-7 M) did not alter the contraction to VIP (lO -v to lO-/M), suggesting that histamine release is not involved in the contractile response of the guinea pig ileum to VIP (data not shown).
VIP
CONTllULCTILE FORCE
10-s
3x10- I
0.22"11.06 1.32"0.0/
104
II.llZ"0Jl?
3xi0-4
10-7
8.13"0.011
!.!!i-*11.11
(emul i.---.,4
REPflESENTATIVE oYIiOORAPH RECOROING
FIG. 1 Concentration-response of the guinea pig ileum to VIP. VIP was added to ileal preparations in the concentrations indicated at the top. Values in the middle column represent mean forces developed to VIP + S.E. for Ig ileal preparations. A representative response of one guinea pig lleum for each concentration of VIP is depicted on the bottom. VIP was added at the arrows and W indicates replacement of the Krebs' buffer with buffer containing no drug,
VIP 10-e M
VIP 1 minute
3x10-SM
VIP 10-7 M
FIG. 2 Concentration-response of the rabbit jejunum to VIP. Dynograph recording of the spontaneous contractions of the rabbit jejunum after increasing concentrations of VIP. W indicates replacement of the Krebs' buffer with buffer containing no drug. Effect of VIP on the Response of the Guinea Pig Ileum to Field Stimulation. In addition to contracting the ileum from baseline force, VIP (I0 -9 to
(6)
O*
0.27_*0.09
O*
0.41+_0.08
10 -8
O*
--
--
0.51_+0.14
O*
1.59_+0.48
0.28+0.15 *
0.87_+0.13
Contractile Force (grams)a
3 x 10 -9
--
--
0.83+_0.14
1.16_+0.14
3 x 10 -8
(VIP in M; Ach in glml)
O*
3.22+_0.92
0 . 9 9 +0.14
1.24_+0-12
10 -7
a Values are means + S.E. for the number of tissues indicated in parentheses. b After control responses to VIP and acetylcholtne were obtained, tissues were incubated with atropine for 20 minutes and responses to VIP and acetylcholtne were repeated. * Values d i f f e r s i g n i f i c a n t l y (p
After Atropine (4) b (4.3 x 10 -b H)
Acetylcholine
After Atropine (5) b (4.3 x 10 -b M)
VIP ( l )
10 -9
Concentration
Effect of Atropine on Contraction of the Guinea Pig Ileum to VIP and Acetylcholine
TABLE I
0*
5.05+1.16
10-6
~n
fD
n
X
0 Tt ~r
g
C/3
m
m Tt
o
rt
Pl P-h
0
Z 0
0
816
Effect of VIP in Intestinal Smooth Muscle
Vol. 26, No. I0, 1980
10-7 M) also increased the response to f i e l d stimulation (Figures 3 and 4). At a time Hen the response to f i e l d stimulation was increased, the contractile response to acetylcholine was unaltered by VIP (Figure 3). Thus, VIP can specifically enhance the guinea pig ileum response to f i e l d stimulation suggesting the VIP exerts a neuronal effect to increase release of acetylcholine.
CONTROLRESPONSE RESPONSETOACETYLCHOUNE TO ACETYLCHOUNE IN THE PRESENCEOF VIP (3xlO4M) A
O I nanlte
t
ACk'TYLC)'iOLIN£ 10 n41/mi
t
1
VI'P AC1ETYLCNOLIN£ 3~IO-OM 10 141/rid
FIG. 3 Effect of VIP on response of the guinea pig ileum to f i e l d stimulation and to the direct application of acetylcholine. Dynograph recording of tissue that was stimulated with square wave Impulses at 40 V, 0.1Hz and for 0.7 msec duration. Stimulation was stopped 30 seconds prior to acetylcholine challenge. A: Control response to acetylcholine (10 ng/ml); B: VIP (3 x lO-8 M) was given approximately 1 minute prior to acetylcholine (10 nglm)) challenge. W indicates replacement of the Krebs' buffer with buffer containing no drugs.
120-
~mMCN~)
8O-
4O"
VAtmAClIMEWalIIIINIIALPOLYIIqEPTIO!Imdarl
FIG. 4 Concentration-response of the field-stimulated guinea pig ileum to VIP in the absence (open circles) and presence (closed circles) of bacitracin (2 x 10-5 M). Points represent mean increases in twitch height + S.E. for the number of tissues indicated in parentheses. To examine the p o s s i b i l i t y that contraction of the guinea pig ileum caused the enhancement of f i e l d stimulated responses, we evaluated the a b i l i t y of histamine and acetylcholine, two known contractile agonists to alter the response to f i e l d stimulation. Neither agent s i g n i f i c a n t l y
Vol. 26, No. i0, 1980
Effect of VIP in I n t e s t i ~ l Smooth Muscle
817
increased the response to field stimulation in the guinea pig ileum (data not shown). Thus, enhancement of acetylcholine release by VIP is independent of its a b i l i t y to induce tone in the guinea pig ileum. Addition of bacitracin (2 x lO-5 M) to the buffer has been suggested to be useful in optimizing responses to peptides (5,27). Although we obtained good responses in the absence of bacitracin, we repeated experiments of VIP enhancement of the responses to field stimulation in the presence and absence of bacitracin (2 x I0 -~ M). The response to VIP was increased about 30 percent when bacitracin was present in the buffer (Figure 4). Role of VIP to Increase Intestinal Levels of C@clic AMP and C~clic GMP. VlP (lO -6 M) elevated both cyclic AMP (30 percent) and cyclic C~MP(24 percent) levels in the rabbit Jejunum (Table 2). In the presence of phosphodiesterase inhibition with isobutylmethylxanthine, VIP elevated cyclic nucleotide levels even further with a small increase in the percent elevation to 44 and 58 percent for cyclic AMP and cyclic GMP, respectively. VIP increased cyclic AMP in mucosal tissue from the rabbit ileum as well (Table 2). In this tissue, however, cyclic GMP levels were unaltered. Similar results were obtained in preliminary experiments with two tissues of longitudinal muscle from the rabbit ileum (data not shown). Since VIP increased the force of spontaneous contractions of the rabbit Jejunum and the height of the response to f i e l d stimulation in ileal smooth muscle, we asked i f such effects might be mediated by the elevations in cyclic nucleotide levels that occur after VIP. We reasoned that i f the iBcrease in ileal twitch height were related to activation of adenylate cyclase by VIP, then phosphodiesterase inhibition should potentiate this response to VIP. For thgse studies, we used the phosphodiesterase inhibitor, papaverine, which at lO-~ M produced a measurable 33 percent (n-g) and 90 percent (n-6) elevation in jejunal cyclic AMP and cyclic GMP levels, respectively. Because papaverine i t s e l f is a smooth muscle relaxant that produced a 14 percent (n.6) and 68 p~rcent (n.7) reduction in the response to f i e l d stimulation at 3 x lO-° M and 3 x lO-OM, respectively, a potential effect on VIP-induced responses was examined at both concentrations. VIP-induced enhancement of the response to f i e l d stimulation in the guinea pig ileum was not potentiated by either concentration of papaverine (Table 3). In fact, papaverine at 3 x lO-5 M co,~)letely inhibited the response to VIP presumably due to a nonspecific relaxant effect of papaverine. Discussion VIP exerts multiple in vitro effects on intestinal smooth muscle that might be related to its p r o p o ~ involvement in certain pathologies of the gastrointestinal tract. The present study describes a contractile effect, a presynaptic action to increase neurotransmitter release and an activation of intestinal adenylate cyclase. VlP increased tone in the guinea pig ileum and increased the rhythmic contractile force of the rabbit jejunum. These results confirm previous reports of VIP-induced contractile responses in intestinal smooth muscle (15,16). In contrast, in vitro relaxant or inhibitory effects of VIP have been reported in other ~ o ~ s c l e preparations such as the guinea pig gallbladder (28), mouse vas deferens (29), guinea pig taenia coli (30,31) and rat portal vein (unpublished observation). In the present experiments, the ileal contraction produced by VIP was p a r t i a l l y antagonized by atropine.
I0.46~0.41 13.3+1.48 19.18+2.15
VIP (lO -6 M) (9)
Isob~tylmethyixanthine (g)c (10 -4 M)
VIP (10.6 M) (g)
19.93÷1.01 19.45+0.88 29.99+1.69
VIP (10 -6 M) (6)
Isobutylmethylxanthine (6) c (10 ~ M)
VIP (10 .6 M) (6)
54.9+6.3 **
52.9+10.0"
43.8+11.3"*
31.3+4.4"
% Increase
0.384+0.11
0.428+0.10
0.181+0.19
0.171+0.04
0.457+0.05
0.292+0.03 -
0.164+0.02
0.130+0.01
Cyclic GMPa (pmoleslmcj protein)
-10.7+18.2
4.1÷10.1
58.4÷17.4"*
23.7+7.2*
% Increase
a Cyclic AMP and cyclic GMP were measured by the radtoimunoassay technique of Steiner et al. (24). Values are means + S. E. b Sections of intact- rabbit jejunum or rabbit ileal mucosa separated from the longitudinal ileal muscle by the procedure of Rang (23) were used. The nugg)er of tissues is indicated in parentheses. c Isobutylmethylxanthine was added to the incubation buffer 5 minutes prior to challenge with VIP. * Values significantly (p
13.19+1.18
Control (6)
Rabbit Ileal l~cosa: b
7.96+0.26
Centrol (9)
Rabbit Jejunum:b
Cyclic AMPa (pmoleslm¢j protein)
Effect of VIP on Cyclic AMP and Cyclic GMP Levels in Rabbit Jejunum and lleal Mucosa
TABLE 2
Oo O
t-J
Z 0
t~
0
f)
0 I'+ =r
k~
g~
rt
0
(3 rt
M t~ t~
O0
O0
m
_0.5+2.4(4)c*
5.3+_2.4(4)*
4.3+_4.0(4)*
40.6+_8.5(4)
48.2+_8.2(8)
10-8
6.3"+2.9(4)*
50.5-+11.0(4)
51.3"+!i.2(8)
3 x 10-8
Values are
-0.3-+Z./(4)c*
54.9+_13.5(4)
85.0+21.6(8)
10-7
b Papav~ine was incorporated into the Krebs' buffer containing bacitracln (2 x 10-5 M) during equilibration and for the duration of the experiment. c Negative sign indicates the percent decrease In twitch height. • Values differ significantly (p
a Tissues were stimulated with square wave impulses (O.1 Hz) at 40 V and 0.7 msec duration. means + S.E. for the number of tissues indicated in parentheses.
(3 x 10-5 M)
Papaverine b
1.34+0.08(20)*
21.7+-4.4(4)
12.1_+1.6(4)
1.85+o.08(2o)*
Papaverineb
(3 x 10 -6 M)
22.3+_4. o(e)
6.5+_4.8(8)
3 x 10 -9
2.20+0.10(40)
10 -9
VIP (Molar)
Percent Increase in Twitch Heighta
Control
Initial Twitch Height (g)a
Effect of VIP on the Field Stimulated Response of the Guinea Pig Ileum in the Presence and Absence of Papaverine
TABLE 3
~D
Oo
rJ l-a ill
o r$
#-m
re It II r~
P-4
o
fo
pl v~
0
~a
;-a 0
ON,
820
Effect of VIP in Intestinal Smooth Muscle
%'ol. 26, No. i0, 1980
Although these data are consistant with a direct interaction of VIP with the muscarinic receptor in the ileum, more l i k e l y , antagonism of the VIP-induced contraction by atropine indicates that VIP may act to release acetylcholine from neural elements in the ileum. A similar conclusion was reached with studies in the ileum using tetrodotoxin (15) and studies on intestinal absorption and blood flow with atropine (32). Since atropine did not completely abolish contractions to VIP, additional mechanisms for VIP-induced contraction must be considered. Studies with the Hl antagonist, pyrilamine, indicate that histamine release is not involved in this contractile response. VIP may be acting to contract the ileum by release of other contractile agents or by a direct mechanism in addition to the neuronally mediated cholinergic action. Further evidence for a stimulatory effect of VIP on neuronal release of acetylcholine in the ileum was derived from studies with the f i e l d stimulated ileum. VIP, in a concentration related fashion enhanced the responses to f i e l d stimulation. This effect was clearly presynaptic since responses to the direct administration of acetylcholine or histamine were unaltered. Consistent with this observation, a small (less than lO percent) potentiation of guinea pig detrusor responses to nerve stimulation has recently been reported for VIP (31). Thus, VIP joins the expanding l i s t of peptides such as the enkephalins (33), angiotensin (34,35) and somatostatin (29,36,37,38) that can modulate presynaptic neurotransmitter release mechanisms. In fact, modulatlon of transmitter release may represent the principle mechanism by which polypeptide hormones affect biological systems. In addition to these effects on smooth muscle function and neuronal release mechanisms, VIP is known to activate adenylate cyclase in broken cell preparations of intestinal origin (18,1g,20,21,22). Our studies confirm this a c t i v i t y and show that activation of adenylate cyclase by VIP occurs in intact intestinal tissue and may be accompanied by increases in cyclic GMP as occurred in rabbit jejunal tissue, although elevations in cyclic GMP may not occur in all tissues. The increases in cyclic AMP induced by VIP in intestinal smooth muscle are unlikely to be related to its effects on neurotransmitter release or ileal contractile mechanisms for the following reasons: 1) Other agents that elevate cyclic nucleotide levels do not produce similar effects; 2) I f cyclic AMP were mediating the effects of VIP, then we would anticipate an enhanced response to VIP after inhibition of phosphodiesterase. This did not occur. Certainly other explanations may be proposed for the i n a b i l i t y of these indirect experiments to reveal a positive correlation to elevations in cyclic AMP. Although further studies must be done in this regard, i t is possible that VIP-induced increases in cyclic AMP more closely relate to its effects on ion transport of intestinal secretion (18,20) than on its a b i l i t y to affect smooth muscle tone or neurotransmitter release. Nevertheless, the present studies document marked effects of VIP in intestinal smooth muscle function to promote increased muscle tone. VIP containing nerves are abundant throughout the gastrointestinal tract (39) and our studies suggest that such nerves may serve to modulate (by enhancement) neurotransmitter release in intestinal smooth muscle. This hypothesis is consistent with an involvement of VIP in the defecation reflex and intestinal pathologies associated with excessive diarrhea such as Crohn's disease (39) and the watery diarrhea syndrome (]O,]l,12).
V o l . 2 6 , No. 10, 1980
Effect
o f VIP i n l n t e s t l n a l
Smooth M u s c l e
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Effect of VIP in Intestinal Smooth Muscle
Vol. 26, No. I0, 1980
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