European Journal o f Pharmacology, 55 (1979) 35--42 © Elsevier/North-Holland Biomedical Press
35
NEUROTENSIN: CONTRACTILE ACTIVITY, SPECIFIC BINDING, AND LACK OF EFFECT ON CYCLIC NUCLEOTIDES IN INTESTINAL SMOOTH MUSCLE PATRICK KITABGI and PIERRE FREYCHET Institut National de la Santd et de la Recherche Mddicale (I.N.S.E.R.M.), Groupe de Recherche sur les Hormones Polypeptidiques et la Physiopathologie Endocrinienne U 145 et Laboratoire de Mddecine Expdrimentale, Universitd de Nice, Facultd de Mddecine (Pasteur), 06034 Nice Cedex, France
Received 19 September 1978, revised MS received 1 December 1978, accepted 11 December 1978
P. KITABGI and P. FREYCHET, Neurotensin: contractile activity, specific binding, and lack o f effect on cyclic nucleotides in intestinal smooth muscle, European J. Pharmacol. 55 (1979) 35--42. Neurotensin contracted the isolated longitudinal smooth muscle strip of the guinea-pig intestine. It induced a biphasic response (relaxation followed by contraction) when the muscle was contracted by histamine. Tetrodotoxin blocked the contraction and the contraction phase of the biphasic response. Dose responses of the relaxing effect of neurotensin and its analogs D-ArgS-neurotensin and D-Arg9-neurotensin on tetrodotoxin-treated muscle gave ECs0 values of 5 nM, 5.5 nM and 110 nM for the three peptides, respectively. [3H]Neurotensin bound specifically to membranes prepared from isolated longitudinal muscle strips of the guinea-pig intestine. In binding studies using [3H]neurotensin and unlabelled peptides, the KDS of neurotensin, D-ArgS-neurotensin and D-Arg9-neurotensin were 4 nM, 2 nM and 300 nM, respectively, values which are in close agreement with the ECs0 of the three peptides. Neurotensin had no effect on the cAMP and cGMP contents in longitudinal intestinal muscle strips of the guinea pig, an observation which suggests that cyclic nucleotides are not involved in the mechanism of action of neurotensin in this muscle preparation. Longitudinal intestinal smooth muscle [ 3H ]Neurotensin binding
D-ArgS-neurotensin D-Arg9-neurotensin
1. I n t r o d u c t i o n Recently, we reported on the pharmacological e f f e c t s o f t h e t r i d e c a p e p t i d e n e u r o t e n s i n ( N T ) on isolated intestinal s m o o t h m u s c l e s (Kitabgi a n d F r e y c h e t , 1 9 7 8 ) . We s h o w e d t h a t N T c o n t r a c t e d t h e guinea-pig i l e u m w h e n in a basal l o w t o n e , a n d t h a t t h e p e p t i d e i n d u c e d a biphasic r e s p o n s e , i.e., r e l a x a t i o n f o l l o w e d b y c o n t r a c t i o n , w h e n t h e ileal m u s c l e was in a c o n t r a c t e d state. F u r t h e r m o r e , we o b s e r v e d t h a t t e t r o d o t o x i n (TTX) a b o l i s h e d t h e c o n t r a c t i o n or t h e c o n t r a c t i o n p h a s e o f t h e biphasic r e s p o n s e i n d u c e d b y N T in t h e guinea-pig ileum. It was c o n c l u d e d t h a t N T c o n t r a c t s t h e guinea-pig i l e u m t h r o u g h a n e r v e - m e d i a t e d p r o c e s s a n d t h a t it relaxes
Neurotensin Cyclic GMP
Cyclic AMP
this p r e p a r a t i o n b y a d i r e c t e f f e c t o n s m o o t h muscle. In t h e p r e s e n t w o r k , we h a v e s t u d i e d s o m e aspects o f t h e m y o g e n i c a c t i o n o f N T o n intestinal s m o o t h m u s c l e . T h e isolated longitudinal s m o o t h m u s c l e o f t h e guinea-pig small i n t e s t i n e was used b e c a u s e this p r e p a r a t i o n c o n t a i n s a high p r o p o r t i o n o f s m o o t h m u s c l e fibers. T h e p h a r m a c o l o g i c a l e f f e c t s o f N T o n t h e c o n t r a c t i l i t y o f this p r e p a r a t i o n were investigated. T h e b i n d i n g o f N T t o m e m b r a n e s p r e p a r e d f r o m isolated l o n g i t u d i n a l s m o o t h m u s c l e was m e a s u r e d a n d t h e doser e s p o n s e o f b i n d i n g was c o m p a r e d t o t h a t o f t h e p h a r m a c o l o g i c a l r e s p o n s e . In a d d i t i o n , t h e ability o f t w o N T analogs, D-ArgS-NT a n d D-Arg9-NT, t o i n h i b i t [ 3 H ] N T b i n d i n g t o
36 muscle membranes was studied and the relative binding potency of these two peptides was compared to their pharmacological potency in the longitudinal muscle preparation. Finally, the contents of adenosine3',5'-cyclic monophosphate (cAMP) and guanosine-3',5'-cyclic monophosphate (cGMP) were measured in pieces of the longitudinal ileal smooth muscle of the guinea-pig exposed to NT; this was done because it has been proposed, although this is controversial, that the mechanism of action on smooth muscle of relaxing drugs such as ~-adrenergic agents involves an increase in the cAMP content of smooth muscle ( B ~ , 1974), and that the action of contracting agents (K ÷, acetylcholine) is mediated by an increase in cGMP levels (Andersson et al., 1975; Lee et al., 1972).
2. Materials and methods
2.1. Drugs Synthetic NT was purchased from Beckman; D-ArgS-NT and D-Argg-NT were gifts from J. Rivier and were synthetized as reported elsewhere (Rivier et al., 1977); [3H]NT (77Ci/ mmol) was obtained as described previously (Kitabgi et al., 1977); acetylcholine chloride (Ach), histamine dihydrochloride, L(-)-isoproterenol hydrochloride, were from Sigma; 3-isobutyl-l-methylxanthine (IBMX) was from Aldrich Chemical; t e t r o d o t o x i n (TTX) was from Sankyo; collagenase (Type 1) was from Sigma.
2.2. Pharmacological studies Male guinea pigs (300--400 g) were used in all studies. The longitudinal muscle was dissected from the ileum according to Paton and Zar (1968), and strips 4--5 cm in length were set up in a 10 ml organ bath containing Tyrode solution (mM) (136.8NAC1; 2.7 KC1; 1 MgSO4; 0.4 NaH2PO4; 11.9 NaHCO3; 3.6 CaC12; 5.5 glucose) maintained at 37°C and
P. KITABGI, P. FREYCHET gassed with 95% and 5% CO2. The resting tension was 0 . 2 - 0 . 3 g and contractions were recorded with an isometric transducer (APELAB, Bagneux, France) connected to a potentiometric recorder (Servotrace, SEFRAM, Paris, France). The tissue was allowed to equilibrate for 45 min and was washed every 10 min. When dose responses of the relaxing effect of NT, D-ArgS-NT and D-Argg-NT were performed, 0 . 3 p M TTX was added to the reservoir of Tyrode solution and the longitudinal muscle was allowed to equilibrate for 20-r-30 min in the TTX-containing Tyrode solution. The muscle was then contracted with 0.1 pM ACh and varying doses of the peptides were added 30 sec later when the contraction was maximal. Muscle was washed 1 min after the addition of peptide and a new cycle started 90 sec after the washout. The peptide-induced relaxation was measured as the drop in tension which occurred within 10 sec following the addition of the peptides. During this 10 sec period, the fading of the contraction elicited by 0.1 pM ACh was negligible.
2. 3. Binding studies 2.3.1. Preparation o f longitudinal smooth muscle membranes The longitudinal smooth muscle was dissected from the entire length of the guineapig intestine and was placed in cold Tyrode solution. Smooth muscle cells were prepared by a modification of the methods of Bagby et al. (1971) and McKenzie et al. {1977). The tissue (about 1 g/animal) was minced in small pieces and was incubated with 5 volumes of Tyrode solution containing 2 mg/ml collagenase at 37°C for 45 min with shaking at 150 cycles/min. After incubation the remaining tissue was sedimented and the supernarant solution removed and kept on ice. An equal volume of T y r o d e solution containing 1 mg/ml collagenase was added to the tissue and the supernatant solution was collected after a 3 0 m i n incubation at 37°C. This operation was repeated twice. The three
NEUROTENSIN AND INTESTINAL LONGITUDINAL SMOOTH MUSCLE supernatant solutions were combined, filtered on nylon gauze, and centrifuged at 1000 X g for 2 min at 4°C. The pellet was gently resuspended in Tyrode solution. Some of the material contained in the pellet coagulated and was discarded. When observed under a phase contrast microscope, the suspension consisted mostly (about 70%) of isolated smooth muscle cells and was contaminated essentially with red blood cells and small debris of capillaries. About 106 smooth muscle cells were obtained from 1 g of tissue. The cell suspension was centrifuged at 1000 X g for 2 min at 4°C and the pellet was resuspended by stirring in Tyrode solution. The material which did not resuspend was discarded. After another centrifugation at 1000 X g for 2 min, the pellet was homogenized in 5 m M Tris-HC1 pH 8 at 4°C in a glass homogenizer with a motor-driven teflon pestle. The homogenate was centrifuged at 3000 × g for 30 min at 4°C and the resulting pellet was stored at --20 ° C. The protein content of this crude membrane preparation was assayed by the m e t h o d of Lowry et al. (1951). About 1 mg of crude membrane protein was obtained from 106 smooth muscle cells.
2.3.2. Binding assay Binding experiments were carried out exactly as described previously for the binding of [3H]NT to synaptic membranes (Kitabgi et al.: 1977). Briefly, [3H]NT at 2 nM was incubated at 24°C in 250 pl of 50 mM Tris • HC1 pH 7.5 containing 1% bovine serum albumin (Sigma) and 0.4 mg/ml of membrane protein. Bound NT was separated from the free by filtration on a Millipore filter (EGWP 025000, 0.2 pm). The filters were placed in scintillation liquid (Unisolve 1, Koch--Light Laboratories) and counted in a Packard scintillation counter. The radioactivity adsorbed on the filters in the absence of smooth muscle membranes (about 0.1% of the total) was subtracted from the data. The specific [3H]NT binding was obtained by subtracting from the total binding the non-specific binding defined as the a m o u n t of radioactivity bound in the presence of 1 pM unlabelled NT.
37
2.4. Measurement o f cyclic nucleotides in longitudinal muscle strips Strips of the longitudinal muscle of the guinea-pig small intestine, about 2 cm in length and weighing 20--30 mg (wet wt), were preincubated for 30 min at 37°C in 2 ml of Tyrode solution gassed with 95% 02 and 5% CO2. After the preincubation time each smooth muscle strip was transferred to a glass homogenizer containing 5 0 0 p l of Tyrode solution and the various drugs to be tested, and was incubated at 37°C for 20 or 60 sec. At the end of the incubation period, 100 pl of 6 N perchloric acid was added to the homogenizer and the strip was immediately homogenized with a motor-driven teflon pestle (full speed). The homogenates were centrifuged (2000 X g for 20 min) and the supernatants were neutralized with NaOH. The cAMP and cGMP levels were measured by radioimmunoassay (Delaage et al., 1978). This technique involves succinylation of the samples to be assayed, which increases the sensitivity of the assay by two orders of magnitude when compared to classical radioimmunoassay for cyclic nucleotides. The results are expressed as pmol of cyclic nucleotide per mg of tissue.
3. Results
3.1. Pharmacological effects of N T a n d analogs on longitudinal muscle strips o f the guinea-pig ileum The isolated longitudinal muscle of the guinea-pig ileum responded to Ach and histamine (not shown) in the same way as did the intact ileum. The ECs0s for the contracting effect of ACh and histamine were 65 nM and 1 5 0 n M , respectively, in the isolated ileal longitudinal muscle, compared to 45 nM and 170 nM in the intact ileum (Kitabgi and Freychet, 1978). Neurotensin, 60 nM, contracted the isolated longitudinal muscle (fig. 1A) and induced a biphasic response when the muscle
38
P. KITABGI, P. FREYCHET
NT 60nM
100
W
1 NT 60nM
0"5g 1
W
~A NT 60nM H lO0nM~
¢
~
50
i
-9
NI60nM H lOOnM '
W
/
J
W
1
05g
1rain Fig. 1. Effect of NT on the longitudinal muscle strips of the guinea-pig ileum. (A) Response of the muscle to 60 nM NT; W, washout. (B) Same as (A) in the presence of 0.3 #M TTX. (C) Effect of 60 nM NT on the muscle contracted by 100 nM histamine (H); W, washout. (D) same as (C) in the presence of 0.3 paM TTX.
was previously contracted b y 100 nM histamine (fig. 1C). Tetrodotoxin, 0.3 ttM, inhibited the contraction (fig. 1B) or the contraction p h a s e but n o t the relaxation phase (fig. 1D), induced b y 6 0 n M NT. The contractile
/~r~D-ARG9NT
i
-8
i
-7
16 -
Fig. 2. Dose---response curves of the relaxation of the longitudinal muscle strips of the guinea-pig ileum by NT (A A), D-ArgS.NT ($ "-) and D-Arg9NT (~' =). All experiments were performed in the presence of 0.3 pM TTX. The results are expressed as the percentage of the maximal relaxation induced by the peptides after contraction of the muscle by 100 nM ACh. Each point is the mean value _+S.E.M. from 3 experiments. Ordinate: percent of maximal relaxation ; abscissa: [ peptide ], log M.
response that followed the NT-induced relaxation in TTX-treated preparations (fig. 1B and D) was always small compared to that seen in the absence of TTX (fig. 1A and C) and was not always observed. These results are similar to those obtained with the intact ileum (Kitabgi and Freychet, 1978). In experiments designed to quantify the dose--response relationships between NT and its relaxing effect on the ACh-contracted longitudinal muscle, the contraction phase that followed the NT-induced relaxing effect was abolished b y incubating the muscle in the presence of 0.3/JM TTX. The dose response thus obtained for NT (fig. 2) was similar to that observed in intact smooth muscle systems (Kitabgi and Freychet, 1978). D-ArgS-NT was almost as potent as NT whereas D-Arg%NT was about 20 times less potent than NT (fig. 2); all three peptides had similar maximal effectiveness. The ECs0s of NT, D-ArgS-NT and D-Arg%NT are indicated in table 1.
NEUROTENSIN AND INTESTINAL LONGITUDINAL SMOOTH MUSCLE TABLE I Comparison of the ECs0s for the relaxing effect of NT, D-ArgS-NT and D-Arg%NT, with the apparent dissociation constants (KD) for the binding of these peptides to membranes from longitudinal muscle strips of the guinea-pig intestine. The ECs0s were derived from fig. 2. The KDS were derived from fig. 3 as explained in the legend to this figure. Peptide
ECs0 (nM)
K D (nM)
NT D-ArgS-NT D-Argg-NT
5.0 5.5 110
4 2 300
1.0
o.s
•
• ~D-ARG 9N~
~
-
T r i t i a t e d N T b o u n d specifically t o c r u d e membranes prepared from the longitudinal s m o o t h m u s c l e o f t h e guinea-pig intestine. When t h e b i n d i n g o f 2 n M [ 3 H ] N T was s t u d i e d at 2 4 ° C as a f u n c t i o n o f t i m e ( n o t s h o w n ) , t h e specific b i n d i n g increased w i t h t i m e a n d r e a c h e d a p l a t e a u a f t e r 10 m i n . A t s t e a d y - s t a t e (30 m i n o f i n c u b a t i o n ) t h e t o t a l b i n d i n g r e p r e s e n t e d 0.3% o f t h e t o t a l radioactivity and the non-specific binding a c c o u n t e d f o r a b o u t 40% o f t h e t o t a l binding. T h e specific b i n d i n g r e p r e s e n t e d 10 f m o l o f [ 3 H ] N T (at 2 nM) b o u n d p e r m g o f m e m b r a n e protein. The inhibition of the binding of 2 nM [ 3 H I N T b y increasing c o n c e n t r a t i o n s o f u n l a b e l l e d N T , D-ArgS-NT a n d D-Arg%NT a f t e r a 30 m i n i n c u b a t i o n at 24 ° C, is s h o w n in fig. 3. T h e values o f t h e a p p a r e n t d i s s o c i a t i o n cons t a n t (KD) derived f o r t h e t h r e e p e p t i d e s are s h o w n in t a b l e 1. T h e y are in close a g r e e m e n t w i t h t h e values o f t h e ECs0 derived f o r t h e three peptides from the dose responses of their relaxing effect on the ACh-contracted longitudinal muscle.
~,,NTi
0
-0,5
3.2. Binding o f N T and relative binding potency o f D-ArgS-NT and D-Arg9-NT in membranes from longitudinal smooth muscle
39
~9
-
18
-'7
-'6
Fig. 3. Inhibition of 2 nM [3H]-NT binding by unlabelled NT (A A), D-ArgS-NT (e =) and D-Argg-NT (-" ---). Data were plotted according to the logit representation, i.e., log[B/(B0--B)] (ordinate) versus [unlabelled peptide], log M (abscissa), where B is the specific binding of [3H]-NT at each given concentration of unlabelled peptide and B0 refers to the specific binding of [3H]-NT in the absence of unlabelled peptide. The dissociation constant K D for NT can be obtained by the relation K D --NTs0--L0• where NTs0 is the concentration of unlabelled NT which produced 50% inhibition of the binding of [3H]-NT and where L0* refers to the total concentration of labelled NT. The dissociation constants K D (A) for the analogs D-ArgS-NT and D-Argg-NT were obtained by the relation: K D (A) = K D × (As0 / NTs0) where As0 is the concentration of analog which inhibited the binding of [3H]-NT by 50%. Data were obtained from three experiments for NT and two experiments for the analogs, with triplicate determinations within each experiment.
3.3. Measurements o f cAMP in longitudinal smooth muscle strips T h e e f f e c t s o f N T a n d o t h e r agents o n t h e c o n t e n t s o f cAMP a n d c G M P in l o n g i t u d i n a l s m o o t h m u s c l e strips o f t h e guinea-pig intestine are s h o w n in t a b l e 2. N e u r o t e n s i n , 1 pM, h a d n o e f f e c t o n t h e basal levels o f t h e t w o cyclic n u c l e o t i d e s a f t e r e i t h e r 20 or 60 sec o f i n c u b a t i o n . I s o p r o t e r e n o l , 1/~M, did n o t c h a n g e t h e c A M P level w i t h e i t h e r o f t h e i n c u b a t i o n times. In t h e p r e s e n c e o f 1/~M a c e t y l c h o l i n e , t h e c G M P level significantly
40
P. KITABGI, P. FREYCHET
TABLE 2 Effects of NT and other agents on cAMP and cGMP levels in longitudinal smooth muscle strips of the guinea-pig intestine, after 20 and 60 sec of incubation. The values are means ± S.E.M. of the number of determinations given in parentheses. Comparisons of means were done using Student's t-test. Additions
Cyclic nucleotide levels (pmol/mg tissue) cAMP
O (Basal) NT 1 ~M Isoproterenol 1 pM ACh 1 pM K ÷ 130 mM IBMX 300 gM
cGMP
20 sec
60 sec
0.38 0.29 0.36 -0.45 --
0.5] ± 0.05 0.50"± 0.07 0.44 ± 0.04 -0.44 ± 0.09 1.59 -+ 0.15
± 0.07 (15) ± 0.06 (7) ± 0.05 (7) ± 0.07 (8)
20 sec (26) (12) (7) (8) (8) :
0.048 0.051 -0.077 0.110 --
60 sec ± 0.004 (18) ± 0.006 (8)
0.048 ± 0.004 (29) 0.051 ± 0.007 (12)
± 0.020 (7) i ± 0.016 (8) 2
0.043 ± 0.007 (7) 0.069 ± 0.017 (8) 0.086 ± 0.007 (8) 2
i p < 0.05; 2 p < 0.001, compared with basal values.
increased a f t e r 2 0 s e c o f i n c u b a t i o n a n d r e t u r n e d to its basal value a f t e r 60 sec. Similarly, w h e n 130 m M K ÷ r e p l a c e d Na + in t h e T y r o d e s o l u t i o n , t h e c G M P level increased t w o - f o l d a f t e r 20 sec o f i n c u b a t i o n a n d r e t u r n e d to its basal value a f t e r 60 sec. T h e p h o s p h o d i e s t e r a s e i n h i b i t o r IBMX at 3 0 0 pM increased t h e cAMP level t h r e e - f o l d a n d t h e c G M P level t w o - f o l d a f t e r a 60 sec i n c u b a t i o n (table 2).
4. Discussion T h e p r e s e n t s t u d y was designed to investigate f u r t h e r t h e e f f e c t o f N T on intestinal s m o o t h m u s c l e , p a r t i c u l a r l y t h e direct, m y o genic a c t i o n o f t h e p e p t i d e as o p p o s e d t o its indirect, n e r v e - m e d i a t e d e f f e c t (Kitabgi a n d F r e y c h e t , 1978). This was a p p r o a c h e d t h r e e w a y s b y investigating: ( 1 ) t h e e f f e c t o f t h e p e p t i d e o n t h e c o n t r a c t i l e a c t i v i t y o f longit u d i n a l m u s c l e strips o f t h e guinea-pig ileum; (2) t h e b i n d i n g o f [ 3 H ] N T to a c r u d e m e m brane fraction prepared from the longitudinal m u s c l e o f t h e guinea-pig small intestine; a n d (3) t h e e f f e c t o f N T o n cAMP a n d c G M P levels in l o n g i t u d i n a l m u s c l e strips o f guineapig small intestine.
O u r results indicate t h a t , in isolated strips o f l o n g i t u d i n a l m u s c l e f r o m t h e guinea-pig ileum, N T e x e r t e d a relaxing e f f e c t which, as in t h e i n t a c t guinea-pig i l e u m (Kitabgi and F r e y c h e t , 1978}, was n o t abolished b y T T X a n d is likely to reflect a direct e f f e c t o f t h e p e p t i d e on s m o o t h muscle. T h e results also s h o w t h a t N T b o u n d to a c r u d e m e m b r a n e p r e p a r a t i o n o f l o n g i t u d i n a l s m o o t h muscle. T w o i t e m s o f d a t a s t r o n g l y suggest, albeit i n d i r e c t l y , t h a t this b i n d i n g involves r e c e p t o r sites w h i c h m e d i a t e t h e e f f e c t o f N T on s m o o t h m u s c l e : (1) t h e a p p a r e n t binding a f f i n i t y o f N T (K D ~ 4 nM) was similar t o t h e ECs0 o f t h e p e p t i d e in e x e r t i n g its relaxing, T T X - r e s i s t a n t e f f e c t (ECso ~- 5 riM); (2) t h e r e was g o o d a g r e e m e n t b e t w e e n t h e relative potencies o f t h e analogs D-ArgS-NT a n d D-Arg 9N T in inhibiting t h e b i n d i n g o f [ 3 H ] N T and t h e d o s e r e s p o n s e s o f t h e p e p t i d e s in e x e r t i n g t h e i r relaxing, T T X - r e s i s t a n t e f f e c t o n s m o o t h m u s c l e strips. It s h o u l d also be n o t e d t h a t t h e d o s e - r e s p o n s e curve a n d a p p a r e n t K D o f [ 3 H ] N T binding t o c r u d e m e m b r a n e s o f l o n g i t u d i n a l s m o o t h m u s c l e o f t h e guinea-pig small intestine w e r e similar t o t h o s e p r e v i o u s l y r e p o r t e d f o r [ 3 H ] N T binding t o s y n a p t i c m e m b r a n e s o f r a t brain (Kitabgi et al., 1 9 7 7 ) . This similarity
NEUROTENSIN AND INTESTINAL LONGITUDINAL SMOOTH MUSCLE also applies t o t h e relative p o t e n c i e s o f D-ArgS-NT and D-Arg9-NT in inhibiting [3H]N T binding: the KDS were 2 nM and 300 nM in s m o o t h muscle m e m b r a n e s , and 5 nM and 400 nM ( d a t a n o t s h o w n ) in brain s y n a p t i c m e m b r a n e s , for D-ArgS-NT and D-Argg-NT, respectively. We were unable to d e t e c t a n y effect o f NT on t h e levels o f cAMP and cGMP in the longitudinal muscle o f the guinea-pig small intestine. I s o p r o t e r e n o l and IBMX were used as c o n t r o l s to increase cAMP, and A C h and K + were used to increase cGMP. We failed to d e t e c t an e f f e c t o f i s o p r o t e r e n o l o n cAMP. This is in c o n t r a s t with the general finding t h a t ~-adrenergic agents increase cAMP levels in s m o o t h muscle tissues (see B~ir, 1 9 7 4 , for review). To test t h e possibility t h a t strips o f longitudinal s m o o t h muscle f r o m the guineapig ileum had lost their responsiveness to fladrenergic agents, the effect o f i s o p r o t e r e n o l on muscle c o n t r a c t i l i t y in such strips was evaluated directly. It was c o n s i s t e n t l y f o u n d t h a t , a l t h o u g h i s o p r o t e r e n o l (1 pM) relaxed the i n t a c t piece o f ileum, it h a d no d e t e c t a b l e e f f e c t o n t h e isolated longitudinal muscle strip (data n o t shown). These findings indicate t h a t the fi-adrenergic effect on longitudinal s m o o t h muscle was lost during the isolation p r o c e d u r e , and m a y explain the lack o f e f f e c t o f i s o p r o t e r e n o l o n t h e c A M P c o n t e n t in this t y p e o f p r e p a r a t i o n . O u r results with t h e effect o f A C h and K ÷ on cGMP, and IBMX o n cAMP and cGMP, are in a g r e e m e n t with t h o s e r e p o r t e d for these agents in s m o o t h muscle (Lee et al., 1 9 7 2 ; A n d e r s s o n et al., 1 9 7 5 ; D i a m o n d a n d Blisard, 1 9 7 6 ; K a t s u k i and Murad, 1977). In c o n c l u s i o n , the results o f the s t u d y r e p o r t e d here do n o t suggest t h a t cyclic n u c l e o t i d e s are involved in the mediation o f NT effects on t h e c o n t r a c t i l i t y o f intestinal s m o o t h muscle.
Acknowledgements We thank Dr. M. Worcel (INSERM U 7, H6pital Necker, Paris, France) for helpful advice during the
41
course of this work; Drs. J. Rivier and M. Brown (Laboratories for Neuroendocrinology, the Salk Institute, San Diego, California, U.S.A.) for the generous gift of D-Arga-neurotensin and D-Arg9neurotensin; Dr. M. Delaage (Centre d'Immunologie, Marseille-Luminy, France) for helpful advice and the generous gift of cyclic nucleotide radioimmunoassay reagents; G. Visciano for excellent technical assistance and illustration work; and J. Duch for expert secretarial assistance. This work was supported in part by grant ATP 71.78.103 from the Institut National de la Sant~ et de la Recherche M~dicale (INSERM), France.
References
Andersson, R., K. Nilsson, J. Wikberg, S. Johansson, E. Mohme-Lundholm and L. Lundholm, 1975, Cyclic nucleotides and the contraction of smooth muscle, Advan. Cycl. Nucl. Res. 5,491. Bagby, R.M., A.M. Young, B.A. Fisher and K. McKinnon, 1971, Contraction of single smooth muscle cells from Bufo marinus stomach, Nature 234, 351. B//r, H.P., 1974, Cyclic nucleotides and smooth muscle, Advan. Cycl. Nucl. Res. 4, 1975. Delaage, M.A., D. Roux and H.L. Cailla, 1978, Recent advances in cyclic nucleotide radioimmunoassay, in: Molecular Biology and Pharmacology of Cyclic Nucleotides, eds. G. Folco and R. Paoletti (NorthHolland Publishing Company, Amsterdam) p. 155. Diamond, J. and K.S. Blisard, 1976, Effects of stimulant and relaxant drugs on tension and cyclic nucleotide levels in canine femoral artery, Mol. Pharmacol. 12, 688. Katsuki, S. and F. Murad, 1977, Regulation of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate levels and contractility in bovine tracheal smooth muscle, Mol. Pharmacol. 13,330. Kitabgi, P., R. Carraway, J. Van Rietschoten, C. Granier, J.L. Morgat, A. Menez, S. Leeman and P. Freychet, 1977, Neurotensin: Specific binding to synaptic membranes from rat brain, Proc. Nat. Acad. Sci. U.S.A. 74, 1846. Kitabgi, P. and P. Freychet, 1978, Effects of neurotensin on isolated intestinal smooth muscles, European J. Pharmacol. 50,349. Lee, T.P., J.F. Kuo and P. Greengard, 1972, Role of muscarinic cholinergic receptors in regulation of guanosine 3',5~-cyclic monophosphate content in mammalian brain, heart muscle and intestinal smooth muscle, Proc. Nat. Acad. Sci. U.S.A. 69, 3287. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J.
42 Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. McKenzie, S.G., R. Frew and H.P. B~r, 1977, Effects of adenosine and related compounds on adenylate cyclase and cyclic AMP levels in smooth muscle, European J. Pharmacol. 4 1 , 1 9 3 . Paton, W.D.M. and M.A. Zar, 1968, The origin of
P. KITABGI, P. FREYCHET acetylcholine released from guinea-pig intestine and longitudinal muscle strips, J. Physiol. 194, 13. Rivier, J.E., L.H. Lazarus, M.H. Perrin and M.R. Brown, 1977, Neurotensin analogues. Structure-activity relationships, J. Med. Chem. 10, 1409.
35
NEUROTENSIN: CONTRACTILE ACTIVITY, SPECIFIC BINDING, AND LACK OF EFFECT ON CYCLIC NUCLEOTIDES IN INTESTINAL SMOOTH MUSCLE PATRICK KITABGI and PIERRE FREYCHET Institut National de la Santd et de la Recherche Mddicale (I.N.S.E.R.M.), Groupe de Recherche sur les Hormones Polypeptidiques et la Physiopathologie Endocrinienne U 145 et Laboratoire de Mddecine Expdrimentale, Universitd de Nice, Facultd de Mddecine (Pasteur), 06034 Nice Cedex, France
Received 19 September 1978, revised MS received 1 December 1978, accepted 11 December 1978
P. KITABGI and P. FREYCHET, Neurotensin: contractile activity, specific binding, and lack o f effect on cyclic nucleotides in intestinal smooth muscle, European J. Pharmacol. 55 (1979) 35--42. Neurotensin contracted the isolated longitudinal smooth muscle strip of the guinea-pig intestine. It induced a biphasic response (relaxation followed by contraction) when the muscle was contracted by histamine. Tetrodotoxin blocked the contraction and the contraction phase of the biphasic response. Dose responses of the relaxing effect of neurotensin and its analogs D-ArgS-neurotensin and D-Arg9-neurotensin on tetrodotoxin-treated muscle gave ECs0 values of 5 nM, 5.5 nM and 110 nM for the three peptides, respectively. [3H]Neurotensin bound specifically to membranes prepared from isolated longitudinal muscle strips of the guinea-pig intestine. In binding studies using [3H]neurotensin and unlabelled peptides, the KDS of neurotensin, D-ArgS-neurotensin and D-Arg9-neurotensin were 4 nM, 2 nM and 300 nM, respectively, values which are in close agreement with the ECs0 of the three peptides. Neurotensin had no effect on the cAMP and cGMP contents in longitudinal intestinal muscle strips of the guinea pig, an observation which suggests that cyclic nucleotides are not involved in the mechanism of action of neurotensin in this muscle preparation. Longitudinal intestinal smooth muscle [ 3H ]Neurotensin binding
D-ArgS-neurotensin D-Arg9-neurotensin
1. I n t r o d u c t i o n Recently, we reported on the pharmacological e f f e c t s o f t h e t r i d e c a p e p t i d e n e u r o t e n s i n ( N T ) on isolated intestinal s m o o t h m u s c l e s (Kitabgi a n d F r e y c h e t , 1 9 7 8 ) . We s h o w e d t h a t N T c o n t r a c t e d t h e guinea-pig i l e u m w h e n in a basal l o w t o n e , a n d t h a t t h e p e p t i d e i n d u c e d a biphasic r e s p o n s e , i.e., r e l a x a t i o n f o l l o w e d b y c o n t r a c t i o n , w h e n t h e ileal m u s c l e was in a c o n t r a c t e d state. F u r t h e r m o r e , we o b s e r v e d t h a t t e t r o d o t o x i n (TTX) a b o l i s h e d t h e c o n t r a c t i o n or t h e c o n t r a c t i o n p h a s e o f t h e biphasic r e s p o n s e i n d u c e d b y N T in t h e guinea-pig ileum. It was c o n c l u d e d t h a t N T c o n t r a c t s t h e guinea-pig i l e u m t h r o u g h a n e r v e - m e d i a t e d p r o c e s s a n d t h a t it relaxes
Neurotensin Cyclic GMP
Cyclic AMP
this p r e p a r a t i o n b y a d i r e c t e f f e c t o n s m o o t h muscle. In t h e p r e s e n t w o r k , we h a v e s t u d i e d s o m e aspects o f t h e m y o g e n i c a c t i o n o f N T o n intestinal s m o o t h m u s c l e . T h e isolated longitudinal s m o o t h m u s c l e o f t h e guinea-pig small i n t e s t i n e was used b e c a u s e this p r e p a r a t i o n c o n t a i n s a high p r o p o r t i o n o f s m o o t h m u s c l e fibers. T h e p h a r m a c o l o g i c a l e f f e c t s o f N T o n t h e c o n t r a c t i l i t y o f this p r e p a r a t i o n were investigated. T h e b i n d i n g o f N T t o m e m b r a n e s p r e p a r e d f r o m isolated l o n g i t u d i n a l s m o o t h m u s c l e was m e a s u r e d a n d t h e doser e s p o n s e o f b i n d i n g was c o m p a r e d t o t h a t o f t h e p h a r m a c o l o g i c a l r e s p o n s e . In a d d i t i o n , t h e ability o f t w o N T analogs, D-ArgS-NT a n d D-Arg9-NT, t o i n h i b i t [ 3 H ] N T b i n d i n g t o
36 muscle membranes was studied and the relative binding potency of these two peptides was compared to their pharmacological potency in the longitudinal muscle preparation. Finally, the contents of adenosine3',5'-cyclic monophosphate (cAMP) and guanosine-3',5'-cyclic monophosphate (cGMP) were measured in pieces of the longitudinal ileal smooth muscle of the guinea-pig exposed to NT; this was done because it has been proposed, although this is controversial, that the mechanism of action on smooth muscle of relaxing drugs such as ~-adrenergic agents involves an increase in the cAMP content of smooth muscle ( B ~ , 1974), and that the action of contracting agents (K ÷, acetylcholine) is mediated by an increase in cGMP levels (Andersson et al., 1975; Lee et al., 1972).
2. Materials and methods
2.1. Drugs Synthetic NT was purchased from Beckman; D-ArgS-NT and D-Argg-NT were gifts from J. Rivier and were synthetized as reported elsewhere (Rivier et al., 1977); [3H]NT (77Ci/ mmol) was obtained as described previously (Kitabgi et al., 1977); acetylcholine chloride (Ach), histamine dihydrochloride, L(-)-isoproterenol hydrochloride, were from Sigma; 3-isobutyl-l-methylxanthine (IBMX) was from Aldrich Chemical; t e t r o d o t o x i n (TTX) was from Sankyo; collagenase (Type 1) was from Sigma.
2.2. Pharmacological studies Male guinea pigs (300--400 g) were used in all studies. The longitudinal muscle was dissected from the ileum according to Paton and Zar (1968), and strips 4--5 cm in length were set up in a 10 ml organ bath containing Tyrode solution (mM) (136.8NAC1; 2.7 KC1; 1 MgSO4; 0.4 NaH2PO4; 11.9 NaHCO3; 3.6 CaC12; 5.5 glucose) maintained at 37°C and
P. KITABGI, P. FREYCHET gassed with 95% and 5% CO2. The resting tension was 0 . 2 - 0 . 3 g and contractions were recorded with an isometric transducer (APELAB, Bagneux, France) connected to a potentiometric recorder (Servotrace, SEFRAM, Paris, France). The tissue was allowed to equilibrate for 45 min and was washed every 10 min. When dose responses of the relaxing effect of NT, D-ArgS-NT and D-Argg-NT were performed, 0 . 3 p M TTX was added to the reservoir of Tyrode solution and the longitudinal muscle was allowed to equilibrate for 20-r-30 min in the TTX-containing Tyrode solution. The muscle was then contracted with 0.1 pM ACh and varying doses of the peptides were added 30 sec later when the contraction was maximal. Muscle was washed 1 min after the addition of peptide and a new cycle started 90 sec after the washout. The peptide-induced relaxation was measured as the drop in tension which occurred within 10 sec following the addition of the peptides. During this 10 sec period, the fading of the contraction elicited by 0.1 pM ACh was negligible.
2. 3. Binding studies 2.3.1. Preparation o f longitudinal smooth muscle membranes The longitudinal smooth muscle was dissected from the entire length of the guineapig intestine and was placed in cold Tyrode solution. Smooth muscle cells were prepared by a modification of the methods of Bagby et al. (1971) and McKenzie et al. {1977). The tissue (about 1 g/animal) was minced in small pieces and was incubated with 5 volumes of Tyrode solution containing 2 mg/ml collagenase at 37°C for 45 min with shaking at 150 cycles/min. After incubation the remaining tissue was sedimented and the supernarant solution removed and kept on ice. An equal volume of T y r o d e solution containing 1 mg/ml collagenase was added to the tissue and the supernatant solution was collected after a 3 0 m i n incubation at 37°C. This operation was repeated twice. The three
NEUROTENSIN AND INTESTINAL LONGITUDINAL SMOOTH MUSCLE supernatant solutions were combined, filtered on nylon gauze, and centrifuged at 1000 X g for 2 min at 4°C. The pellet was gently resuspended in Tyrode solution. Some of the material contained in the pellet coagulated and was discarded. When observed under a phase contrast microscope, the suspension consisted mostly (about 70%) of isolated smooth muscle cells and was contaminated essentially with red blood cells and small debris of capillaries. About 106 smooth muscle cells were obtained from 1 g of tissue. The cell suspension was centrifuged at 1000 X g for 2 min at 4°C and the pellet was resuspended by stirring in Tyrode solution. The material which did not resuspend was discarded. After another centrifugation at 1000 X g for 2 min, the pellet was homogenized in 5 m M Tris-HC1 pH 8 at 4°C in a glass homogenizer with a motor-driven teflon pestle. The homogenate was centrifuged at 3000 × g for 30 min at 4°C and the resulting pellet was stored at --20 ° C. The protein content of this crude membrane preparation was assayed by the m e t h o d of Lowry et al. (1951). About 1 mg of crude membrane protein was obtained from 106 smooth muscle cells.
2.3.2. Binding assay Binding experiments were carried out exactly as described previously for the binding of [3H]NT to synaptic membranes (Kitabgi et al.: 1977). Briefly, [3H]NT at 2 nM was incubated at 24°C in 250 pl of 50 mM Tris • HC1 pH 7.5 containing 1% bovine serum albumin (Sigma) and 0.4 mg/ml of membrane protein. Bound NT was separated from the free by filtration on a Millipore filter (EGWP 025000, 0.2 pm). The filters were placed in scintillation liquid (Unisolve 1, Koch--Light Laboratories) and counted in a Packard scintillation counter. The radioactivity adsorbed on the filters in the absence of smooth muscle membranes (about 0.1% of the total) was subtracted from the data. The specific [3H]NT binding was obtained by subtracting from the total binding the non-specific binding defined as the a m o u n t of radioactivity bound in the presence of 1 pM unlabelled NT.
37
2.4. Measurement o f cyclic nucleotides in longitudinal muscle strips Strips of the longitudinal muscle of the guinea-pig small intestine, about 2 cm in length and weighing 20--30 mg (wet wt), were preincubated for 30 min at 37°C in 2 ml of Tyrode solution gassed with 95% 02 and 5% CO2. After the preincubation time each smooth muscle strip was transferred to a glass homogenizer containing 5 0 0 p l of Tyrode solution and the various drugs to be tested, and was incubated at 37°C for 20 or 60 sec. At the end of the incubation period, 100 pl of 6 N perchloric acid was added to the homogenizer and the strip was immediately homogenized with a motor-driven teflon pestle (full speed). The homogenates were centrifuged (2000 X g for 20 min) and the supernatants were neutralized with NaOH. The cAMP and cGMP levels were measured by radioimmunoassay (Delaage et al., 1978). This technique involves succinylation of the samples to be assayed, which increases the sensitivity of the assay by two orders of magnitude when compared to classical radioimmunoassay for cyclic nucleotides. The results are expressed as pmol of cyclic nucleotide per mg of tissue.
3. Results
3.1. Pharmacological effects of N T a n d analogs on longitudinal muscle strips o f the guinea-pig ileum The isolated longitudinal muscle of the guinea-pig ileum responded to Ach and histamine (not shown) in the same way as did the intact ileum. The ECs0s for the contracting effect of ACh and histamine were 65 nM and 1 5 0 n M , respectively, in the isolated ileal longitudinal muscle, compared to 45 nM and 170 nM in the intact ileum (Kitabgi and Freychet, 1978). Neurotensin, 60 nM, contracted the isolated longitudinal muscle (fig. 1A) and induced a biphasic response when the muscle
38
P. KITABGI, P. FREYCHET
NT 60nM
100
W
1 NT 60nM
0"5g 1
W
~A NT 60nM H lO0nM~
¢
~
50
i
-9
NI60nM H lOOnM '
W
/
J
W
1
05g
1rain Fig. 1. Effect of NT on the longitudinal muscle strips of the guinea-pig ileum. (A) Response of the muscle to 60 nM NT; W, washout. (B) Same as (A) in the presence of 0.3 #M TTX. (C) Effect of 60 nM NT on the muscle contracted by 100 nM histamine (H); W, washout. (D) same as (C) in the presence of 0.3 paM TTX.
was previously contracted b y 100 nM histamine (fig. 1C). Tetrodotoxin, 0.3 ttM, inhibited the contraction (fig. 1B) or the contraction p h a s e but n o t the relaxation phase (fig. 1D), induced b y 6 0 n M NT. The contractile
/~r~D-ARG9NT
i
-8
i
-7
16 -
Fig. 2. Dose---response curves of the relaxation of the longitudinal muscle strips of the guinea-pig ileum by NT (A A), D-ArgS.NT ($ "-) and D-Arg9NT (~' =). All experiments were performed in the presence of 0.3 pM TTX. The results are expressed as the percentage of the maximal relaxation induced by the peptides after contraction of the muscle by 100 nM ACh. Each point is the mean value _+S.E.M. from 3 experiments. Ordinate: percent of maximal relaxation ; abscissa: [ peptide ], log M.
response that followed the NT-induced relaxation in TTX-treated preparations (fig. 1B and D) was always small compared to that seen in the absence of TTX (fig. 1A and C) and was not always observed. These results are similar to those obtained with the intact ileum (Kitabgi and Freychet, 1978). In experiments designed to quantify the dose--response relationships between NT and its relaxing effect on the ACh-contracted longitudinal muscle, the contraction phase that followed the NT-induced relaxing effect was abolished b y incubating the muscle in the presence of 0.3/JM TTX. The dose response thus obtained for NT (fig. 2) was similar to that observed in intact smooth muscle systems (Kitabgi and Freychet, 1978). D-ArgS-NT was almost as potent as NT whereas D-Arg%NT was about 20 times less potent than NT (fig. 2); all three peptides had similar maximal effectiveness. The ECs0s of NT, D-ArgS-NT and D-Arg%NT are indicated in table 1.
NEUROTENSIN AND INTESTINAL LONGITUDINAL SMOOTH MUSCLE TABLE I Comparison of the ECs0s for the relaxing effect of NT, D-ArgS-NT and D-Arg%NT, with the apparent dissociation constants (KD) for the binding of these peptides to membranes from longitudinal muscle strips of the guinea-pig intestine. The ECs0s were derived from fig. 2. The KDS were derived from fig. 3 as explained in the legend to this figure. Peptide
ECs0 (nM)
K D (nM)
NT D-ArgS-NT D-Argg-NT
5.0 5.5 110
4 2 300
1.0
o.s
•
• ~D-ARG 9N~
~
-
T r i t i a t e d N T b o u n d specifically t o c r u d e membranes prepared from the longitudinal s m o o t h m u s c l e o f t h e guinea-pig intestine. When t h e b i n d i n g o f 2 n M [ 3 H ] N T was s t u d i e d at 2 4 ° C as a f u n c t i o n o f t i m e ( n o t s h o w n ) , t h e specific b i n d i n g increased w i t h t i m e a n d r e a c h e d a p l a t e a u a f t e r 10 m i n . A t s t e a d y - s t a t e (30 m i n o f i n c u b a t i o n ) t h e t o t a l b i n d i n g r e p r e s e n t e d 0.3% o f t h e t o t a l radioactivity and the non-specific binding a c c o u n t e d f o r a b o u t 40% o f t h e t o t a l binding. T h e specific b i n d i n g r e p r e s e n t e d 10 f m o l o f [ 3 H ] N T (at 2 nM) b o u n d p e r m g o f m e m b r a n e protein. The inhibition of the binding of 2 nM [ 3 H I N T b y increasing c o n c e n t r a t i o n s o f u n l a b e l l e d N T , D-ArgS-NT a n d D-Arg%NT a f t e r a 30 m i n i n c u b a t i o n at 24 ° C, is s h o w n in fig. 3. T h e values o f t h e a p p a r e n t d i s s o c i a t i o n cons t a n t (KD) derived f o r t h e t h r e e p e p t i d e s are s h o w n in t a b l e 1. T h e y are in close a g r e e m e n t w i t h t h e values o f t h e ECs0 derived f o r t h e three peptides from the dose responses of their relaxing effect on the ACh-contracted longitudinal muscle.
~,,NTi
0
-0,5
3.2. Binding o f N T and relative binding potency o f D-ArgS-NT and D-Arg9-NT in membranes from longitudinal smooth muscle
39
~9
-
18
-'7
-'6
Fig. 3. Inhibition of 2 nM [3H]-NT binding by unlabelled NT (A A), D-ArgS-NT (e =) and D-Argg-NT (-" ---). Data were plotted according to the logit representation, i.e., log[B/(B0--B)] (ordinate) versus [unlabelled peptide], log M (abscissa), where B is the specific binding of [3H]-NT at each given concentration of unlabelled peptide and B0 refers to the specific binding of [3H]-NT in the absence of unlabelled peptide. The dissociation constant K D for NT can be obtained by the relation K D --NTs0--L0• where NTs0 is the concentration of unlabelled NT which produced 50% inhibition of the binding of [3H]-NT and where L0* refers to the total concentration of labelled NT. The dissociation constants K D (A) for the analogs D-ArgS-NT and D-Argg-NT were obtained by the relation: K D (A) = K D × (As0 / NTs0) where As0 is the concentration of analog which inhibited the binding of [3H]-NT by 50%. Data were obtained from three experiments for NT and two experiments for the analogs, with triplicate determinations within each experiment.
3.3. Measurements o f cAMP in longitudinal smooth muscle strips T h e e f f e c t s o f N T a n d o t h e r agents o n t h e c o n t e n t s o f cAMP a n d c G M P in l o n g i t u d i n a l s m o o t h m u s c l e strips o f t h e guinea-pig intestine are s h o w n in t a b l e 2. N e u r o t e n s i n , 1 pM, h a d n o e f f e c t o n t h e basal levels o f t h e t w o cyclic n u c l e o t i d e s a f t e r e i t h e r 20 or 60 sec o f i n c u b a t i o n . I s o p r o t e r e n o l , 1/~M, did n o t c h a n g e t h e c A M P level w i t h e i t h e r o f t h e i n c u b a t i o n times. In t h e p r e s e n c e o f 1/~M a c e t y l c h o l i n e , t h e c G M P level significantly
40
P. KITABGI, P. FREYCHET
TABLE 2 Effects of NT and other agents on cAMP and cGMP levels in longitudinal smooth muscle strips of the guinea-pig intestine, after 20 and 60 sec of incubation. The values are means ± S.E.M. of the number of determinations given in parentheses. Comparisons of means were done using Student's t-test. Additions
Cyclic nucleotide levels (pmol/mg tissue) cAMP
O (Basal) NT 1 ~M Isoproterenol 1 pM ACh 1 pM K ÷ 130 mM IBMX 300 gM
cGMP
20 sec
60 sec
0.38 0.29 0.36 -0.45 --
0.5] ± 0.05 0.50"± 0.07 0.44 ± 0.04 -0.44 ± 0.09 1.59 -+ 0.15
± 0.07 (15) ± 0.06 (7) ± 0.05 (7) ± 0.07 (8)
20 sec (26) (12) (7) (8) (8) :
0.048 0.051 -0.077 0.110 --
60 sec ± 0.004 (18) ± 0.006 (8)
0.048 ± 0.004 (29) 0.051 ± 0.007 (12)
± 0.020 (7) i ± 0.016 (8) 2
0.043 ± 0.007 (7) 0.069 ± 0.017 (8) 0.086 ± 0.007 (8) 2
i p < 0.05; 2 p < 0.001, compared with basal values.
increased a f t e r 2 0 s e c o f i n c u b a t i o n a n d r e t u r n e d to its basal value a f t e r 60 sec. Similarly, w h e n 130 m M K ÷ r e p l a c e d Na + in t h e T y r o d e s o l u t i o n , t h e c G M P level increased t w o - f o l d a f t e r 20 sec o f i n c u b a t i o n a n d r e t u r n e d to its basal value a f t e r 60 sec. T h e p h o s p h o d i e s t e r a s e i n h i b i t o r IBMX at 3 0 0 pM increased t h e cAMP level t h r e e - f o l d a n d t h e c G M P level t w o - f o l d a f t e r a 60 sec i n c u b a t i o n (table 2).
4. Discussion T h e p r e s e n t s t u d y was designed to investigate f u r t h e r t h e e f f e c t o f N T on intestinal s m o o t h m u s c l e , p a r t i c u l a r l y t h e direct, m y o genic a c t i o n o f t h e p e p t i d e as o p p o s e d t o its indirect, n e r v e - m e d i a t e d e f f e c t (Kitabgi a n d F r e y c h e t , 1978). This was a p p r o a c h e d t h r e e w a y s b y investigating: ( 1 ) t h e e f f e c t o f t h e p e p t i d e o n t h e c o n t r a c t i l e a c t i v i t y o f longit u d i n a l m u s c l e strips o f t h e guinea-pig ileum; (2) t h e b i n d i n g o f [ 3 H ] N T to a c r u d e m e m brane fraction prepared from the longitudinal m u s c l e o f t h e guinea-pig small intestine; a n d (3) t h e e f f e c t o f N T o n cAMP a n d c G M P levels in l o n g i t u d i n a l m u s c l e strips o f guineapig small intestine.
O u r results indicate t h a t , in isolated strips o f l o n g i t u d i n a l m u s c l e f r o m t h e guinea-pig ileum, N T e x e r t e d a relaxing e f f e c t which, as in t h e i n t a c t guinea-pig i l e u m (Kitabgi and F r e y c h e t , 1978}, was n o t abolished b y T T X a n d is likely to reflect a direct e f f e c t o f t h e p e p t i d e on s m o o t h muscle. T h e results also s h o w t h a t N T b o u n d to a c r u d e m e m b r a n e p r e p a r a t i o n o f l o n g i t u d i n a l s m o o t h muscle. T w o i t e m s o f d a t a s t r o n g l y suggest, albeit i n d i r e c t l y , t h a t this b i n d i n g involves r e c e p t o r sites w h i c h m e d i a t e t h e e f f e c t o f N T on s m o o t h m u s c l e : (1) t h e a p p a r e n t binding a f f i n i t y o f N T (K D ~ 4 nM) was similar t o t h e ECs0 o f t h e p e p t i d e in e x e r t i n g its relaxing, T T X - r e s i s t a n t e f f e c t (ECso ~- 5 riM); (2) t h e r e was g o o d a g r e e m e n t b e t w e e n t h e relative potencies o f t h e analogs D-ArgS-NT a n d D-Arg 9N T in inhibiting t h e b i n d i n g o f [ 3 H ] N T and t h e d o s e r e s p o n s e s o f t h e p e p t i d e s in e x e r t i n g t h e i r relaxing, T T X - r e s i s t a n t e f f e c t o n s m o o t h m u s c l e strips. It s h o u l d also be n o t e d t h a t t h e d o s e - r e s p o n s e curve a n d a p p a r e n t K D o f [ 3 H ] N T binding t o c r u d e m e m b r a n e s o f l o n g i t u d i n a l s m o o t h m u s c l e o f t h e guinea-pig small intestine w e r e similar t o t h o s e p r e v i o u s l y r e p o r t e d f o r [ 3 H ] N T binding t o s y n a p t i c m e m b r a n e s o f r a t brain (Kitabgi et al., 1 9 7 7 ) . This similarity
NEUROTENSIN AND INTESTINAL LONGITUDINAL SMOOTH MUSCLE also applies t o t h e relative p o t e n c i e s o f D-ArgS-NT and D-Arg9-NT in inhibiting [3H]N T binding: the KDS were 2 nM and 300 nM in s m o o t h muscle m e m b r a n e s , and 5 nM and 400 nM ( d a t a n o t s h o w n ) in brain s y n a p t i c m e m b r a n e s , for D-ArgS-NT and D-Argg-NT, respectively. We were unable to d e t e c t a n y effect o f NT on t h e levels o f cAMP and cGMP in the longitudinal muscle o f the guinea-pig small intestine. I s o p r o t e r e n o l and IBMX were used as c o n t r o l s to increase cAMP, and A C h and K + were used to increase cGMP. We failed to d e t e c t an e f f e c t o f i s o p r o t e r e n o l o n cAMP. This is in c o n t r a s t with the general finding t h a t ~-adrenergic agents increase cAMP levels in s m o o t h muscle tissues (see B~ir, 1 9 7 4 , for review). To test t h e possibility t h a t strips o f longitudinal s m o o t h muscle f r o m the guineapig ileum had lost their responsiveness to fladrenergic agents, the effect o f i s o p r o t e r e n o l on muscle c o n t r a c t i l i t y in such strips was evaluated directly. It was c o n s i s t e n t l y f o u n d t h a t , a l t h o u g h i s o p r o t e r e n o l (1 pM) relaxed the i n t a c t piece o f ileum, it h a d no d e t e c t a b l e e f f e c t o n t h e isolated longitudinal muscle strip (data n o t shown). These findings indicate t h a t the fi-adrenergic effect on longitudinal s m o o t h muscle was lost during the isolation p r o c e d u r e , and m a y explain the lack o f e f f e c t o f i s o p r o t e r e n o l o n t h e c A M P c o n t e n t in this t y p e o f p r e p a r a t i o n . O u r results with t h e effect o f A C h and K ÷ on cGMP, and IBMX o n cAMP and cGMP, are in a g r e e m e n t with t h o s e r e p o r t e d for these agents in s m o o t h muscle (Lee et al., 1 9 7 2 ; A n d e r s s o n et al., 1 9 7 5 ; D i a m o n d a n d Blisard, 1 9 7 6 ; K a t s u k i and Murad, 1977). In c o n c l u s i o n , the results o f the s t u d y r e p o r t e d here do n o t suggest t h a t cyclic n u c l e o t i d e s are involved in the mediation o f NT effects on t h e c o n t r a c t i l i t y o f intestinal s m o o t h muscle.
Acknowledgements We thank Dr. M. Worcel (INSERM U 7, H6pital Necker, Paris, France) for helpful advice during the
41
course of this work; Drs. J. Rivier and M. Brown (Laboratories for Neuroendocrinology, the Salk Institute, San Diego, California, U.S.A.) for the generous gift of D-Arga-neurotensin and D-Arg9neurotensin; Dr. M. Delaage (Centre d'Immunologie, Marseille-Luminy, France) for helpful advice and the generous gift of cyclic nucleotide radioimmunoassay reagents; G. Visciano for excellent technical assistance and illustration work; and J. Duch for expert secretarial assistance. This work was supported in part by grant ATP 71.78.103 from the Institut National de la Sant~ et de la Recherche M~dicale (INSERM), France.
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