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Expression of functional receptors for vasoactive intestinal peptide in freshly isolated and cultured gastric muscle cells
Regulatory Peptides, 47 (1993) 223-232
223
© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00
REGPEP 01524
Expression of functional receptors for vasoactive intestinal peptide in freshly isolated and cultured gastric muscle cells Y. Chijiiwa l, K.S. Murthy, J.R. Grider and G.M. Makhlouf Departments of Physiology and Medicine, Medical College of Virginia, Richmond, VA (USA) (Received 8 February 1993; revised version received and accepted 26 April 1993)
Key words." Peptide histidine isoleucine (PHI); Secretin; VIP antagonist; VIP binding; Muscle relaxation; Gastric smooth muscle
Summary Vasoactive intestinal peptide (VIP) receptors were characterized in freshly isolated and cultured smooth muscle cells from guinea pig stomach by radioligand binding and by measurement of relaxation in single isolated and cultured cells. 125I-VIP bound to both freshly isolated and cultured muscle cells: binding was rapid, specific, saturable and temperature-dependent, and was inhibited in a concentration-dependent fashion by VIP, VIP10-28, PHI and secretin, in this order. Competition curves for VIP could be resolved into high- and lowaffinity components, yielding similar binding constants in freshly isolated and cultured cells (high-affinity K d 0.11 and 0.22 nM; low-affinity K a 59 and 37 nM; high-affinity binding sites: 1183 and 1021 per cell, representing about 1 ~o of total binding sites). VIP10-28 inhibited a25I-VIP binding completely and acted as potent competitive antagonist of VIP-induced relaxation (Ki 0.5 nM). PHI and secretin, however, inhibited partly 125I-VIP binding: the pattern of inhibition implied that VIP interacts with VIP-preferring receptors that are recognized by PHI and secretin as well as with VIP-specific receptors. The pattern of binding is consistent with recent evidence indicating that VIP activates two signalling pathways, a VIP-specific, nitric oxide/cGMP-dependent pathway and a common cAMP-dependent pathway shared by all three peptides. PHI and secretin were relatively more potent as relaxant agents than as inhibitors of ~25I-VIP binding raising the possibility that PHI and secretin could interact additionally with PHI- and secretin-preferring receptors in mediating relaxation.
Correspondence to: G.M. Makhlouf, Box 711, MCV Station, Medical College of Virginia, Richmond, VA 23298-0711, USA. Abbreviations: VIP, vasoactive intestinal peptide; PHI, peptide histidine isoleucine; PHM, peptide histidine methionine; CCK-8, cholecystokinin octapeptide. ~Dr. Y. Chijiiwa was a Visiting Scientist from Kyushu University, Japan.
224 Introduction
Vasoactive intestinal peptide and its homologues, peptide histidine isoleucine (PHI) in animals and peptide histidine methionine (PHM) in humans, are synthesized in the same precursor and co-released from neurons of the enteric nervous system [9,26,30]. VIP fulfills many of the criteria required to establish it as a nonadrenergic, noncholinergic inhibitory neurotransmitter, responsible for relaxation in various regions of the gut [13]. VIP neurons innervate circular smooth muscle in all regions of the gut [7,10]; release of VIP from these neurons during electrical or reflex stimulation is accompanied by a stoichiometric increase in relaxation that can be blocked by specific VIP antiserum and selective VIP antagonists (e.g., VIP10-28) [2,11-14]. Recent evidence suggests that nitric oxide also is involved in neurallymediated relaxation in the gut [29] and that its release and action are closely linked to the release and action of VIP [15-17,19]. In the myenteric plexus, NO synthase is present exclusively in VIP neurons [9]; NO produced in these neurons regulates VIP release [17]. VIP, in turn, acts on muscle cells to re-generate NO [15,16,19]. Consistent with this notion, NO synthase inhibitors abolish NO production induced by nerve stimulation of muscle strips and partially inhibit VIP release and muscle relaxation [15]. In isolated muscle cells, VIP but not PHI, stimulates NO production; NO synthase inhibitors abolish VIP-induced NO production and partly inhibit relaxation [ 15,19]. The disparate effects of VIP and PHI suggest that VIP may interact with receptors coupled to different signalling pathways. VIP receptors have been characterized extensively in neural, epithelial, exocrine and vascular tissues by radioligand binding [ 18,2025 ]. Competition curves are often wide-spanned suggesting the presence of multiple receptors [18,2325]. In studies on heterogeneous membranes, however, it is not possible to determine whether multiple receptors are located on membranes derived from one cell type or from different cell types.
In earlier studies on muscle strips [12-14] and isolated muscle cells [3] of guinea pig stomach, VIP was shown to cause relaxation that was accompanied by increase in intracellular levels of cAMP. More recent studies on muscle cells isolated from various regions of the gut in several species have yielded similar results [ 15,16]. In contrast, Ennes et al. [8] detected VIP binding and increase in cAMP in cultured but not in freshly isolated muscle cells from rabbit colon; no measurements of relaxation were reported. In the present study, we have characterized VIP binding and VIP-induced relaxation in freshly isolated muscle cells devoid of neural elements and in cultured smooth muscle cells from guinea pig stomach, and determined the relative potencies of PHI, secretin and the VIP antagonist, VIP10-28, so as to gain further insight into the mechanisms of action of VIP in smooth muscle. The results demonstrate identical binding and relaxation in freshly isolated and cultured muscle cells and provide evidence for binding of VIP to more than one receptor type consistent with its coupling to distinct signalling pathways.
Materials and Methods
Dispersion of gastric muscle cells Smooth muscle cells were isolated from the circular muscle layer of guinea pig stomach as described previously [3-5]. Muscle strips were incubated for two 45-min periods at 31 °C in 25 ml of Hepes medium containing 150 units/ml collagenase (Type II) and 0.01 ~o soybean trypsin inhibitor. The composition of the medium was (in mM): NaC1 120, KC1 4, KH2PO 4 2.6, CaC12 2, MgC12 0.6, glucose 14, and Eagle's essential amino acids mixture 2.1 ~o- The partially digested strips were washed and re-incubated in enzyme-free medium to allow the cells to dissociate spontaneously. The cells were harvested by filtration through 500-#m Nitex mesh and centrifuged twice at 350 g for 10 min to eliminate cell fragments and organelles.
225
Culture of gastric muscle cells For cell culture, cells harvested by filtration were centrifuged at 350 g for 10 min and re-suspended in Dulbecco's modified Eagle's medium (DMEM-0) containing penicillin (200 units/ml), streptomycin (200/~g/ml, gentamycin (100 ~g/ml), fungizone (0.5 #g/ml), and amphotericin B (2.5 ~g/ml). The cells were centrifuged again and re-suspended in the same medium with 10~o fetal bovine serum (DMEM-10). Each stomach yielded about 3.106 cells which were plated in 24-well clusters (Costar, Cambridge, MA) at a concentration of 105 cells/0.5 ml and placed in a CO 2 incubator at 37 ° C. The cells adhered to the bottom of the well within 24-48 h, began dividing within 72 h and attained full confluence in 10-14 days whereupon each well contained 0.26 + 0.02 mg protein; this amount of protein is present in about 10 6 freshly isolated muscle cells. During culture, the DMEM-10 medium was replaced every 3 days. Cells in culture (Fig. 1) assumed typical spindle-like shapes and a hill-and-valley architecture. The cells stained for smooth muscle-specific 7-actin with monoclonal antibody CGA7 and responded to contractile and relaxant agonists (see Results).
Fig. 1. Confluentcultures of smooth muscle cells from guinea pig stomach showing characteristic spindle-shaped cells and cell architecture. Bar = 100/~m.
Measurement of contraction and relaxation in freshly &olated and cultured muscle cells In freshly isolated muscle cells contraction and relaxation were measured by scanning micrometry as described previously [3,4]. Briefly, 0.5 ml of cell suspension (10 4 cells/ml) was added to 0.2 ml of medium containing a contractile agonist and the reaction terminated at 30 s when peak contraction was attained. The lengths of 50 cells treated with and without agonist were measured by scanning micrometry in sequential microscopic fields and contractile response expressed as the mean decrease in cell length from control. For measurement of relaxation, the cells were preincubated for 60 s with the relaxant agent and then for 30 s with a maximally effective concentration of the contractile agent, cholecystokinin octapeptide (CCK-8 1 nM). Relaxation was expressed as percent inhibition of maximal contraction. In cultured muscle cells, contraction and relaxation were measured by scanning micrometry in individual cells located at the edge of semi-confluent monolayers. For this purpose, cells were grown on coverslips which were positioned so as to form the ceiling of a perfusion chamber. The volume of the chamber was 0.1 ml and the perfusion rate was 1 ml/min. The ratio of perfusion rate to chamber volume (10:1) made it possible to stimulate the cells rapidly with agonists and insure rapid termination of response. A similar approach was previously used to make measurements on single, freshly isolated muscle cells anchored electrostatically to the ceiling of the chamber [ 5 ]. Measurement of binding in isolated muscle cells Muscle cells were suspended in Hepes medium which contained also 1~o bovine serum albumin (BSA), amastatin (10/~M), phosphoramidon (1/~M) and bacitracin (0.7 mM). Triplicate aliquots (0.3 ml) of cell suspension (10 6 cells/ml) were incubated with 125I-VIP (50 pM), alone or in the presence of the unlabeled VIP (10/zM). Bound and free radioligand were separated by rapid filtration under reduced
226 pressure through 5 #m polycarbonate Nucleopore filters followed by repeated washing (four times) with 3 ml of ice-cold Hepes medium containing 0.2~o BSA. Non-specific binding was measured as the amount of radioactivity associated with the muscle cells in the presence of 10 # M unlabeled VIP. Specific binding was calculated as the difference between total and non-specific binding. Non-specific binding was 22.6 + 1.8~o oftotat binding. The time course of binding was determined at 1, 2, 5, 10, 20 and 40 rain and temperature dependence at 4 °, 21 ° and 31 ° C. The ability of VIP, PHI, secretin and VIP10-28 to inhibit binding of 125I-VIP (50 pM) was measured after 5 min incubation at 21 ° C.
Measurement of binding in cultured muscle cells Each well was washed twice with 1 ml of Hepes medium containing 0.01% soybean trypsin inhibitor, 0.2~o bovine serum albumin and 0.1 ~o bacitracin. 125I-VIP (40 pM in 10/A) was added to 0.5 ml of binding medium in each well, alone or in the presence of unlabeled VIP (10/~M). At the end of the incubation period, the medium was aspirated and the wells washed four times with ice-cold Hepes medium. The cell layer was scraped off and solubilized with 1 ml of 0.1 M NaOH. Radioactivity was counted and protein content measured by the Micro BCA (bicinchoninic acid) method (Pierce Chemical Co., Rockford, IL). Non-specific binding was determined as the amount of radioactivity associated with cultured cells in the presence of 10 # M VIP. Nonspecific binding was 33.5 +4.0~o of total 125I-VIP bound. The time course of binding was determined in separate wells at 2, 5, 10, 20, 30 and 40 min and temperature dependence at 4 °, 21 ° and 37°C. The ability of VIP, PHI, secretin and VIP10-28 to inhibit binding of 125I-VIP (40 pM) was measured after 20 min incubation at 21°C. Data analysis Results were calculated as means + S.E. of n experiments. Statistical significance was tested using Student's t-test for paired or unpaired values. Bind-
ing curves were resolved into high- and low-affinity binding sites using L I G A N D program. The P.fit program was used to compute ICso and ECso values for concentration-response curves. K i for VIP10-28 was calculated from the fit of a linear Schild plot.
Materials 1zsI-VIP (2200 Ci/mmol) was obtained from N E N - D U P O N T , Wilmington, DE; CCK-8, VIP, PHI, secretin and VIP10-28 were obtained from Bachem, Torrance, CA; fungizone, streptomycin, penicillin from Hazelton Biologics, Lenexa, KS; fetal bovine serum from GIBCO-BRL, Grand Island, NY; Hepes from Research Organics, Inc. Cleveland, OH; collagenase type II and soybean trypsin inhibitor from Worthington, Freehold, NJ; Dulbecco's modified Eagle's medium from Mediatech, Washington DC; gentamycin, amphotericin B, bovine serum albumin, bacitracin and all other chemicals from Sigma Chemicals, St. Louis, MO.
Results
Binding of I:5I-VIP to freshly isolated gastric muscle cells Specific binding of ~25I-VIP to suspensions of guinea pig gastric muscle cells was rapid, attained a peak in 5 min and declined progressively to 60 + 8 ~o of peak in 40 rain (Fig. 2). Binding was optimal at 21 °C (range 4 o to 31 ° C) and reversible upon exposure to unlabeled VIP. In competition studies, VIP, PHI, secretin and VIP10-28 inhibited the binding of azsI-VIP in a concentration-dependent fashion (Fig. 3). The order of potency based on the ICso values listed in Table I was VIP > VIP 10-28 > PHI > secretin. VIP10-28 inhibited completely 125I-VIP binding and the competition curve was acute. Unlike VIP10-28, neither PHI nor secretin inhibited completely 125I-VIP binding: residual binding was 30 + 7 ~o ( P < 0.01) with 10 /~M PHI and 50+2~o (P<0.001) with 10 /~M secretin.
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MINUTES Fig. 2. Time course of specific binding of 125I-VIPto freshly isolated and cultured guinea pig gastric smooth muscle cells. Muscle ceils were incubated at 21°C with ~25I-VIP in the presence or absence of 10 #M unlabeled VIP. Maximal specific binding at 5 min was 1.92 + 0.25~o of added radioactivity in freshly isolated muscle cells and 2.05 + 0.5~o in cultured muscle cells. Results are expressed as percent of binding at 5 min. Values are means + S.E. of 4-6 experiments each done in duplicate.
~0 60 J
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0 -11 -9 -7 -5 CONCENTRATION (log M}
Binding of 125I"VIP to cultured gastric muscle cells
Fig. 3. Inhibition of ~251-VIP binding to freshly isolated (upper panel) and cultured (lower panel) gastric muscle cells by unlabeled VIP, PHI and secretin. Muscle cells were incubated for 5 min (freshly isolated cells) or 20 min (cultured cells) at 21°C with ~25I-VIP in the presence or absence of 10 /~M unlabeled VIP. Results are expressed as percent of control specific binding. Binding constants were determined from the fit of the curves using LIGAND program. Values are means + S.E. of 4-8 experiments each done in duplicate.
The characteristics o f binding o f 125I-VIP to cultured gastric muscle cells were similar to those o f binding to suspensions o f freshly isolated cells. The time course o f binding was rapid, attained a p e a k in 5 min but unlike binding to cell suspension did not decline during the subsequent 40 rain (Fig. 2). Binding was optimal at 2 1 ° C (range 4 ° to 3 7 ° C ) a n d reversible u p o n exposure to unlabeled VIP. The c o m p e t i t i o n curves for VIP, P H I , secretin a n d V I P 1 0 - 2 8 were closely similar to those o b t a i n e d in freshly isolated muscle cells (Fig. 3). A s shown in T a b l e I, the ord6r o f potency, V I P > V I P 1 0 2 8 > P H I > s e c r e t i n , a n d the absolute ICs0 values were similar to those o b t a i n e d in freshly isolated
muscle cells. H e r e also, V I P 1 0 - 2 8 inhibited completely 125I-VIP binding a n d the c o m p e t i t i o n curve was acute. In contrast, neither P H I nor secretin inhibited completely 1ESI-VIP binding: residual binding was 25 + 6~o ( P < 0.01)with 10 # M P H I and 34 + 8 ~o ( P < 0 . 0 1 ) with 10/~M secretin. T h e c o m p e t i t i o n curve for V I P in cultured muscle cells was also b r o a d a n d could be resolved into highaffinity a n d low-affinity c o m p o n e n t s with K d values (0.22 n M and 37 n M , respectively) similar to those o b t a i n e d in freshly isolated muscle cells. T h e n u m b e r o f high-affinity binding sites, 1021 per cell ( b a s e d on
The c o m p e t i t i o n curve for V I P was b r o a d a n d could be resolved into high-affinity a n d low-affinity c o m p o n e n t s with K d values o f 0.11 n M a n d 59 n M , respectively. T h e n u m b e r o f high-affinity sites, 1183 per cell, was a b o u t 1 ~o o f total binding sites.
228 TABLEI (A)
Freshly isolated muscle cells Binding
Cultured muscle cells Binding
IC50
potency
ICso
potency
VIP PHI Secretin VIP10-28
0.96 + 0.15 nM 210 + 70 nM 6930 + 3140 nM 160 _+20 nM
1 219 7187 167
0.78 + 0.12 nM 270 _+80 nM 2140 + 840 nM 160 + 20 nM
1 346 2692 205
(B)
Relaxation
VIP PHI Secretin
Relaxation
ECs0
potency
ECs0
potency
0.64 + 0.16 nM 6.3 + 0.7 nM 30.0 + 7.4 nM
1 10 47
0.17 + 0.03 nM 1.3 + 0.4 nM 22.5 + 8.8 nM
1 8 132
ICso values were calculated from the fit of binding competition curves and EC~0 from the fit of concentration-response curves. Values are means _+S.E. of 4-8 for binding and 4-6 experiments for relaxation.
an estimate o f 4.106 cells/mg protein) was a b o u t 1 ~o o f total binding sites.
Relaxation induced by VIP, PHI and secretin in freshly isolated muscle cells VIP, P H I and secretin elicited c o n c e n t r a t i o n d e p e n d e n t relaxation in freshly isolated suspensions o f muscle cells (Fig. 4). The ECs0 values listed in Table I show that the order o f p o t e n c y for relaxation (VIP > P H I > secretin) was similar to that for inhibition o f 125I-VIP binding, but that b o t h P H I and secretin were relatively m o r e potent in causing relaxation than in inhibiting 125I-VIP binding. V I P 1 0 - 2 8 inhibited V I P - i n d u c e d relaxation in a c o n c e n t r a t i o n - d e p e n d e n t fashion (ICs0 35 + 7 n M ) (Fig. 5). A t a c o n c e n t r a t i o n o f 10 # M , V I P 1 0 - 2 8 virtually abolished relaxation i n d u c e d by VIP, P H I a n d secretin. A t lower c o n c e n t r a t i o n s in the range o f 1-100 n M , V I P 1 0 - 2 8 shifted the concentrationr e s p o n s e curves for V I P to the right (Fig. 6). Schild analysis o f the d a t a yielded an inhibitory dissociation c o n s t a n t (Ki) for V I P 1 0 - 2 8 o f 0.5 + 0.3 n M .
Relaxation induced by VIP, PHI and secretin in cultured muscle cells VIP, P H I and secretin elicited concentrationd e p e n d e n t relaxation in cultured muscle cells. The m e a s u r e m e n t s were m a d e on single cells at the edge o f semi-confluent cultures. The ECs0 values listed in Table I showed that the o r d e r o f p o t e n c y for relaxation, V I P > P H I > secretin, was similar to that for freshly isolated muscle cells. In cultured muscle cells also, P H I and secretin were relatively more p o t e n t in causing relaxation than in inhibiting 125I-V1P binding.
Discussion The present study shows that VIP binds to and induces relaxation o f freshly isolated and cultured muscle cells from guinea pig stomach. The characteristics o f binding and relaxation were identical in the two p r e p a r a t i o n s implying full expression of VIP receptors and signalling p a t h w a y s both before and after culture.
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Fig. 5. Dose-dependent inhibition by VIP10-28 of relaxation induced by a submaximal concentration of VIP (10 nM) in freshly isolated gastric muscle cells. Relaxation was measured by scanning micrometry as described in Methods and expressed as percent decrease in maximal CCK-induced contraction. Values are means + S.E. of 3 experiments.
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-12 -10 -8 -6 PEPTIDE (log M) Fig. 4. Concentration-response curves for relaxant effects of VIP, PHI and secretin in isolated (upper panel) and cultured (lower panel) gastric muscle cells. Relaxation was measured by scanning micrometry as described in Methods and expressed as percent decrease in maximal CCK-induced contraction (31.8+_1.7 #m decrease from control cell length). Values are means + S.E. of 4-6 experiments.
The identical patterns of VIP binding in freshly isolated and cultured gastric muscle cells of the guinea pig differed from those reported by Ennes et al. [8] for freshly isolated and cultured colonic muscle cells of the rabbit where binding was observed only after cell culture. The difference was not due to the method of dispersion which was similar in both studies, but was probably due to the low density of receptors which confounds detection in freshly isolated muscle cells. Measurements of VIP-induced relaxation in freshly isolated muscle cells from various regions of the gut (stomach, intestine, and colon)
in several species, including human, dog, rabbit and guinea pig confirm the presence of VIP receptors and their ability to mediate relaxation [ 16]. Muscle cells from all these regions exhibited great sensitivity to VIP (threshold < 10-12 M) implying considerable amplification of the signal by a relatively small number of receptors. It is worth noting that measurements of relaxation could be made on single cultured muscle cells and that these measurements were similar to those obtained on suspensions of freshly isolated muscle cells (Table I). Previous studies had shown that measurements on freshly isolated single cells and suspensions of cells were similar [5]. Several features distinguished 125I-VIP binding to muscle cells. (1) Binding was rapid, attained 33~o (freshly isolated cells) to 66~o (cultured muscle cells) of peak binding within 2 min and was maximal within 5 rain. Binding declined progressively in freshly isolated muscle cells suggesting the presence of peptidase activity following cell dispersion that was not susceptible to inhibition by phosphoramidon or amastatin.
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VIP (log M) Fig. 6. Concentration-responsecurves for the relaxant effect of VIP alone(closedcircles)and in the presenceof VIP 10-28 (1 nM, closedtriangles; 10nM closed squares; 100 nM, closeddiamonds) in freshlyisolated gastric muscle cells. Relaxationwas measured by scanning micrometryas described in Methods and expressed as percentdecreasein maximalCCK-inducedcontraction.Values are means+ S.E. of 3 experiments.
Similar rapid binding was previously reported for contractile peptides (substance P and neurokinin A) [28] and may be a feature of binding to rapidly responding, excitable cells. (2) The competition curves for VIP were broad with low Hill coefficients (n H 0.6) similar to those reported for VIP in other cell types (e.g., canine intestinal mucosal membranes [23 ], rat brain, pituitary and mesenteric artery membranes [ 18,25], and cultured endothelial cells [24]). The curves could be resolved into high- and low-affinity binding sites yielding binding constants similar to those reported for other cell types [6,20,21,23-25]. (3) PHI and secretin only partially inhibited 1251VIP binding implying the existence of VIP-preferring receptors that are recognized by PHI and secretin as well as VIP-specific receptors that are not recognized by either PHI or secretin. This pattern of competition is distinctly different from that observed in other cell
types where PHI completely displaces VIP binding and appears to interact only with VIP receptors [ 18,20,21,22-25]. (4) VIP10-28 was a potent inhibitor of ~25I-VIP binding and VIP-induced relaxation. Both the slope of the competition curves (Hill coefficient of 1) (Fig. 3) and the slope of the Schild plot (1.05) (Fig. 6) were not significantly different from unity consistent with competitive antagonism by VIP10-28. Comparison of the inhibitory constants, however, showed VIP10-28 to be relatively more potent as an inhibitor of relaxation (K i 0.5 + 0.3 nM) than of binding (IC50 160 + 20 nM). One explanation of the difference is that inhibition of relaxation reflects antagonism of high-affinity receptors (K a binding 0.1-0.2 nM) whereas inhibition of binding reflects displacement of 125I-VIP from both high-affinity (1~o) and low-affinity (99 ~o) receptors. (5) The relative potencies of PHI and secretin as relaxant agents differed from their relative potencies as competitors of 125I-VIP binding. In freshly isolated and cultured muscle cells, PHI was 8-10-fold, and secretin 47-132-fold less potent than VIP in causing relaxation. In contrast, PHI was 219-346fold, and secretin 2692-7187-fold less potent than VIP in inhibiting ~25I-VIP binding. While the relative potencies of PHI and secretin as inhibitors of 1251VIP binding reflected their affinity for VIP receptors, their potencies as relaxant agents may reflect additionally their ability to interact with PHI- and secretin-specific receptors. The functional significance of VIP-specific receptors should be viewed in light of recent studies in which VIP was shown to interact with two signalling pathways in guinea pig gastric muscle cells [ 15,16,19]. One pathway involves interaction of VIP with receptors coupled to adenylate cyclase leading to generation of cAMP and activation of cAMPdependent protein kinase. A separate pathway involves interaction of VIP with receptors that mediate sequentially activation of nitric oxide synthase and generation of NO, activation of soluble guanylate cyclase and generation of cGMP, and activation
231 o f c G M P - d e p e n d e n t p r o t e i n kinase. S i n c e o n l y V I P , b u t n o t o t h e r r e l a x a n t agents, i n c l u d i n g its h o m o logues, P H I
a n d secretin, i s o p r o t e r e n o l , forskolin,
a n d p e r m e a n t d e r i v a t i v e s o f cyclic 3 ' , 5 ' - a d e n o s i n e monophosphate, stimulates NO and cGMP generation, the N O - d e p e n d e n t
p a t h w a y a p p e a r s to repre-
sent i n t e r a c t i o n o f V I P w i t h V I P - s p e c i f i c r e c e p t o r s . V I P - p r e f e r r i n g r e c e p t o r s w h i c h are r e c o g n i z e d by P H I are c o u p l e d p r e s u m a b l y to a c t i v a t i o n o f a d e n y late cyclase. P r e l i m i n a r y studies ( J - G . Jin, K . S . M u r thy, J . R . G r i d e r a n d G . M . M a k h l o u f , u n p u b l i s h e d studies) suggest t h a t P i t u i t a r y A d e n y l a t e C y c l a s e A c tivating Peptide (PACAP) activates both pathways m e d i a t e d by V I P c o n s i s t e n t with i n t e r a c t i o n o f V I P and PACAP
with a common
r e c e p t o r o n gastric
m u s c l e cells similar to t h a t r e p o r t e d for t h e s e t w o p e p t i d e s in o t h e r cell types [ 1,27].
Acknowledgement T h i s w o r k w a s s u p p o r t e d by g r a n t D K - 2 8 3 0 0 f r o m the N a t i o n a l I n s t i t u t e o f D i a b e t e s a n d D i g e s t i v e a n d Kidney Diseases.
References 1 Arimura, A., Receptors for pituitary adenylate cyclaseactivating polypeptide: comparison with vasoactive intestinal peptide receptors, Trends Endocilnol. Metab., 3 (1992)288294. 2 Biancani, P., Walsh, J.H. and Behar, J., Vasoactive intestinal polypeptide: A neurotransmitter for lower esophageal sphincter relaxation, J. Clin. Invest., 73 (1984) 963-967. 3 Bitar, K.N. and Makhlouf, G.M., Relaxation of isolated gastric smooth muscle cells by vasoactive intestinal peptide, Science, 216 (1982) 531-533. 4 Bitar, K.N. and Makhlouf, G.M., Receptors on smooth muscle cells: characterization by contraction and specific antagonists, Am. J. Physiol., 242 (1982) G400-G407. 5 Bitar, K.N. and Makhlouf, G.M., Measurement of function in isolated single smooth muscle cells, Am. J. Physiol., 250 (1986) G357-360. 6 Christophe, J.P., Conlon, T.P. and Gardner, J.D., Interaction of porcine vasoactive intestinal peptide with dispersed acinar
cells from the guinea pig, J. Biol. Chem., 251 (1976) 46294634. 7 Costa, M. and Furness, J.B., The origins, pathways and terminations of neurons with VIP-like immunoreactivity in the guinea pig small intestine, Neuroscience, 8 (1983) 665676. 8 Ennes, H.S., McRoberts, J.A., Hyman, P.E. and Snape, W.J., Characterization of colonic circular smooth muscle cells in culture, Am. J. Physiol., 263 (1992) G365-G370. 9 Fahrenkrug, J., Vasoactive intestinal peptide, In G.M. Makhlouf, Handbook of Physiology, The Gastrointestinal System, Am. Physiol. Soc., Washington DC, 1989, Vol. 2, pp. 611-629. 10 Furness, J.B., Bornstein, J.C., Murphy, R. and Pompolo, S,, Roles of peptides in transmission in the enteric nervous system. Trends Neurosci., 15 (1992)66-71. 11 Goyal, R.K., Rattan, S. and Said, S.I., VIP as a possible neurotransmitter of non-cholinergic, non-adrenergic inhibitory neurones, Nature, 288 (1985) 378-380. 12 Grider, J.R. and Makhlouf, G.M., Colonic peristaltic reflex: identification of vasoactive intestinal peptide as mediator of descending relaxation, Am. J. Physiol., 251 (1986) G40-G45. 13 Gilder, J.R. and Makhlouf, G.M., Vasoactive intestinal peptide: transmitter of inhibitory motor neurons of the gut, Ann. N.Y. Acad. Sci., 527 (1988) 369-377. 14 Grider, J.R. and Rivier, J.J., Vasoactive intestinal peptide (VIP) as transmitter of inhibitory motor neurons of the gut: Evidence from the use of selective VIP antagonists and VIP antiserum, J. Pharmacol. Exp. Ther., 253 (1990) 738-742. 15 Grider, J.R., Murthy, K.S., Jin, J-G. and Makhlouf, G.M., Stimulation of nitric oxide from muscle cells by VIP: prejunctional enhancement of VIP release, Am. J. Physiol., 262 (1992) G774-G778. 16 Grider, J.R. and Jin, J-G., VIP-induced nitric oxide production and relaxation in isolated muscle cells of the gut in human and other mammalian species, Gastroenterology, 104 (1993) A515. 17 Grider, J.R. and Jin, J-G., VIP release and L-citrulline production from isolated ganglia of the myenteric plexus: evidence for regulation of VIP release by nitric oxide, Neuroscience, 54 (1993) 521-526. 18 Huang, M. and Rorstad, O.P., PHI preferentially binds to VIP receptors in normal rat tissues, Peptides, 11 (1990) 10151020. 19 Jin, J-G., Murthy, K.S., Gilder, J.R. and Makhlouf, G.M., Activation of distinct cAMP- and cGMP-dependent pathways by relaxant agents in isolated gastric muscle cells, Am. J. Physiol., 264 (1993) G470-G477. 20 Jensen, R.T., Tatemoto, K., Mutt, V., Lemp, G.F. and Gardner, J.D., Actions of a newly isolated intestinal peptide PHI on pancreatic acini, Am. J. Physiol., 241 (1981) G498-G502.
232 21 Jensen, R.T., C.G. Charlton, Adachi, H., Jones, S.W., O'Donohue, T.L. and Gardner, J.D., Use of ~25I-secretin to identify and characterize high- affinity secretin receptors on pancreatic acini, Am. J. Physiol., 245 (1983) G186-G195. 22 Laburthe, M. and Amiranoff, B., Peptide receptors in intestinal epithelium. In G.M. Makhlouf, Handbook of Physiology, Vol. 2, American Physiological Society, Washington DC, 1991, pp. 215-243. 23 Mao, Y-K., Barnett, W., Coy, D.H., Tougas, G. and Daniel, E.E., Distribution of vasoactive intestinal polypeptide (VIP) binding in circular muscle and characterization of VIP binding in canine small intestinal mucosa, J. Pharmacol. Exp. Ther., 258 (1991) 986-991. 24 Pasyk, E., Mao, Y-K., Ahmad, S., Shen, S-H. and Daniel, E.E., An endothelial cell-line contains functional vasoactive intestinal polypeptide receptors: they control inwardly rectifying K ÷ channels, Eur. J. Pharmacol., 221 (1992) 209-214. 25 Rorstad, O.P., Wanke, I., Coy, D.H., Fournier, A. and Huang, M., Selectivity for binding of peptide analogues to vascular
26
27
28
29
30
receptors for vasoactive intestinal peptide, Mol. Pharmacol. 37 (1990) 971-977. Said, S.I. and Mutt, V., Polypeptide with broad biological activity: Isolation from small intestine, Science, 169 (1970) 1217-1218. Shivers, B.D., Gorcs, T.J., Gottschall, P.E. and Arimura, A., Two high affinity binding sites for pituitary adenylate cyclaseactivating polypeptide have different tissue distributions, Endocrinology, 128 (1991) 3055-3065. Souquet, J-C, Bitar, K.N., Grider, J.R. and Makhlouf, G.M., Receptors for substance P on isolated smooth muscle cells of the guinea pig, Am. J. Physiol., 253 (1987) G666-672. Stark, M.E. and Szurzewski, J.H., Role of nitric oxide in gastrointestinal and hepatic function and disease, Gastroenterology, 103 (1992) 1928-1949. Tatemoto, K. and Mutt, V., Isolation and characterization of the intestinal peptide porcine PHI (PHI-27), a new member of the glucagon- secretin family, Proc. Natl. Acad. Sci. USA, 78 (1981) 6603-6607.
223
© 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00
REGPEP 01524
Expression of functional receptors for vasoactive intestinal peptide in freshly isolated and cultured gastric muscle cells Y. Chijiiwa l, K.S. Murthy, J.R. Grider and G.M. Makhlouf Departments of Physiology and Medicine, Medical College of Virginia, Richmond, VA (USA) (Received 8 February 1993; revised version received and accepted 26 April 1993)
Key words." Peptide histidine isoleucine (PHI); Secretin; VIP antagonist; VIP binding; Muscle relaxation; Gastric smooth muscle
Summary Vasoactive intestinal peptide (VIP) receptors were characterized in freshly isolated and cultured smooth muscle cells from guinea pig stomach by radioligand binding and by measurement of relaxation in single isolated and cultured cells. 125I-VIP bound to both freshly isolated and cultured muscle cells: binding was rapid, specific, saturable and temperature-dependent, and was inhibited in a concentration-dependent fashion by VIP, VIP10-28, PHI and secretin, in this order. Competition curves for VIP could be resolved into high- and lowaffinity components, yielding similar binding constants in freshly isolated and cultured cells (high-affinity K d 0.11 and 0.22 nM; low-affinity K a 59 and 37 nM; high-affinity binding sites: 1183 and 1021 per cell, representing about 1 ~o of total binding sites). VIP10-28 inhibited a25I-VIP binding completely and acted as potent competitive antagonist of VIP-induced relaxation (Ki 0.5 nM). PHI and secretin, however, inhibited partly 125I-VIP binding: the pattern of inhibition implied that VIP interacts with VIP-preferring receptors that are recognized by PHI and secretin as well as with VIP-specific receptors. The pattern of binding is consistent with recent evidence indicating that VIP activates two signalling pathways, a VIP-specific, nitric oxide/cGMP-dependent pathway and a common cAMP-dependent pathway shared by all three peptides. PHI and secretin were relatively more potent as relaxant agents than as inhibitors of ~25I-VIP binding raising the possibility that PHI and secretin could interact additionally with PHI- and secretin-preferring receptors in mediating relaxation.
Correspondence to: G.M. Makhlouf, Box 711, MCV Station, Medical College of Virginia, Richmond, VA 23298-0711, USA. Abbreviations: VIP, vasoactive intestinal peptide; PHI, peptide histidine isoleucine; PHM, peptide histidine methionine; CCK-8, cholecystokinin octapeptide. ~Dr. Y. Chijiiwa was a Visiting Scientist from Kyushu University, Japan.
224 Introduction
Vasoactive intestinal peptide and its homologues, peptide histidine isoleucine (PHI) in animals and peptide histidine methionine (PHM) in humans, are synthesized in the same precursor and co-released from neurons of the enteric nervous system [9,26,30]. VIP fulfills many of the criteria required to establish it as a nonadrenergic, noncholinergic inhibitory neurotransmitter, responsible for relaxation in various regions of the gut [13]. VIP neurons innervate circular smooth muscle in all regions of the gut [7,10]; release of VIP from these neurons during electrical or reflex stimulation is accompanied by a stoichiometric increase in relaxation that can be blocked by specific VIP antiserum and selective VIP antagonists (e.g., VIP10-28) [2,11-14]. Recent evidence suggests that nitric oxide also is involved in neurallymediated relaxation in the gut [29] and that its release and action are closely linked to the release and action of VIP [15-17,19]. In the myenteric plexus, NO synthase is present exclusively in VIP neurons [9]; NO produced in these neurons regulates VIP release [17]. VIP, in turn, acts on muscle cells to re-generate NO [15,16,19]. Consistent with this notion, NO synthase inhibitors abolish NO production induced by nerve stimulation of muscle strips and partially inhibit VIP release and muscle relaxation [15]. In isolated muscle cells, VIP but not PHI, stimulates NO production; NO synthase inhibitors abolish VIP-induced NO production and partly inhibit relaxation [ 15,19]. The disparate effects of VIP and PHI suggest that VIP may interact with receptors coupled to different signalling pathways. VIP receptors have been characterized extensively in neural, epithelial, exocrine and vascular tissues by radioligand binding [ 18,2025 ]. Competition curves are often wide-spanned suggesting the presence of multiple receptors [18,2325]. In studies on heterogeneous membranes, however, it is not possible to determine whether multiple receptors are located on membranes derived from one cell type or from different cell types.
In earlier studies on muscle strips [12-14] and isolated muscle cells [3] of guinea pig stomach, VIP was shown to cause relaxation that was accompanied by increase in intracellular levels of cAMP. More recent studies on muscle cells isolated from various regions of the gut in several species have yielded similar results [ 15,16]. In contrast, Ennes et al. [8] detected VIP binding and increase in cAMP in cultured but not in freshly isolated muscle cells from rabbit colon; no measurements of relaxation were reported. In the present study, we have characterized VIP binding and VIP-induced relaxation in freshly isolated muscle cells devoid of neural elements and in cultured smooth muscle cells from guinea pig stomach, and determined the relative potencies of PHI, secretin and the VIP antagonist, VIP10-28, so as to gain further insight into the mechanisms of action of VIP in smooth muscle. The results demonstrate identical binding and relaxation in freshly isolated and cultured muscle cells and provide evidence for binding of VIP to more than one receptor type consistent with its coupling to distinct signalling pathways.
Materials and Methods
Dispersion of gastric muscle cells Smooth muscle cells were isolated from the circular muscle layer of guinea pig stomach as described previously [3-5]. Muscle strips were incubated for two 45-min periods at 31 °C in 25 ml of Hepes medium containing 150 units/ml collagenase (Type II) and 0.01 ~o soybean trypsin inhibitor. The composition of the medium was (in mM): NaC1 120, KC1 4, KH2PO 4 2.6, CaC12 2, MgC12 0.6, glucose 14, and Eagle's essential amino acids mixture 2.1 ~o- The partially digested strips were washed and re-incubated in enzyme-free medium to allow the cells to dissociate spontaneously. The cells were harvested by filtration through 500-#m Nitex mesh and centrifuged twice at 350 g for 10 min to eliminate cell fragments and organelles.
225
Culture of gastric muscle cells For cell culture, cells harvested by filtration were centrifuged at 350 g for 10 min and re-suspended in Dulbecco's modified Eagle's medium (DMEM-0) containing penicillin (200 units/ml), streptomycin (200/~g/ml, gentamycin (100 ~g/ml), fungizone (0.5 #g/ml), and amphotericin B (2.5 ~g/ml). The cells were centrifuged again and re-suspended in the same medium with 10~o fetal bovine serum (DMEM-10). Each stomach yielded about 3.106 cells which were plated in 24-well clusters (Costar, Cambridge, MA) at a concentration of 105 cells/0.5 ml and placed in a CO 2 incubator at 37 ° C. The cells adhered to the bottom of the well within 24-48 h, began dividing within 72 h and attained full confluence in 10-14 days whereupon each well contained 0.26 + 0.02 mg protein; this amount of protein is present in about 10 6 freshly isolated muscle cells. During culture, the DMEM-10 medium was replaced every 3 days. Cells in culture (Fig. 1) assumed typical spindle-like shapes and a hill-and-valley architecture. The cells stained for smooth muscle-specific 7-actin with monoclonal antibody CGA7 and responded to contractile and relaxant agonists (see Results).
Fig. 1. Confluentcultures of smooth muscle cells from guinea pig stomach showing characteristic spindle-shaped cells and cell architecture. Bar = 100/~m.
Measurement of contraction and relaxation in freshly &olated and cultured muscle cells In freshly isolated muscle cells contraction and relaxation were measured by scanning micrometry as described previously [3,4]. Briefly, 0.5 ml of cell suspension (10 4 cells/ml) was added to 0.2 ml of medium containing a contractile agonist and the reaction terminated at 30 s when peak contraction was attained. The lengths of 50 cells treated with and without agonist were measured by scanning micrometry in sequential microscopic fields and contractile response expressed as the mean decrease in cell length from control. For measurement of relaxation, the cells were preincubated for 60 s with the relaxant agent and then for 30 s with a maximally effective concentration of the contractile agent, cholecystokinin octapeptide (CCK-8 1 nM). Relaxation was expressed as percent inhibition of maximal contraction. In cultured muscle cells, contraction and relaxation were measured by scanning micrometry in individual cells located at the edge of semi-confluent monolayers. For this purpose, cells were grown on coverslips which were positioned so as to form the ceiling of a perfusion chamber. The volume of the chamber was 0.1 ml and the perfusion rate was 1 ml/min. The ratio of perfusion rate to chamber volume (10:1) made it possible to stimulate the cells rapidly with agonists and insure rapid termination of response. A similar approach was previously used to make measurements on single, freshly isolated muscle cells anchored electrostatically to the ceiling of the chamber [ 5 ]. Measurement of binding in isolated muscle cells Muscle cells were suspended in Hepes medium which contained also 1~o bovine serum albumin (BSA), amastatin (10/~M), phosphoramidon (1/~M) and bacitracin (0.7 mM). Triplicate aliquots (0.3 ml) of cell suspension (10 6 cells/ml) were incubated with 125I-VIP (50 pM), alone or in the presence of the unlabeled VIP (10/zM). Bound and free radioligand were separated by rapid filtration under reduced
226 pressure through 5 #m polycarbonate Nucleopore filters followed by repeated washing (four times) with 3 ml of ice-cold Hepes medium containing 0.2~o BSA. Non-specific binding was measured as the amount of radioactivity associated with the muscle cells in the presence of 10 # M unlabeled VIP. Specific binding was calculated as the difference between total and non-specific binding. Non-specific binding was 22.6 + 1.8~o oftotat binding. The time course of binding was determined at 1, 2, 5, 10, 20 and 40 rain and temperature dependence at 4 °, 21 ° and 31 ° C. The ability of VIP, PHI, secretin and VIP10-28 to inhibit binding of 125I-VIP (50 pM) was measured after 5 min incubation at 21 ° C.
Measurement of binding in cultured muscle cells Each well was washed twice with 1 ml of Hepes medium containing 0.01% soybean trypsin inhibitor, 0.2~o bovine serum albumin and 0.1 ~o bacitracin. 125I-VIP (40 pM in 10/A) was added to 0.5 ml of binding medium in each well, alone or in the presence of unlabeled VIP (10/~M). At the end of the incubation period, the medium was aspirated and the wells washed four times with ice-cold Hepes medium. The cell layer was scraped off and solubilized with 1 ml of 0.1 M NaOH. Radioactivity was counted and protein content measured by the Micro BCA (bicinchoninic acid) method (Pierce Chemical Co., Rockford, IL). Non-specific binding was determined as the amount of radioactivity associated with cultured cells in the presence of 10 # M VIP. Nonspecific binding was 33.5 +4.0~o of total 125I-VIP bound. The time course of binding was determined in separate wells at 2, 5, 10, 20, 30 and 40 min and temperature dependence at 4 °, 21 ° and 37°C. The ability of VIP, PHI, secretin and VIP10-28 to inhibit binding of 125I-VIP (40 pM) was measured after 20 min incubation at 21°C. Data analysis Results were calculated as means + S.E. of n experiments. Statistical significance was tested using Student's t-test for paired or unpaired values. Bind-
ing curves were resolved into high- and low-affinity binding sites using L I G A N D program. The P.fit program was used to compute ICso and ECso values for concentration-response curves. K i for VIP10-28 was calculated from the fit of a linear Schild plot.
Materials 1zsI-VIP (2200 Ci/mmol) was obtained from N E N - D U P O N T , Wilmington, DE; CCK-8, VIP, PHI, secretin and VIP10-28 were obtained from Bachem, Torrance, CA; fungizone, streptomycin, penicillin from Hazelton Biologics, Lenexa, KS; fetal bovine serum from GIBCO-BRL, Grand Island, NY; Hepes from Research Organics, Inc. Cleveland, OH; collagenase type II and soybean trypsin inhibitor from Worthington, Freehold, NJ; Dulbecco's modified Eagle's medium from Mediatech, Washington DC; gentamycin, amphotericin B, bovine serum albumin, bacitracin and all other chemicals from Sigma Chemicals, St. Louis, MO.
Results
Binding of I:5I-VIP to freshly isolated gastric muscle cells Specific binding of ~25I-VIP to suspensions of guinea pig gastric muscle cells was rapid, attained a peak in 5 min and declined progressively to 60 + 8 ~o of peak in 40 rain (Fig. 2). Binding was optimal at 21 °C (range 4 o to 31 ° C) and reversible upon exposure to unlabeled VIP. In competition studies, VIP, PHI, secretin and VIP10-28 inhibited the binding of azsI-VIP in a concentration-dependent fashion (Fig. 3). The order of potency based on the ICso values listed in Table I was VIP > VIP 10-28 > PHI > secretin. VIP10-28 inhibited completely 125I-VIP binding and the competition curve was acute. Unlike VIP10-28, neither PHI nor secretin inhibited completely 125I-VIP binding: residual binding was 30 + 7 ~o ( P < 0.01) with 10 /~M PHI and 50+2~o (P<0.001) with 10 /~M secretin.
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MINUTES Fig. 2. Time course of specific binding of 125I-VIPto freshly isolated and cultured guinea pig gastric smooth muscle cells. Muscle ceils were incubated at 21°C with ~25I-VIP in the presence or absence of 10 #M unlabeled VIP. Maximal specific binding at 5 min was 1.92 + 0.25~o of added radioactivity in freshly isolated muscle cells and 2.05 + 0.5~o in cultured muscle cells. Results are expressed as percent of binding at 5 min. Values are means + S.E. of 4-6 experiments each done in duplicate.
~0 60 J
eeretin
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P10-28
0 -11 -9 -7 -5 CONCENTRATION (log M}
Binding of 125I"VIP to cultured gastric muscle cells
Fig. 3. Inhibition of ~251-VIP binding to freshly isolated (upper panel) and cultured (lower panel) gastric muscle cells by unlabeled VIP, PHI and secretin. Muscle cells were incubated for 5 min (freshly isolated cells) or 20 min (cultured cells) at 21°C with ~25I-VIP in the presence or absence of 10 /~M unlabeled VIP. Results are expressed as percent of control specific binding. Binding constants were determined from the fit of the curves using LIGAND program. Values are means + S.E. of 4-8 experiments each done in duplicate.
The characteristics o f binding o f 125I-VIP to cultured gastric muscle cells were similar to those o f binding to suspensions o f freshly isolated cells. The time course o f binding was rapid, attained a p e a k in 5 min but unlike binding to cell suspension did not decline during the subsequent 40 rain (Fig. 2). Binding was optimal at 2 1 ° C (range 4 ° to 3 7 ° C ) a n d reversible u p o n exposure to unlabeled VIP. The c o m p e t i t i o n curves for VIP, P H I , secretin a n d V I P 1 0 - 2 8 were closely similar to those o b t a i n e d in freshly isolated muscle cells (Fig. 3). A s shown in T a b l e I, the ord6r o f potency, V I P > V I P 1 0 2 8 > P H I > s e c r e t i n , a n d the absolute ICs0 values were similar to those o b t a i n e d in freshly isolated
muscle cells. H e r e also, V I P 1 0 - 2 8 inhibited completely 125I-VIP binding a n d the c o m p e t i t i o n curve was acute. In contrast, neither P H I nor secretin inhibited completely 1ESI-VIP binding: residual binding was 25 + 6~o ( P < 0.01)with 10 # M P H I and 34 + 8 ~o ( P < 0 . 0 1 ) with 10/~M secretin. T h e c o m p e t i t i o n curve for V I P in cultured muscle cells was also b r o a d a n d could be resolved into highaffinity a n d low-affinity c o m p o n e n t s with K d values (0.22 n M and 37 n M , respectively) similar to those o b t a i n e d in freshly isolated muscle cells. T h e n u m b e r o f high-affinity binding sites, 1021 per cell ( b a s e d on
The c o m p e t i t i o n curve for V I P was b r o a d a n d could be resolved into high-affinity a n d low-affinity c o m p o n e n t s with K d values o f 0.11 n M a n d 59 n M , respectively. T h e n u m b e r o f high-affinity sites, 1183 per cell, was a b o u t 1 ~o o f total binding sites.
228 TABLEI (A)
Freshly isolated muscle cells Binding
Cultured muscle cells Binding
IC50
potency
ICso
potency
VIP PHI Secretin VIP10-28
0.96 + 0.15 nM 210 + 70 nM 6930 + 3140 nM 160 _+20 nM
1 219 7187 167
0.78 + 0.12 nM 270 _+80 nM 2140 + 840 nM 160 + 20 nM
1 346 2692 205
(B)
Relaxation
VIP PHI Secretin
Relaxation
ECs0
potency
ECs0
potency
0.64 + 0.16 nM 6.3 + 0.7 nM 30.0 + 7.4 nM
1 10 47
0.17 + 0.03 nM 1.3 + 0.4 nM 22.5 + 8.8 nM
1 8 132
ICso values were calculated from the fit of binding competition curves and EC~0 from the fit of concentration-response curves. Values are means _+S.E. of 4-8 for binding and 4-6 experiments for relaxation.
an estimate o f 4.106 cells/mg protein) was a b o u t 1 ~o o f total binding sites.
Relaxation induced by VIP, PHI and secretin in freshly isolated muscle cells VIP, P H I and secretin elicited c o n c e n t r a t i o n d e p e n d e n t relaxation in freshly isolated suspensions o f muscle cells (Fig. 4). The ECs0 values listed in Table I show that the order o f p o t e n c y for relaxation (VIP > P H I > secretin) was similar to that for inhibition o f 125I-VIP binding, but that b o t h P H I and secretin were relatively m o r e potent in causing relaxation than in inhibiting 125I-VIP binding. V I P 1 0 - 2 8 inhibited V I P - i n d u c e d relaxation in a c o n c e n t r a t i o n - d e p e n d e n t fashion (ICs0 35 + 7 n M ) (Fig. 5). A t a c o n c e n t r a t i o n o f 10 # M , V I P 1 0 - 2 8 virtually abolished relaxation i n d u c e d by VIP, P H I a n d secretin. A t lower c o n c e n t r a t i o n s in the range o f 1-100 n M , V I P 1 0 - 2 8 shifted the concentrationr e s p o n s e curves for V I P to the right (Fig. 6). Schild analysis o f the d a t a yielded an inhibitory dissociation c o n s t a n t (Ki) for V I P 1 0 - 2 8 o f 0.5 + 0.3 n M .
Relaxation induced by VIP, PHI and secretin in cultured muscle cells VIP, P H I and secretin elicited concentrationd e p e n d e n t relaxation in cultured muscle cells. The m e a s u r e m e n t s were m a d e on single cells at the edge o f semi-confluent cultures. The ECs0 values listed in Table I showed that the o r d e r o f p o t e n c y for relaxation, V I P > P H I > secretin, was similar to that for freshly isolated muscle cells. In cultured muscle cells also, P H I and secretin were relatively more p o t e n t in causing relaxation than in inhibiting 125I-V1P binding.
Discussion The present study shows that VIP binds to and induces relaxation o f freshly isolated and cultured muscle cells from guinea pig stomach. The characteristics o f binding and relaxation were identical in the two p r e p a r a t i o n s implying full expression of VIP receptors and signalling p a t h w a y s both before and after culture.
229
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Fig. 5. Dose-dependent inhibition by VIP10-28 of relaxation induced by a submaximal concentration of VIP (10 nM) in freshly isolated gastric muscle cells. Relaxation was measured by scanning micrometry as described in Methods and expressed as percent decrease in maximal CCK-induced contraction. Values are means + S.E. of 3 experiments.
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-12 -10 -8 -6 PEPTIDE (log M) Fig. 4. Concentration-response curves for relaxant effects of VIP, PHI and secretin in isolated (upper panel) and cultured (lower panel) gastric muscle cells. Relaxation was measured by scanning micrometry as described in Methods and expressed as percent decrease in maximal CCK-induced contraction (31.8+_1.7 #m decrease from control cell length). Values are means + S.E. of 4-6 experiments.
The identical patterns of VIP binding in freshly isolated and cultured gastric muscle cells of the guinea pig differed from those reported by Ennes et al. [8] for freshly isolated and cultured colonic muscle cells of the rabbit where binding was observed only after cell culture. The difference was not due to the method of dispersion which was similar in both studies, but was probably due to the low density of receptors which confounds detection in freshly isolated muscle cells. Measurements of VIP-induced relaxation in freshly isolated muscle cells from various regions of the gut (stomach, intestine, and colon)
in several species, including human, dog, rabbit and guinea pig confirm the presence of VIP receptors and their ability to mediate relaxation [ 16]. Muscle cells from all these regions exhibited great sensitivity to VIP (threshold < 10-12 M) implying considerable amplification of the signal by a relatively small number of receptors. It is worth noting that measurements of relaxation could be made on single cultured muscle cells and that these measurements were similar to those obtained on suspensions of freshly isolated muscle cells (Table I). Previous studies had shown that measurements on freshly isolated single cells and suspensions of cells were similar [5]. Several features distinguished 125I-VIP binding to muscle cells. (1) Binding was rapid, attained 33~o (freshly isolated cells) to 66~o (cultured muscle cells) of peak binding within 2 min and was maximal within 5 rain. Binding declined progressively in freshly isolated muscle cells suggesting the presence of peptidase activity following cell dispersion that was not susceptible to inhibition by phosphoramidon or amastatin.
230 80
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VIP (log M) Fig. 6. Concentration-responsecurves for the relaxant effect of VIP alone(closedcircles)and in the presenceof VIP 10-28 (1 nM, closedtriangles; 10nM closed squares; 100 nM, closeddiamonds) in freshlyisolated gastric muscle cells. Relaxationwas measured by scanning micrometryas described in Methods and expressed as percentdecreasein maximalCCK-inducedcontraction.Values are means+ S.E. of 3 experiments.
Similar rapid binding was previously reported for contractile peptides (substance P and neurokinin A) [28] and may be a feature of binding to rapidly responding, excitable cells. (2) The competition curves for VIP were broad with low Hill coefficients (n H 0.6) similar to those reported for VIP in other cell types (e.g., canine intestinal mucosal membranes [23 ], rat brain, pituitary and mesenteric artery membranes [ 18,25], and cultured endothelial cells [24]). The curves could be resolved into high- and low-affinity binding sites yielding binding constants similar to those reported for other cell types [6,20,21,23-25]. (3) PHI and secretin only partially inhibited 1251VIP binding implying the existence of VIP-preferring receptors that are recognized by PHI and secretin as well as VIP-specific receptors that are not recognized by either PHI or secretin. This pattern of competition is distinctly different from that observed in other cell
types where PHI completely displaces VIP binding and appears to interact only with VIP receptors [ 18,20,21,22-25]. (4) VIP10-28 was a potent inhibitor of ~25I-VIP binding and VIP-induced relaxation. Both the slope of the competition curves (Hill coefficient of 1) (Fig. 3) and the slope of the Schild plot (1.05) (Fig. 6) were not significantly different from unity consistent with competitive antagonism by VIP10-28. Comparison of the inhibitory constants, however, showed VIP10-28 to be relatively more potent as an inhibitor of relaxation (K i 0.5 + 0.3 nM) than of binding (IC50 160 + 20 nM). One explanation of the difference is that inhibition of relaxation reflects antagonism of high-affinity receptors (K a binding 0.1-0.2 nM) whereas inhibition of binding reflects displacement of 125I-VIP from both high-affinity (1~o) and low-affinity (99 ~o) receptors. (5) The relative potencies of PHI and secretin as relaxant agents differed from their relative potencies as competitors of 125I-VIP binding. In freshly isolated and cultured muscle cells, PHI was 8-10-fold, and secretin 47-132-fold less potent than VIP in causing relaxation. In contrast, PHI was 219-346fold, and secretin 2692-7187-fold less potent than VIP in inhibiting ~25I-VIP binding. While the relative potencies of PHI and secretin as inhibitors of 1251VIP binding reflected their affinity for VIP receptors, their potencies as relaxant agents may reflect additionally their ability to interact with PHI- and secretin-specific receptors. The functional significance of VIP-specific receptors should be viewed in light of recent studies in which VIP was shown to interact with two signalling pathways in guinea pig gastric muscle cells [ 15,16,19]. One pathway involves interaction of VIP with receptors coupled to adenylate cyclase leading to generation of cAMP and activation of cAMPdependent protein kinase. A separate pathway involves interaction of VIP with receptors that mediate sequentially activation of nitric oxide synthase and generation of NO, activation of soluble guanylate cyclase and generation of cGMP, and activation
231 o f c G M P - d e p e n d e n t p r o t e i n kinase. S i n c e o n l y V I P , b u t n o t o t h e r r e l a x a n t agents, i n c l u d i n g its h o m o logues, P H I
a n d secretin, i s o p r o t e r e n o l , forskolin,
a n d p e r m e a n t d e r i v a t i v e s o f cyclic 3 ' , 5 ' - a d e n o s i n e monophosphate, stimulates NO and cGMP generation, the N O - d e p e n d e n t
p a t h w a y a p p e a r s to repre-
sent i n t e r a c t i o n o f V I P w i t h V I P - s p e c i f i c r e c e p t o r s . V I P - p r e f e r r i n g r e c e p t o r s w h i c h are r e c o g n i z e d by P H I are c o u p l e d p r e s u m a b l y to a c t i v a t i o n o f a d e n y late cyclase. P r e l i m i n a r y studies ( J - G . Jin, K . S . M u r thy, J . R . G r i d e r a n d G . M . M a k h l o u f , u n p u b l i s h e d studies) suggest t h a t P i t u i t a r y A d e n y l a t e C y c l a s e A c tivating Peptide (PACAP) activates both pathways m e d i a t e d by V I P c o n s i s t e n t with i n t e r a c t i o n o f V I P and PACAP
with a common
r e c e p t o r o n gastric
m u s c l e cells similar to t h a t r e p o r t e d for t h e s e t w o p e p t i d e s in o t h e r cell types [ 1,27].
Acknowledgement T h i s w o r k w a s s u p p o r t e d by g r a n t D K - 2 8 3 0 0 f r o m the N a t i o n a l I n s t i t u t e o f D i a b e t e s a n d D i g e s t i v e a n d Kidney Diseases.
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