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Interactive Mechanisms among Pituitary Adenylate Cyclase-Activating Peptide, Vasoactive Intestinal Peptide, and Parathyroid Hormone Receptors in Guinea Pig Cecal Circular Smooth Muscle Cells

Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812–82, Japan

Address all correspondence and requests for reprints to: Yasuaki Motomura, M.D., Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-82, Japan.

	Abstract

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Vasoactive intestinal peptide (VIP) causes relaxation of smooth muscle cells via both VIP-specific receptor coupled to nitric oxide synthase and VIP-preferring receptor coupled to adenylate cyclase. Because the mechanism of interaction among VIP, pituitary adenylate cyclase-activating peptide (PACAP), and PTH is still unclear, the characteristics of the receptors for PACAP and PTH in circular muscle cells obtained from the guinea pig cecum were investigated. The effects of an inhibitor of cAMP-dependent protein kinase [cyclic adenosine 3',5'-monophosphorothioate (Rp-cAMPS)], guanylate cyclase inhibitors, antagonists of these peptides, and the selective receptor protection on the relaxing effect produced by PACAP, VIP, and PTH were examined. PACAP-induced relaxation was significantly inhibited by a VIP antagonist, a PTH antagonist, Rp-cAMPS, and an inhibitor of particulate guanylate cyclase. VIP-induced relaxation was significantly inhibited by a PACAP antagonist and a PTH antagonist. PTH-induced relaxation was significantly inhibited by a VIP-specific receptor antagonist and Rp-cAMPS, but not by a PACAP antagonist. A PTH antagonist significantly inhibited a VIP-preferring receptor agonist-induced relaxation. The muscle cells in which cholecystokinin octapeptide and PTH receptors were protected completely abolished the inhibitory responses to VIP and PACAP. The muscle cells in which cholecystokinin octapeptide and VIP or PACAP receptors were protected completely abolished the inhibitory response to PTH. This study shows that PACAP induces relaxation of these muscle cells via both VIP-preferring receptor coupled to adenylate cyclase and PACAP-specific receptor, and that PTH induces relaxation of the muscle cells via PTH-specific receptor coupled to adenylate cyclase. In addition, the results of a selective receptor protection show that PTH does not bind to VIP receptors, and that VIP does not bind to PTH receptor. Therefore, this study first demonstrates the presence of one-way inhibitory mechanisms from the PTH-specific receptor to the VIP-preferring receptor, and from the VIP-specific receptor to the PTH-specific receptor in the mechanisms of interaction between VIP and PTH.

	Introduction

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PITUITARY adenylate cyclase-activating peptide (PACAP), which was first isolated from ovine hypothalamus, exists in two biologically active forms; PACAP-(1–27) and PACAP-(1–38) (1, 2). The N-terminal subsequence, 1–28, of PACAP-(1–38) exhibits 68% similarity to vasoactive intestinal peptide (VIP) (2).

PACAP interacts with at least two receptor subtypes coupled to adenylate cyclase: PACAP type I receptor and VIP/PACAP type II receptor. The PACAP type I receptor with a high affinity for PACAP and a low affinity for VIP is typically found in the hypothalamus, the anterior pituitary, and the rat pancreatic carcinoma cell line (3, 4, 5, 6). It has been also reported that the PACAP type I receptor was coupled to phospholipase C (7, 8, 9). The VIP/PACAP type II receptor with a high affinity for both PACAP and VIP distributes in the rat lung, liver, mouse cultured splenocytes, and human jejunal epithelial plasma membrane (10, 11, 12). Recently, the PACAP type II receptor has been subdivided into VIP/PACAP₁ (VIP₁) receptor with a high affinity for secretin and VIP/PACAP₂ (VIP₂) receptor with a low affinity for secretin (13, 14, 15, 16). This VIP/PACAP₁ receptor seems to be identical to the VIP-preferring receptor coupled with adenylate cyclase, which is recognized by VIP, peptide histidine isoleucine (PHI), and secretin (17). Although the pharmacological properties of PACAP in the gastrointestinal smooth muscle are similar to those of VIP (10, 18, 19, 20, 21, 22, 23, 24), the mechanisms of the effect seem to be different in some species and regions (19, 21, 24). PACAP relaxes smooth muscle cells of the guinea pig taenia coli via apamin-sensitive receptor, which is distinct from that for VIP and is considered the third receptor for PACAP (19). The inhibition of PACAP-induced relaxation by apamin was also observed in the human and rat colonic longitudinal muscle strip (21, 24).

Recently, nitric oxide (NO) has been described as a major inhibitory mediator in the gastrointestinal smooth muscle (25). VIP stimulates NO production in gastric muscle cells of the guinea pig and rabbit (17, 26, 27) and in cecal muscle cells of the guinea pig (28) via VIP-specific receptor, which is distinct from VIP/PACAP type II receptor (29, 30). This NO production activates soluble guanylate cyclase, leading to the generation of cGMP and muscle relaxation ultimately. On the other hand, it has been reported that in the rabbit stomach, VIP and PACAP activate membrane-bound NO synthase via VIP/PACAP type II receptor coupled to pertussis toxin-sensitive G_i1–2 (31).

PTH with 84 amino acid residues, which is secreted from parathyroid glands, plays an important role in calcium and phosphorus homeostasis. The C-terminally truncated form of PTH-(1–34) was shown to retain the complete activity of the full-length hormone (32). In addition to its classical calcium metabolic effects, PTH causes relaxation of smooth muscle in the cardiovascular system, gastrointestinal tract, trachea, uterus, and vas deferens (33). The cross-interaction between PTH and VIP on their respective receptors in the ileal smooth muscle cells was reported previously (34); however, the mechanisms of the interaction and the role of VIP receptor subtypes in the interaction are unclear. PTH interacts with at least two receptor subtypes: PTH/PTH-related peptide (PTHrP) receptor and PTH2 receptor. The PTH/PTHrP receptor is the classical heptahelical membrane-bound receptor expressed predominantly in bone and kidney and is coupled through two guanyl nucleotide regulatory proteins: G_s, to the adenylate cyclase-cAMP-protein kinase A pathway, and G_q, to the inositol triphosphate-cytosolic calcium-protein kinase C signal transduction pathway (35, 36, 37). The other receptor, PTH2 receptor, is abundantly expressed in the rat brain and pancreas and is a G protein-coupled receptor (38). PTH-(1–34), but not PTHrP-(1–34), binds to PTH2 receptor. PTH-(1–34) stimulates cAMP accumulation and the release of cytosolic free calcium in the human embryonic kidney cell line stably expressing the recombinant human PTH2 receptors (39).

Although PACAP, VIP, and PTH relax smooth muscle directly, the mechanisms of interaction of these peptides in smooth muscle cells are still unclear. The present study was designed to investigate the relationship among PACAP-(1–38), VIP-(1–28), and PTH-(1–34); the roles of VIP receptor subtypes in the interaction; and the characteristics of the receptors for PACAP and PTH in circular muscle cells obtained from guinea pig cecum using antagonists of these peptides and a selective receptor protection method.

	Materials and Methods

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Materials
PACAP-(1–38), PACAP-(6–38), VIP-(1–28), VIP-(10–28), PTH-(1–34), [Tyr³⁴]bovine (b) PTH-(7–34), atrial natriuretic peptide (ANP), ANP-(1–11), PHI, and cholecystokinin octapeptide (CCK-8) were obtained from Peptide Institute (Osaka, Japan); collagenase (CLS type II) was obtained from Worthington Biochemical Corp. (Freehold, NJ); trypsin inhibitor (type I-S), BSA, phorbol 12-myristate 13-acetate (PMA), and N-ethylmaleimide (NEM) were purchased from Sigma Chemical Co. (St. Louis, MO); cyclic adenosine 3',5'-monophosphorothioate (Rp-cAMPS) was purchased from Biolog Life Science Institute (Bremen, Germany); 6-amino-5,8-quinolinedione (LY83583) was obtained from Calbiochem (San Diego, CA); HEPES was obtained from Wako Pure Chemical (Osaka, Japan); and acrolein was purchased from Tokyo Kasei (Tokyo, Japan).

Preparation of dispersed muscle cells
Circular muscle cells were isolated from the cecal circular muscle layer of the guinea pig by a method similar to that used by Bitar and Makhlouf for preparing smooth muscle cells from the guinea pig stomach (40). In brief, male Hartley guinea pigs (300–600 g) fed a standard diet at the Animal Center of Kyushu University (Fukuoka, Japan) were fasted overnight and killed by the intracardiac injection of pentobarbital sodium. The cecum was removed and placed in ice-cold HEPES buffer (120 mM NaCl, 2.6 mM KH₂PO₄, 4 mM KCl, 2 mM CaCl₂, 0.6 mM MgCl₂, 25 mM HEPES, 14 mM glucose, and 0.01% trypsin inhibitor, pH 7.4). The taenia coli was removed, then the circular muscle layer was prepared by scraping off the mucosa. The muscle layer was cut into pieces and incubated for two successive 40-min periods at 31 C in 15 ml HEPES medium containing 150 U/ml collagenase. After incubation, the partly digested strips were washed with 50 ml enzyme-free HEPES medium and reincubated in 15 ml fresh HEPES medium to allow the cells to disperse spontaneously. Cells were then harvested by filtration through a 500-µm polyester mesh.

Measurement of contraction and relaxation in dispersed cells
Dispersed cells were stimulated by adding a 0.5-ml aliquot of cell suspension to 0.2 ml of a solution containing the test agent and were incubated at 22 C for 30 sec, because we previously found that CCK-8 induced the maximal contractile response in cecal circular smooth muscle cells after 30 sec of incubation (41). With longer periods of incubation, the contractile response decreased. To examine the inhibitory effects of PACAP-(1–38), VIP-(1–28), and PTH-(1–34) on muscle cell contraction stimulated by 1 nM CCK-8, various concentrations of each peptide and 1 nM CCK-8 were added simultaneously to aliquots of cells. The inhibitory effects of these peptides on CCK-8-stimulated contraction were regarded as having a relaxing effect. To investigate the effects of VIP-(10–28), a VIP antagonist (30, 42, 43), and [Tyr³⁴]bPTH-(7–34), a PTH antagonist (44), on PACAP-induced relaxation, various concentrations of VIP-(10–28) or [Tyr³⁴]bPTH-(7–34), 1 nM CCK-8, and 0.1 µM PACAP-(1–38) were added simultaneously to aliquots of cells. To investigate the effects of PACAP-(6–38), a PACAP antagonist (45), and [Tyr³⁴]bPTH-(7–34) on VIP-induced relaxation, various concentrations of PACAP-(6–38) or [Tyr³⁴]bPTH-(7–34), 1 nM CCK-8, and 0.1 µM VIP-(1–28) were added simultaneously to cells. To investigate the effects of VIP-(10–28) and PACAP-(6–38) on PTH-induced relaxation, various concentrations of VIP-(10–28) or PACAP-(6–38), 1 nM CCK-8, and 0.1µM PTH-(1–34) were added simultaneously to cells. To examine the roles of the adenylate cyclase system and the guanylate cyclase system in the PACAP- and PTH-induced relaxation, we added Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase (46); 6-amino-5,8-quinolinedione (LY83583), an inhibitor of soluble guanylate cyclase (47, 48); and PMA, an inhibitor of particulate guanylate cyclase (49, 50), to separate aliquots of cells simultaneously with 1 nM CCK-8 and 0.1µM PACAP-(1–38) or 0.1 µM PTH-(1–34). In addition, to investigate the action of PACAP-(6–38) or [Tyr³⁴]bPTH-(7–34) on the VIP-specific receptor, various concentrations of PACAP-(6–38) or [Tyr³⁴]bPTH-(7–34), 1 nM CCK-8, and 1 µM ANP, which induces relaxation of cecal circular smooth muscle cells from the guinea pig via VIP-specific, but not via VIP-preferring, receptor (28), were added simultaneously to cells. Moreover, to examine whether PTH acts on the VIP-preferring receptor, various concentrations of [Tyr³⁴]bPTH-(7–34), 1 nM CCK-8, and 0.1 µM PHI, which interacts with VIP-preferring receptor and causes relaxation of muscle cells (42), were added simultaneously to cells. To investigate the effect of ANP-(1–11), an ANP antagonist (28), on PTH-induced relaxation, we added various concentrations of ANP-(1–11), 1 nM CCK-8, and 0.1 µM PTH-(1–34) to cells simultaneously. At the end of the incubation, acrolein at a final concentration of 1% was added to interrupt the reaction. The length of 50 cells in microscopic fields was measured by image-splitting micrometry, and the percent decrease in mean cell length was determined by comparison with the control.

Selective receptor protection
Cells from each suspension were treated as previously described (51). Receptor protection was accomplished by adding 1 nM CCK-8 and 1 µM of one of the following peptides to the cell suspension for 2 min: PACAP-(1–38) to protect PACAP receptors, VIP to protect VIP receptors, and PTH to protect PTH receptors. Next, 0.5 mM NEM was added to inactivate all unprotected receptors, and the incubation was continued for an additional 20 min at 31 C. It is presumed that alkylation of sulfhydryl groups by NEM treatment inactivates the unoccupied receptors. The cells were centrifuged twice at 150 x g for 10 min each time to remove NEM and the protective peptides and were resuspended in fresh medium. Treatment with NEM had no effect on the length of the cells (74.7 ± 1.6 µm in the absence of NEM vs. 74.9 ± 1.4 µm in the presence of NEM). To confirm that the selective receptor protection method is serviceable, we examined whether the contractile effect of CCK-8 on muscle cells, in which CCK receptors only were protected, can be preserved, and whether the inhibitory effects of PACAP, VIP, and PTH on the CCK-8-induced contraction of these muscle cells can be completely abolished. To examine the relationship among the receptors for PACAP, VIP, and PTH in cecal circular smooth muscle cells, we investigated the effects of these peptides on CCK-8-induced contraction of NEM-treated muscle cells in which only two kinds of receptors (CCK and one of these peptides) were protected.

Data analysis
Contraction was expressed as the percent decrease in cell length from the control value. Values are the mean ± SEM of n experiments. Statistical analysis was performed using Student’s t test or Welch’s method after inspection of variances. P < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Contraction and relaxation of dispersed cells
PACAP-(6–38), VIP-(10–28), [Tyr³⁴]bPTH-(7–34), and ANP-(1–11) had no effect by themselves on cecal circular smooth muscle cells and had no effect on 1 nM CCK-8-induced contraction. PACAP-(1–38), VIP-(1–28), and PTH-(1–34) inhibited the contractile response produced by 1 nM CCK-8 in a concentration-dependent manner, with IC₅₀ values of 120, 38, and 40 pM, respectively (Fig. 1).

View larger version (20K): [in this window] [in a new window]   Figure 1. Inhibitory effects of PACAP-(1–38) (open circle), VIP (closed circle), and PTH (open square) on the 1 nM CCK-8-stimulated contraction of isolated cecal circular smooth muscle cells. Each value shows the mean ± SEM (n = 4–6 experiments). The vertical axis shows the percent decrease in mean cell length compared with the control value.

View larger version (43K): [in this window] [in a new window]   Figure 2. A, Inhibitory effect of VIP-(10–28), a VIP antagonist, on the PACAP-induced relaxation of isolated cecal circular smooth muscle cells. B, Inhibitory effect of [Tyr34]bPTH-(7–34), a PTH antagonist, on the PACAP-induced relaxation of isolated cecal circular smooth muscle cells. Each value shows the mean ± SEM (n = 4–5 experiments). N.S., Not significant. PTH-(7–34) = [Tyr34]bPTH-(7–34).

View larger version (40K): [in this window] [in a new window]   Figure 3. A, Inhibitory effect of PACAP-(6–38), a PACAP antagonist, on the VIP-induced relaxation of isolated cecal circular smooth muscle cells. B, Inhibitory effect of [Tyr34]bPTH-(7–34), a PTH antagonist, on the VIP-induced relaxation of isolated cecal circular smooth muscle cells. Each value shows the mean ± SEM (n = 4–6 experiments). N.S., Not significant. PTH-(7–34) = [Tyr34]bPTH-(7–34).

View larger version (34K): [in this window] [in a new window]   Figure 4. A, Inhibitory effect of VIP-(10–28), a VIP antagonist, on the PTH-induced relaxation of isolated cecal circular smooth muscle cells. B, Effect of PACAP-(6–38), a PACAP antagonist, on the PTH-induced relaxation of isolated cecal circular smooth muscle cells. Each value shows the mean ± SEM (n = 5 experiments). N.S., Not significant.

View larger version (34K): [in this window] [in a new window]   Figure 5. A, Inhibitory effect of Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, on the PACAP-induced relaxation of isolated cecal circular smooth muscle cells. B, Inhibitory effect of PMA, an inhibitor of particulate guanylate cyclase, on the PACAP-induced relaxation of isolated cecal circular smooth muscle cells. C, Effect of LY83583, an inhibitor of soluble guanylate cyclase, on the PACAP-induced relaxation of isolated cecal circular smooth muscle cells. Each value shows the mean ± SEM (n = 5–6 experiments). N.S., Not significant.

View larger version (31K): [in this window] [in a new window]   Figure 6. A, Inhibitory effect of Rp-cAMPS, an inhibitor of cAMP-dependent protein kinase, on the PTH-induced relaxation of isolated cecal circular smooth muscle cells. B, Effect of PMA, an inhibitor of particulate guanylate cyclase, on the PTH-induced relaxation of isolated cecal circular smooth muscle cells. C, Effect of LY83583, an inhibitor of soluble guanylate cyclase, on the PTH-induced relaxation of isolated cecal circular smooth muscle cells. Each value shows the mean ± SEM (n = 4 experiments). N.S., Not significant.

View larger version (31K): [in this window] [in a new window]   Figure 7. Inhibitory effect of ANP-(1–11), a VIP-specific receptor antagonist, on the PTH-induced relaxation of isolated cecal circular smooth muscle cells. Each value shows the mean ± SEM (n = 4 experiments). N.S., Not significant.

View larger version (36K): [in this window] [in a new window]   Figure 8. Inhibitory effect of [Tyr34]bPTH-(7–34), a PTH antagonist, on the PHI-induced relaxation of isolated cecal circular smooth muscle cells. Each value shows the mean ± SEM (n = 4 experiments). N.S., Not significant. PTH-(7–34) = [Tyr34]bPTH-(7–34).

View larger version (22K): [in this window] [in a new window]   Figure 9. Effect of selective receptor protection on the relaxing potency induced by PTH, VIP, and PACAP-(1–38) in cecal circular smooth muscle cells. Muscle cells were incubated for 2 min in the presence of 1 nM CCK-8 with 1 µM PTH (A), 1 nM CCK-8 with 1 µM VIP (B), or 1 nM CCK-8 with 1 µM PACAP (C) as protective agents and another 20 min with 0.5 mM NEM to inactivate unprotected receptors. The cells were washed and resuspended, and the contractile response to CCK-8 and relaxing responses to PTH, VIP, and PACAP were examined. Each value shows the mean ± SEM (n = 4 experiments). N.S., Not significant.

	Discussion

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In gastric muscle cells, VIP interacts with two signaling pathways (27); VIP-preferring receptor coupled to adenylate cyclase, leading to generation of cAMP and activation of cAMP-dependent protein kinase, which is also recognized by PHI and secretin, and VIP-specific receptor coupled to NO synthase, leading to generation of NO, which is not recognized by either PHI or secretin, have been identified (17). The relationship between PACAP and VIP in the gastrointestinal smooth muscle seems to be different in different species and regions of the gut. PACAP induces relaxation of the smooth muscle cells from guinea pig taenia coli without increasing the cAMP level via the apamin-sensitive receptor, which is not recognized by VIP (19). In some experiments, PACAP was also shown to induce relaxation via the apamin-sensitive receptor (21, 24). Apamin, which selectively blocks Ca²⁺-activated K⁺ channels, inhibited the effect of PACAP, but not the effect of (Bu)₂cAMP, a cAMP analog (24). These reports indicated that the PACAP receptor is coupled to Ca²⁺-dependent K⁺ channels, but not to adenylate cyclase system. In the taenia coli muscle cells of guinea pig, PACAP-induced relaxation via the apamin-sensitive receptor was not inhibited by protein kinase G inhibitor (19), which suggests that PACAP does not activate the guanylate cyclase-cGMP-protein kinase G signal transduction pathway. This apamin-sensitive receptor is considered the third receptor for PACAP (19, 24). Grider et al. (18) reported that PACAP and VIP induced relaxation of the rat colonic circular muscle cells via the VIP-PACAP common apamin-insensitive receptor coupled to NO generation in muscle cells. This type of receptor was also observed in gastric muscle cells (31). On the other hand, VIP stimulates NO production in gastric muscle cells of the guinea pig and rabbit (17, 26, 27) and in cecal muscle cells of the guinea pig (28) via VIP-specific receptor, which is distinct from VIP/PACAP type II receptor (42). Most recently, Murthy et al. (53) reported that PACAP and VIP interact with ANP clearance receptor coupled to NO synthase in dispersed rabbit gastric muscle cells. PTH interacts with at least two receptor subtypes: PTH/PTHrP receptor and PTH2 receptor. In the gastrointestinal tract, immunoreactive PTH and PTHrP have been detected in extracts of the rat myenteric plexus (54). It would be expected that VIP and PTH interact with each other from the fact that the PTH/PTHrP receptor has a relative homology with the VIP receptor (55). Indeed, in the guinea pig ileum, cross-interaction between VIP and PTH on their respective receptors in smooth muscle cells was reported (34).

The present study suggests that the inhibitory effect of PACAP on cecal circular smooth muscle cells is mediated by both cAMP-dependent protein kinase and particulate guanylate cyclase. In addition, VIP-(10–28), a VIP antagonist, significantly inhibited PACAP-induced relaxation in a concentration-dependent manner. PACAP-(6–38), a PACAP antagonist, significantly inhibited VIP-induced relaxation. On the other hand, PACAP-(6–38) could not inhibit the effect of ANP, which binds to VIP-specific receptor on cecal circular muscle cells. These results suggest that PACAP interacts with two types of receptors on cecal circular smooth muscle cells, one of which is the VIP-preferring receptor coupled to adenylate cyclase, and the other of which may be PACAP-specific receptor coupled to particulate guanylate cyclase, which is recognized by PACAP, but not by VIP (Fig. 10). In selective receptor protection, the muscle cells where CCK-8 and PACAP receptors were protected completely preserved the inhibitory response to PACAP, but partially preserved the inhibitory response to VIP. Treatment of the cells with CCK-8 and VIP completely preserved the inhibitory response to VIP, but partially preserved the inhibitory response to PACAP. These results also support the presence of a common receptor to which both PACAP and VIP bind.

View larger version (24K): [in this window] [in a new window]   Figure 10. Hypothetical inhibitory mechanisms of PACAP and PTH in cecal circular smooth muscle cells. PACAP induces the relaxation of muscle cells via both VIP-preferring receptor coupled to adenylate cyclase and PACAP-specific receptor coupled to particulate guanylate cyclase. PTH induces relaxation of muscle cells via PTH-specific receptor coupled to adenylate cyclase. The one-way inhibitory mechanisms from the VIP-specific receptor to the PTH-specific receptor and from the PTH-specific receptor to the VIP-preferring receptor are suggested. PACAP-R, PACAP receptor; PTH-R, PTH receptor; AC, adenylate cyclase; GC, guanylate cyclase; NOS, NO synthase; PKA, cAMP dependent protein kinase; PKG, cGMP-dependent protein kinase.

In the central nervous system, some evidence of intramembrane interactions between physically distinct receptors have been reported. Several modulators, such as CCK, neurotensin, and adenosine, reduce the affinity of dopamine D2, but not the affinity of dopamine D1 receptors in vitro and in vivo (56, 57, 58). The interaction between receptors for chemical signals occurred at the level of the neuronal membrane receptor-receptor interaction and was divided into four types: binding site-binding site interaction inside a receptor macromolecule, intramembrane interaction between physically distinct but adjacent receptors, intramembrane interaction between physically distinct and nonadjacent receptors involving the activation of mobile membrane-associated proteins such as G proteins, and interaction between physically distinct and nonadjacent receptors through intracellular processes. Although in the present study, the presence of one-way inhibitory mechanisms from PTH receptor to VIP-preferring receptor and from VIP-specific receptor to PTH receptor was suggested, the inhibitory mechanisms are still unclear. Additional studies are needed to elucidate the physiological role and mechanisms of the interactions between these receptors.

Received October 23, 1997.

	References

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