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The spontaneous activity of the rat isolated colon is suppressed by prostacyclin analogues such as cicaprost (IC50=4.0 nM). Activation of prostanoid IP1-receptors located on NANC inhibitory neurones is involved. However, several non-prostanoids, which show medium to high IP1 agonist potency on platelet and vascular preparations, exhibit very weak inhibitory activity on the colon. The aim of the study was to investigate this discrepancy. Firstly, we have demonstrated the very high depolarizing potency of cicaprost on the rat isolated vagus nerve (EC50=0.23 nM). Iloprost, taprostene and carbacyclin were 7.9, 66, and 81 fold less potent than cicaprost, indicating the presence of IP1 as opposed to IP2-receptors. Three non-prostanoid prostacyclin mimetics, BMY 45778, BMY 42393 and ONO-1301, although much less potent than cicaprost (195, 990 and 1660 fold respectively), behaved as full agonists on the vagus nerve. On re-investigating the rat colon, we found that BMY 45778 (0.1–3 μM), BMY 42393 (3 μM) and ONO-1301 (3 μM) behaved as specific IP1 partial agonists, but their actions required 30–60 min to reach steady-state and only slowly reversed on washing. This profile contrasted sharply with the rapid and readily reversible contractions elicited by a related non-prostanoid ONO-AP-324, which is an EP3-receptor agonist. The full versus partial agonism of the non-prostanoid prostacyclin mimetics may be explained by the markedly different IP1 agonist sensitivities of the two rat neuronal preparations. However, the slow kinetics of the non-prostanoids on the NANC system of the colon remain unexplained, and must be taken into account when characterizing neuronal IP-receptors. Keywords: Prostacyclin, prostanoid IP-receptors, prostanoid EP3-receptors, cicaprost, iloprost, BMY 45778, ONO-1301, vagus nerve, colon, NANC innervation Introduction The non-prostanoid prostacyclin mimetics activate prostanoid IP1-receptors on platelets and vascular smooth muscle to elicit inhibitory effects characteristic of prostacyclin (see Jones et al., 1993; Meanwell et al., 1994 for reviews). One of the most potent of these agents is BMY 45778 (Figure 1), which completely inhibits aggregation in human platelet-rich plasma and has a Kd of 7 nM for the IP1-receptor on human platelet membranes (Seiler et al., 1997). It also fully relaxes human pulmonary artery rings with an IC50 of about 3 nM (Jones et al., 1997). The non-prostanoids tend to be less potent inhibitors on rat and rabbit platelet suspensions (Armstrong et al., 1989; Merritt et al., 1991). However, we were surprised by the very weak inhibitory effects of BMY 45778 on the spontaneous motility of the rat isolated colon (10–15% inhibition at 2–10 μM) in comparison to the complete inhibition induced by the prostacyclin analogue cicaprost (IC50=4 nM) (Wise et al., 1995). In addition, two other non-prostanoids, BMY 42393 (Figure 1, Seiler et al., 1994) and EP 185 (Jones et al., 1993) had minimal inhibitory activity at a concentration of 10 μM. Cicaprost's action on the rat colon is abolished by tetrodotoxin (TTX), and it appears that activation of IP-receptors on non-adrenergic non-cholinergic (NANC) neurones in the colon leads to the release of transmitters that inhibit the pacemaker/smooth muscle system (Figure 3a) (Qian & Jones, 1995). Figure 1 6a-Carba analogues of prostacyclin (upper row) and non-prostanoid prostacyclin mimetics (lower row) with differing agonist specificities. IP1/IP2 receptor selectivity is derived from ligand binding measurements in rat brain (Takechi et al., 1996) and ... Figure 3 Rat colon: (a) Schematic arrangement of the NANC neurone/pacemaker/smooth muscle system and the proposed sites of action of agonists for IP1, EP3, nicotinic (N) and β3-adrenergic receptors. β3-Adrenoceptors may also be located on the smooth ... The question therefore arose as to whether non-prostanoid prostacyclin mimetics are genuinely poor agonists at IP-receptors on rat peripheral neurones, or have other actions that interfere with IP agonist activity. There are two related reasons for considering the latter possibility. Firstly, the high lipophilicity of the non-prostanoids may affect cell membrane function, and their diaryl-heteroatomic units resemble moieties found in other pharmacologically active agents (e.g. methadone analogues and diphenylmethane antihistamines; see Foye, 1990). Secondly, the control of colonic motility through the interaction of enteric neurones, pacemaker cells (interstitial cells of Cajal) and smooth muscle cells is known to be complex (Huizinga & Thuneberg, 1997). In the case of IP agonist action, at least two inhibitory neurotransmitter systems are involved (Figure 3a); nitric oxide (NO) is one transmitter while the other remains unidentified, but does not appear to be ATP, VIP or pituitary adenylate cyclase activating peptide (PACAP) (Qian & Jones, 1995). As a result, there are a number of steps at which a non-prostanoid might exert an additional action. Consequently, we have looked for an anatomically and mechanistically simpler neuronal preparation from the rat. The rat cervical vagus nerve is depolarized by 5-HT and has been used previously to characterize excitatory 5-HT-receptors (mainly 5-HT3) involved in vagal afferent nerve activity in emesis (Ireland & Tyers, 1987; Rhodes et al., 1992; Minami et al., 1997). We now report that this preparation is depolarized by cicaprost at subnanomolar concentrations, and that BMY 45778, BMY 42393, and a third non-prostanoid prostacyclin mimetic, ONO-1301 (Figure 1, Kondo & Hamanaka, 1995; Kondo et al., 1995) also show strong depolarizing activity in the nanomolar range. These findings prompted us to reinvestigate the actions of the nonprostanoids on the rat colon preparation.

Contraction of guinea-pig isolated aorta induced by the prostaglandin E analogue sulprostone (1-400 nM) has a lower maximum response (40%) than that of phenylephrine or U-46619 (TP-receptor agonist). A prostanoid EP3-receptor subtype is involved based on agonist potency ranking: equi-effective molar ratios (EMR) are sulprostone (EC50 approximately equal to 23 nM) 1.0, SC-46275 0.11, misoprostol 2.2, gemeprost 3.3, PGE2 5.4, 17-phenyl PGE2 6.0, GR-63799 8.9. GR-63799, which contains a bulky ester group, is relatively more potent on neuronal EP3 preparations than on the aorta. ONO-AP-324, a relative of the non-prostanoid prostacyclin mimetic series, behaves as an EP3 partial agonist on the aorta, inhibiting sulprostone responses but acting synergistically (in a similar manner to sulprostone) with phenylephrine; it may be a useful pharmacological tool for studying EP3-receptors. Sulprostone contractions are markedly suppressed in zero-Ca2+ bathing fluid containing either 2 mM EDTA or 50 microM EGTA, and by Cd2+ (500 microM), but are usually unaffected by nifedipine (0.3 microM) and verapamil (4.44 microM). Influx of Ca2+, but not through L-type Ca2+-channels, appears to be the major contractile mechanism. The guinea-pig aorta is a valuable addition to the vascular EP3 preparations available and may increase our knowledge of the mechanisms whereby Gi-coupled receptors mediate vasoconstriction (c.f. 5-HT1B/D- and alpha2-receptors). The possibility of certain EP3 agonists distinguishing EP3-receptor isoforms is discussed.

1. The possibility that prostacyclin (IP-) receptor agonists inhibit spontaneous contractions of the rat isolated colon by activating enteric neurones has been investigated. Cicaprost was used as the test agonist because of its high stability, selectivity and potency (IC50 = 3.8 nM). 2. The Na+ channel blockers saxitoxin (STX, 1 nM) and tetrodotoxin (TTX, 1 microM), whilst having little effect on resting spontaneous activity, virtually abolished the inhibitory actions of cicaprost (10 nM) and nicotine (3 microM); inhibitory responses to isoprenaline (20 nM) were not affected. Phentolamine (1 microM), propranolol (1 microM) and atropine (1 microM) had no effect on cicaprost inhibition. These data are compatible with release of inhibitory NANC transmitter(s) by cicaprost. 3. A transmitter role for nitric oxide was investigated. The nitric oxide synthase (NOS) inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 100 microM) inhibited the actions of both cicaprost (10 nM) and nicotine (3 microM) by 50-60%, but did not affect responses to isoprenaline (20 nM) or sodium nitroprusside (1-5 microM). The enantiomeric D-NAME (100 microM), which has negligible NOS inhibitory activity, had no effect on the action of cicaprost. 4. The involvement of purinergic transmitters was also investigated. Desensitization to the inhibitory action of ATP did not affect cicaprost responses. The P2x/P2y-receptor antagonist, suramin, at 300 microM blocked ATP responses, but not those due to adenosine; it did not affect cicaprost inhibition. The selective adenosine A1-receptor antagonist, DPCPX, used at a sufficiently high concentration (5 microM) to block adenosine A2-receptors, did not affect cicaprost inhibition. Apamin (25 nM), a blocker of calcium activated K+ channels on smooth muscle, abolished or markedly reduced the inhibitory actions of ATP and adenosine, and partially inhibited cicaprost and nicotine responses. The combination of L-NAME(100 microM) and apamin (25 nM) abolished cicaprost and nicotine responses.5. Investigation of vasoactive intestinal peptide (VIP) as a potential transmitter showed that its inhibitory action on the colon (IC50 = 50 nM) was partially inhibited by TTX (1 microM). alpha-Chymotrypsin abolished the effect of VIP but had no effect on cicaprost inhibition. Attempts to inhibit VIP responses using peptide antagonists and by agonist desensitization were unsuccessful.6. KCI (40 mM) contracted the colon and abolished spontaneous activity. Under these conditions,isoprenaline, sodium nitroprusside and ATP induced relaxation, whereas cicaprost (10-3 10 nM) had no effect. Cicaprost inhibited both the tone and the spontaneous activity induced by the EP1/EP3-receptor agonist, sulprostone (8.6 nM) but not when either TTX (1 microM) or KC1 (40 mM) was also present. On KCl-treated preparations, the prostacyclin analogue, iloprost (10-500 nM), induced contraction,presumably due to activation of EP-receptors.7. It is concluded that IP-receptor agonists inhibit the contractility of rat colon by stimulating the release of at least two transmitters from NANC enteric neurones. Nitric oxide appears to be one of the transmitters. The second transmitter mechanism is apamin-sensitive; the experimental results do not support ATP, adenosine or VIP as transmitter candidates. However, further studies using more potent and selective receptor antagonists are required.