Pharmacological characterization of a rat 5-hydroxytryptamine type3 receptor subunit (r5-HT3A(b)) expressed in Xenopus laevis oocytes

1. The present study has utilized the two electrode voltage-clamp technique to examine the pharmacological profile of a splice variant of the rat orthologue of the 5-hydroxytryptamine type 3A subunit (5-HT_3A(b)) heterologously expressed in Xenopus laevis oocytes.
2. At negative holding potentials, bath applied 5-HT (300 nM–10 μM) evoked a transient, concentration-dependent (EC₅₀=1.1±0.1 μM), inward current. The response reversed in sign at a holding potential of −2.1±1.6 mV.
3. The response to 5-HT was mimicked by the 5-HT₃ receptor selective agonists 2-methyl-5-HT (EC₅₀=4.1±0.2 μM), 1-phenylbiguanide (EC₅₀=3.0±0.1 μM), 3-chlorophenylbiguanide (EC₅₀=140± 10 nM), 3,5-dichlorophenylbiguanide (EC₅₀=14.5±0.4 nM) and 2,5-dichlorophenylbiguanide (EC₅₀= 10.2±0.6 nM). With the exception of 2-methyl-5-HT, all of the agonists tested elicited maximal current responses comparable to those produced by a saturating concentration (10 μM) of 5-HT.
4. Responses evoked by 5-HT at EC₅₀ were blocked by the 5-HT₃ receptor selective antagonist ondansetron (IC₅₀=231±22 pM) and by the less selective agents (+)-tubocurarine (IC₅₀=31.9± 0.01 nM) and cocaine (IC₅₀=2.1±0.2 μM).
5. The data are discussed in the context of results previously obtained with the human and mouse orthologues of the 5-HT_3A subunit. Overall, the study reinforces the conclusion that species differences detected for native 5-HT₃ receptors extend to, and appear largely explained by, differences in the properties of homo-oligomeric receptors formed from 5-HT_3A subunit orthologues.

     

Investigation into the contractile response of melatonin in the guinea-pig isolated proximal colon: the role of 5-HT4 and melatonin receptors

1. The interaction of melatonin (N-acetyl-5-methoxytryptamine) with 5-hydroxytryptamine₄ (5-HT₄) receptors and/or with melatonin receptors (ML₁, ML₂ sites) has been assessed in isolated strips of the guinea-pig proximal colon. In the same preparation, the pharmacological profile of a series of melatonin agonists (2-iodomelatonin, 6-chloromelatonin, N-acetyl-5-hydroxytryptamine (N-acetyl-5-HT), 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT)) was investigated.
2. In the presence of 5-HT_1/2/3 receptor blockade with methysergide (1 μM) and ondansetron (10 μM), melatonin (0.1 nM–10 μM), 5-HT (1 nM–1 μM) and the 5-HT₄ receptor agonist, 5-methoxytryptamine (5-MeOT: 1 nM–1 μM) caused concentration-dependent contractile responses. 5-HT and 5-MeOT acted as full agonists with a potency (−log EC₅₀) of 7.8 and 8.0, respectively. The potency value for melatonin was 8.7, but its maximum effect was only 58% of that elicited by 5-HT.
3. Melatonin responses were resistant to atropine (0.1 μM), tetrodotoxin (0.3 μM), and to blockade of 5-HT₄ receptors by SDZ 205,557 (0.3 μM) and GR 125487 (3, 30 and 300 nM). The latter antagonist (3 nM) inhibited 5-HT-induced contractions with an apparent pA₂ value of 9.6. GR 125487 antagonism was associated with 30% reduction of the 5-HT response maximum. Contractions elicited by 5-HT were not modified when melatonin (1 and 10 nM) was used as an antagonist.
4. Like melatonin, the four melatonin analogues concentration-dependently contracted colonic strips. The rank order of agonist potency was: 2-iodomelatonin (10.8) >6-chloromelatonin (9.9) ⩾ N-acetyl-5-HT (9.8) ⩾5-MCA-NAT (9.6) >melatonin (8.7), an order typical for ML₂ sites. In comparison with the other agonists, 5-MCA-NAT had the highest intrinsic activity.
5. The melatonin ML_1B receptor antagonist luzindole (0.3, 1 and 3 μM) had no effect on the concentration-response curve to melatonin. Prazosin, an α-adrenoceptor antagonist possessing moderate/high affinity for melatonin ML₂ sites did not affect melatonin-induced contractions at 0.1 μM. Higher prazosin concentrations (0.3 and 1 μM) caused a non-concentration-dependent depression of the maximal response to melatonin without changing its potency. Prazosin (0.1 and 1 μM) showed a similar depressant behaviour towards the contractile responses to 5-MCA-NAT.
6. In the guinea-pig proximal colon, melatonin despite some structural similarity with the 5-HT₄ receptor agonist 5-MeOT, does not interact with 5-HT₄ receptors (or with 5-HT_1/2/3 receptors). As indicated by the rank order of agonist potencies and by the inefficacy of luzindole, the most likely sites of action of melatonin are postjunctional ML₂ receptors. However, this assumption could not be corroborated with the use of prazosin as this ‘ML₂ receptor antagonist'' showed only a non-concentration-dependent depression of the maximal contractile response to both melatonin and 5-MCA-NAT. Further investigation with the use of truly selective antagonists at melatonin ML₂ receptors is required to clarify this issue.

     

Modulation by 5-HT1A receptors of the 5-HT2 receptor-mediated tachykinin-induced contraction of the rat trachea in vitro

1. In the Fisher 344 rat, tachykinins have been shown to cause the release of 5-hydroxytryptamine (5-HT) from airway mast cells, which then causes direct smooth muscle activation as well as the release of acetylcholine from cholinergic nerves. The aim of the present study was to examine the modulatory effects of 5-HT receptors on the neurokinin A (NKA)-induced release of endogenous 5-HT and airway smooth muscle contraction in the isolated Fisher 344 rat trachea.
2. The selective 5-HT₂ receptor antagonist ketanserin (0.1 μM) produced an almost complete inhibition of the contractions caused by NKA (n=4, P<0.0001, two-way ANOVA), and a significant rightward shift of the concentration-response curve to 5-HT (n=8, P<0.001, two-way ANOVA).
3. The partial agonist for 5-HT_1A receptors, 8-OH-DPAT (1 μM), and the full agonist for 5-HT₁ receptors, 5-CT (0.3 μM), potentiated the submaximal contractions induced by the 5-HT₂ receptor agonist α-methyl-5-HT (0.1 μM) (n=4; P<0.005 and P<0.05, respectively). 8-OH-DPAT (1 μM), as well as the 5-HT_1A receptor antagonists pMPPI, SDZ 216525 and NAN-190 (0.1 μM each), caused significant inhibition of the tracheal contractions induced both by NKA (10  nM–3  μM) and 5-HT (10 nM–10 μM) (n=4–10). This suggests that activation of 5-HT_1A receptors potentiates the 5-HT₂ receptor-mediated contractions.
4. SDZ 216525 (0.1 μM) significantly reduced the maximal contraction produced by 1 μM NKA (n=10, P<0.001), without affecting the release of endogenous 5-HT. These data rule out the involvement of a 5-HT_1A receptor-mediated positive feedback mechanism of the 5-HT release from mast cells.
5. Even in the presence of atropine (1 μM), 8-OH-DPAT (1 μM) further reduced the maximal NKA-induced contraction (n=4, P<0.0001), while the contractions of the rat isolated trachea induced by electrical field stimulation and the concentration-response curve to carbachol were unaffected by pMPPI (0.1 μM), SDZ 216525 (0.1 μM), NAN-190 (0.1 μM) and 8-OH-DPAT (1 μM) (n=4–6). These data demonstrate that the 5-HT_1A receptor-mediated potentiation of contractile responses is not due to non-specific inhibition of airway smooth muscle contraction or to modulation of postganglionic nerve activation.
6. The selective 5-HT_1B/1D receptor antagonist GR 127935, the selective 5-HT₃ receptor antagonist tropisetron and the selective 5-HT₄ receptor antagonists SB 204070 and GR 113808 (0.1 μM each) had no effect on the concentration-response curve for NKA (n=6–10), ruling out the involvement of 5-HT_1B/1D, 5-HT₃ and 5-HT₄ receptors.
7. The α-adrenoreceptor antagonist phentolamine (1 μM) had no effect on the 5-HT-induced contractions (n=4), ruling out the involvement of α-adrenoreceptors.
8. In conclusion, the tachykinin-induced contraction of the F334 rat isolated trachea is mediated by the stimulation of 5-HT₂ receptors. Activation of 5-HT_1A receptors located on airway smooth muscle potentiates the direct contractile effects of 5-HT₂ receptor activation. The 5-HT_1B/1D, 5-HT₃ and 5-HT₄ receptors are not involved in the NKA-induced contraction of rat airways.

     

Characterization of the contraction to 5-HT in the canine colon longitudinal muscle

1. Although conscious dogs have often been used for colonic motility studies with 5-hydroxytryptamine (5-HT), the effects of 5-HT on the isolated colon have not been thoroughly characterized yet. The current study was undertaken to characterize the response to 5-HT of the canine isolated colon longitudinal muscle.
2. Longitudinal strips of canine midcolon deprived of (sub)mucosa were prepared for isotonic measurement. 5-HT induced contractions from 3 nM onwards, which were not affected by selective inhibition of 5-HT re-uptake, monoamine oxidase or blockade of α-adrenoceptors. Tetrodotoxin (0.3 μM) did not affect the responses to 5-HT, suggesting that smooth muscle 5-HT receptors are involved. The selective 5-HT₄ receptor antagonist SB 204070 (10 nM) slightly enhanced contractions to 5-HT and therefore it was included in the organ bath solution in all further experiments. The 5-HT₁ and 5-HT₂ receptor antagonist methysergide (0.1 μM) depressed the curve to 5-HT, but the selective 5-HT₃ receptor antagonist granisetron (0.3 μM) had no effect.
3. Besides 5-HT, α-methyl-5-HT (α-Me-5-HT), 5-methoxytryptamine (5-MeOT), 2-methyl-5-HT (2-Me-5-HT) and 5-carboxamidotryptamine (5-CT) also induced contractions, with the following rank order of potency (pEC₅₀ values in parentheses): 5-HT (6.9)=α-methyl-5-HT (6.9)>2-Me-5-HT (5.8)=5-MeOT (5.7)=5-CT (5.6), indicative of 5-HT₂ receptor involvement. α-Me-5-HT produced a bell-shaped curve, which was not affected by α-adrenoceptor blockade. 5-HT, 5-MeOT, 2-Me-5-HT and 5-CT produced a monophasic concentration-response curve, consistent with an interaction with a single receptor site. 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and tryptamine only induced contractions at a concentration exceeding 1 μM.
4. The selective 5-HT_2B receptor antagonist SB 204741 (0.3 μM) did not affect the curve to 5-HT. Ketanserin, cisapride and spiroxatrine behaved as competitive antagonists with pK_b values of, respectively, 8.4, 8.1 and 6.7. Spiroxatrine (1 μM) shifted the curve to 5-MeOT rightward yielding an apparent pA₂ of 7.1. Other antagonists at 5-HT_2A receptors also surmountably inhibited the contractions to 5-HT (apparent pA₂ value in parentheses): mesulergine (8.2), cinanserin (8.2), yohimbine (6.2) and mianserin (8.6). However, as well as a rightward shift, methiothepin (8.3), pizotifen (8.6) and spiperone (8.8) also caused a depression of the curve, indicative of ‘pseudo-irreversible'' antagonism. Taken together, the above mentioned affinity estimates most closely corresponded to literature affinity values for 5-HT_2A receptors.
5. It was concluded that 5-HT induces contractions of the canine midcolon longitudinal muscle primarily by stimulation of smooth muscle 5-HT_2A receptors. The presence of inhibitory 5-HT₄ receptors cannot be ruled out.

     

Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

1. Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit γ-aminobutyric acid (GABA)_A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels.
2. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique.
3. GABA (50 μM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3–10 μM) and picrotoxin (0.3–10 μM), with IC₅₀ values and 95% confidence intervals (CI) of 1.2 μM (1.1–1.4) and 3.6 μM (3.0–4.3), respectively, and were potentiated by sodium pentobarbitone (30 μM) and diazepam (1 μM) to (mean±s.e.mean) 168±18% and 117±4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 μM)-evoked responses were inhibited by MDL 72222 (1 μM) to 10±4% of control; DMPP (10 μM)-evoked responses were inhibited by hexamethonium (100 μM) to 12±5% of control, and αbMeATP (30 μM)-evoked responses were inhibited by PPADS (10 μM) to 21±5% of control. Together, these data are consistent with activation of GABA_A, 5-HT₃, nicotinic ACh and P2_X receptors, respectively.
4. Ciprofloxacin (10–3000 μM) inhibited GABA_A-mediated responses in the vagus nerve with an IC₅₀ (and 95% CI) of 202 μM (148–275). BPAA (1–1000 μM) had little or no effect on the GABA_A-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times.
5. Responses mediated by 5-HT₃, nicotinic ACh and P2_X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 μM), BPAA (100 μM) or the combination of these drugs (both at 100 μM).
6. GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC₅₀ of 3.6 μM (2.8–4.5), a value not significantly different from that determined in the vagus nerve. Ciprofloxacin also inhibited the GABA-evoked response with an IC₅₀ of 334 μM (256–437) and BPAA (100 μM) potentiated these antagonist effects. However, the magnitude of the synergy was 48 times less than that seen in the vagus nerve.
7. These data indicate that ciprofloxacin and BPAA are selective antagonists of GABA_A receptors, an action that may contribute to their excitatory effects in vivo. Additionally, our data suggest that the molecular properties of GABA_A receptors in different regions of the CNS influence the extent to which these drugs synergistically inhibit the GABA_A receptor.

     

Pharmacological modulation of GABAA receptor-mediated postsynaptic potentials in the CA1 region of the rat hippocampus

1. It is unclear whether GABA_A receptor-mediated hyperpolarizing and depolarizing synaptic potentials (IPSP_As and DPSP_As, respectively) are evoked by (a) the same populations of GABAergic interneurones and (b) exhibit similar regulation by allosteric modulators of GABA_A receptor function. We have attempted to address these questions by investigating the effects of (a) known agonists for presynaptic receptors on GABAergic terminals, and (b) a range of GABA_A receptor ligands, on each response.
2. The GABA uptake inhibitor NNC 05-711 (10 μM) enhanced whereas bicuculline (10 μM) inhibited both IPSP_As and DPSP_As.
3. (−)-Baclofen (5 μM), [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAGO; 0.5 μM), and carbachol (10 μM) caused substantial depressions (up to 99%) of DPSP_As that were reversed by CGP 55845A (1 μM), naloxone (10 μM) and atropine (5 μM), respectively. In contrast, 2-chloroadenosine (CADO; 10 μM) only slightly depressed DPSP_As. Quantitatively, the effect of each agonist was similar to that reported for IPSP_As.
4. The neurosteroid ORG 21465 (1–10 μM), the anaesthetic propofol (50–500 μM), the barbiturate pentobarbitone (100–300 μM) and zinc (50 μM) all enhanced DPSP_As and IPSP_As.
5. The benzodiazepine (BZ) agonist flunitrazepam (10–50 μM) and inverse agonist DMCM (1 μM) caused a respective enhancement and inhibition of both IPSP_As and DPSP_As. The BZω₁ site agonist zolpidem (10–30 μM) produced similar effects to flunitrazepam.
6. The anticonvulsant loreclezole (1–100 μM) did not affect either response.
7. These data demonstrate that similar populations of inhibitory interneurones can generate both IPSP_As and DPSP_As by activating GABA_A receptors that are subject to similar allosteric modulation.

     

Evidence that ATP acts at two sites to evoke contraction in the rat isolated tail artery

1. The site(s) at which P2-receptor agonists act to evoke contractions of the rat isolated tail artery was studied by use of P2-receptor antagonists and the extracellular ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
2. Suramin (1 μM–1 mM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (0.3–300 μM) inhibited contractions evoked by equi-effective concentrations of α,β-methyleneATP (α,β-meATP) (5 μM), 2-methylthioATP (2-meSATP) (100 μM) and adenosine 5′-triphosphate (ATP) (1 mM) in a concentration-dependent manner. Responses to α,β-meATP and 2-meSATP were abolished, but approximately one third of the peak response to ATP was resistant to suramin and PPADS.
3. Contractions evoked by uridine 5′-triphosphate (UTP) (1 mM) were slightly inhibited by suramin (100 and 300 μM) and potentiated by PPADS (300 μM).
4. Desensitization of the P2X₁-receptor by α,β-meATP abolished contractions evoked by 2-meSATP (100 μM) and reduced those to ATP (1 mM) and UTP (1 mM) to 15±3% and 68±4% of control.
5. Responses to α,β-meATP (5 μM) and 2-meSATP (100 μM) were abolished when tissues were bathed in nominally calcium-free solution, while the peak contractions to ATP (1 mM) and UTP (1 mM) were reduced to 24±6% and 61±13%, respectively, of their control response.
6. ARL 67156 (3–100 μM) potentiated contractions elicited by UTP (1 mM), but inhibited responses to α,β-meATP (5 μM), 2-meSATP (100 μM) and ATP (1 mM) in a concentration-dependent manner.
7. These results suggest that two populations of P2-receptors are present in the rat tail artery; ligand-gated P2X₁-receptors and G-protein-coupled P2Y-receptors.

     

5-HT1B receptor-mediated contractions in human temporal artery: evidence from selective antagonists and 5-HT receptor mRNA expression

1. In the human temporal artery both 5-HT_1-like and 5-HT_2A receptors mediate the contractile effects of 5-hydroxytryptamine (5-HT) and we have suggested that the 5-HT_1-like receptors resemble more closely recombinant 5-HT_1B than 5-HT_1D receptors. To investigate further which subtype is involved, we investigated the blockade of 5-HT-induced contractions by the 5-HT_1B-selective antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2-methyl-4′[(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-yl] carbonyl} furo[2,3-f]indole-3-spiro-4′-piperidine oxalate) and the 5-HT_1D-selective antagonist BRL-15572 (1-phenyl-3[4-3-chlorophenyl piperazin-1-yl] phenylpropan-2-ol). We also used RT-PCR to search for the mRNA of 5-HT_1B, 5-HT_1D and other 5-HT receptors.
2. The contractile effects of 5-HT in temporal artery rings were partially antagonized by SB-224289 (20, 200 nM) (apparent K_B=1 nM) and ketanserin (1 μM) but not by BRL-15572 (500 nM).
3. Sumatriptan evoked contractions (EC₅₀, 170 nM) that were resistant to blockade by BRL-15572 (500 nM) but antagonized by SB-224289 (20, 200 nM).
4. The potency of 5-HT (EC₅₀) was estimated to be 94 nM for the ketanserin-sensitive receptor and 34 nM for the SB-224289-sensitive receptor. The fraction of maximal 5-HT response mediated through SB-224289-sensitive receptors was 0.20–0.67, the remainder being mediated through ketanserin-sensitive receptors.
5. We detected arterial receptor mRNA for the following receptors (incidence): 5-HT_1B (8/8), 5-HT_1D (2/8), 5-HT_1F (0/4), 5-HT_2A (0/8), 5-HT_2B (0/8), 5-HT_2C (0/8), 5-HT₄ (4/8) and 5-HT₇ (4/8).
6. We conclude that the ketanserin-resistant fraction of the 5-HT effects and the effects of sumatriptan are mediated by 5-HT_1B receptors. The lack of antagonism by BRL-15572 rules out 5-HT_1D receptors as mediators of the contractile effects of 5-HT and sumatriptan.

     

Evidence that the substance P-induced enhancement of pacemaking in lymphatics of the guinea-pig mesentery occurs through endothelial release of thromboxane A2

1. In vitro studies were performed to examine the mechanisms underlying substance P-induced enhancement of constriction rate in guinea-pig mesenteric lymphatic vessels.
2. Substance P caused an endothelium-dependent increase in lymphatic constriction frequency which was first significant at a concentration of 1 nM (115±3% of control, n=11) with 1 μM, the highest concentration tested, increasing the rate to 153±4% of control (n=9).
3. Repetitive 5 min applications of substance P (1 μM) caused tachyphylaxis with tissue responsiveness tending to decrease (by an average of 23%) and significantly decreasing (by 72%) for application at intervals of 30 and 10 min, respectively.
4. The competitive antagonist of tachykinin receptors, spantide (5 μM) and the specific NK₁ receptor antagonist, WIN51708 (10 μM) both prevented the enhancement of constriction rate induced by 1 μM substance P.
5. Endothelial cells loaded with the Ca²⁺ sensing fluophore, fluo 3/AM did not display a detectable change in [Ca²⁺]_i upon application of 1 μM substance P.
6. Inhibition of nitric oxide synthase by N^G nitro-L-arginine (L-NOARG; 100 μM) had no significant effect on the response induced by 1 μM substance P.
7. The enhancement of constriction rate induced by 1 μM substance P was prevented by the cyclo-oxygenase inhibitor, indomethacin (3 μM), the thromboxane A₂ synthase inhibitor, imidazole (50 μM), and the thromboxane A₂ receptor antagonist, SQ29548 (0.3 μM).
8. The stable analogue of thromboxane A₂, U46619 (0.1 μM) significantly increased the constriction rate of lymphangions with or without endothelium, an effect which was prevented by SQ29548 (0.3 μM).
9. Treatment with pertussis toxin (PTx; 100 ng ml⁻¹) completely abolished the response to 1 μM substance P without inhibiting either the perfusion-induced constriction or the U46619-induced enhancement of constriction rate.
10. Application of the phospholipase A₂ inhibitor, antiflammin-1 (1 nM) prevented the enhancement of lymphatic pumping induced by substance P (1 μM), without inhibiting the response to either U46619 (0.1 μM) or acetylcholine (10 μM).
11. The data support the hypothesis that the substance P-induced increase in pumping rate is mediated via the endothelium through NK₁ receptors coupled by a PTx sensitive G-protein to phospholipase A₂ and resulting in generation of the arachidonic acid metabolite, thromboxane A₂, this serving as the diffusible activator.

     

Evidence for the involvement of 5-HT4 receptors in the 5-hydroxytryptamine-induced pattern of migrating myoelectric complex in sheep

1. The effects induced by 5-hydroxytryptamine (5-HT) on gastrointestinal myoelectric activity in conscious sheep were recorded through electrodes chronically implanted and analysed by computer. The 5-HT receptors and the cholinergic neuronal pathways involved in these actions were investigated.
2. The intravenous (i.v.) administration of 5-HT (2, 4 and 8 μg kg⁻¹ min⁻¹, 5 min) induced an antral inhibition concomitant with a duodenal activity front that migrated to the jejunum, followed by a period of intestinal inactivity. This myoelectric pattern closely resembled that observed in the phases III and I of the migrating myoelectric complex (MMC) in sheep. The 0.5 μg kg⁻¹ min⁻¹ dose evoked the same pattern in only two out of the six animals used. Likewise, the 1 μg kg⁻¹ min⁻¹ dose similarly affected four of the six animals. In addition, a transient stimulation was observed in the antrum and jejunum when the two highest doses were used.
3. The 5-HT₁ antagonist, methiothepin (0.1 mg kg⁻¹), the 5-HT₂ antagonists, ritanserin (0.1 mg  kg⁻¹) and ketanserin (0.3 mg  kg⁻¹), the 5-HT₃ antagonists, granisetron (0.2 mg kg⁻¹) and ondansetron (0.5 mg kg⁻¹), as well as the 5-HT₄ antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808 (0.2 mg kg⁻¹), did not modify the spontaneous gastrointestinal myoelectric activity. However, the cholinoceptor antagonists, atropine (0.2 mg kg⁻¹) and hexamethonium (2 mg kg⁻¹), inhibited gastrointestinal activity.
4. When these antagonists were injected i.v. 10 min before 5-HT (2 or 4 μg kg⁻¹ min⁻¹, 5 min), only {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808, atropine and hexamethonium were able to modify the 5-HT-induced actions, all of them being completely blocked by the three antagonists.
5. Our data show that 5-HT initiates a MMC-like pattern in the gastrointestinal area in sheep through 5-HT₄ receptors. Furthermore, these actions are mediated by cholinergic neural pathways involving muscarinic and nicotinic receptors. However, our results do not indicate a role for either 5-HT₁, 5-HT₂ or 5-HT₃ receptors in the 5-HT-induced effects.

     

The interaction of diadenosine polyphosphates with P2X-receptors in the guinea-pig isolated vas deferens

1. The site(s) at which diadenosine 5′,5′′′-P¹,P⁴-tetraphosphate (AP₄A) and diadenosine 5′, 5′′′-P¹,P⁵-pentaphosphate (AP₅A) act to evoke contraction of the guinea-pig isolated vas deferens was studied by use of a series of P₂-receptor antagonists and the ecto-ATPase inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP (ARL 67156).
2. Pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (300 nM–30 μM), suramin (3–100 μM) and pyridoxal-5′-phosphate (P-5-P) (3–1000 μM) inhibited contractions evoked by equi-effective concentrations of AP₅A (3 μM), AP₄A (30 μM) and α,β-methyleneATP (α,β-meATP) (1 μM), in a concentration-dependent manner and abolished them at the highest concentrations used.
3. PPADS was more potent than suramin, which in turn was more potent than P-5-P. PPADS inhibited AP₅A, AP₄A and α,β-meATP with similar IC₅₀ values. No significant difference was found between IC₅₀ values for suramin against α,β-meATP and AP₅A or α,β-meATP and AP₄A, but suramin was more than 2.5 times more potent against AP₄A than AP₅A. P-5-P showed the same pattern of antagonism.
4. Desensitization of the P_2X1-receptor by α,β-meATP abolished contractions evoked by AP₅A (3 μM) and AP₄A (30 μM), but had no effect on those elicited by noradrenaline (100 μM).
5. ARL 67156 (100 μM) reversibly potentiated contractions evoked by AP₄A (30 μM) by 61%, but caused a small, significant decrease in the mean response to AP₅A (3 μM).
6. It is concluded that AP₄A and AP₅A act at the P_2X1-receptor, or a site similar to the P_2X1-receptor, to evoke contraction of the guinea-pig isolated vas deferens. Furthermore, the potency of AP₄A, but not AP₅A, appears to be inhibited by an ecto-enzyme which is sensitive to ARL 67156.

     

Diazoxide- and leptin-activated KATP currents exhibit differential sensitivity to englitazone and ciclazindol in the rat CRI-G1 insulin-secreting cell line

1. The effects of the antidiabetic agent englitazone and the anorectic drug ciclazindol on ATP-sensitive K⁺ (K_ATP) channels activated by diazoxide and leptin were examined in the CRI-G1 insulin-secreting cell line using whole cell and single channel recording techniques.
2. In whole cell current clamp mode, the hyperglycaemic agent diazoxide (200 μM) and the ob gene product leptin (10 nM) hyperpolarised CRI-G1 cells by activation of K_ATP currents. K_ATP currents activated by either agent were inhibited by tolbutamide, with an IC₅₀ for leptin-activated currents of 9.0 μM.
3. Application of englitazone produced a concentration-dependent inhibition of K_ATP currents activated by diazoxide (200 μM) with an IC₅₀ value of 7.7 μM and a Hill coefficient of 0.87. In inside-out patches englitazone (30 μM) also inhibited K_ATP channel currents activated by diazoxide by 90.8±4.1%.
4. In contrast, englitazone (1–30 μM) failed to inhibit K_ATP channels activated by leptin, although higher concentrations (>30 μM) did inhibit leptin actions. The englitazone concentration inhibition curve in the presence of leptin resulted in an IC₅₀ value and Hill coefficient of 52 μM and 3.2, respectively. Similarly, in inside-out patches englitazone (30 μM) failed to inhibit the activity of K_ATP channels in the presence of leptin.
5. Ciclazindol also inhibited K_ATP currents activated by diazoxide (200 μM) in a concentration-dependent manner, with an IC₅₀ and Hill coefficient of 127 nM and 0.33, respectively. Furthermore, application of ciclazindol (1 μM) to the intracellular surface of inside-out patches inhibited K_ATP channel currents activated by diazoxide (200 μM) by 86.6±8.1%.
6. However, ciclazindol was much less effective at inhibiting K_ATP currents activated by leptin (10 nM). Ciclazindol (0.1–10 μM) had no effect on K_ATP currents activated by leptin, whereas higher concentrations (>10 μM) did cause inhibition with an IC₅₀ value of 40 μM and an associated Hill coefficient of 2.7. Similarly, ciclazindol (1 μM) had no significant effect on K_ATP channel activity following leptin addition in excised inside-out patches.
7. In conclusion, K_ATP currents activated by diazoxide and leptin show different sensitivity to englitazone and ciclazindol. This may be due to differences in the mechanism of activation of K_ATP channels by diazoxide and leptin.

     

Involvement of 5-HT1B/1D and 5-HT2A receptors in 5-HT-induced contraction of endothelium-denuded rabbit epicardial coronary arteries

1. The receptors responsible for 5-hydroxytryptamine (5-HT)-mediated contraction of rabbit isolated epicardial coronary artery denuded of endothelium was examined by bioassay.
2. A variety of 5-HT mimetics caused concentration-dependent contractions. The rank order of agonist potency was 5-carboxamidotryptamine (5-CT)>5-HT>(±)-α-methyl-5-hydroxytryptamine ((±)-α-me-5-HT)=sumatriptan. This was not consistent with relative potencies at any single recognized 5-HT receptor, suggesting the presence of a mixed receptor population. In one subset of preparations precontracted with U46619 (10–30 nM) with the endothelium intact, none of the agonists caused a relaxation.
3. Contractions to 5-HT were antagonized by ketanserin, a 5-HT_2A-selective antagonist, but the displacement of concentration-response curves was inconsistent with an interaction between 5-HT and a single receptor population; the slope of regression between antagonist log M concentration and agonist log (concentration-ratio −1) was shallow (0.57). Responses to 5-HT were also antagonized by the 5-HT_1B/1D-receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 and, again, the slope of regression was shallow (0.68). These data suggest a possible involvement of 5-HT_2A and 5-HT_1B or 5-HT_1D receptors in the response to 5-HT.
4. Contractions to (±)-α-me-5-HT, which is selective for 5-HT_2A over 5-HT_1B and 5-HT_1D receptors, were competitively antagonized by low concentrations of ketanserin. The regression between antagonist log M concentration and agonist log (concentration-ratio −1) fitted the Schild equation with a slope that was not significantly different from unity (0.95), giving a pA₂ value of 9.0. {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (3–30 nM), had no effect on the contractile response to (±)-α-me-5-HT. These data establish, unequivocally, the presence of 5-HT_2A receptors in the tissue.
5. Sumatriptan, a relatively selective 5-HT_1B/1D-receptor agonist, induced contractions that were antagonized competitively by {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (3–30 nM), although there was a reduction in the maximum response when concentrations of {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 exceeded 3 nM. The apparent pA₂ (estimated by imposing a unit slope on the log agonist (concentration-ratio −1) value in the presence of 3 nM {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935) was 8.92. Contractions to sumatriptan were not affected by low (5-HT_2A receptor-selective) concentrations of ketanserin, but were antagonized in a competitive manner at higher concentrations (pA₂ 6.5). These data appear to confirm the presence of 5-HT_1B and/or 5-HT_1D receptors in the tissue.
6. Antagonism of 5-HT responses by {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 was reassessed after blockade of 5-HT_2A receptors with 1 μM ketanserin. Under these conditions, {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 was able to antagonize 5-HT-induced contractions fully. The slope of regression between log M antagonist concentration and log agonist (concentration-ratio −1) fitted the Schild equation with a slope not significantly different from unity (1.1) (albeit there was still a reduction in maximum response when {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 concentration exceeded 3 nM). The apparent pA₂ value was 8.8. This reinforces the evidence that 5-HT_1B and/or 5-HT_1D receptors contribute to the effects of 5-HT in the tissue.
7. In conclusion, in endothelium denuded rabbit epicardial coronary arteries, 5-HT activates 5-HT_2A and 5-HT_1D and/or 5-HT_1B receptors to cause contraction. This appears to be similar to the situation in man.

     

Comparative desensitization of the human 5-HT2A and 5-HT2C receptors expressed in the human neuroblastoma cell line SH-SY5Y

1. We have used previously characterized clones of the human neuroblastoma cell line, SH-SY5Y, constitutively expressing either the human 5-HT_2A or 5-HT_2C receptor to compare their desensitization profiles after exposure to 5-HT.
2. 5-HT stimulated [³H]-inositol phosphate ([³H]-IP_x) production at both the 5-HT_2C (pEC₅₀=8.03±0.15) and 5-HT_2A receptors (pEC₅₀=7.15±0.08), with maximal responses occurring after exposure to 1 μM and 10 μM 5-HT, respectively.
3. Exposure of cells to maximally effective concentrations of 5-HT caused time- and concentration-dependent desensitization of [³H]-IP_x formation. The 5-HT_2A response desensitized slower (t_1/2=110 min) and with lower sensitivity than that of the 5-HT_2C receptor (t_1/2=12.5 min). In each case, desensitization was blocked by co-administration of a specific receptor antagonist. Following exposure to 10 μM 5-HT for 2 h, both receptors exhibited extensive desensitization, with subsequent responses to 5-HT reduced by more than 80%.
4. 5-HT stimulated Ins(1,4,5)P₃ production with a potency similar to that for [³H]-IP_x production at each receptor. In both cases Ins(1,4,5)P₃ levels peaked rapidly then returned to basal level within a short time. This peak consistently occurred earlier for the 5-HT_2C receptor (5 s) than for the 5-HT_2A receptor (20 s).
5. Prior exposure of SH-SY5Y/5-HT_2C cells to 5-HT (1 μM/15 min) caused a significant decrease in the 5-HT-stimulated peak in Ins(1,4,5)P₃ levels whereas no such change occurred in SH-SY5Y/5-HT_2A cells following exposure to 10 μM 5-HT for 15 min.
6. These results indicate that the human 5-HT_2A and 5-HT_2C receptors both exhibit desensitization at the level of inositol phosphate formation when expressed in the same cellular environment, with the 5-HT_2C receptor being more sensitive to 5-HT-mediated desensitization than the 5-HT_2A receptor.

     

Modulation by general anaesthetics of rat GABAA receptors comprised of α1β3 and β3 subunits expressed in human embryonic kidney 293 cells

1. Radioligand binding and patch-clamp techniques were used to study the actions of γ-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5α-pregnan-3α-ol-20-one on rat α1 and β3 GABA_A receptor subunits, expressed either alone or in combination.
2. Membranes from HEK293 cells after transfection with α1 cDNA did not bind significant levels of [³⁵S]-tert-butyl bicyclophosphorothionate ([³⁵S]-TBPS) (<0.03 pmol mg⁻¹ protein). GABA (100 μM) applied to whole-cells transfected with α1 cDNA and clamped at −60 mV, also failed to activate discernible currents.
3. The membranes of cells expressing β3 cDNAs bound [³⁵S]-TBPS (∼1 pmol mg⁻¹ protein). However, the binding was not influenced by GABA (10 nM–100 μM). Neither GABA (100 μM) nor picrotoxin (10 μM) affected currents recorded from cells expressing β3 cDNA, suggesting that β3 subunits do not form functional GABA^A receptors or spontaneously active ion channels.
4. GABA (10 nM–100 μM) modulated [³⁵S]-TBPS binding to the membranes of cells transfected with both α1 and β3 cDNAs. GABA (0.1 μM–1 mM) also dose-dependently activated inward currents with an EC₅₀ of 9 μM recorded from cells transfected with α1 and β3 cDNAs, clamped at −60 mV.
5. Propofol (10 nM–100 μM), pentobarbitone (10 nM–100 μM) and 5α-pregnan-3α-ol-20-one (1 nM–30 μM) modulated [³⁵S]-TBPS binding to the membranes of cells expressing either α1β3 or β3 receptors. Propofol (100 μM), pentobarbitone (1 mM) and 5α-pregnan-3α-ol-20-one (10 μM) also activated currents recorded from cells expressing α1β3 receptors.
6. Propofol (1 μM–1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing β3 homomers. In contrast, application of 5α-pregnan-3α-ol-20-one (10 μM) failed to activate detectable currents.
7. Propofol (100 μM)-activated currents recorded from cells expressing either α1β3 or β3 receptors reversed at the C1⁻ equilibrium potential and were inhibited to 34±13% and 39±10% of control, respectively, by picrotoxin (10 μM). 5α-Pregnan-3α-ol-20-one (100 nM) enhanced propofol (100 μM)-evoked currents mediated by α1β3 receptors to 1101±299% of control. In contrast, even at high concentration 5α-pregnan-3α-ol-20-one (10 μM) caused only a modest facilitation (to 128±12% of control) of propofol (100 μM)-evoked currents mediated by β3 homomers.
8. Propofol (3–100 μM) activated α1β3 and β3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 μM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a ‘surge'' current on cessation of propofol (300 μM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5α-pregnan-3α-ol-20-one (10 μM) to cells expressing α1β3 receptors.
9. These observations demonstrate that rat β3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABA_A receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

     

Inhibitory action of niflumic acid on noradrenaline- and 5-hydroxytryptamine-induced pressor responses in the isolated mesenteric vascular bed of the rat

1. The effects of niflumic acid, an inhibitor of calcium-activated chloride currents, were compared with the actions of the calcium channel blocker nifedipine on noradrenaline- and 5-hydroxytryptamine (5-HT)-induced pressor responses of the rat perfused isolated mesenteric vascular bed.
2. Bolus injections of noradrenaline (1 and 10 nmol) increased the perfusion pressure in a dose-dependent manner. Nifedipine (1 μM) inhibited the increase in pressure produced by 1 nmol noradrenaline by 31±5%. Niflumic acid (10 and 30 μM) also inhibited the noradrenaline-induced increase in perfusion pressure and 30 μM niflumic acid reduced the pressor response to 1 nmol noradrenaline by 34±6%.
3. The increases in perfusion elicited by 5-HT (0.3 and 3 nmol) were reduced by niflumic acid (10 and 30 μM) in a concentration-dependent manner and 30 μM niflumic acid inhibited responses to 0.3 and 3 nmol 5-HT by, respectively, 49±8% and 50±7%. Nifedipine (1 μM) decreased the pressor response to 3 nmol 5-HT by 44±9%.
4. In the presence of a combination of 30 μM niflumic acid and 1 μM nifedipine the inhibition of the pressor effects of noradrenaline (10 nmol) and 5-HT (3 nmol) was not significantly greater than with niflumic acid (30 μM) alone. Thus the effects of niflumic acid and nifedipine were not additive.
5. In Ca-free conditions the transient contractions induced by 5-HT (3 nmol) were not reduced by 30 μM niflumic acid, suggesting that this agent does not inhibit calcium release from the intracellular store or the binding of 5-HT to its receptor.
6. Niflumic acid 30 μM did not inhibit the pressor responses induced by KCl (20 and 60 μmol) which were markedly reduced by 1 μM nifedipine. In addition, 1 μM levcromakalim decreased pressor responses produced by 20 μmol KCl. These data suggest that niflumic acid does not block directly calcium channels or activate potassium channels.
7. It is concluded that niflumic acid selectively reduces a component of noradrenaline- and 5-HT-induced pressor responses by inhibiting a mechanism which leads to the opening of voltage-gated calcium channels. Our data suggest that the Ca²⁺-activated chloride conductance may play a pivotal role in the activation of voltage-gated calcium channels in agonist-induced constriction of resistance blood vessels.

     

Effect of some cyclooxygenase inhibitors on the increase in guanosine 3′:5′-cyclic monophosphate induced by NO-donors in human whole platelets

1. The effect of the NSAIDs indomethacin, indoprofen, diclofenac and acetylsalicylic acid on the increase in guanosine 3′:5′-cyclic monophosphate (cyclic GMP) induced by nitric oxide-donor agents was tested in human whole platelets and in platelet crude homogenate.
2. In whole platelets, indomethacin reduced the increase in cyclic GMP induced by the nitric oxide-donors (NO-donors) sodium nitroprusside (NaNP) and S-nitroso-N-acetylpenicillamine (SNAP) in a dose-dependent way, its IC₅₀ being 13.7 μM and 15.8 μM, respectively.
3. Of the other cyclooxygenase inhibitors tested, only indoprofen reduced the increase in cyclic GMP induced by both NO-donors in a dose-dependent way (IC₅₀=32.7 μM, NaNP and 25.0 μM, SNAP), while acetylsalicylic acid (up to 1000 μM) and diclofenac (up to 100 μM) were ineffective.
4. However, in platelet crude homogenate neither indomethacin nor indoprofen reduced the cyclic GMP production.
5. Indomethacin (10 μM), indoprofen (30 μM), diclofenac (100 μM) and acetylsalicylic acid (1000 μM) showed a comparable efficacy in inhibiting platelet thromboxane B₂ (TXB₂) production, suggesting that the inhibitory effect of indomethacin and indoprofen on the increase in cyclic GMP induced by both NO-donors was not mediated by inhibition of cyclooxygenase.
6. In vitro, the NSAIDs analysed did not interfere with nitrite production of SNAP.
7. The unhomogeneous behaviour of NSAIDs on the increase in cyclic GMP induced by NO-donors in whole platelets may contribute to the different pharmacological and toxicological characteristics of the drugs, providing new knowledge on the effect of indomethacin and indoprofen.

     

Potentiation of adenosine A1 receptor-mediated inositol phospholipid hydrolysis by tyrosine kinase inhibitors in CHO cells

1. The effect of protein tyrosine kinase inhibitors on human adenosine A₁ receptor-mediated [³H]-inositol phosphate ([³H]-IP) accumulation has been studied in transfected Chinese hamster ovary cells (CHO-A1) cells.
2. In agreement with our previous studies the selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated the accumulation of [³H]-IPs in CHO-A1 cells. Pre-treatment with the broad spectrum tyrosine kinase inhibitor genistein (100 μM; 30 min) potentiated the responses elicited by 1 μM (199±17% of control CPA response) and 10 μM CPA (234±15%). Similarly, tyrphostin A47 (100 μM) potentiated the accumulation of [³H]-IPs elicited by 1 μM CPA (280±32%).
3. Genistein (EC₅₀=13.7±1.2 μM) and tyrphostin A47 (EC₅₀=10.4±3.9 μM) potentiated the [³H]-IP response to 1 μM CPA in a concentration-dependent manner.
4. Pre-incubation with the inactive analogues of genistein and tyrphostin A47, daidzein (100 μM; 30 min) and tyrphostin A1 (100 μM; 30 min), respectively, had no significant effect on the accumulation of [³H]-IPs elicited by 1 μM CPA.
5. Genistein (100 μM) had no significant effect on the accumulation of [³H]-IPs produced by the endogenous thrombin receptor (1 u ml⁻¹; 100±10% of control response). In contrast, tyrphostin A47 produced a small augmentation of the thrombin [³H]-IP response (148±13%).
6. Genistein (100 μM) had no effect on the [³H]-IP response produced by activation of the endogenous G_q-protein coupled CCK_A receptor with the sulphated C-terminal octapeptide of cholecystokinin (1 μM CCK-8; 96±6% of control). In contrast, tyrphostin A47 (100 μM) caused a small but significant increase in the response to 1 μM CCK-8 (113±3% of control).
7. The phosphatidylinositol 3-kinase inhibitor LY 294002 (30 μM) and the MAP kinase kinase inhibitor PD 98059 (50 μM) had no significant effect on the [³H]-IP responses produced by 1 μM CPA and 1 μM CCK-8.
8. These observations suggest that a tyrosine kinase-dependent pathway may be involved in the regulation of human adenosine A₁ receptor mediated [³H]-IP responses in CHO-A1 cells.

     

The mechanisms of enhancement and inhibition of field stimulation responses of guinea-pig vas deferens by prostacyclin analogues

1. In the guinea-pig isolated vas deferens preparation bathed in Tyrode''s solution, the prostacyclin analogues, cicaprost, TEI-9063, iloprost, taprostene and benzodioxane-prostacyclin, enhanced twitch responses to submaximal electrical field stimulation (20%-EFS). The high potency of cicaprost (EC₁₅₀=1.3 nM) and the relative potencies of the analogues (equi-effective molar ratios=1.0, 0.85, 1.6, 17 and 82, respectively) suggest the involvement of a prostacyclin (IP-) receptor.
2. Maximum enhancement induced by cicaprost in 2.5 mM K⁺ Krebs-Henseleit solution was similar to that in Tyrode solution (2.7 mM K⁺), but was progressively reduced as the K⁺ concentration was increased to 3.9, 5.9 and 11.9 mM. There was also a greater tendency for the other prostacyclin analogues to inhibit EFS responses in 5.9 mM standard K⁺ Krebs-Henseleit solution; this may be attributed to their agonist actions on presynaptic EP₃-receptors resulting in inhibition of transmitter release.
3. The EFS enhancing action of cicaprost was not affected by the α₁-adrenoceptor antagonist prazosin (100 and 1000 nM). Cicaprost (20 and 200 nM) did not affect contractile responses of the vas deferens to either ATP (5 μM) or α,β-methylene ATP (1 μM) in the presence of tetrodotoxin (TTX, 100 nM). In addition, enhancement by cicaprost of responses to higher concentrations of ATP (30 and 300 μM) in the absence of TTX, as shown previously by others, was not seen. Prostaglandin E₂ (PGE₂, 10 nM) and another prostacyclin analogue TEI-3356 (20 nM) enhanced purinoceptor agonist responses. Unexpectedly, TTX (0.1 and 1 μM) partially inhibited contractions elicited by 10–1000 μM ATP; contractions elicited by 1–3 μM ATP were unaffected. Further studies are required to establish whether a pre- or post-synaptic mechanism is involved.
4. In a separate series of experiments, cicaprost (5–250 nM), TEI-9063 (3–300 nM), 4-aminopyridine (10–100 μM) and tetraethylammonium (100–1000 μM) enhanced both 20%-EFS responses and the accompanying overflow of noradrenaline to a similar extent. In further experiments with the EP₁-receptor antagonist AH 6809, TEI-3356 (1.0–100 nM) and the EP₃-receptor agonist, sulprostone (0.1–1.0 nM) inhibited both maximal EFS responses and noradrenaline overflow, thus confirming previous reports of the high activity of TEI-3356 at the EP₃-receptor. Cicaprost had no significant effect on noradrenaline overflow at 10 and 100 nM, but produced a modest inhibition at 640 nM.
5. In conclusion, our studies show that prostacyclin analogues (particularly TEI-3356) can inhibit EFS responses of the guinea-pig vas deferens by acting as agonists at presynaptic EP₃-receptors. Prostacyclin analogues (particularly cicaprost and TEI-9063) can also enhance EFS responses through activation of IP-receptors. The mechanism of the enhancement has not been rigorously established but from our results we favour a presynaptic action to increase transmitter release.