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Notes: Abstract The aim of this study was to assess at which extent an even co-release of the tachykinins, substance P (SP) and neurokinin A (NKA), occurs from enteric neurons/nerves of the guinea-pig proximal colon during graded depolarization. In this preparation, a sharply diverging NK1/NK2 receptor pattern of tachykininergic co-transmission has been observed in physiological studies. The experiments were performed in capsaicin-pretreated (10μM for 15min) mucosa-free smooth muscle of guinea-pig proximal colon, to exclude the mucosa and the peripheral endings of primary afferent nerves as possible sources of released tachykinins. The content of extractable tachykinins was measured as SP- and NKA-like immunoreactivities (-LI) by radioimmunoassay. Chromatographic characterization of aqueous acetic acid extracts showed one peak of SP-LI corresponding to authentic SP, whereas there were multiple peaks of NKA-LI, the major one co-eluting with authentic NKA. An increased outflow of both SP- and NKA-LI was evenly produced in a concentration-dependent manner when the preparations were superfused with a high potassium (K) medium in which NaCl had been replaced with equimolar amounts (20–100mM) of KCl. The high K-evoked release of SP- and NKA-LI was dependent upon the presence of extracellular calcium and was inhibited by about 50% in the presence of the N-type voltage-dependent calcium channel blocker, ω-conotoxin GVIA (0.1μM). ω-Conotoxin MVIIC (1μM), a non-selective blocker of N-, P- and Q-type voltage-dependent calcium channels, likewise produced about 40% inhibition of evoked release of both peptides. No evidence for a role of L-type channels in tachykinin release was obtained, since the addition of nifedipine (1μM) or Bay K8644 (1μM) did not significantly affect the response to high K. Neither NK1 receptor agonist (septide, 0.1μM) or antagonist(SR 140333, 10nM) nor NK2 receptor agonists ([βAla8]NKA(4-10) and GR 64349, 0.1μM each) or antagonist (SR 48968, 10nM) did affect the high K-evoked release of tachykinins. We conclude that SP and NKA are evenly co-released in response to graded depolarization of enteric nerves in the guinea-pig colon. Therefore, the specialization of tachykininergic transmission observed in functional studies does not originate at the prejunctional level. The co-release of tachykinins involves the influx of extracellular calcium via N-type but not L-type calcium channels. No evidence for the presence of NK1 or NK2 autoreceptors affecting tachykinin release from enteric neurons was obtained.

Notes: Abstract The human tachykinin NK2 receptor stably expressed in Chinese hamster ovary cells (CHO-hNK2R cells) was characterized by studying the effect of neurokinin A (NKA), the preferred natural ligand, and that of other agonists and antagonists in both binding experiments and functional assays. Competition experiments using [125I]NKA showed that CHO-hNK2R cells express binding sites which have high affinity for NKA (K i=3.4±0.9 nM), GR 64349 (K i=12±3 nM) and [βAla8]NKA(4–10) (K i=21±8 nM) and for the antagonists MEN 10627 (K i=0.55±0.2 nM), and MEN 11420 (K i=2.4±0.8 nM). In contrast, the tachykinin NK1 and NK3 receptor agonists [Sar9,Met(O2)11]SP and senktide, respectively, were recognized with low affinity (K i〉10 μM). NKA (EC50=68±18 nM) induced a rapid and concentration-dependent increase in the intracellular level of inositoltrisphosphate (IP3). The concentration-response curve to GR 64349 (EC50=155±14 nM) was close to that of NKA, whereas [βAla8]NKA(4–10) (EC50=445±78 nM) and SP (EC50=3197±669 nM) were 7- and 50-fold less potent, respectively. In addition, NKA stimulated the release of arachidonic acid and the production of prostaglandin E2 (PGE2) in a concentration-dependent manner. Also in this assay, NKA was found to be more potent than the other agonists tested (the EC50 values were 3±0.3, 9±3, 7.8±0.9 and 217±37 nM for NKA, GR 64349, [βAla8]NKA(4–10) and SP, respectively). MEN 10627 and MEN 11420 were potent and competitive antagonists in blocking NKA-induced IP3 formation and PGE2 release: MEN 10627 and MEN 11420 displayed comparable potencies in blocking the two functional responses initiated by occupancy of the NK2 receptor by NKA. Pretreatment of the cells with pertussis toxin (500 ng/ml for 18 h) did not significantly modify the basal or stimulated phosphatidylinositol turnover but reduced the basal and NKA-induced PGE2 release by about 35%. The phospholipase C inhibitor U-73122 (10 μM) prevented the NKA-induced formation of IP3 but did not affect PGE2 release. Conversely, the phospholipase A2 inhibitor quinacrine (100 μM) blocked the release of arachidonic acid and PGE2 without affecting the NKA-stimulated formation of IP3. Chelation of extracellular calcium with 3 mM EGTA inhibited the NKA-induced PGE2 release by 81% but was without effect on basal and NKA-stimulated IP3 production. The calcium channel blockers verapamil (10 μM) and ω-conotoxin GVIA (0.1 μM) did not modify the basal PGE2 production and had no significant effect on the response to tachykinins while the blocker of non-selective cation channels, SKF-96365 (10 μM), inhibited the response to NKA by about 74%. SKF-96365 did not affect the basal or the NKA-induced IP3 formation. In conclusion, our data demonstrate that the human tachykinin NK2 receptor expressed in CHO cells displays binding affinity and functional properties which are those of a native NK2 receptor. No pharmacological evidence for heterogeneity of the human NK2 receptor was obtained in this study. Our findings indicate that the human tachykinin NK2 receptor is independently coupled to both PLC and PLA2 signaling pathways. Activation of the PLA2 pathway may be linked to the opening of a voltage-independent cation channel which activates a Ca2+-dependent PLA2.

Notes: Abstract This study investigates the role of tachykinin NK1 and NK2 receptors in motor responses induced by the intravesical instillation of capsaicin in urethane-anaesthetized rats. SR 140,333 (1 µmol/kg, i.v.), a non-peptide NK1 receptor antagonist, abolished urinary bladder contractions induced by the selective NK1 receptor agonist [Sar9]SP-sulfone (0.1-100 nmol/kg, i.v.) without affecting those induced by the NK2 receptor agonist [ßAla8]NKA(4-10). MEN 11,420 (100 nmol/kg, i.v.), a cyclic peptide NK2 receptor antagonist, abolished bladder contractions induced by [ßAla8]NKA(4-10) (0.3-300 nmol/kg, i.v.) without modifying those induced by [Sar9]SP-sulfone. Intravesical instillation of capsaicin (6 nmol/0.6 ml/rat) produced a motor response consisting in a primary contraction followed by a series of high amplitude phasic contractions. The intravesical instillation of saline (0.6 ml/rat) produced a primary contraction of lower amplitude with respect to that induced by capsaicin and the total area under the curve was also lower in saline-instilled rats, however the number and the amplitude of phasic contractions was similar to that induced by capsaicin. MEN 11,420 (100 nmol/kg, i.v.) did not modify motor responses induced by the intravesical administration of saline. In contrast, in capsaicin-instilled rats, MEN 11,420 (100 nmol/kg, i.v.) reduced the primary contraction, the area under the curve and also the number of phasic contractions. SR 140,333 (1 µmol/kg, i.v.) reduced the primary contraction but not other parameters. The combination of SR 140,333 (1 µmol/kg, i.v.) and MEN 11,420 (100 nmol/kg, i.v.) produced an additive inhibitory effect on the primary contraction but not a further inhibition on other parameters with respect to that observed with MEN 11,420 alone. In hexamethonium (110 µmol/kg, i.v.)-pretreated animals the intravesical instillation of capsaicin produced a tonic contraction having greater amplitude and area than that induced by saline. MEN 11,420, but not SR 140,333, significantly reduced the bladder response to capsaicin in hexamethonium-pretreated rats. Again, the combined administration of MEN 11,420 and SR 140,333 did not produce further inhibitory effect in comparison to MEN 11,420 alone. It is concluded that the motor responses induced by the intravesical instillation of capsaicin are mediated by the activation of peripheral tachykinin NK2 receptors.

Notes: Abstract We have characterized the action of the novel, water-soluble, tachykinin NK2 receptor antagonist MEN 11420 ([Asn(2-AcNH-β-D-Glc)-Asp-Trp-Phe-Dap-Leu] c(2β–5β)) on the circular muscle of the guinea-pig and human colon in vitro and on the guinea-pig colon in vivo. In organ bath experiments on guinea-pig colon MEN 11420 produced a concentration-dependent rightward shift of the concentration-response curve to the NK2 receptor selective agonist, [βAla8]neurokinin A (NKA) (4–10) with a pKB value of 8.1. Up to 1 μM MEN 11420 had no effect on the concentration-response curve to methacholine, to the NK1 receptor selective agonist, [Sar9]substance P (SP) sulfone, to the NK3 receptor selective agonist, senktide, or on the response to exogenous SP. The response to exogenous NKA was inhibited, although the shift of the concentration-response curve to NKA produced by MEN 11420 at 1 μM (dose ratio 5.3) was much smaller than that produced against [βAla8]NKA (4–10) (dose ratio 102), presumably because NKA also stimulates NK1 receptors at relatively low concentrations. In sucrose gap, MEN 11420 concentration-dependently inhibited both depolarization (IC50 0.34 μM) and contraction (IC50 = 0.32 μM) produced by [βAla8]NKA (4–10) (0.3 μM for 10 s) in the guinea-pig colon without affecting the corresponding responses produced by [Sar9]SP sulfone. When similar experiments were performed in the circular muscle of the human colon MEN 11420 concentration-dependently inhibited both depolarization and contraction induced by [βAla8]NKA(4– 10) with IC50s of 99 and 75 nM, respectively. MEN 11420 (1 μM) had no effect on the nonadrenergic noncholinergic (NANC) depolarization and contraction produced by a short period of electrical field stimulation (EFS, 10 Hz for 1 s) in the guinea-pig colon and selectively inhibited the sustained component of depolarization produced during a prolonged period of EFS (3 Hz for 3 min), without affecting the concomitant depolarization. Nifedipine (1 μM) eliminated the NANC contraction to a short period of EFS and the phasic contraction in response to a prolonged period of EFS. MEN 11420 (1 μM) abolished the nifedipine-resistant NANC contraction produced by prolonged period of electrical field stimulation (EFS, 3 Hz for 3 min). All electrical and mechanical NANC responses to EFS which were resistant to MEN 11420, either in the absence or presence of nifedipine, were abolished by the subsequent application of the NK1 receptor antagonist, SR 140333 (1 μM). Up to 3 μM, MEN 11420 had no significant effect on the cholinergic excitatory junction potential or the NANC inhibitory junction potential evoked by single pulse EFS, nor did it affect membrane conductance. In urethane-anaesthetized guinea-pigs MEN 11420 (10– 100 nmol/kg i.v.) produced a dose-dependent and long lasting (〉 3 h) inhibition of the contractile response (15 ± 2 mmHg) of the proximal colon induced by [βAla8]NKA (4–10) (3 nmol/kg i.v.). MEN 11420 (300 nmol/kg i.v.) did not affect the contraction produced by [Sar9]SP sulfone. MEN 11420 (300 nmol/kg) produced a limited (Emax about 40% inhibition) and transient (recovery within 60 min) inhibition of the atropine- and hexamethonium-sensitive phasic contractions of the proximal colon induced by threshold distension of a colonic balloon. On the other hand, MEN 11420 (10–300 nmol/kg i.v.) produced a dose-dependent complete and prolonged (〉 2 h from administration) inhibition of the atropine-resistant and hexamethonium-sensitive phasic contraction induced by suprathreshold distension of the colonic balloon. We conclude that MEN 11420 is a potent and selective tachykinin NK2 receptor antagonist devoid of significant inhibitory activity toward excitatory transmission mediated via tachykinin NK1 or muscarinic receptors. The present findings indicate that SP and NKA are likely involved in the preferential activation of NK1 and NK2 receptors during tachykininergic transmission, although NKA can act as an effective NK1 receptor ligand.

Notes: Abstract. The effects of an i.v. administration of endothelin-1, -2 and -3 (0.25–3 nmol kg–1) or their corresponding proendothelins (1–20 nmol kg–1) on blood pressure and 6 keto-prostaglandin F1α (6 keto-PGF1α) release in the anaesthetized ganglion-blocked rat were evaluated. The same peptides were tested for their ability to release 6 keto-PGF1α from the rat vas deferens in vitro. Endothelins and proendothelins showed a transient hypotensive effect followed by a potent, long lasting vasopressor response. Blood pressure increase induced by endothelins was found to be dose-dependently correlated with 6 keto-PGF1α plasma level increases. On the other hand proendothelins produced similar pressor responses, but their effect on 6 keto-PGF1α plasma levels was much less intense at equipressor doses. The effects of endothelins on arterial pressure and 6 keto-PGF1α release were phosphoramidon-insensitive, while the activities of proendothelins were reduced by phosphoramidon (10 mg kg–1 i.v.). Both endothelins (5–15 nmol/l) and proendothelins (100–300 nmol/l) were able to increase to a similar extent 6 keto-PGF1α levels in the rat vas deferens incubation buffer. The releasing activity of endothelins was not modified by the pretreatment with phosphoramidon (50 μmol/l). This pretreatment strongly inhibited proendothelin-1 and -2 effects, but not that of proendothelin-3. In conclusion, the results presented in this study indicate that all tested peptides induce 6 keto-PGF1α release in both vascular and non vascular tissue; all three proendothelins are activated by the same phosphoramidon-sensitive endothelin converting enzyme in our in vivomodel, while proendothelin-3 may be processed by a different enzyme(s) in the rat vas deferens, in vitro. They also suggest a different localization of the sites where the peptides are activated and/or exert pressor activity and the sites where they induce 6 keto-PGF1α release. Finally, prostacyclin release could have a general counter-regulatory activity on effects of endothelin peptides.

Notes: Abstract The effects of an iv. administration of endothelin-1, −2 and −3 (0.25–3 nmol kg−1) or their corresponding proendothelins (1–20 nmol kg−1) on blood pressure and 6 keto-prostaglandin F1α (6 keto-PGF1α) release in the anaesthetized ganglion-blocked rat were evaluated. The same peptides were tested for their ability to release 6 keto-PGF1a from the rat vas deferens in vitro. Endothelins and proendothelins showed a transient hypotensive effect followed by a potent, long lasting vasopressor response. Blood pressure increase induced by endothelins was found to be dose-dependently correlated with 6 keto-PGF1α plasma level increases. On the other hand proendothelins produced similar pressor responses, but their effect on 6 keto-PGF1α plasma levels was much less intense at equipressor doses. The effects of endothelins on arterial pressure and 6 keto-PGF1α release were phosphoramidon-insensitive, while the activities of proendothelins were reduced by phosphoramidon (10 mg kg−1 i.v.). Both endothelins (5–15 nmol/l) and proendothelins (100–300 nmol/l) were able to increase to a similar extent 6 keto-PGF1α levels in the rat vas deferens incubation buffer. The releasing activity of endothelins was not modified by the pretreatment with phosphoramidon (50 μmol/l). This pretreatment strongly inhibited proendothelin-1 and −2 effects, but not that of proendothelin-3. In conclusion, the results presented in this study indicate that all tested peptides induce 6 keto-PGF1α release in both vascular and non vascular tissue; all three proendothelins are activated by the same phosphoramidon-sensitive endothelin converting enzyme in our in vivo model, while proendothelin-3 may be processed by a different enzyme(s) in the rat vas deferens, in vitro. They also suggest a different localization of the sites where the peptides are activated and/or exert pressor activity and the sites where they induce 6 keto-PGF1α release. Finally, prostacyclin release could have a general counter-regulatory activity on effects of endothelin peptides.