Receptors In Guinea-pig Ileum Research Articles

HBK-14 and HBK-15 Do Not Influence Blood Pressure, Lipid Profile, Glucose Level, or Liver Enzymes Activity after Chronic Treatment in Rats.

Older and even new antidepressants cause adverse effects, such as orthostatic hypotension, hyper- or hypoglycemia, liver injury or lipid disorders. In our previous experiments we showed significant antidepressant- and anxiolytic-like activities of dual 5-HT1A and 5-HT7 antagonists with α1-adrenolitic properties i.e. 1-[(2,6-dimethylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-14) and 1-[(2-chloro-6-methylphenoxy)ethoxyethyl]-4-(2-methoxyphenyl)piperazine hydrochloride (HBK-15). Here, we evaluated the influence of chronic administration of HBK-14 and HBK-15 on blood pressure (non-invasive blood pressure measurement system for rodents), lipid profile (total cholesterol, low density lipoproteins—LDL, high density lipoproteins—HDL, triglycerides), glucose level, and liver enzymes activity (aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase). We determined potential antihistaminic (isolated guinea pig ileum) and antioxidant properties (ferric reducing ability of plasma–FRAP, non-protein thiols–NPSH, stable free radical diphenylpicrylhydrazyl—DPPH) cytotoxicity. Our experiments revealed that HBK-14 and HBK-15 did not influence blood pressure, lipid profile, glucose level or liver enzymes activity in rats after 2-week treatment. We also showed that none of the compounds possessed antioxidant or cytotoxic properties at antidepressant- and anxiolytic-like doses. HBK-14 and HBK-15 very weakly blocked H1 receptors in guinea pig ileum. Positive results of our preliminary experiments on the safety of HBK-14 and HBK-15 encourage further studies concerning their effectiveness in the treatment of depression and/or anxiety disorders.

Differential effects of R-isovaline and the GABAB agonist, baclofen, in the guinea pig ileum

R-isovaline is a non-proteinogenic amino acid which produces analgesia in a range of nociceptive assays. Mediation of this effect by metabotropic receptors for γ-aminobutyric acid (GABA) and glutamate, demonstrated by previous work, may depend on the type of tissue or receptor system. The objective of this study was to assess the activity of R-isovaline acting at GABAB and group II metabotropic glutamate receptors in guinea pig ileum, which is known to exhibit well-defined responses to GABAB agonists such as baclofen. The effects of bath-applied R-isovaline and RS-baclofen were examined on electrically evoked contractions of guinea pig ileum and during GABAB antagonism by CGP52432. In separate experiments, the group II metabotropic glutamate receptor agonist, LY354740 was applied to determine the functional presence of these receptors. R-isovaline (1–100mM) decreased the amplitude of ileal muscle contractions and increased tension. RS-baclofen reduced contraction amplitude, but decreased tension. CGP52432 did not prevent the effects of R-isovaline on contraction amplitude, but antagonized effects of RS-baclofen on contraction amplitude. The group II metabotropic glutamate receptor agonist, LY354740, produced no detectable effects on evoked contractions. R-isovaline differed significantly from RS-baclofen in its actions in the guinea pig ileum, indicated in particular by the finding that CGP52432 blocked only the effects of RS-baclofen. The ileal tissue did not respond to a group II metabotropic glutamate receptor agonist, previously shown to co-mediate R-isovaline analgesia. These findings raise the possibility of a novel therapeutic target at unknown receptors for R-isovaline-like compounds in the guinea pig ileum.

Antihistamine activity of the new peptidepreparation from the cod liver

The effect of the peptide substance on the induced production of histamine by culture of basophile, interaction of the preparation with the H1-histamine receptors and possible sedative effect on animal models were studied. The introduction of the peptide substance in cell culture of basophile didn’t have a significant effect on the histamine release. Study of the interaction of the polypeptide substance with H1-histamine receptors of guinea pig ileum showed that the preparation inhibits the response of unstriated muscles to histamine. The effect was similar to antihistamine drug - ketotifen. In the models of “open field” and “elevated criss-cross labyrinth” it was found that the peptide preparation hasn’t a sedative effect.

ChemInform Abstract: Synthesis of Antagonists of Muscarinic (M3) Receptors.

AbstractA series of α‐hydroxyamides of type (VII) and (VIII) are prepared and evaluated as antagonists of the M3 muscarinic receptor in guinea pig ileum.

Synthesis of antagonists of muscarinic (M3) receptors

Several α-hydroxyamides with (2,6-dialkoxyphenoxy)methyl substituents have been prepared and their activities as antagonists of the M3 muscarinic receptor in guinea pig ileum have been evaluated. N-{1-[(Phenyl)methyl]piperidin-4-yl}-2-{2-[(2,6-dimethoxyphenoxy)-methyl]phenyl}-2-hydroxypropanamide and N-(1-[{6-amino-4-[(1-propylpiperidin-4-yl)methyl]pyridin-2-yl}methyl]piperidin-4-yl)-2-cyclopentyl-2-hydroxy-2-phenylacetamide were the most potent compounds prepared, the micromolar potency of the latter indicating that it may be worth further investigation.

Estimation of endogenous adenosine activity at adenosine receptors in guinea‐pig ileum using a new pharmacological method

Adenosine modulates neurotransmission and in the intestine adenosine is continuously released both from nerves and from smooth muscle. The main effect is modulation of contractile activity by inhibition of neurotransmitter release and by direct smooth muscle relaxation. Estimation of adenosine concentration at the receptors is difficult due to metabolic inactivation. We hypothesized that endogenous adenosine concentrations can be calculated by using adenosine receptor antagonist and agonist and dose ratio (DR) equations. Plexus-containing guinea-pig ileum longitudinal smooth muscle preparations were made to contract intermittently by electrical field stimulation in organ baths. Schild plot regressions were constructed with 2-chloroadenosine (agonist) and 8-(p-sulfophenyl)theophylline (8-PST; antagonist). In separate experiments the reversing or enhancing effect of 8-PST and the inhibiting effect of 2-chloroadenosine (CADO) were analysed in the absence or presence of an adenosine uptake inhibitor (dilazep), and nucleoside overflow was measured by HPLC. Using the obtained DR, baseline adenosine concentration was calculated to 28 nm expressed as CADO activity, which increased dose dependently after addition of 10(-6) m dilazep to 150 nm (P < 0.05). HPLC measurements yielded a lower fractional increment (80%) in adenosine during dilazep, than found in the pharmacological determination (440%). Endogenous adenosine is an important modulator of intestinal neuro-effector activity, operating in the linear part of the dose-response curve. Other adenosine-like agonists might contribute to neuromodulation and the derived formulas can be used to calculate endogenous agonist activity, which is markedly affected by nucleoside uptake inhibition. The method described should be suitable for other endogenous signalling molecules in many biological systems.

In vitro pharmacological investigations of Sapindus trifoliatus in various migraine targets

Phytotherapies have offered alternative sources of therapy for migraine and gained much importance in prophylactic treatment. The aqueous extract of pericarp of fruits of Sapindus trifoliatus Linn (ST), family Sapindaceae was evaluated for its affinity for 5-HT 1B/1D receptors in rabbit saphenous vein, α-adrenoceptors in rabbit aorta, GABA receptors in guinea pig ileum, 5-HT 2B receptors in rat fundus and vanilloid receptors in guinea pig trachea. The calcium blocking effect was studied in rabbit aorta while the modulatory role of ST on platelet serotonin release was evaluated in human platelets. The aqueous extract of Sapindus trifoliatus exhibited significant 5-HT 2B receptor inhibition and moderate platelet serotonin release inhibition.

Nα-Imidazolylalkyl and Pyridylalkyl Derivatives of Histaprodifen: Synthesis and in Vitro Evaluation of Highly Potent Histamine H1-Receptor Agonists

A novel series of N(alpha)()-imidazolylalkyl and pyridylalkyl derivatives of histaprodifen (6, 2-[2-(3,3-diphenylpropyl)imidazol-4-yl]ethanamine) was synthesized and evaluated as histamine H(1)-receptor agonists. The title compounds displayed partial agonism at contractile H(1)-receptors of guinea pig ileum and were at least equipotent with histamine. Agonist effects of the new derivatives were susceptible to blockade by the H(1)-receptor antagonist mepyramine (2-100 nM). In the imidazole series, suprahistaprodifen (51, [2-[2-(3,3-diphenylpropyl)-1H-imidazol-4-yl]ethyl]-[2-(1H-imidazol-4-yl)ethyl]amine, N(alpha)-2-[(1H-imidazol-4-yl)ethyl]histaprodifen) showed the highest H(1)-receptor agonist potency ever reported in the literature (pEC(50) 8.26, efficacy E(max) 96%). Elongation of the alkyl spacer from ethyl to butyl decreased activity from 3630% (ethyl, 51) to 163% (butyl, 53) of histamine potency. The exchange of the terminal imidazole nucleus for a pyridine ring resulted in compounds with comparably high potency. A decrease in agonist potency and efficacy was observed when the attachment of the alkyl spacer was consecutively changed from the ortho to the meta and the para position, respectively, of the pyridine ring. The pyridine series that contained a butyl chain possessed the highest potency and affinity. N(alpha)-[4-(2-pyridyl)butyl]histaprodifen (56) emerged as a strong partial agonist, being almost equipotent with 51 (pEC(50) 8.16, E(max) 89%). Compounds 51 and 56 also showed potent partial agonism at contractile H(1) receptors in guinea pig aorta and potently activated H(1)-receptor-mediated endothelium-dependent relaxation in the rat aorta. Compounds 51-65 displayed low to moderate affinity at H(2), H(3), and M(3) receptors in functional models of guinea pig. Collectively, N(alpha)-imidazolylalkyl- and N(alpha)-pyridylalkyl-substituted histaprodifens represent a novel class of potent H(1)-receptor agonists. These compounds may be useful to define the (patho)physiological role of the H(1)-receptor and refine molecular models of H(1)-receptor activation.

NF449: a subnanomolar potency antagonist at recombinant rat P2X1 receptors.

Antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alphabetameATP; mediated by P2X1 receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by alphabetameATP (mediated by P2X3 receptors) or adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y1 receptors), ATP-induced increases of [Ca2+]i in human embryonic kidney (HEK) 293 cells (mediated by P2Y2 receptors), inward currents evoked by ATP in follicle cell-free Xenopus laevis oocytes expressing rP2X1 or rP2X3 receptors and degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (alpha1A-adrenoceptors) and guinea-pig ileum (histamine H1 and muscarinic M3 receptors). At native (pIC50=7.15) and recombinant (pIC50=9.54) P2X1 receptors, NF449 was a highly potent antagonist. The P2X3 receptors present in guinea-pig ileum (pIC50=5.04) or expressed in oocytes (pIC50 approximately 5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y1 receptors in guinea-pig ileum (pIC50=4.85) and P2Y2 receptors in HEK 293 cells (pIC50=3.86), and showed very low inhibitory potency on ecto-nucleotidases (pIC50<3.5). NF449 (100 microM) did not interact with alpha1A-adrenoceptors or histamine H1 and muscarinic M3 receptors. Thus, the antagonism by NF449 is highly specific for P2 receptors. In conclusion, the subnanomolar potency at rP2X1 receptors and the rank order of potency, P2X1 >> P2X3 > P2Y1 > P2Y2 > ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X1-selective radioligand.

The novel pyridoxal-5′-phosphate derivative PPNDS potently antagonizes activation of P2X 1 receptors

Pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate) (PPNDS) potently antagonized P2X 1 receptor-mediated responses in rat vas deferens (p K B=7.43) and Xenopus laevis oocytes (pIC 50=7.84). It showed lower (up to 20,000-fold) inhibitory potency on ecto-nucleotidase in Xenopus oocytes and on P2Y 1 receptors in guinea-pig ileum (p A 2=6.13). PPNDS did not interact with α 1A-adrenoceptors, adenosine A 1 and A 2B, histamine H 1 and muscarinic M 3 receptors. Thus, PPNDS is a novel, specific P2 receptor antagonist and represents the pyridoxal-5′-phosphate derivative with the highest potency at P2X 1 receptors.

Neurons bearing NK(3) tachykinin receptors in the guinea-pig ileum revealed by specific binding of fluorescently labelled agonists.

The localisation of NK(3) tachykinin receptors in guinea-pig ileum was studied using the fluorescently labelled agonists, Cy3. 5-neurokinin A and Cy3.5-kassinin. Binding to nerve cell bodies in the myenteric and submucosal plexuses was visualised using confocal microscopy. Binding to NK(1) receptors was blocked by the NK(1) receptor antagonist, CP-99994. NK(3) receptors, demonstrated by binding in the presence of CP-99994, occurred in 72% of myenteric and 38% of submucosal neurons. Colocalisation with other markers was examined to deduce the classes of neurons with NK(3) receptors. In myenteric ganglia, NK(3) receptors were present on the following: 73% of calbindin-immunoreactive (IR) intrinsic primary afferent neurons, 63% of calretinin-IR excitatory motor neurons and ascending interneurons, 63% of nitric oxide synthase-IR inhibitory motor neurons and descending interneurons, 79% of strongly neuropeptide Y (NPY)-IR secretomotor neurons, 67% of weakly NPY-IR descending interneurons and motor neurons, and 46% of NK(1) receptor-IR neurons. In submucosal ganglia, NK(3) receptors were on 65% of calretinin-IR secretomotor/vasodilator neurons, 81% of NPY-IR cholinergic secretomotor neurons, 2% of vasoactive intestinal peptide-IR non-cholinergic secretomotor neurons and were completely absent from substance P-IR intrinsic primary afferent neurons. The results support physiological studies suggesting that NK(3) receptors mediate tachykinin transmission between myenteric sensory neurons and to interneurons and/or motor neurons in descending inhibitory and ascending excitatory pathways.

Opioid Receptor Assays

AbstractOpioid receptors mediate the pharmacologic actions of morphine‐like analgesics, a class of drugs that are important clinical agents which are also widely abused socially as a result of their euphorogenic and addictive properties. Cloning of the opioid receptors has confirmed the original postulate that there are only three classes of opioid receptors: µ, δ, and κ. The most commonly used isolated tissue preparations for measuring activity at these receptors are the guinea pig ileum and the mouse vas deferens. This unit describes preparation of these tissues to measure agonist and antagonist properties of test compounds at the µ‐ and κ‐opioid receptors in guinea pig ileum and the d‐opioid receptor in mouse vas deferens.

M2 receptor binding of the selective antagonist AF-DX 384: possible involvement of the common allosteric site.

The hypothesis was tested that M2-selective antagonists partially utilize the allosteric site of muscarinic M2 receptors. The interactions of the allosteric agent W84 (hexane-1, 6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide]) were studied with the M2/M4-selective AF-DX 384 [(+/-)-5, 11-dihydro-11-([(2-(2-[(dipropylamino)methyl]-1-piperidinyl)ethyl)amino]carbonyl)-6H-pyrido(2,3-b)(1,4)-benzodiazepine-6-one], the nonselective N-methylscopolamine (NMS), and a number of other muscarinic antagonists. In isolated paced guinea pig atria, the antagonistic effect of W84 against oxotremorine- and arecaidine propargyl ester-induced negative inotropic actions reached a limiting value at higher W84 concentrations, revealing negative cooperativity (factors of cooperativity alpha = 311 and alpha = 495, respectively). The antagonistic potency of W84 in this M2 receptor model (W84 binding constant KA approximately 160 nM) was higher than at M1/M4-like receptors of rabbit vas deferens (KB approximately 800 nM) and at M3 receptors of guinea pig ileum (KB approximately 4,000 nM). In paced atria, combinations of W84 with muscarinic antagonists yielded more-than-additive antagonistic effects against oxotremorine in case of conventional antagonists such as NMS (alpha = 18) but less-than-additive effects with the M2-preferring AF-DX 384 (alpha = 444). In guinea pig heart homogenates, the equilibrium binding of [3H]NMS was only partially inhibited by W84 (alpha = 2.4), whereas [3H]AF-DX 384 binding could be suppressed completely (alpha = 194). The difference in cooperativity reflects that W84 inhibits [3H]NMS dissociation with a approximately 40-fold higher potency (ECdiss = 900 nM) than [3H]AF-DX 384 dissociation (ECdiss = 33,300 nM). [3H]NMS dissociation also could be retarded by AF-DX 384 (ECdiss = 22,000 nM), probably via an interaction with the site used by W84. The results suggest that the binding domain of AF-DX 384 partially overlaps with the common allosteric site of the M2 receptor protein.

Affinity of Mosapride Citrate, a New Gastroprokinetic Agent, for 5-HT4 Receptors in Guinea Pig Ileum

We examined the binding affinity of mosapride citrate (mosapride) (4-amino-5-chloro-2-ethoxy-N-{[4-(4-fluorobenzyl)-2-morpholinyl]methyl}benzamide citrate), a novel gastroprokinetic agent, for the 5-hydroxytryptamine (5-HT) 4 receptors in guinea pig ileum using a selective 5-HT4-receptor radioligand, [3H]GR 113808. In membrane preparations from longitudinal muscle with myenteric plexus in guinea pig ileum, specific [3H]GR113808 binding revealed a single saturable site of high affinity (Kd = 0.28±0.02 nM, Bmax = 45±3 fmol/mg protein). Mosapride and other 5-HT4-receptor agonists inhibited the specific binding of [3H]GR113808 in guinea pig ileum. The 5-HT4 agonists examined displayed the following inhibition potency order: BIMU-8 > cisapride > mosapride > renzapride > 5-HT > zacopride > metoclopramide. Mosapride exhibited monophasic inhibition of the specific [3H]GR 113808 binding in the ileum (Ki value: 84.2 nM). The presence of mosapride (30 nM) significantly increased the Kd value to 0.44±0.05 nM in the Scatchard analysis of [3H]GR113808 binding. Bmax of [3H]GR113808, however, was not affected (48±4 fmol/mg protein) by mosapride. As for the affinity of mosapride, the addition of GppNHp (100 μM) slightly increased the Ki value to 104 nM. These results indicate that mosapride has an affinity for 5-HT4 receptors in guinea pig ileum in the radioligand binding study.

O-62 - Antagonistic effect of corynanatheidine, an alkaloid from Thai medicinal plant Mitragyna speciosa, on opioid receptors in guinea-pig ileum

O-62 - Antagonistic effect of corynanatheidine, an alkaloid from Thai medicinal plant Mitragyna speciosa, on opioid receptors in guinea-pig ileum

Interaction of local anaesthetics with histamine H1 receptors in guinea-pig ileum.

The interaction of amine local anaesthetics and related compounds with histamine H1 receptors was investigated in guinea-pig ileal longitudinal muscle. Quinacrine, chloroquine, tetracaine and procaine inhibited [3H]mepyramine binding to solubilized membrane from ileal muscle with pKi values of 5.27 +/- 0.11, 5.66 +/- 0.01, 4.28 +/- 0.08 and 3.97 +/- 0.11, respectively. The pKB values obtained from the initial parallel shift of the dose-response curves for histamine in the presence of these drugs were 5.49 +/- 0.11, 6.14 +/- 0.09, 4.86 +/- 0.06 and 4.58 +/- 0.06, respectively, in reasonable agreement with the pKi values. The combined dose-ratio test with both local anaesthetics and antagonist (mepyramine) present showed that tetracaine and procaine were competitive and chloroquine was partially competitive, but that quinacrine was not competitive at histamine H1 receptors. These local anaesthetics inhibited histamine-induced desensitization in guinea-pig ileum. Receptor occupancy (%) by agonist decreased from 95.2 (without inhibitor) to 73.9, 42.8, 35.9 and 33.9 in the presence of quinacrine, chloroquine, tetracaine or procaine, respectively, under the conditions where each inhibitor drug induced half maximum inhibition of desensitization. The results suggested that most of these local anaesthetics interacted competitively at histamine H1 receptors and inhibited desensitization through their antagonizing actions, whereas quinacrine interacted allosterically and inhibited desensitization through a separate action.

Activation of M2 muscarinic receptors in guinea-pig ileum opens cationic channels modulated by M3 muscarinic receptors.

In longitudinal muscle of guinea-pig ileum, activation of muscarinic receptors causes contraction antagonised by M3 receptor subtype antagonists despite a preponderance of M2 receptor subtype binding sites. Experiments on single smooth muscle cells under voltage-clamp described here show that the cationic current evoked by carbachol which normally causes depolarization of the muscle is inhibited competitively by M2 antagonists with affinities typical of antagonism at a M2 receptor. However, M3 antagonists strongly reduced the maximum cationic current which could be evoked by carbachol in a non-competitive manner with affinities typical for an action at M3 receptors. Thus cation channels are gated by M2 receptor activation but strongly modulated by activation of M3 receptors.

Evidence for the Presence of Regional Differences in the Subtype Specificity of Muscarinic Receptors in Rabbit Lower Urinary Tract

To elucidate the subtype specificity of muscarinic cholinergic receptors in mediating contractile responses in the lower urinary tract, we investigated contractile and biochemical properties of muscarinic receptors in bladder dome, bladder base and urethra of the rabbit. Isometric contractile response curves to increasing concentrations of carbachol were constructed in the absence and presence of various concentrations of subtype selective muscarinic antagonists. Bladder dome, bladder base, and urethra demonstrate different characteristics in terms of efficacy and potency with respect to carbachol-induced contractile responses. Emax values are significantly larger and ED50 values are significantly smaller in bladder dome and bladder base than in urethra. Calculation of the pA2 values, the negative logarithm of the antagonist affinity constant (KB), for a series of muscarinic antagonists, i.e., atropine (nonselective), pirenzepine (M1 selective), methoctramine (M2 selective), and 4-DAMP (M1/M3 selective) indicate that the carbachol-induced contractile response in bladder dome and bladder base is mediated through the M3 receptor subtype whereas the carbachol-induced contractile response in urethra is probably mediated through the M1 and/or M3 and possibly M2 subtypes. Muscarinic cholinergic antagonists inhibit [3H]quinulidinyl benzilate binding to bladder dome, bladder base and urethra with the following rank order of affinities: atropine > 4-DAMP > methoctramine > pirenzepine. The binding data indicate the predominance of the M2 receptor subtype in all three regions.

Functional evidence for NO-synthase activation by substance P through a mechanism not involving classical tachykinin receptors in guinea-pig ileum in vitro.

1. This study tested the hypothesis that a nitric oxide synthase (NOS) was activated in guinea-pig ileum in vitro in response to substance P (SP), and attempted to characterize the tachykinin receptor involved in this activation by the use of selective receptor agonists and antagonists. 2. Strips of guinea-pig ileum (8 x 2 mm) were superfused (Krebs, 37 degrees C, 2 ml min-1) with: (i) tachykinin receptor agonists: SP, GR 73,632 (NK1), GR 64,349 (NK2), senktide (NK3), and neuropeptide (NP) gamma; (ii) tachykinin receptor antagonists: CP 99,994 (NK1), SR 48,968 (NK2), SR 142,801 (NK3); (iii) nerve-related agents: carbachol (CCh), atropine, tetrodotoxin (TTX), hexamethonium; (iv) NOS inhibitors: N omega-nitro-L-arginine-methyl-ester (L-NAME), N omega-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG); (v) NO-related agents, L-arginine (L-Arg), D-arginine (D-Arg), sodium nitroprusside (NaNP) and methaemoglobin. Muscle contractility was recorded isometrically and quantified as integrated area of activity. 3. SP, tachykinin receptor agonists and NP gamma (10 pM to 10 microM), produced concentration-dependent contractions of ileal strips, with EC50s in the nanomolar range, and maximal responses (Emax) attained at 0.1 microM for SP and 1 microM for the other agonists. The Emax response to SP equalled that to KCl (60 mM) taken as a 100% control (99.3% [93.0-105.7]; mean and 95% CI; n = 12); a comparable Emax contraction was obtained with the other tachykinin receptor agonists (1 microM) as well as with CCh (1 microM). 4. Under baseline conditions, L-NAME (1 microM), L-NMMA (1 microM) and AG (1 microM), failed to contract the muscle strip. In contrast, when superfused for 3 min, 10 min after SP (0.1 microM), they induced a transient contraction of the strip (e.g. for 1 microM L-NAME: 50 to 70 s duration; amplitude 73 +/- 12%, n = 24). 5. The NOS inhibitor-induced contractile response was not obtained after KCl (60 mM), GR 73,632, GR 64,349, senktide or CCh (all up to 1 microM). In contrast, this contractile response was obtained after NP gamma (1 microM). 6. Blockade of tachykinin NK1, NK2 and NK3 receptors by continuous superfusion of CP 99,994, SR 48,968 and SR 142,801 (1 microM) respectively, starting 5 min before SP, did not modify the response to L-NAME, superfused 10 min after SP (0.1 microM). The contractile response to L-NAME (1 microM) was blocked by atropine (1 microM), superfused either before or after SP. In contrast, it persisted after TTX or hexamethonium (1 microM) superfused in the same conditions. 7. The amplitude of NOS inhibitor-induced contraction (1 microM) was dependent on the concentration of priming SP (1 pM to 1 microM). In contrast, the contractile response to NOS inhibitors (1 nM to 10 microM) of the ileum strip primed with SP (0.1 microM) was not concentration-related. 8. L-NAME-induced contraction was prevented by continuous superfusion of L-Arg (1 microM), but not D-Arg (1 microM). In addition, the NO donor, sodium nitroprusside (1 microM) and the NO scavenger, methaemoglobin (10 micrograms ml-1), both prevented the contractile response to L-NAME. 9. In summary, SP and to a lesser extent NP gamma, exert a permissive action allowing contractile stimulating effects of L-NAME, L-NMMA and AG, in guinea-pig ileum in vitro, by a mechanism which apparently does not involve tachykinin NK1, NK2 and NK3 receptors. This action is likely to result from the activation of a NO-synthase by SP in the vicinity of intestinal myocytes. Thus, L-NAME, L-NMMA or AG, by blocking this SP-induced NO production, unveiled a smooth muscle contraction which involves a cholinoceptor (atropine-sensitive) mechanism.

Synthesis and biological activity of tachykinin analogs containing the adamantane moiety

The adamantane moiety was introduced in the tachykinin NK2 receptor-selective agonist [β-Ala8]-NKA(4–10) (H-Asp-Ser-Phe-Val-β-Ala-Leu-Met-NH2, MEN 10210) and in different positions of the NK2 receptor antagonist MEN 10376 (H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2) in order to investigate how this substitution affects their biological activity at tachykinin NK1, NK2 and NK3 receptors. 1-Adamantaneacetic acid (1-Ada-CH2COOH) was directly conjugated in the solid phase as the preformed OBt active ester to the N-terminal position of MEN 10210, obtaining MEN 10586 (1-Ada-CH2CO-Asp-Ser-Phe-Val-β-Ala-Leu-Met-NH2). The Pfp ester of adamantaneacetic acid (1) was prepared and used for the acylation of the N-terminal position of MEN 10376, yielding MEN 10606 (1-Ada-CH2CO-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2). Compound 1 was then used to obtain the building block Fmoc-Lys(1-Ada-CH2CO)-OH as a modified amino acid for the synthesis of MEN 10818 [H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys(1-Ada-CH2CO)-NH2]. In order to investigate the biological activity of the peptide bearing the adamantane group together with the free N-terminal amino function, we synthesised MEN 10676 [H-Asp(O-2-Ada)-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2] using Fmoc-Asp(O-2-Ada)-OH, in which 2-adamantanole was the protecting group of the aspartate β-COOH moiety during the peptide synthesis and survived the final peptide cleavage and deprotection carried out under controlled conditions. MEN 10586 showed an agonist activity comparable to that of the parent compound MEN 10210 at NK1 and NK2 receptors of guinea pig ileum, rabbit isolated pulmonary artery and hamster isolated trachea preparations, while it showed a 25-fold higher agonist activity at NK3 receptors of rat isolated portal vein. The three modified antagonist analogs displayed similar or reduced affinity at NK1, NK2 and NK3 receptors as compared to MEN 10376. The drop was particularly evident (>2 log units) at the NK2 receptors of the rabbit isolated pulmonay artery.