neurokinin-a and amastatin

Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2-activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsaicin, which stimulates and ablates specific sensory neurons, but it was resistant to cyclo-oxygenase inhibition. In contrast, capsaicin treatment failed to block PAR-2-mediated secretion from the salivary glands. Intravenous calcitonin gene-related peptide (CGRP) and neurokinin A markedly elicited gastric mucus secretion, as did substance P to a lesser extent. Specific antagonists of the CGRP1 and NK2, but not the NK1, receptors inhibited PAR-2-mediated mucus secretion. Pretreatment with the PAR-2 agonist strongly prevented gastric injury caused by HCl-ethanol or indomethacin. Thus, PAR-2 activation triggers the cytoprotective secretion of gastric mucus by stimulating the release of CGRP and tachykinins from sensory neurons. In contrast, the PAR-2-mediated salivary exocrine secretion appears to be independent of capsaicin-sensitive sensory neurons.

Topics: Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents, Non-Steroidal; Anti-Ulcer Agents; Calcitonin Gene-Related Peptide; Capsaicin; Diclofenac; Duodenum; Gastric Mucins; Male; Misoprostol; Neurokinin A; Oligopeptides; Peptides; Protease Inhibitors; Rats; Rats, Wistar; Receptor, PAR-2; Receptors, Thrombin; Saliva; Stomach; Substance P

We examined tachykinin-induced contractions of uteri from rats during the oestrous cycle. The potencies of substance P, neurokinin A, neurokinin B and the tachykinin NK2 receptor-selective agonist, [Lys5, MeLeu9, Nle10] neurokinin A-(4-10), and of the non-peptide tachykinin NK1, NK2 and NK3 receptor antagonists (S)1-[2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)pip eridin-3-yl]ethyl]-4phenyl-1-azonia-bicyclo[2.2.2]octane (SR 140333), (S)-N-methyl-N [4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzam ide (SR 48968) and (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)prop yl)-4-phenylpiperidin-4-yl)-N-methylacetamide (SR 142801), were examined. The relative agonist potencies, i.e., [Lys5, MeLeu9, Nle10] neurokinin A-(4-10) > or = neurokinin A > neurokinin B > or = substance P were similar in preparations from rats in dioestrus/metoestrus and those in proestrus/oestrus. Apparent pK(B) values for SR 48968 versus neurokinin A and [Lys5, MeLeu9, Nle10] neurokinin A-(4-10), were 9.9 and 9.2, respectively, indicating activation of an NK2 receptor. SR 140333 (10 nM) produced only a small rightward shift of the log concentration-response curve to substance P. SR 48968 (3 nM), but not SR 142801 (100-300 nM) reduced the effect of neurokinin B. These data indicate that in the rat tachykinin-induced contractions of the uteri during the oestrous cycle are mediated primarily by tachykinin NK2 receptors, and that fluctuations in ovarian hormonal levels during the oestrous cycle have little influence on the uterine response to tachykinins.

Topics: Animals; Anti-Bacterial Agents; Benzamides; Diestrus; Estrus; Female; Methacholine Chloride; Neurokinin A; Neurokinin-1 Receptor Antagonists; Peptides; Piperidines; Proestrus; Quinuclidines; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Tachykinins; Uterine Contraction

1. The aim of this study was to characterize the tachykinin NK2 receptor subtype mediating the spasmogenic response in the human isolated bronchus. The motor response to neurokinin A (NKA) and the selective NK2 agonist [beta Ala8]NKA(4-10), as well as the antagonistic effects of cyclic (L659,877) and linear (MEN 10376) peptide NK2 antagonists were assessed in the presence or absence of amastatin (an inhibitor of aminopeptidases A and M). 2. NKA was more potent than [beta Ala8]NKA(4-10) in eliciting bronchoconstriction (pD2 being 7,43 and 6,87 respectively). In the presence of amastatin (1 microM), the estimated affinity of [beta Ala8]NKA(4-10), but not that of NKA, was significantly increased to yield a pD2 of 7,44. 3. L659,877 and MEN 10376 inhibited [beta Ala8]NKA(4-10)-induced contraction with similar affinities; pA2 values were 5.7 +/- 0.22 and 6.3 +/- 0.32, respectively. Amastatin (1 microM) increased the potency of MEN 10376 to 7.28 +/- 0.46, whereas that of L659,877 was unaffected. 4. In the presence of amastatin the pseudopeptide MDL 28,564 behaved as a partial agonist. 5. We conclude that the NK2 receptor subtype present in the human bronchus has properties similar to those described for the circular muscle of the human colon and thus may be classified as a 'NK2A' subtype. We show that the apparent potency of peptides, bearing N-terminal acidic residues, is influenced by an amastatin-sensitive peptidase, possibly aminopeptidase A.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Bronchi; Bronchial Spasm; Female; Humans; In Vitro Techniques; Male; Molecular Sequence Data; Neurokinin A; Peptide Fragments; Peptides; Peptides, Cyclic; Receptors, Neurokinin-2; Tachykinins

Tachykinin receptors mediating uterotonic effects were examined in preparations from oestrogen-primed rats. In the absence of peptidase inhibitors [Lys5-MeLeu9-Nle10] NKA (4-10) was 14-fold more potent than neurokinin A (NKA), but the two peptides were equipotent in the presence of phosphoramidon alone and in combination with amastatin. The NK-2 receptor antagonist SR 48968 antagonised responses to the tachykinins. These findings indicate that an NK-2 receptor is present in the oestrogen-primed rat uterus and that endopeptidase 24.11 plays a major role to inactivate NKA in this tissue.

Topics: Animals; Anti-Bacterial Agents; Benzamides; Carbachol; Estradiol; Female; Glycopeptides; Kinetics; Neurokinin A; Neurokinin-1 Receptor Antagonists; Peptide Fragments; Peptides; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Uterus

The tachykinin (NK2) receptor-mediating contraction of the human isolated bladder to NKA was investigated by studying the affinities of eight structurally different receptor-selective antagonists (linear peptides, cyclic peptides and pseudopeptides, nonpeptide NK2 receptor antagonists). The affinities of the antagonists were compared to those measured for the same ligands at the NK2 receptors previously characterized in the rabbit pulmonary artery and hamster trachea. In the presence of a cocktail of peptidase inhibitors (bestatin captopril and thiorphan, 1 microM each) no significant correlation was found between pA2 values measured in the human bladder vs. those measured in the other two NK2 receptor-bearing preparation. In the presence of the aminopeptidase inhibitor amastatin, however, pA2 values of linear antagonists bearing an N-terminal Asp residue MEN 10,207 and MEN 10,376 were significantly enhanced and these pA2 values were used for analysis; a significant correlation was found between pA2 values measured in the human urinary bladder and rabbit pulmonary artery. The pseudopeptide analog of NKA (4-10), MDL 28,564 which also bears a N-terminal Asp residue behaved as an agonist and its action was enhanced by amastatin. We conclude that the NK2 receptor-mediating contraction of the human urinary bladder smooth muscle is similar to that previously characterized in the rabbit pulmonary artery (NK2A receptor category); in the human bladder smooth muscle an amastatin-sensitive peptidase (possibly aminopeptidase A) limits biological activity of linear peptide derivatives of NKA bearing a N-terminal Asp residue.

Topics: Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Anti-Bacterial Agents; Drug Interactions; Humans; In Vitro Techniques; Middle Aged; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Neurokinin A; Peptide Fragments; Peptides; Protease Inhibitors; Receptors, Neurokinin-2; Tachykinins; Urinary Bladder

The tachykinin peptide agonists neurokinin A and [beta Ala8]neurokinin A-(4-10), and the NK2 tachykinin receptor-selective antagonists MEN 10,208, MEN 10,207, MEN 10,282, MEN 10,376 and R396 were assayed in the isolated rabbit pulmonary artery and isolated hamster trachea in the absence and in the presence of the aminopeptidase inhibitor amastatin (10 microM for 30 min). The affinity of MEN 10,208 in the rabbit pulmonary artery was markedly reduced in the presence of amastatin (pKB values from 7.47 to 5.94), while it was unchanged in the hamster trachea. Neither neurokinin A, [beta Ala8]neurokinin A-(4-10), nor the other antagonists were affected by pretreatment with amastatin in either bioassay. The results obtained in the rabbit pulmonary artery show that MEN 10,208 is degraded by local amastatin-sensitive enzymes (possibly aminopeptidase M), which may convert the linear octapeptide MEN 10,208 to the heptapeptide MEN 10,207 by removing the N-terminal Thr from the amino acid sequence of MEN 10,208. The present results are discussed in relation to a previously reported heterogeneity between NK2 receptors of the rabbit and bovine species, and show amastatin to be a new tool for the classification of tachykinin receptors with peptide ligands.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Cricetinae; In Vitro Techniques; Male; Mesocricetus; Molecular Sequence Data; Muscle, Smooth; Muscle, Smooth, Vascular; Neurokinin A; Oligopeptides; Peptides; Pulmonary Artery; Rabbits; Receptors, Neurokinin-2; Receptors, Neurotransmitter; Trachea