Characterization of species-related differences in the pharmacology of tachykinin NK receptors 1, 2 and 3

Tachykinin NK receptors (NKRs) differ to a large degree among species with respect to their affinities for small molecule antagonists. The aims of the present study were to clone NKRs from gerbil (NK₂R and NK₃R) and dog (NK₁R, NK₂R and NK₃R) in which the sequence was previously unknown and to investigate the potency of several NKR antagonists at all known human, dog, gerbil and rat NKRs.The NKR protein coding sequences were cloned and expressed in CHO cells. The inhibitory concentrations of selective and non-selective NKR antagonists were determined by inhibition of agonist-induced mobilization of intracellular Ca²⁺. Receptor homology models were constructed based on the rhodopsin crystal structure to investigate and identify the antagonist binding sites and interaction points in the transmembrane (TM) regions of the NKRs.Data collected using the cloned dog NK₁R confirmed that the dog NK₁R displays similar pharmacology as the human and the gerbil NK₁R, but differs greatly from the mouse and the rat NK₁R. Despite species-related amino acid (AA) differences located close to the antagonist binding pocket of the NK₂R, they did not affect the potency of the antagonists ZD6021 and saredutant. Two AA differences located close to the antagonist binding site of NK₃R likely influence the NK₃R antagonist potency, explaining the 3-10-fold decrease in potency observed for the rat NK₃R. For the first time, detailed pharmacological experiments in vitro with cloned NKRs demonstrate that not only human, but also dog and gerbil NKR displays similar antagonist pharmacology while rat diverges significantly with respect to NK₁R and NK₃R.     

Localization of tachykinin receptors and Fos-like immunoreactivity induced by substance P in guinea-pig brain

1. In the present study, a comparison was made between the distribution of tachykinin NK1 and NK3 receptor immunoreactivity and the distribution of Fos-like immunoreactivity induced by the tachykinin agonist substance P (SP) in the guinea-pig brain. 2. In agreement with results from previous studies in rat brain, NK1 receptor-immunoreactive neurons were found to be widely distributed throughout the brain in the striatum and in diencephalic and mesencephalic structures, while NK3 receptor-immunoreactive neurons were mainly in telencephalic structures. Considerable overlap was observed between NK1 and NK3 receptor distributions. 3. Substance P induced Fos-like immunoreactivity (Fos-LI) in extensive areas of the guinea-pig brain. The induction of Fos-LI was markedly inhibited in many areas by pretreatment with the NK1 receptor antagonist SR 140333. The NK3 receptor antagonist SR 142801 reduced Fos-LI staining in fewer areas, although a reduction was observed in the cortex, striatum and hypothalamus. 4. In general, tachykinin receptors were located at sites corresponding to areas of functional activation by SP, as shown by Fos-LI. These results extend previous studies by adding a functional dimension to tachykinin receptor localization studies.     

Structure–activity studies at the rat tachykinin NK2 receptor: effect of substitution at position 5 of neurokinin A

A series of analogues of neurokinin A(4–10) was synthesized using solid phase techniques with Chiron pins, and purified by HPLC. The potencies of 10 peptides with substitution at Ser⁵ were assessed at rat fundus NK2 receptors. In membrane binding studies with [¹²⁵I]-[Lys⁵,Tyr(I₂)⁷,MeLeu⁹,Nle¹⁰]-NKA(4–10), all compounds except [Asp⁵]NKA(4–10) showed reasonable affinity, and analogues with Lys and Arg substitutions were five-fold more potent than NKA(4–10). In functional studies, all peptides were able to contract the rat isolated fundus strips. Analogues with Phe, His and Asn substitutions were substantially weaker in functional than in binding studies, whereas there was an excellent correlation (r = 0.95) between binding and functional potency for the remaining seven peptides. [Phe⁵]NKA(4–10) is in fact neurokinin B(4–10) and this residue may be critical in determining selectivity between NK2 and NK3 receptors. Analogues with a basic residue (Lys, Arg) at position 5 showed both increased affinity and functional potency, whereas the neutral [Asn⁵]NKA(4–10) was equally as weak in contractile studies as the acidic [Asp⁵]NKA(4–10). However, [Glu⁵]NKA(4–10) and [Gln⁵]NKA(4–10) were no different from NKA(4–10). Our results could indicate the presence of a negative charge on the NK2 receptor, close to position 5 of NKA. This would facilitate interaction with positively charged side chains and impede interaction with negatively charged side chains, particularly the inflexible side chain of aspartic acid. Thus, not only the charge, but also the length of the side chain of the residue at position 5, seems to be important for interaction with the rat NK2 receptor.     

Tachykinin NK1 receptor antagonists for the control of chemotherapy-induced nausea and vomiting

The treatment of neoplastic disease with chemotherapeutic cytotoxic drugs has long been associated with profound nausea and vomiting (emesis). This became the most feared side effect of this type of treatment and was so severe that some patients would withdraw from further treatment, thus jeopardising their clinical outcome and possibly life expectancy. The introduction of the 5-HT₃ receptor antagonists had a significant impact in this area, offering substantial reductions in emesis, largely through prophylactic treatment. Unfortunately, some forms of emesis were resistant to treatment with these drugs, so the search has continued to identify new chemical entities with a higher level of efficacy and a broader spectrum of activity. Data generated in animals has identified tachykinin NK₁ receptors as highly important in the emetic reflex and experimental evidence strongly supports NK₁ receptor antagonists as highly efficacious anti-emetic agents, with unparalleled broad spectrum activity. Several novel antagonists have recently entered clinical development and data are emerging to support their anti-emetic activity. This area continues to attract substantial medicinal chemical research effort. Most major pharmaceutical companies are seeking new matter through structural refinement of early leads or discovery of novel compounds from library screening. The scope of chemical lead matter has advanced from early piperidine and quinuclidines to a spectrum of templates that improve expectations for a well-tolerated therapeutic agent. Recent success in combining 5-HT₃ and NK₁ antagonists in emesis treatment is expected to greatly advance clinical outcomes with newer and safer agents.     

Peripheral tachykinin receptors as potential therapeutic targets in visceral diseases

More than 10 years of intensive preclinical investigation of selective tachykinin (TK) receptor antagonists has provided a rationale to the speculation that peripheral neurokinin (NK)-1, -2 and -3 receptors may be involved in the pathophysiology of various human diseases at the visceral level. In the airways, despite promising effects in animal models of asthma, pilot clinical trials with selective NK-1 or -2 receptor antagonists in asthmatics have been ambiguous, whereas the potential antitussive effects of NK-1, -2 or -3 antagonists have not yet been verified in humans. In the gastrointestinal (GI) tract, irritable bowel syndrome (IBS) and pancreatitis are appealing targets for peripherally-acting NK-1 and -2 antagonists, respectively. In the genito-urinary tract, NK-1 receptor antagonists could offer some protection against nephrotoxicity and cytotoxicity induced by chemotherapeutic agents, whereas NK-2 receptor antagonists appear to be promising new agents for the treatment of neurogenic bladder hyperreflexia. Finally, there is preclinical evidence for hypothesising an effect of NK-3 receptor antagonists on the cardiovascular disturbance that characterises pre-eclampsia. Other more speculative applications are also mentioned.     

速激肽受体拮抗剂对白三烯C_4诱导豚鼠气道收缩和微血管渗漏的作用

本实验探讨了内源性速激肽是否参与白三烯Ｃ４（ＬＴＣ４）的气道效应．ＬＴＣ４（０．５μｇｋｇ－１，ｉｖ）可增高豚鼠肺内压（ＩＰＰ）和气道内依文思蓝渗出。速激肽ＮＫ－１受体拮抗剂ＣＰ－９６３４５｛（２Ｓ，３Ｓ）－顺式－２－（二苯甲基）－Ｎ－［（２－甲氧苯）－甲基］－１－杂氮双环［２．２．２］辛烷－３－胺｝１ｍｇｋｇ－１，ｉｖ，可减弱ＬＴＣ４诱导的依文思蓝渗出；ＮＫ－２受体拮抗剂ＳＲ－４８９６８｛（Ｓ）－Ｎ－甲基－Ｎ－［４－（４－乙酰氨基－４－苯基哌啶）－２－（３，４－二氯苯基）丁基］苯甲酰胺｝，１ｍｇｋｇ－１，ｉｖ，可抑制ＩＰＰ的增高．白三烯拮抗剂ＯＮＯ－１０７８（０．０３ｍｇｋｇ－１，ｉｖ）可阻断这两种反应．结果说明内源性速激肽增强ＬＴＣ４的气道作用，其中ＮＫ－１受体介导微血管渗漏，ＮＫ－２受体介导支气管收缩．     

Tachykinin NK1 and NK3 receptors in the prefrontal cortex of the human brain

1. The tachykinins are neuropeptides found in both the central and peripheral nervous systems that play a role in inflammation and pain mechanisms and some autonomic reflexes and behaviours. 2. Although the distribution of the tachykinin receptors has been described in the brains of various animal species, little is known about the distribution of the NK1 and NK3 receptors in the human brain. 3. The present paper examines the distribution of the NK1 and NK3 receptors in the prefrontal cortex of formalin-fixed postmortem human brain tissue by immunohistochemical techniques. 4. The majority of NK1 receptor immunoreactivity appeared as a thin band of punctate staining at the pial surface, with dark brown dots of NK1 receptor immunoreactivity predominantly scattered across the mid to upper cortical layers. 5. The NK3 receptor immunoreactivity was found in the glia limitans at the pial surface, where astrocytes and beaded fibres were intensely stained. Dots of NK3 receptor immunoreactivity were scattered across all cortical layers. In the white matter, astrocytes and beaded fibres displayed NK3 receptor immunoreactivity, particularly in areas surrounding blood vessels.     

Effects of tachykinin receptor agonists and antagonists on the guinea-pig isolated oesophagus

1. Vagal nerve stimulation of the guinea-pig isolated oesophagus produced a triphasic tetrodotoxin (TTX)-sensitive contractile response. The third phase, which was resistant to ganglion blocking drugs, was selectively abolished by capsaicin, suggesting the involvement of one or more neuropeptides released from afferent neurons. Receptors on cholinergic neurons were subsequently activated because the response was atropine sensitive. Contractile responses resulting from exogenous substance P were abolished by atropine and TTX and enhanced by physostigmine. These findings suggest that the third phase may be mediated by the action of a substance P-like neuropeptide released from sensory nerve endings that subsequently activated cholinergic neurons. 2. The tachykinin receptors in the body of the guinea-pig oesophagus were characterized by determining the relative agonist potencies of natural tachykinins as well as tachykinin receptor-selective analogues. Antagonist affinities were also determined. The results indicated the presence of both NK2 and NK3 receptors. In addition, the effects of a cocktail of peptidase inhibitors (captopril, thiorphan and amastatin) on responses to various tachykinins and synthetic analogues were determined. The results indicate that one or more peptidases are present in this preparation. 3. Experiments using various tachykinin receptor antagonists were performed to determine whether the activation of tachykinin receptors played a role in the mediation of the third phase of the response to vagal nerve stimulation. While this response was unaffected by NK1 and NK2 receptor-selective antagonists, it was only partially inhibited (23%) by the NK3 receptor antagonist SR 142801. Thus, in the guinea-pig oesophagus, it appears that NK3 receptors play only a minor role in mediating a contractile response when afferent neurons are excited by vagal nerve stimulation.     

Proinflammatory and Antiinflammatory Peptides

edited by Sami I. Said, Marcel Dekker, 1998. $195.00 (xxviii+711 pages) ISBN 0 8247 0120 8     

Human tachykinin NK2 receptor: a comparative study of the colon and urinary bladder

1. The present study compared the binding and functional characteristics of tachykinin NK2 receptors in human detrusor muscle with those in human colon circular muscle. 2. In radioligand binding studies, similar KD values were observed for tachykinin NK2 receptor radioligands [125I]-neurokinin (NK) A, [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10,]NKA(4-10) and [3H]-SR48968 in both human colon circular muscle (0.28-1.1 nmol/L) and human bladder detrusor (0.49-0.91 nmol/L), suggesting binding was primarily to tachykinin NK2 receptors. Receptor capacity (Bmax) was greater in colon compared with detrusor muscle. 3. In functional studies of isolated smooth muscle contraction, there was an excellent positive correlation between human bladder detrusor and colon circular muscle with respect to in vitro contractile potency (r = 0.97) and maximum responses (r = 0.98) to tachykinins, selective tachykinin receptor ligands and l-Ala-substituted NKA(4-10) analogues. 4. Species differences between the human and rat tachykinin NK2 receptors were apparent as observed by a low correlation for potency (r = 0.77) and efficacy (r = 0.32) of l-Ala-substituted analogues in isolated smooth muscle contractile studies. 5. Minor differences observed in the affinity and potency of NK2 receptor agonists between colon and bladder are dependent on the tissue of interest, the receptor-effector coupling and the presence of other tachykinin receptors. Overall, the NK2 receptors of human colon and urinary bladder smooth muscle appear pharmacologically identical.