Types Of Tachykinin Receptors Research Articles

G-Protein-Coupled Receptors: Next Generation Therapeutic Targets in Head and Neck Cancer?

Therapeutic outcome in head and neck squamous cell carcinoma (HNSCC) is poor in most advanced cases. To improve therapeutic efficiency, novel therapeutic targets and prognostic factors must be discovered. Our studies have identified several G protein-coupled receptors (GPCRs) as promising candidates. Significant epigenetic silencing of GPCR expression occurs in HNSCC compared with normal tissue, and is significantly correlated with clinical behavior. Together with the finding that GPCR activity can suppress tumor cell growth, this indicates that GPCR expression has potential utility as a prognostic factor. In this review, we discuss the roles that galanin receptor type 1 (GALR1) and type 2 (GALR2), tachykinin receptor type 1 (TACR1), and somatostatin receptor type 1 (SST1) play in HNSCC. GALR1 inhibits proliferation of HNSCC cells though ERK1/2-mediated effects on cell cycle control proteins such as p27, p57, and cyclin D1, whereas GALR2 inhibits cell proliferation and induces apoptosis in HNSCC cells. Hypermethylation of GALR1, GALR2, TACR1, and SST1 is associated with significantly reduced disease-free survival and a higher recurrence rate. Although their overall activities varies, each of these GPCRs has value as both a prognostic factor and a therapeutic target. These data indicate that further study of GPCRs is a promising strategy that will enrich pharmacogenomics and prognostic research in HNSCC.

Aberrant methylation inactivates somatostatin and somatostatin receptor type 1 in head and neck squamous cell carcinoma.

PurposeThe aim of this study was to define somatostatin (SST) and somatostatin receptor type 1 (SSTR1) methylation profiles for head and neck squamous cell carcinoma (HNSCC) tumors at diagnosis and follow up and to evaluate their prognostic significance and value as a biomarker.MethodsGene expression was measured by quantitative RT-PCR. Promoter methylation status was determined by quantitative methylation-specific PCR (Q-MSP) in HNSCC.ResultsMethylation was associated with transcription inhibition. SST methylation in 81% of HNSCC tumor specimens significantly correlated with tumor size (P = 0.043), stage (P = 0.008), galanin receptor type 2 (GALR2) methylation (P = 0.041), and tachykinin-1 (TAC1) (P = 0.040). SSTR1 hypermethylation in 64% of cases was correlated with tumor size (P = 0.037), stage (P = 0.037), SST methylation (P < 0.001), and expression of galanin (P = 0.03), GALR2 (P = 0.014), TAC1 (P = 0.023), and tachykinin receptor type 1 (TACR1) (P = 0.003). SST and SSTR1 promoter hypermethylation showed highly discriminating receiver operator characteristic curve profiles, which clearly distinguished HNSCC from adjacent normal mucosal tissues. Concurrent hypermethylation of galanin and SSTR1 promoters correlated with reduced disease-free survival (log-rank test, P = 0.0001). Among patients with oral cavity and oropharynx cancer, methylation of both SST and SSTR1 promoters correlated with reduced disease-free survival (log-rank test, P = 0.028). In multivariate logistic-regression analysis, concomitant methylation of galanin and SSTR1 was associated with an odds ratio for recurrence of 12.53 (95% CI, 2.62 to 59.8; P = 0.002).ConclusionsCpG hypermethylation is a likely mechanism of SST and SSTR1 gene inactivation, supporting the hypothesis that SST and SSTR1 play a role in the tumorigenesis of HNSCC and that this hypermethylation may serve as an important biomarker.

Frequent promoter hypermethylation of tachykinin-1 and tachykinin receptor type 1 is a potential biomarker for head and neck cancer

The aim of this study was to define TAC1 and TACR1 methylation profiles for head and neck squamous cell carcinoma (HNSCC) tumors at diagnosis and follow-up and to evaluate their prognostic significance and value as a biomarker of recurrence. TAC1 and TACR1 expression was measured in a panel of cell lines by quantitative RT-PCR. The TAC1 and TACR1 promoter methylation status was determined by quantitative methylation-specific PCR. Methylation was associated with TAC1 and TACR1 transcription inhibition. TAC1 methylation in 49/100 (49 %) of HNSCC tumor specimens significantly correlated with p16 methylation (P = 0.010), E-cadherin methylation (P = 0.041), galanin methylation (P = 0.037), and disease-free survival (P = 0.002). Stage III and IV patients manifesting TAC1 hypermethylation had significantly shorter survivals than did patients without TAC1 methylation (P = 0.022). TACR1 methylation in 34/100 (34 %) cases was significantly correlated with galanin methylation (P = 0.014) and GALR1 methylation (P = 0.004). TAC1 promoter hypermethylation was statistically correlated with reduced disease-free survival (log-rank test, P = 0.002). In multivariate logistic-regression analysis, methylation of TAC1 and of the gene pair TAC1 and TACR1 was associated with an odds ratio for recurrence of 3.35 (95 % CI, 1.37-8.19; P = 0.008) and 5.09 (95 % CI, 1.44-18.02; P = 0.011), respectively. CpG hypermethylation is a likely mechanism of TAC1 and TACR1 gene inactivation, supporting the hypothesis that TAC1 and TACR1 play a role in the tumorigenesis of HNSCC and that this hypermethylation may serve as an important biomarker.

Identification of a tachykinin NK 2 receptor splice variant and its expression in human and rat tissues

The tachykinins substance P, neurokinin A and neurokinin B are implicated in different diseases and play an important role in neuroimmunomodulation. These peptides interact with three distinct types of tachykinin receptors termed NK 1, NK 2 and NK 3. While most mammalian genes encoding G protein-coupling cell membrane receptors are intron-less, the three tachykinin receptors contain introns in their genomic structures. In the present study, we have identified a splice variant of the tachykinin NK 2 receptor that results from skipping of exon 2 in the processing of the tachykinin NK 2 receptor mRNA. By using reverse transcription-polymerase chain reaction analysis, we observed that the tachykinin NK 2 receptor splice variant, that we named NK 2β, appeared in different human and rat tissues that also express the wild type, tachykinin NK 2α isoform. Compared to tachykinin receptor NK 2α isoform mRNA levels, the NK 2β isoform was strongly expressed in human and rat uteri, expressed in a moderate degree in the rat urinary bladder, colon, duodenum and stomach and unexpressed in the rat cerebral cortex, kidney, thoracic aorta, skeletal muscle and heart. These data describe the first known tachykinin receptor splice variant and suggest that the variety of tachykinin receptors may be further expanded through the generation of splicing isoforms. The presence of the truncated isoform may have a physiological significance in the regulation of tachykinin NK 2 receptor protein levels.

Comparative distribution of NK1, NK2, and NK3 receptors in the rat brainstem auditory nuclei

While the distribution of substance P in the auditory system is well illustrated, the localization of its receptors has not yet been documented. The goal of our study was to characterize the distribution of the tachykinin receptors NK1-R, NK2-R and NK3-R in the brainstem auditory nuclei of the adult rat using immunohistochemical techniques. The immunoreactivity of the neurokinin receptors was found to be widely distributed in most neurons of the cochlear nucleus (CN), the lateral superior olive (LSO), the medial nucleus of the trapezoid body (MNTB) and in the inferior colliculus (IC). Immunoreactivity was generally confined to post-synaptic targets (neuronal cell body and proximal or primary dendrites) in all auditory nuclei. However, unlike brainstem nuclei, the IC showed, in addition to neuronal cell body staining, a positive axonal immunolabeling (axons and pre-synaptic terminals) with the anti-NK1-R antibody. This axonal staining, revealing a pre-synaptic expression of NK1-R, is in good agreement with the known presence of substance P in the IC neurons. The absence of axonal staining in the superior olivary complex nuclei which projects afferent to the IC indicated that the NK1-R labeled axons are rather intrinsic IC fibers or descending thalamic projections to the IC. Overall, the wide distribution of the three types of tachykinin receptors observed in the present study argues for an important role of tachykinin neuropeptides in the central auditory system.

Tachykinin activation of human monocytes from patients with rheumatoid arthritis: in vitro and ex-vivo effects of cyclosporin A

Three types of tachykinin receptors, namely NK1, NK2and NK3, are known to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), respectively. We previously demonstrated that NK1and NK2receptors are present on human monocytes, SP and NKA inducing superoxide anion production and tumor necrosis factor- alpha (TNF-α) mRNA expression. NK2receptor stimulation also triggered an enhanced respiratory burst in monocytes isolated from rheumatoid arthritis (RA) patients. This study was aimed to evaluate the in vitro and ex-vivo effects of cyclosporin A (CsA) on tachykinins-evoked TNF-α release from monocytes of healthy donors and RA patients. CsA (100ng/ml) potently inhibited phorbol ester- and tachykinin-evoked TNF-α secretion. In RA patients treated with CsA (SandimmunRNeoralR) 2.5mg/kg/die, a significant time-dependent reduction in TNF-α secretion from monocytes was measured. This may contribute to the CsA therapeutic activity in RA.

Tachykinin activation of human monocytes from patients with interstitial lung disease, healthy smokers or healthy volunteers

Three types of tachykinin receptors, NK1, NK2and NK3, have been described to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) respectively. Experimental evidence indicates that SP and NKA modulate the activity of inflammatory and immune cells, including mononuclear ones, and points to their involvement in lung pathophysiology. We previously reported that NK1and NK2receptors are present on monocytes (MO) isolated from healthy donors or rheumatoid patients – a greater sensitivity to NK2receptor stimulation was observed in the latter condition. This study evaluated the effects of SP and NKA, as well as NK1and NK2selective agonists and antagonists, on MO obtained from healthy volunteers, healthy smokers or patients with interstitial lung diseases (e.g. sarcoidosis and idiopathic pulmonary fibrosis). Superoxide anion (O2–) production was chosen as a parameter of cell activation. SP and NKA dose-dependently evoked O2–production from MO in all the conditions evaluated, their effects being competitively antagonized by selective antagonists (CP 96 345 and MEN 10 627, respectively). When selective NK1and NK2agonists were used, [Sar9Met(O2)11]SP, a selective NK1agonist, induced a more than doubled O2production in MO obtained from patients with interstitial lung diseases as compared to healthy volunteers, whereas MO isolated from healthy volunteers were more sensitive to NK2receptor stimulation.

Tachykinin receptors on human monocytes: their involvement in rheumatoid arthritis

Three types of tachykinin receptors, namely NK1, NK2 and NK3, are known to preferentially interact with substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), respectively. Experimental evidence indicates that SP and NKA modulate the activity of inflammatory and immune cells, including mononuclear ones. This study evaluated the effects of mammalian tachykinins and selective tachykinin agonists and antagonists on human monocytes isolated from healthy donors: SP, NKA and NKB all evoked a dose-dependent superoxide anion (O2-) production and the NK2 selective agonist [beta-Ala8]-NKA(4-10) induced a full response. The NK3 selective agonist senktide was inactive, while the NK1 selective agonists septide and [Sar9Met(O2)11]SP displayed some effects. These results indicate that NK2 and also some NK1 receptors are present in monocytes isolated from healthy donors. The role of tachykinin receptor activation in rheumatoid arthritis was also investigated, by measuring O2- production and TNF-alpha mRNA expression in monocytes isolated from rheumatoid patients. Tachykinins enhanced the expression of this cytokine in both control and rheumatoid monocytes and NK2 receptor stimulation was shown to trigger an enhanced respiratory burst in monocytes from rheumatoid patients. In conclusion, these results indicate that NK2 and NK1 receptors are present on human monocytes, the former being preferentially involved in rheumatoid arthritis.

Expression of tachykinin receptors inXenopus oocytes injected with poly (A)+ RNA from cat dorsal root ganglion

The expression of the types of tachykinin receptors in the dorsal root ganglion (DRG) neurons by means ofXenopus oocyte expressing system was studied. Poly(A)(+) RNAs were extracted from cat cervical and lumbar DRG. Two days after injection of Poly (A)(+) RNAs, the oocytes were recorded with the two-electrode voltage clamp technique. In the oocytes injected with DRG poly(A)(+) RNA, [Sar(9), Met(O(2))(11)]-substance P(Sar -SP, 1 mumol/L), neurokinin A (NKA, 1 mumol/L) or [beta-Ala(8)]-neurokinin A((4-10)) (Ala-NKA, 1 mumol/L) produced an inward current comprising a rapid spike and a long sustained oscillatory component for several minutes. Sar-SP induced response was blocked by NK-1 antagonist L-668, 169 (1 mumol/L), but not by NK-2 antagonist L-659, 877(1mumol/L). In contrast, Ala-NKA and NKA responses were only blocked by L-659, 877. The oocytes injected with DH Poly(A)(+)RNA also responded to Sar-SP and NKA with similar inward currents, which were selectively blocked by L-668, 169 and L-659, 877, respectively. These tachykinins-induced responses had a potent desensitization. The present data indicate expression of NK-1 and NK-2 receptors in DRG neurons, suggesting that there may be tachykinin autoreceptors on the nociceptive primary afferent terminals.

What are the roles of substance p and neurokinin-1 receptors in the control of negative chronotropic or negative dromotropic vagal motoneurons? a physiological and ultrastructural analysis

Recent data indicate that there is a cardiotopic organization of negative chronotropic and negative dromotropic neurons in the nucleus ambiguus (NA). Negative dromotropic neurons are found in the rostral ventrolateral NA (rNA-VL), negative chronotropic neurons are found in the caudal ventrolateral NA (cNA-VL), and both types of neurons are found in an intermediate level of the ventrolateral NA (iNA-VL). Substance P (SP) immunoreactive nerve terminals synapse upon negative chronotropic vagal motoneurons in the iNA-VL, and SP microinjections in the NA cause bradycardia. In the present report we have attempted to: (1) define the type of tachykinin receptor which mediates the negative chronotropic effect of SP microinjections into the iNA-VL; (2) define the physiological effect of microinjections of a selective SP agonist into the rNA-VL on atrioventricular (AV) conduction; and (3) find ultrastructural evidence for synaptic interactions of SP-immunoreactive nerve terminals with negative dromotropic vagal motoneurons in the rNA-VL. Microinjections of the excitatory amino acid glutamate (Glu) into the iNA-VL to activate all local vagal preganglionic neurons caused both bradycardia and a decrease in the rate of AV conduction. Injections of the selective neurokinin-1 (NK-1) receptor agonist drug GR-73632 also caused bradycardia, however the rapid onset of agonist induced desensitization prevented an evaluation of potential effects on AV conduction in the iNA-VL. These data suggest that the SP-induced bradycardia which can be elicited from the NA is mediated, at least in part, by NK-1 receptors. Microinjections of Glu into the rNA-VL caused a decrease in AV conduction without an effect on cardiac rate. On the other hand, GR-73632 microinjections into rNA-VL did not affect AV conduction. Following injections of the β subunit of cholera toxin conjugated to horseradish peroxidase (CTB-HRP) into the left atrial fat pad ganglion which selectively mediates changes in AV conduction, retrogradely labeled neurons were histochemically visualized in the rNA-VL. These tissues were subsequently processed for the simultaneous immunocytochemical visualization of SP, and examined by electron microflapy. Histochemically labeled neurons were large, multipolar, with abundant cytoplasm containing large masses of rough endoplasmic reticulum, and exhibited distinctive dendritic and somatic spines. Unlabeled nerve terminals were noted to form either asymmetric or symmetric synapses with dendrites, dendritic spines, and perikarya of histochemically labeled neurons. SP-immunoreactive nerve terminals were also detected in the rNA-VL. SP terminals typically contained numerous small pleomorphic vesicles, multiple large dense core vesicles, and several mitochondria, and they synapsed upon unlabeled dendritic profiles. A total of 154 SP-immunoreactive nerve terminals were observed on photomicrographs of tissues which also contained histochemically labeled profiles. None made an identifiable synapse with a retrogradely labeled profile on the sections examined. In summary, both physiological and ultrastructural data indicate that SP terminals in the iNA-VL do modify the output of negative chronotropic vagal motoneurons. This effect is mediated by NK-1 receptors. On the other hand both physiological and ultrastructural data indicate that SP terminals in the rNA-VL do not modify the output of negative dromotropic vagal motoneurons. Therefore different mechanisms (neurotransmitters or receptors) mediate the central vagal control of cardiac rate and AV conduction.

Autoradiographic localization of NK1 and NK3 tachykinin receptors in rat kidney.

The distribution of neurokinin receptors in rat kidney, renal artery, renal vein, and proximal ureter was evaluated by autoradiography after in vitro labeling of NK1 sites with [125I]Bolton-Hunter substance P (BHSP) or NK3 sites with [125I][MePhe7]neurokinin B ([MePhe7]NKB). Film autoradiography using [125I][MePhe7]NKB revealed specific binding sites associated with the renal vein and its large branches, the renal pelvis, the inner strip of outer renal medulla, and the proximal ureter. High-resolution autoradiograms demonstrated that these sites were localized to the smooth muscle layer in the veins, pelvis, and ureter. Neither the renal arterial system nor the renal cortex contained specific [125I][MePhe7]NKB binding sites although a high level of nonspecific binding was associated with the renal artery. Specific binding of [125I]BHSP was associated with the renal artery and renal pelvis but not the renal veins. Arterial NK1 receptors appeared to be localized to the adventitia. The results indicate that at least two types of tachykinin receptor are present in the rat kidney. The distinct localization observed for most of the NK1 and NK3 receptors suggests that they have different functions.

Synergistic role of muscarinic acetylcholine and tachykinin NK-2 receptors in intestinal peristalsis.

It is known that tachykinins (substance P, neurokinin A) participate in the excitatory neural pathways subserving peristaltic motor activity in the intestine. The aim of the present study was to elucidate the types of tachykinin receptor (NK-1 or NK-2) involved in peristalsis by the use of receptor subtype-selective antagonists. Peristaltic motility in isolated segments of the guinea-pig ileum was induced by pumping fluid into the oral end of the intestinal segment. By way of the intraluminal pressure the compliance of the intestinal wall during the preparatory phase and the pressure threshold to trigger the emptying phase of peristalsis were recorded. The tachykinin antagonists were used at concentrations that were at least 30 times in excess of the equilibrium dissociation constants which had previously been evaluated with receptor subtype-selective agonists on the guinea-pig ileum circular muscle. The NK-1 selective antagonist CP-96,345 (0.3 microM) had a slight stimulant influence on peristalsis, whereas the NK-2 selective antagonists MEN-10,376 (10 microM), GR-94,800 (0.3 microM) and SR-48,968 (0.1 microM) led to a small inhibition of motor activity. However, when given after exposure of the ileum to a threshold concentration of atropine (5-20 nM) causing little depression of peristalsis, the tachykinin NK-2 receptor antagonists invariably abolished peristalsis. This synergistic interaction was not seen when SR-48,968 was administered after the ileal segments had been exposed to concentrations of hexamethonium, isoproterenol or calcitonin gene-related peptide that by themselves caused a slight inhibition of peristalsis only.(ABSTRACT TRUNCATED AT 250 WORDS)

Facilitatory effects of selective agonists for tachykinin receptors on cholinergic neurotransmission: evidence for species differences.

1. Exogenous tachykinins modulate cholinergic neurotransmission in rabbit and guinea-pig airways. We have investigated the effect of selective tachykinin receptor agonists and antagonists on cholinergic neurotransmission evoked by electrical field stimulation (EFS) of bronchial rings in rabbit, guinea-pig and human airways in vitro to assess which type of tachykinin receptor is mediating this facilitatory effect. 2. Bronchial rings were set up for isometric tension recording. Contractile responses to EFS (60 V, 0.4 ms, 2 Hz for 10 s every min) and exogenous acetylcholine (ACh) were obtained and the effects of selective tachykinin agonists and antagonists were investigated. 3. In rabbit bronchi the endogenous tachykinins, substance P (SP) and neurokinin A (NKA) (10 nM) potentiated cholinergic responses to EFS (by 287.6 +/- 121%, P < 0.01 and 181.4 +/- 56.5%, P < 0.001 respectively). 4. The NK1 receptor selective agonist, [Sar9]SP sulphone (10 nM) evoked a maximal facilitatory action on cholinergic responses of 334.9 +/- 63% (P < 0.01) (pD2 = 8.5 +/- 0.06) an effect which was blocked by the selective NK1-receptor antagonist, CP 96,345 (100 nM) (P < 0.05) but not by the NK2 receptor antagonist, MEN 10,376 (100 nM). The NK2 receptor selective agonist, [beta Ala8]NKA(4-10) (10 nM), produced a maximum enhancement of 278 +/- 83.5% (P < 0.01) (pD2 = 8.7 +/- 0.1) an effect which was blocked by MEN 10,376 (100 nM) (P < 0.05) and not by CP 96,345. [MePhe7]NKB, an NK3 receptor selective agonist was without effect. 5. The rank order of potency of NK2 receptor antagonists against enhancement of cholinergic responses by [Beta Ala8]NKA(4-10) was MEN 10,376> L 659,877> R 396. This pattern together with the observation of the full agonist activity of MDL 28,564 indicates that the NK2 receptors in the rabbit bronchus are similar to those which are present in the rabbit pulmonary artery.6. Neither [Sar9]SP sulphone (5 nM) nor [Beta Ala8]NKA(4- 10) (1 nM) had any effect on contractile responses to ACh (10 MicroM) suggesting a pre-junctional mechanism of action.7. By contrast, in guinea-pig bronchi only the NK1-receptor agonist [Sar9]SP sulphone (3 nM) was effective in enhancing cholinergic neurotransmission but the effect was relatively small (maximal enhancement 25.7 +/- 5.5%, P<0.01). In human bronchial rings all the selective neurokinin agonists were without effect on cholinergic neurotransmission.8. These results suggest that tachykinins may play an important role in modulating cholinergic neurotransmission in rabbit (via NK1 and NK2 receptors) and guinea-pig airways (via NK1 receptor) but have no demonstrable effect on human airways

Multiple types of tachykinin receptor mediate a slow excitation of rat spinal motoneurones in vitro

Using intracellular current clamp recording from motoneurones of the neonatal rat spinal cord in vitro, the action of tachykinin receptor agonists was investigated. Test drugs included the endogenously occurring neuropeptide substance P and synthetic compounds, such as substance P methylester (SPMeO), [βAla 8]neurokinin A 4·;10 ([Ala]NKA), [MePhe 7]neurokinin B ([MePhe]NKB) and senktide. SPMeO and [Ala]NKA were used as selective agonists at NK 1 and NK 2 receptors, respectively, while [MePhe]NKB or senktide were employed to activate NK 3 receptors. In control solution, all compounds produced sustained depolarization with increase in input resistance although at comparable levels of membrane depolarization different patterns of motoneuronal firing were observed dependent on the type of agonist tested. In tetrodotoxin (TTX; 1 μM) solution, the depolarization caused by substance P or SPMeO largely persisted while in the majority of cells the effect of [Ala]NKA, [MePhe]NKB or senktide was blocked. It is suggested that NK 1 receptors primarily mediated the actions of substance P on spinal motoneurones and that activation of NK 2 or NK 3 receptors, predominantly found on interneurones, induced motoneuronal depolarization with different firing patterns.

NK1 receptors mediate tachykinin-induced plasma extravasation in the rat knee joint.

The tachykinin receptor type that mediates tachykinin-induced plasma extravasation in the rat knee joint was identified by using selective antagonists as well as natural or synthetic agonists. Substance P (SP) and neurokinin (NK) A induced plasma extravasation with almost the same potency and the maximum response was obtained at 5 nmol/knee. NKB was about ten times less potent than SP or NKA. The NK1 selective agonist, [Sar9, Met(O2)11]-SP, was about ten times more potent than SP, and the NK2 selective agonist, [Nle10]-NKA4-10, was about fifty times less potent than NK1 agonist. The NK3 agonist, Senktide, was totally ineffective at 0.5-50 nmol/knee. All responses induced by SP (5 nmol/knee), NKA (5 nmol/knee), NKB (50 nmol/knee), NK1 agonist (0.5 nmol/knee) or NK2 agonist (25 nmol/knee) were significantly and profoundly inhibited by the NK1 selective antagonist, RP67580, but not by the NK2 selective antagonist, SR48968. Taken together, we conclude that tachykinin-induced plasma extravasation in the rat knee joint is mediated via NK1 receptors.

Substance P and neurokinin A induced desentization to cardiovascular and behavioral effects: evidence for the involvement of different tachykinin receptors

Desensitization and cross-desensitization to the cardiovascular and behavioral effects elicited by intracerebroventricular (i.c.v.) substance P (SP) and neurokinin A (NKA) injections were examined in conscious, freely moving rats. The cardiovascular responses to equimolar doses of both peptides were identical, however, the pattern of the behavioral responses differed. Relative to SP, NKA was weaker in eliciting hindquarter grooming but more effective in eliciting wet dog shakes. SP pretreatment (50 pmol) desensitized the cardiovascular and behavioral responses to both, subsequent injections of SP (50 pmol) as well as of NKA (50 or 500 pmol) injected 30 or 60 min after SP, indicating cross-desensitization. NKA pretreatment (50 pmol) partly reduced the cardiovascular but not the behavioral responses to subsequent equimolar doses of NKA. The cardiovascular responses to SP (50 pmol) were reduced only 30 min but not 60 min after pretreatment with a 10 times higher dose of NKA (500 pmol). Of all behavioral manifestations to i.c.v. SP, only hindquarter grooming was attenuated by pretreatment with either dose of NKA. The equal potency of SP and NKA in eliciting the cardiovascular effects but different pattern of behavioral responses to these peptides suggest an involvement of different types of tachykinin receptors in mediating the central effects of the two peptides. The fact that NKA induced cross-desensitization selectively to one type of behavioral manifestations elicited by SP, indicates the existence of two subtypes of SP (NK 1) receptors in the rat brain.

Agonists selective for tachykinin NK1 and NK3 receptors excite subpopulations of neurons in the rat medial habenula nucleus in vitro.

The purpose of the present study was to determine the tachykinin receptor types present on neurones of the rat medial habenula nucleus. Extracellular recordings were made from spontaneously active medial habenula neurones in tissue slices of rat brain. The tachykinin analogues [Sar9,Met(O2)11]substance P, [beta-Ala8]neurokinin A-(4-10) and senktide were chosen for their potency and selectivity at NK1, NK2 and NK3 receptors, respectively. No neurone was observed to respond to [beta-Ala8]neurokinin A-(4-10). Neurones did respond to [Sar9,Met(O2)11]substance P and to senktide with a rapid and concentration-dependent increase in firing rate. Of the neurones where recordings were made, responses were obtained to one, both or neither of the agonists. These data suggest that receptors similar to NK1 and NK3, but not NK2 receptors, are differentially expressed on subpopulations of neurones of this nucleus.

NK 1 receptors mediate the increase in mucociliary activity produced by tachykinins

The mucocilisry activity of the rabbit maxillary sinus is increased after exposure to airway irritants such as cigarette smoke and capsaicin. This effect is partly due to a cholinergic reflex but involves an atropine-resistant response probably mediated by the release of tachykinins such as substance P or neurokinin A from sensory nerve endings. The aim of the present investigation was to evaluate the type of tachykinin receptor which mediates this increase in mucociliary activity. The mucociliary activity of the rabbit maxillary sinus was studied photoelectrically in vivo. It was found that a selective NK 1 receptor agonist, [Sar 9,Met(O 2) 11]substance P, dose dependently stimulated mucociliary activity, the maximum increase being 43.74 ± 6.07% at a dose of 1 nmol/kg. A selective NK 2 receptor agonist, [N]e 10]neurokinin A-(4–10), produced a much weaker response, the maximum increase being 15.23 ± 3.86% at a dose of 10 nmol/kg, whereas an NK 3 receptor agonist, [Pro 7]neurokinin B, was without effect. When the effects of the selective agonists were compared with the responses elecited by naturally occurring tachykinins at a dose of 1 pmol/kg, the order of the magnitude of the responses was [Sar 9, Met(O 2) 11]substance P > substance P > neurokinin A. At this dosage the NK 2 and NK 3 receptor agonists did not have a significant effect. Pretreatment with the endopeptidase inhibitor phosphoramidon did not influence the magnitude of the responses but increased their duration. It is concluded that the NK 1 receptor is responsible for the increase in mucociliary activity elicited by tachykinins released from sensory afferents in the upper airways.

Use of selective antagonists to dissociate the central cardiovascular and behavioural effects of tachykinins on NK1 and NK2 receptors in the rat.

1. The effects of intracerebroventricular (i.c.v.) pretreatment with selective NK1 ((+/-)-CP 96,345), NK2a (MEN 10,207; MEN 10,376) and NK2b (R 396) tachykinin receptor antagonists on the cardiovascular and behavioural responses to i.c.v. substance P (SP) and neurokinin A (NKA) were studied in conscious rats. 2. SP and NKA (25 pmol) induced mean arterial blood pressure and heart rate increases of the same magnitude and duration. The cardiovascular responses to both peptides were accompanied by excessive face washing, sniffing, grooming and wet dog shakes. 3. The cardiovascular responses to SP but not to NKA were attenuated by pretreatment with a NK1 receptor antagonist, (+/-)-CP 96,345. Of the behavioural responses, only face washing was significantly inhibited. 4. The cardiovascular and behavioural effects of NKA but not of SP were significantly attenuated by pretreatment with the selective NK2b receptor antagonist, R 396. 5. The selective NK2a receptor antagonists, MEN 10,207 and MEN 10,376, did not affect the cardiovascular and behavioural responses to either SP or NKA. 6. These results suggest, firstly, that the cardiovascular and behavioural effects of i.c.v. SP are mediated by NK1 receptors; secondly, that NKA injected i.c.v. does not interact with NK1 receptors but with another type of tachykinin receptor which may belong to the NK2b subclass. These findings provide pharmacological evidence for the existence of functionally active NK2 receptors in the rat brain.

Tachykinin receptors in rabbit airways--characterization by functional, autoradiographic and binding studies.

1. In many species, both NK1 and NK2 tachykinin receptors appear to be important in mediating the contraction of airway smooth muscle. We have examined the distribution and characterization of receptors for tachykinins in rabbit airways using functional length tension studies, autoradiography and radioligand binding studies. 2. Contractile responses to tachykinins were elicited in four different areas of the respiratory tree--trachea, and three progressively more distal areas of the right bronchus. The NK2 receptor-preferring agonists, neurokinin A (NKA), neuropeptide gamma (NP gamma) and the NK2-selective [Lys5 MeLeu9, Nle10]-NKA(4-10) [NKA (4-10) analogue] produced similar contraction in all four areas. Substance P (SP) and the NK1-selective [Sar9,Met(O2)11]-SP (Sar-SP) exhibited a marked location-dependence in the magnitude of contraction, producing minimal contraction in the trachea and more proximal bronchi with contractions becoming progressively larger in the more distal airways. Senktide (which is selective for the NK3 receptor) produced negligible contraction in all areas. 3. The NK2-selective antagonist, MDL29,913, was a weak antagonist of NKA and NKA(4-10) analogue. At a concentration of 2 microM, it produced a small but significant shift in the response curve to NKA and a greater shift (8 fold) in the curve to NKA(4-10) analogue, but it had no effect on responses to Sar-SP. The non peptide NK1 receptor antagonist, CP-96,345, was also unexpectedly weak in this preparation. The pD2 value for Sar-SP was decreased 27 fold by CP-96,345 at a concentration of 1 microM, without alteration in the maximum response.4. Autoradiographic binding sites to ['251I]-NKA were sparse over smooth muscle in proximal airway preparations and markedly increased in density in the more distal airways. There was negligible binding over vascular smooth muscle and epithelium.5. Radioligand binding studies revealed binding to ['251I]-NKA which was 82% specific. The order of potency for inhibition of ['251I]-NKA binding was SP> = Sar-SP> NKA = NPy>CP-96,345> NKA(4-10) analogue >NKB>>>MEN 10207 (the NK2 subtype selective antagonist) >MDL 29,913> senktide. This profile indicates binding predominantly to NK, receptors.6. These results suggest that there are at least two types of tachykinin receptors in rabbit airways, a population of NK, receptors, the density of which is greatest in the periphery and, in addition, NK2 receptors which are uniformly distributed throughout the airways. These receptors have unusual characteristics in that the NK, antagonist, CP-96,345 and the NK2 antagonist, MDL 29,913 respectively exhibited only weak potency.