Intra-raphe neurokinin-induced hyperactivity: effects of 5,7-dihydroxytryptamine lesions

Rats were implanted with cannulae in the median raphe nucleus (MR). 5,7-Dihydroxytryptamine (5,7-DHT) or vehicle was infused either directly through the MR cannula, or bilaterally into the medial forebrain bundle (MFB). The MR 5,7-DHT lesions completely blocked the hyperactivity elicited by injections into the MR of the neurokinin (NK) 3 agonists, DiMe-C7 and senktide, and the NK-2 agonist, neurokinin A. In contrast, the MFB 5,7-DHT lesions did not affect the locomotor hyperactivity produced by intra-MR administration of DiMe-C7 and senktide, but appeared to attenuate the effects of NKA. The data indicate that intra-raphe neurokinin-induced hyperactivity is mediated by 5-HT neurons, and that 5-HT projections to the forebrain may be involved in the behavioral activation induced by intra-raphe neurokinin A administration, but not that induced by intra-MR NK-3 agonists.     

The N-terminal domain of substance P is required for complete homologous desensitization but not phosphorylation of the rat neurokinin-1 receptor

The agonist activity of substance P (SP) is a function of the C-terminal domain of the peptide. A C-terminal SP fragment (SP(6-11)) and analog (septide) and neurokinin A (NKA; a related tachykinin with a divergent N-terminal amino acid sequence) were found to be full neurokinin-1 receptor (NK-1R) agonists, but were not able to desensitize the receptor maximally as much as SP. Substance P caused 95.6 +/- 0.9% maximal desensitization of the NK-1R whereas SP(6-11), septide, and NKA(only)caused 74 +/- 3.5, 50.6 +/- 8, and 71.5 +/- 4.4% maximal desensitization, respectively (mean +/- SEM; P < 0.001 vs SP). When a series of SP C-terminal fragment peptides were tested for their NK-1R desensitizing activity, it was found that SP(5-11)and SP(6-11)caused significantly less maximal NK-1R desensitization than SP. SP N-terminal fragment peptides had no effect on the ability of SP(6-11)to compete with(3)H-SP binding, generate an IP(3)response, or cause NK-1R desensitization when tested with or without SP(6-11). SP, SP(6-11), septide, and NKA all maximally stimulated 8-9-fold increases in NK-1R phosphorylation. When attached to the C-terminal domain of SP responsible for NK-1R binding and agonism, the N-terminus of SP is responsible for 25-50% of homologous desensitization and this may occur via a mechanism other than NK-1R phosphorylation.     

Effects of neurokinins on human colonic motility

Abstract Neurokinins have been implicated as excitatory neuromessengers involved in the mediation of different reflexes in the mammalian gastrointestinal tract. However, marked interspecies variations in reported receptor distribution and in regional peptide content do not allow the extrapolation of results obtained in animals to the human gastrointestinal tract. To characterize the myogenic and neurogenic mechanical response of human colonic muscle to neurokinins, we studied the inotropic response of muscle strips from the proximal and distal human colon, and the rectum to the NK-1 receptor agonists substance P (SP) and substance P methylester (SPME), to the NK-2 receptor agonists neurokinin A (NKA) and neurokinin A 4–10 (NKA4–10) and to the NK-3 receptor agonist neurokinin B (NKB). Even though all neurokinins caused a dose-dependent inotropic response, NKA was 15–20 times more potent than SP or SPME in all areas of the colon. The efficacy and potency of NKA was highest in distal circular colon. The response to exogenous SP and NKA was partially mediated by actions of these peptides on myenteric nerves, as indicated by the sensitivity of the mechanical response to atropine, tetrodotoxin and hexamethonium. Densensitization to NKA, but not to SP significantly increased the atropine-resistant part of the off response to electrical field simulation. These results suggest the following: (a) NKA is a potent agonist in human colon with a proximal to distal gradient in potency and in efficacy; (b) the response to NK-I, NK-2 and NK-3 agonists involves cholinergic and nicotinic mechanisms; (c) the increase in the atropine resistant off-response after desensitization with NKA is consistent with the existence of inhibitory NK-2 receptors on non-cholinergic myenteric neurons.     

The behavioural effects of a novel substance P analogue following infusion into the ventral tegmental area or substantia nigra of rat brain

The behavioural response following infusion of a novel, stable substance P (SP) analogue, DiMe-C7, into the ventral tegmental area (VTA) of rats was characterized and contrasted with the response to an equal dose of the parent compound SP. DiMe-C7 produced a longer-lasting behavioural stimulation than SP as evidenced in several behaviours, including locomotor activity, wet dog shakes, rearing and grooming. DiMe-C7-induced locomotor activity and rearing were potentiated by concurrent peripheral administration of D-amphetamine and blocked by pretreatment with haloperidol. Such responses to DiMe-C7 may thus be dependent upon dopaminergic activity. When given immediately following VTA infusion of DiMe-C7, morphine decreased, while naloxone had no effect upon most behavioural measures. The effect of methysergide on DiMe-C7 or SP into the substantia nigra reticulata produced a pattern of responses similar to nature to those produced by VTA infusion but different with respect to time course. These findings suggest that DiMe-C7 is a metabolically stable analogue of substance P which manifests prolonged actions on behaviour when centrally administered. Further, a role for central dopaminergic mechanisms is implicated in DiMe-C7-induced behavioural action.     

Contractile effect of tachykinins on Suncus murinus (house musk shrew) isolated ileum

Recent studies used Suncus murinus to investigate the anti-emetic potential of NK(1) tachykinin receptor antagonists. However, the pharmacology of tachykinin receptors in this species has not been fully characterized. In the present studies, therefore, we examined a range of tachykinin receptor agonists for a capacity to induce contractions of the isolated ileum. The tachykinin NK1 receptor preferring agonists substance P, septide and [Sar9Met(O2)11] substance P, and the tachykinin NK2 preferring agonists neurokinin A and GR 64349 (Lys-Asp-Ser-Phe-Val-Gly-R-gamma-lactam-Leu-Met-NH2) caused concentration dependent contractions with EC50 values in the nanomolar range. However, the tachykinin NK3 preferring agonists neurokinin B and senktide (1nM-1microM) induced only weak contractions. The action of senktide, but not [Sar9Met(O2)11] substance P, septide, or GR 64349, was antagonized significantly by atropine (P<0.05); tetrodotoxin and hexamethonium were inactive. The tachykinin NK1 receptor antagonist CP-99,994 ((+)-[(2S,3S)-3-(2-methoxy-benzyl-amino)-2-phenylpiperidine]) (10-100nM) inhibited substance P- and septide-induced contractions non-competitively. The pA2 value estimated for CP-99,994 against septide was 7.3+/-0.1. It also non-competitively antagonized the contractile responses induced by [Sar9Met(O2)11] substance P with a pA2 of 7.4+/-0.1. CP-99,994 also had a slight inhibitory action on neurokinin A-induced contractions, but did not modify the action of GR 64349. Conversely, the tachykinin NK2 receptor antagonist, saredutant, competitively antagonized GR 64349-induced contractions with a pA2 of 7.34+/-0.02. On the other hand, the presence of both CP-99,994 and saredutant competitively antagonized substance P-induced contraction. The present studies indicate that tachykininNK1 and NK2 receptors exist in the ileum of S. murinus and are involved in mediating contractions directly on smooth muscle, whereas tachykinin NK3 receptors may play a minor role involving a release of acetylcholine.     

Activation of neurokinin receptors modulates K and Cl channel activity in cultured astrocytes from rat cortex

Short application of the neurokinin receptor agonist substance P (SP) leads to a biphasic depolarization of astrocytes cultured from rat cortex. The rapid and transient depolarizing event lasted few seconds, the slow one several minutes. In some cells, only the slow depolarizing component was observed. During the slow depolarizing event, the sensitivity of the membrane potential for a change in the K+ gradient decreased, indicating a decrease in the relative K+ permeability of the membrane. The rapid SP-induced depolarization could be reversed, when the membrane potential was depolarized to about 0 mV by elevation of the extracellular K+ concentration, indicating a reversal potential close to the Cl- equilibrium potential. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, SP induced a biphasic inward current with a similar time course as the SP-induced membrane depolarization. Evaluating current-to-voltage curves indicated a conductance decrease during the slow inward current with a reversal potential of the SP-dependent current close to the K+ equilibrium potential. The mean open time of single K+ channels, measured in the cell-attached configuration of the patch-clamp technique, decreased after application of SP. In contrast, the mean open time of single Cl- channels increased. We conclude that activation of neurokinin receptors in astrocytes modulates the activity of K+ and Cl- channels, leading to a complex depolarization of the membrane potential.     

The involvement of neurokinin receptor subtypes in somatosensory processing in the superficial dorsal horn of the cat.

As well as substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) have recently been found in the superficial dorsal horn of the spinal cord; NKA originating mainly in fine primary afferents. We have investigated the effects of these tachykinins and a range of analogues on somatosensory responses of single identified dorsal horn neurons, when applied ionophoretically to the region of the substantia gelatinosa. Behavioural reflex tests of thermal nociception were carried out in parallel. The role of NK-1, NK-2 and NK-3 receptors was addressed. NK-1-selective agonists attenuated the non-nociceptive responses of identified multireceptive spinocervical tract (SCT) neurons. Of the endogenous tachykinins, both SP and NKB (a weak NK-1 agonist) showed this effect. No role for NK-3 receptors was identified in our experiments. NK-2-selective agonists (including NKA) caused a unique and selective facilitation of thermal nociceptive responses. NKA also reduced reflex response latency in tail-flick and hot plate tests. NKA as a primary afferent transmitter may thus be involved in mediating or facilitating the expression of thermal nociceptive inputs in the substantia gelatinosa. NKA and SP could be considered as acting in concert in the superficial dorsal horn in an effectively pro-nociceptive modulatory role. Evidence from receptor-selective antagonists supports that obtained with agonists for the roles of particular NK receptors in somatosensory processing. NK-2, but not NK-1 or NK-3 antagonists attenuated endogenous thermal nociceptive responses, supporting the hypothesis that an NK-2 agonist (such as NKA) may normally participate in expression of thermal nociception in the superficial dorsal horn. Behavioural experiments showing increased response latencies with a putative NK-2 selective antagonist further supported the involvement of NK-2 receptors in thermal nociception.     

A selective and extremely potent antagonist of the neurokinin-1 receptor

Sendide [Tyr6,D-Phe7,D-His9]-substance P(6-11) has been examined by measurements of ligand binding to crude membrane fractions and by functional tests on the spinally mediated behavioral response. Sendide potently displaced [3H]-labeled substance P (SP) binding to mouse spinal cord membranes in a competitive manner. In vivo, sendide, intrathecally co-injected with SP, competitively antagonized SP-induced scratching, biting and licking. The behaviors elicited by physalaemin, septide and [Sar9, Met(O2)11]-SP were also reduced by co-administration of sendide. Large doses of sendide were needed to reduce the action of neurokinin A, D-septide, neurokinin B and eledoisin. The in vitro and in vivo pharmacological profile of sendide demonstrated that it is a selective and extremely potent antagonist of the neurokinin-1 receptor.     

Behavioural effects of tachykinins and related peptides

Substance P (SP) and related tachykinins administered either intracerebroventricularly or directly into the ventral tegmental area of the mesencephalon of rat brain caused increased locomotor activity, grooming behaviour and wet dog shakes. Kassinin, eledoisin, neurokinin A and DiMe-C7, agonists with some selectivity for the SP-E-receptor elicited the greatest locomotor activity and wet dog shake responses, whereas SP and physalaemin which are more selective for the SP-P-receptor were most effective in eliciting the grooming response.     

Differential sensitivity to phenoxybenzamine alkylation among types of neurokinin binding sites

The effect of phenoxybenzamine (PBZ) treatment of crude membranes was examined on binding to neurokinin/tachykinin NK-1, NK-2, and NK-3 binding sites. PBZ in concentrations up to 300 microM resulted in only a slight reduction in Bmax in binding of iodinated substance P to NK-1 sites in rat submaxillary gland and in rat urinary bladder. PBZ in concentrations as low as 30 microM resulted in a decrease in Bmax or in affinity in binding of iodinated neurokinin A (substance K) to NK-2 sites in hamster bladder and in rat bladder, and a decrease in affinity in binding of iodinated eledoisin to NK-3 sites in rat cerebral cortex. The results indicate a parallelism in differential sensitivity to PBZ alkylation between neurokinin biological receptors and neurokinin ligand binding sites.     

Electrophysiological Evidence That Neurokinin A Acts Via NK-1 Receptors in the Cat Dorsal Horn

The aim of the present study was to investigate the effects of the non-peptide NK-2 receptor antagonist, SR 48968 on the responses of dorsal horn neurons to iontophoretic application of the endogenous NK-2 receptor ligand, neurokinin A, and on synaptically elicited responses in chloralose-anaesthetized cats. The effect of iontophoretic application of neurokinin A was tested on 51 dorsal horn neurons. Of these, 43 were wide dynamic range and the rest non-nociceptive neurons. Neurokinin A induced a slow, prolonged excitation of 25 of the wide dynamic range neurons. All remaining neurons were unaffected. SR 48968 (50 pg to 1.0 mg/kg, i.v.) did not affect the on-going basal activity (n= 8) or the slow excitation induced by neurokinin A in any of the nine wide dynamic range neurons tested. To eliminate the possibility that systemically administered SR 48968 may not be reaching central sites, SR 48968 was also applied iontophoretically (70–120 nA) to five neurons and tested against excitatory responses to iontophoretically applied neurokinin A. The on-going activity of these cells were unaffected by SR 48968. The responses to neurokinin A were also unaffected suggesting that neurokinin A did not mediate its effects via NK-2 receptors. SR 48968 also had no effect on the excitatory responses of seven neurons to iontophoretic application of the NK-1 receptor agonist, substance P indicating that substance P actions are not mediated via NK-2 receptors and that SR 48968 did not react with NK-1 receptors. Responses of the neurons to non-noxious (hair) stimulation (n = 10), noxious mechanical (n= 5) and noxious thermal (n= 8) stimulation of the receptive field were also unaffected by SR 48968, suggesting a lack of participation of NK-2 receptors in these responses. However, responses of wide dynamic range neurons to neurokinin A were totally blocked by i.v. administration (0.5 mg/kg) of the NK-1 receptor antagonists CP-96,345 (n= 7) and CP-99,994 (n= 5) but not by CP-96,344 (n= 4), the inactive enantiomer of CP-96,345. These data suggest that neurokinin A, like substance P may be acting via NK-1, rather than NK-2 receptors, to produce excitation of wide dynamic range neurons in the dorsal horn of the cat spinal cord.     

Behavioural and biochemical responses following activation of midbrain dopamine pathways by receptor selective neurokinin agonists.

Preferential activation of mesolimbic and nigro-striatal dopamine (DA) pathways by receptor-selective and peptidase-resistant neurokinin (NK) agonists is reported. The DA cell body region of the mesolimbic pathway appears to be activated by NK agonists selective for NK-1 and NK-3 receptors whereas the DA cell bodies in the substantia nigra are under an excitatory NK-2 receptor-mediated influence. Stimulation of the mesolimbic DA pathway by NK-1 (Ava[L-Pro9,N-Me-Leu10]SP (7-11) [GR73632]) or NK-3 (Senktide) agonists increase locomotor activity. Additional studies showed that this elevated motor response observed after intra-VTA infusion of GR73632 was accompanied by a corresponding increase in DA turnover in the terminal fields of this pathway. Similarly, unilateral activation of the nigro-striatal DA pathway by NK-2 selective agonists (Ava (D-Pro9) SP (7-11) [GR51667] or [Lys3,Gly8,R-Lac-Leu9]NKA (3-10) [GR64349]) elicit contralateral rotational activity and an increase in DA turnover in the ipsilateral striatum. The rotational response was attenuated by prior administration of an NK-2 antagonist (cyclo (Gln, Trp, Phe, Gly, Leu, Met)] L-659877]) into the nigra. Peripheral injection of haloperidol, a DA antagonist, also blocked the NK-2 agonist induced rotations.     

Receptor-selective, peptidase-resistant agonists at neurokinin NK-1 and NK-2 receptors: new tools for investigating neurokinin function.

The pharmacological profiles of two novel neurokinin agonists have been investigated. delta Ava[L-Pro9,N-MeLeu10]SP(7-11) (GR73632) and [Lys3,Gly8-R-gamma-lactam-Leu9] NKA(3-10) (GR64349) are potent and selective agonists at NK-1 and NK-2 receptors respectively. In the guinea-pig isolated trachea preparation, contractions induced by these agonists were largely unaffected by inclusion of peptidase inhibitors in the bathing medium, indicating that these agonists are resistant to metabolism by peptidases. In the anaesthetised guinea-pig, both agonists were more potent bronchoconstrictor agents than either NKA or the SP analogue, SP methylester. In the anaesthetised rat, the NK-1 agonist, GR73632 was more potent than SP, NKA or NKB at causing the histamine-independent extravasation of plasma proteins into the skin after intradermal administration. The NK-2 agonist, GR64349 and the NK-3 agonist, senktide were without significant effect in this model. These agonists are useful tools for characterizing neurokinin receptor-mediated actions both in vitro and in vivo.     

Differences in circular muscle contraction and peristaltic motor inhibition caused by tachykinin NK1 receptor agonists in the guinea-pig small intestine

The tachykinin NK1 receptor agonist substance P methyl ester (SPOME) impedes intestinal peristalsis by releasing nitric oxide (NO) from inhibitory motor neurones. Since NK1 receptor agonists differ in their receptor interaction, we set out to compare a range of NK1 receptor agonists including SPOME, septide and GR-73 632 in their effects on propulsive peristalsis and circular muscle activity in the guinea-pig isolated small intestine. SPOME (100-300 nM) inhibited peristalsis by a rise of the pressure threshold at which peristaltic waves were triggered, whereas septide and GR-73 632 (30-300 nM) interrupted peristalsis by causing circular muscle spasms. Separate experiments showed that all three NK1 receptor agonists caused contraction of the circular muscle, which was enhanced by the NO synthase inhibitor NG-nitro-L-arginine methyl ester (300 mM) and the P2X purinoceptor antagonist suramin (300 mM). In contrast, tetrodotoxin (300 nM) augmented the contractile effect of septide and GR-73 632 but not that of SPOME. It is concluded that the motor response to NK1 receptor agonists involves release of NO and adenosine triphosphate from inhibitory motor neurones. However, the NK1 receptor agonists differ in the mechanism by which they cause inhibitory transmitter release, which corresponds to differences in their antiperistaltic action.     

Ionic dependence of a P2-purinoceptor mediated depolarization of cultured astrocytes.

The membrane potential of cultured mouse astrocytes was recorded to assess the effects of extracellular adenosine 5'-triphosphate (ATP) and related H purines on astrocyte electrophysiology. The purines were applied with or without the presence of barium, which blocks the high resting K+ conductance in astrocytes. The response to ATP alone was a moderate depolarization; however, the response to ATP in the presence of barium was a large, dose dependent depolarization. The ED50 was approximately 10 microM. The effect of adenosine 5'-diphosphate (ADP) or adenosine 5'-monophosphate (AMP), in the presence of barium, on membrane potential was less than that of ATP. Adenosine, with or without barium, had no effect on membrane potential; furthermore, adenosine agonists in barium produced no response. The results of applying various ATP analogues indicate that the response is mediated via a P2-purinoceptor. Ion replacement studies reveal a complicated response to ATP that has several components and involves Na+ and K+. These results show that astrocytes respond with ionic changes to very small, physiological concentrations of extracellular ATP. We suggest that ATP plays a role in interactions between neurons/endothelial cells and glial cells.     

P2Y receptor-activating nucleotides modulate cellular reactive oxygen species production in dissociated hippocampal astrocytes and neurons in culture independent of parallel cytosolic Ca(2+) rise and change in mitochondrial potential

With mixed cultures of hippocampal astrocytes and neurons, we investigated the influence of nucleotides on cytosolic Ca(2+) level, generation of reactive oxygen species (ROS), and mitochondrial potential. We employed ATP and four purine/pyrimidine derivates, which are P2Y receptor subtype-preferring agonists. Stimulation with ATP, a P2Y(1/2/4) receptor agonist in rat, caused a large cytosolic Ca(2+) increase in astrocytes and a considerably smaller Ca(2+) response in neighboring neurons. The P2Y(1) receptor antagonist MRS2179 completely blocked the ATP-induced Ca(2+) response in astrocytes and neurons. Application of ATP significantly reduced the mitochondrial potential in neurons, which was not inhibited by MRS2179. Interestingly, MRS2179 mediated a mitochondrial depolarization without affecting the cytosolic Ca(2+) level. Stimulation with UDP, a P2Y(6) receptor agonist; UTP, a P2Y(2/4) receptor agonist; 2MeSATP, a P2Y(1) receptor agonist; or 2MeSADP, a P2Y(1/12/13) receptor agonist, evoked significant Ca(2+) responses in astrocytes but small Ca(2+) responses in neurons. In astrocytes, there was an inverse relationship between the amplitude of the cytosolic Ca(2+) peak and the rate of ROS generation in response to nucleotide application. Activation with UDP resulted in the highest ROS generation that we detected, whereas 2MeSADP and 2MeSATP reduced the ROS generation below the basal level. 2MeSADP and UDP caused mitochondrial depolarization of comparable size. Thus, neither in astrocytes nor in neurons did the degree of mitochondrial depolarization correlate with ROS generation. Nucleotides acting via P2Y receptors can modulate ROS generation of hippocampal neurons without acutely changing the cytosolic Ca(2+) level. Thus, ROS might function as a signaling molecule upon nucleotide-induced P2Y receptor activation in brain.     

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.     

The effect of K+ and glutamate receptor agonists on the membrane potential of suspensions of primary cultures of rat astrocytes as measured with a cyanine dye, DiS-C2-(5)

The cyanine dye DiS-C2-(5) was used to investigate the effect of K+ and glutamate receptor agonists on the membrane potential of whole populations of primary rat astrocytes in suspension. Increasing the external K+ concentration from 5 to 40 mM caused a depolarization of the cells. Ba2+ blocked the response to K+, whereas 4-aminopyridine had no effect on the depolarization. The effect of added external K+ was enhanced by the addition of the neutral K+ ionophore valinomycin. This supports the view that the membrane potential of primary astrocytes is dependent of the K+ gradient, and suggests that the membrane is not ideally permeable to K+ ions. Glutamate caused a depolarization of the cells which was not affected by Ba2+. In the presence of veratridine and ouabain no effect of glutamate was seen. The cells were also depolarized by the glutamate receptor agonists quisqualate, kainate and N-methyl-D-aspartate (NMDA). The response to kainate was blocked by kynurenate, which also diminished the depolarization caused by glutamate. NMDA was effective when added after kainate. The effect of the glutamate receptor agonists tested was generally smaller than that of glutamate itself, and a prior addition of one of the agonists diminished the response to glutamate. The results obtained suggest that cyanine dyes are well suited for investigating the behavior of whole populations of cultured primary astrocytes.