Effects of intracavernous injection of P2X3 and NK1 receptor antagonists on erectile dysfunction induced by spinal cord transection in rats.

This study aimed to explore the effects of intracavernous injection (ICI) of P2X3 and NK1 receptor antagonists on erectile dysfunction (ED) induced by spinal cord transection in rats. Sixty male Sprague-Dawley (SD) rats were randomly divided into the following three groups (20 rats each group): sham operation group (C group), thoracic spinal cord transection group (T group) and sacral spinal cord transection group (S group). An ED model was established through complete transection of the thoracic or sacral spinal cord. Intracavernous pressure (ICP) with and without injection of P2X3 (Suramin) or NK1 (GR82334) receptor antagonists was recorded 3 weeks after surgery. Immunohistochemistry was employed to detect the expression of P2X3 and NK1 receptors in the dorsal root ganglion (DRG) and smooth muscle of corpus cavernosum. Data were processed with SPSS 17.0. ICI with Suramin (0.1, 0.3 and 1 mm) or GR82334 (0.1, 0.3 and 1 mm) increased ICP dose dependently in the T and S groups. The expression of P2X3 and NK1 receptors in DRG and smooth muscle of corpus cavernosum was up-regulated in the T and S groups. It is concluded that ICI of P2X3 and NK1 receptor antagonists may improve the recovery of erectile function in a rat model with ED after spinal cord transection.

The function of P2X3 receptor and NK1 receptor antagonists on cyclophosphamide-induced cystitis in rats.

PURPOSE: The purpose of the study is to explore the function of P2X3 and NK1 receptors antagonists on cyclophosphamide (CYP)-induced cystitis in rats. METHODS: Sixty female Sprague-Dawley (SD) rats were randomly divided into three groups. The rats in the control group were intraperitoneally (i.p.) injected with 0.9% saline (4 ml/kg); the rats in the model group were i.p. injected with CYP (150 mg/kg); and the rats in the intervention group were i.p. injected with CYP with subsequently perfusion of bladder with P2X3 and NK1 receptors' antagonists, Suramin and GR 82334. Spontaneous pain behaviors following the administration of CYP were observed. Urodynamic parameters, bladder pressure-volume curve, maximum voiding pressure (MVP), and maximum cystometric capacity (MCC), were recorded. Pathological changes in bladder tissue were observed. Immunofluorescence was used to detect the expression of P2X3 and NK1 receptors in bladder. RESULTS: Cyclophosphamide treatment increased the spontaneous pain behaviors scores. The incidence of bladder instability during urine storage period of model group was significantly higher than intervention group (χ(2) = 7.619, P = 0.007) and control group (χ(2) = 13.755, P = 0.000). MCC in the model group was lower than the control and intervention groups (P < 0.01). Histological changes evident in model and intervention groups rats' bladder included edema, vasodilation, and infiltration of inflammatory cells. In model group, the expression of P2X3 receptor increased in urothelium and suburothelium, and NK1 receptor increased in suburothelium, while the expression of them in intervention group was lower. CONCLUSIONS: In CYP-induced cystitis, the expression of P2X3 and NK1 receptors increased in urothelium and/or suburothelium. Perfusion of bladder with P2X3 and NK1 receptors antagonists ameliorated the bladder function.

NK1 receptor activation by geniohyoid primary afferents modulates parasympathetic postganglionic neuronal excitability in the rat.

Previous studies have indicated that the geniohyoid (GH) muscle receives innervation via both the hypoglossal nerve (CNXII) and the ansa cervicalis. Our recent studies revealed that the efferent root that contributes to the ansa cervicalis is a parasympathetic pathway and contains postganglionic cell bodies. Afferent axons from the GH muscle also travel via the ansa cervicalis, and afferent cell bodies are located in spinal ganglia. The present study attempts to locate the central terminations of these afferents. From the peripheral cut end of the ansa cervicalis, we recorded afferent discharges that coincided with inspiration and these were elicited by stretch of the GH muscle. After cutting CNXII proximal to its union with the ansa cervicalis, we applied horseradish peroxidase to the branch of CNXII that innervates the GH muscle. This procedure labeled cells ipsilaterally in the C2 spinal ganglia but not in the brainstem or upper spinal cord. Substance P-reactive terminals in the peripheral CNXII trunk were in apparent contact with vasoactive intestinal peptide-reactive cell bodies. Addition of the NK1 receptor agonist SP(NK1) excited parasympathetic postganglionic neurons and the specific NK1 receptor antagonist GR82334 blocked these effects in vitro. These results suggest that GH primary afferents synapse on parasympathetic postganglionic neurons in the CNXII trunk and that activation of SP(NK1) receptors modulates activity in these neurons.

Effects of central neurokinin-1 receptor antagonism on cocaine- and opiate-induced locomotor activity and self-administration behaviour in rats.

The neuropeptide substance P (SP) and its preferred receptor, the neurokinin-1 (NK-1) receptor, have been implicated in some of the reward-related behavioural effects of abused drugs, including psychostimulants and opiates. The first objective of the present series of experiments was to assess the role of the NK-1 receptor in two reward-related behavioural effects of cocaine: locomotor activity and self-administration. In tests for locomotor activity, rats were given intracerebroventricular (ICV) infusions of the selective NK-1 receptor antagonist, GR82334 (0, 10, 50 pmol), prior to systemic injections of cocaine. In self-administration experiments, rats were trained to self-administer cocaine on a fixed-ratio 5 (FR5) schedule of reinforcement. Following acquisition of stable responding, animals were pretreated with GR82334 (0, 2, 10, 50 pmol; ICV) prior to subsequent self-administration sessions. Based on evidence suggesting a potentially selective role for NK-1 receptors in opiate reward, we also examined the effects of GR82334 on morphine-induced locomotor activity and heroin self-administration. Results showed that GR82334 had no effect on cocaine-induced locomotor activity or cocaine self-administration, but attenuated morphine-induced locomotor activity and increased heroin self-administration. These findings suggest that endogenous activity at NK-1 receptors may play a specific role in opiate-induced, but not cocaine-induced, locomotor activation and reinforcement.

Regulation of surfactant secretion from isolated Type II pneumocytes by substance P.

Substance P, an eleven amino acid neuropeptide, significantly inhibited release of [3H]phosphatidylcholine from pulmonary Type II epithelial cells in vitro. Basal release and release in response to the beta-adrenergic agonist, terbutaline and 12-O-tetradecanoylphorbol 13-acetate (TPA) were significantly decreased in the presence of substance P. Inhibitory effects of substance P were noted following a 1 h exposure of primary cultures of Type II cells in vitro and persisted up to 3 h in the presence of the secretagogues, TPA and terbutaline. The IC50 values for substance P inhibition of [3H]PC release were 10 microM for basal release, 40 microM for TPA-induced release and 50 microM for terbutaline-induced release. The related neuropeptide, physalaemin and the stable active analog of substance P, [pGlu5, MePhe8, MeGly9]substance P [5-11], had no significant inhibitory effects on surfactant release whether in the presence or absence of TPA or terbutaline. These data support the hypothesis that NH2-terminal basic groups of substance P are necessary for inhibition of surfactant secretion from isolated Type II cells and support the concept that an inhibitory system contributes to mediation of surfactant secretion from Type II epithelial cells.

Potassium release from submandibular salivary gland in vitro.

Rat submandibular gland slices, incubated in continuously-gassed Krebs-Ringer bicarbonate buffer, were shown to release K+ in response to alpha-adrenergic and muscarinic cholinergic stimulation. The system employed the specific alpha-, beta-adrenergic and cholinergic receptor-blocking agents phentolamine, propranolol and atropine, respectively, in combination with the agonists L-epinephrine and carbamylcholine both of which required the presence of Ca2+ for their effect. The introduction of Ca2+ into the cell via the ionophore A23187, with all neurotransmitter receptors blocked, resulted in K+ release. Ouabain also allowed extensive K+ release which was in addition to, and hence independent of, that elicited by epinephrine and carbamylcholine. Ethacrynic acid, a potent inhibitor of salivary secretion in vivo, had no influence on K+ movement. K+ was released by both physalaemin and an eledoisin-related peptide independently of normal neurotransmitter receptors. The activity of the eledoisin-related peptide did not require the presence of extracellular Ca2+. The implication of cyclic GMP at some stage of K+ release was suggested by experiments with a phosphodiesterase inhibitor. The results support an hypothesis where the initial stimulus at either alpha-adrenergic or muscarinic cholinergic receptors causes an immediate permeability change such that Ca2+ enters the cells resulting in K+ release. The loss of K+ is quickly countered by the ouabain-sensitive (Na+ + K+) ATPase which would be activated by the lowered intracellular K+ levels.

Substance P, injected intrathecally, antagonizes the spinal antinociceptive effect of morphine, baclofen and noradrenaline.

The effect of substance P on the antinociceptive effect of morphine, baclofen and noradrenaline in the spinal cord was examined in the tail-flick and hot plate tests, after intrathecal administration. Substance P (5-20 micrograms) produced a dose-related antagonism of the effect of all three agents, which persisted for the entire time-course of the antinociceptive effect in each case. The rank order of potency of substance P and related peptides in antagonizing the antinociceptive effect of morphine, was substance P greater than physalaemin greater than eledoisin greater than eledoisin-related peptide. A similar order of potency was observed against noradrenaline, except that physalaemin appeared to be the most potent. The intrathecal administration of these peptides did not alter reaction latencies in the tail-flick test when baseline values were 2-3 sec, but produced a significant decrease in latency (hyperalgesia) when baseline values were 6-8 sec. There was a slight increase in reaction latency in the hot plate test. The specificity of the antagonism was examined by determining the effect of phentolamine on the antinociceptive effect of morphine and baclofen. Although phentolamine (30 micrograms) produced comparable hyperalgesia to substance P, it did not significantly alter the antinociceptive effect of morphine or baclofen. These results suggest that alterations in the function of substance P in the spinal cord may contribute to the spinal antinociceptive effects of morphine, baclofen and noradrenaline. The effects of substance P on nociception in the spinal cord appear to be mediated by substance P type receptors.

The effects of neurokinin-1 receptor agonists on spinal motoneurones of the neonatal rat.

The effects of substance P (SP) and the selective NK1 receptor agonist [Sar9Met(O2)11] substance P on neonate rat spinal motoneurones were examined using intracellular recordings. Bath-administration of SP (0.1-3 microM) or [Sar9Met(O2)11] substance P (0.01-3 microM) induced a tetrodotoxin (TTX)-insensitive (10 microM) depolarization and a tetraethylammoniumchloride (TEA)-sensitive (3 mM) decrease in membrane conductance. The duration of the slow afterhyperpolarizations (AHPs) following the action potentials were significantly reduced (p = 0.003) by both NK1 receptor agonists. The mean duration of the sAHPs (+/- SEM) in control was 67.8 +/- 6.3 ms whereas in the presence of SP and [Sar9Met(O2)11] substance P their duration was reduced to 41.7 +/- 4.6 ms. Low Ca2+ (0.2 mM)-containing artificial cerebrospinal fluid (ACSF) or addition of BaCl2 or CdCl2 (2 mM) reduced the durations of the slow AHPs by 55%. In the presence of these agents SP and [Sar9Met(O2)11] substance P practically abolished the remaining slow AHPs, suggesting that the agonists also reduce a calcium-independent current. None of the effects induced by the NK1 receptor agonists were antagonized by the NK1 receptor antagonists (+/-)-CP-96,345 (10 microM), RP 67580 (1 microM) or GR 82334 (3-5 microM). In conclusion this study demonstrates that SP and [Sar9Met(O2)11] substance P elicit their effects on NK1 receptors by modulating at least two potassium currents, namely IK and ICa(K).

Responses of functionally identified neurones in the dorsal horn of the cat spinal cord to substance P, neurokinin A and physalaemin.

The mammalian tachykinins, substance P and neurokinin A, and the non-mammalian tachykinin, physalaemin, were tested on functionally identified dorsal horn neurones in vivo. The experiments were done on cats which were anaesthetized with sodium pentobarbital or were anaemically decerebrated. Extracellular single-unit recordings were made in the lumbar spinal cord and the tachykinins were applied by iontophoresis. Each neurone was classified functionally as wide dynamic range, non-nociceptive, nociceptive specific or proprioceptive. The response to tachykinin application was determined for each neurone. Application of each of the tachykinins evoked a characteristic excitatory response which was delayed in onset, slow in developing and prolonged: physalaemin excited 99/131 neurones tested, neurokinin A excited 45/63 neurones and substance P excited 32/49 neurones. With two neurones physalaemin evoked a depression of the rate of firing, which may have been caused indirectly by excitation of a neighbouring neurone. Such depression was not elicited by either substance P or by neurokinin A. Physalaemin had a preferential excitatory effect on nociceptive neurones evoking excitation of 76/94 nociceptive neurones compared with 12/23 non-nociceptive neurones (chi 2 = 7.9, 1 d.f., P = 0.005). Substance P also caused a preferential excitation, with 30/40 nociceptive neurones being excited while all of the non-nociceptive neurones (n = 7) were unaffected (chi 2 = 11.5, 1 d.f., P = 0.0007). In contrast, neurokinin A failed to have a preferential effect; 32/46 nociceptive and 9/10 non-nociceptive neurones were excited (chi 2 = 1.0, 1 d.f., P = 0.40). Comparing the proportions of nociceptive neurones excited by the different tachykinins indicated that this type of neurone was not differently sensitive to any of the three peptides (chi 2 = 3.2, 2 d.f., P = 0.20). On the other hand, non-nociceptive neurones were preferentially excited by neurokinin A and physalaemin compared with substance P (chi 2 = 13.4, 2 d.f., P = 0.001). With regard to the endogenous tachykinins the results of this study may be interpreted in the following ways. The differential excitatory effect of substance P on nociceptive neurones supports the proposed role for this peptide in the transmission specifically of nociceptive inputs at the first afferent synapse. On the other hand, as neurokinin A excited non-nociceptive as well as nociceptive neurones, there may be a functional role for neurokinin A distinct from that of substance P.

Substance P inhibits the acute stimulation of ganglionic tyrosine hydroxylase activity by a nicotinic agonist.

Incubation of rat superior cervical ganglia with dimethylphenylpiperazinium (30 microM) for 30 min resulted in a two-fold increase in tyrosine hydroxylase activity. This effect was completely inhibited by substance P (30 microM) but not by substance P-free acid, kassinin or physalaemin. Neither of these four peptides alone produced any change in the activity of tyrosine hydroxylase. The IC50 for the inhibitory effect of substance P was approximately 3 microM. Substance P did not inhibit the stimulatory effects of bethanechol or vasoactive intestinal peptide on this enzyme activity. Thus substance P, acting at a site which has a different pharmacology than previously characterized substance P receptors, selectively inhibits nicotinic stimulation of tyrosine hydroxylase activity. These data raise the possibility that substance P may modulate the nicotinic regulation of catecholamine synthesis in sympathetic ganglia in vivo.

Sequential activation of the triple excitatory transmission to the circular muscle of guinea-pig colon.

The aim of this study was to resolve the temporal relationships of the triple excitation of the circular muscle of guinea-pig colon that occurs in response to activation of the intrinsic excitatory nerves by using atropine and tachykinin NK1 and NK2 receptor selective antagonists to define the relative contribution of the transmitters involved. In organ bath experiments, performed in the presence of blockers of inhibitory innervation, a train of electrical pulses at 5 Hz for 300 s produced a sustained contraction of the circular muscle of guinea-pig colon: the sequential addition of atropine (1 microM), of the tachykinin NK1 receptor antagonist, SR 140333 (0.3 microM) and of the tachykinin NK2 receptor antagonist, MEN 11420 (1 microM) produced a cumulative inhibitory effect and progressively delayed the onset of the contractile response to nerve stimulation. In the presence of peptidase inhibitors, atropine was less effective in inhibiting the contractile response for prolonged periods of stimulation: however, the pattern of inhibition of the evoked response produced by the sequential addition of blocker drugs was not substantially affected. The selective tachykinin NK3 receptor agonist, senktide, produced a concentration-dependent contraction of guinea-pig colon. The sequential addition of atropine (1 microM), SR 140333 (0.3 microM) and MEN 11420 (1 microM) reproduced the effect of the same drugs on the response to electrical nerve stimulation. The peptide blocker of N-type voltage-dependent calcium channels, omega-conotoxin (0.1 microM) produced a partial inhibitory effect of the response to senktide. The omega-conotoxin-resistant response to 1 microM senktide was inhibited and delayed by the progressive application of atropine, SR 140333 and MEN 11420, similar to the effect observed in the absence of omega-conotoxin. In sucrose gap, single-pulse electrical field stimulation produced a fast excitatory junction potential which was largely (90%) inhibited by atropine; application of a low concentration of the potassium channel blocker, 4-aminopyridine (30 microM), markedly enhanced the atropine-resistant excitatory junction potential which was abolished by the NK1 receptor antagonist, GR 82334. We conclude that, during prolonged electrical or chemical stimulation of excitatory motorneurons, there is a sequential, time-dependent activation of the three excitatory mechanisms in the circular muscle of guinea-pig colon: the pattern of activation is relatively independent of the intensity of stimulation and/or the mechanisms of secretion of released transmitters. Postjunctional factors predominate in determining the relative contribution of the three transmitters, acetylcholine, substance P and neurokinin A, in producing excitation of the circular muscle.

Tachykinins enhance the depression of spinal nociceptive neurons caused by cutaneously applied vibration in the cat.

The present investigation was prompted by previous studies in our laboratory which have indicated that tachykinins enhance depressant effects of purines and that the purine adenosine mediates a vibration-induced depression of nociceptive dorsal horn neurons. Extracellular recordings were made from single nociceptive neurons in the lower lumbar segments of anaesthetized cats. Vibration (80 Hz; 2.5-3.5 s every 20-25 s) was applied to the hindlimb using a feedback-controlled mechanical stimulator. The tachykinins physalaemin, substance P and neurokinin A were administered by iontophoresis. Physalaemin was tested on vibration-induced responses of 29 neurons; each neuron was excited by this tachykinin. To control for possible changes in the response to vibration caused by the excitation per se, statistical comparisons were made of the vibration-induced responses during excitation by tachykinins and during excitation by glutamate. In 16 cases the magnitude of the vibration-induced depression was significantly greater during the excitation caused by physalaemin. With the remaining neurons the response to vibration failed to differ during the excitation by physalaemin compared with the excitation by glutamate. In four of the 16 cases subthreshold applications of vibration caused depression after administration of physalaemin. The P1-purinergic (adenosine) antagonist, caffeine, was administered in three cases where vibration caused depression only with application of physalaemin. In each of these cases the depression was reversibly blocked by caffeine (10 or 40 mg kg-1 i.v.). The magnitude of vibration-induced depression was significantly increased during excitation by neurokinin A (5/14 neurons) or by substance P (1/9 neurons). From the results of the present study we suggest that tachykinins enhance the vibration-induced depression. This enhancement may be due to enhanced depression by adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Purine-induced depression of dorsal horn neurons in the cat spinal cord: enhancement by tachykinins.

The neurokinins, physalaemin, substance P, neurokinin A and bradykinin, were tested on the responses of single spinal neurons to the purines, adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate and to GABA. Experiments were done on anaesthetized cats, recording extracellularly from functionally identified sensory neurons in the lumbar dorsal horn. All compounds were administered by iontophoresis. Neurokinins caused a slow, prolonged excitation which outlasted the period of application. Physalaemin was tested on responses to ATP in 24 units. In each case application of ATP caused either depression, excitation or a biphasic response when the application was not pre-conditioned by ejection of physalaemin. For 11 units, with ATP applications subthreshold to alter the on-going firing rate, such applications caused depression when they were preceded by administration of physalaemin. Three units were tested with ATP applications which caused the excitatory response; when the applications of ATP were preceded by ejection of physalaemin, there was then a depressant component in the response. In these 14 cases, the magnitude of the depression or of the depressant component of the response, was measured using currents which failed to produce depression in the absence of physalaemin ejection; the mean magnitude of this depression was 34.7 +/- 1.6% (+/- S.E.M.). With the 10 remaining units, responses to ATP were unaffected by application of physalaemin. The early components of the biphasic and excitatory responses were unaffected by physalaemin and hence it appeared to have a differential effect, enhancing only the depressant effects of ATP. The enhancement of depression was reversible, lasting up to 30 min following a single ejection. Neither control current nor glutamate mimicked the effect of physalaemin in the responses to application of ATP. The depressant response to adenosine 5'-monophosphate was also enhanced by physalaemin: ejections of adenosine 5'-monophosphate subthreshold to affect the on-going firing rate caused depression after physalaemin application in 3 of 8 units (average depression: 35.0 +/- 3.3%). On the other hand, depression induced by GABA was unaffected by physalaemin in every case (n = 8); in 4 of these cases GABA was tested on units for which purine-induced depression was enhanced by physalaemin. Thus, physalaemin preferentially affected depressant responses to ATP and to adenosine 5'-monophosphate.(ABSTRACT TRUNCATED AT 400 WORDS)

Specialization of tachykinin NK1 and NK2 receptors in producing fast and slow atropine-resistant neurotransmission to the circular muscle of the guinea-pig colon.

We studied the relative contribution of tachykinin NK1 and NK2 receptors in producing nonadrenergic noncholinergic excitation of the circular muscle of the guinea-pig proximal colon in response to electrical field stimulation. All experiments were performed in the presence of atropine, guanethidine, indomethacin, apamin and L-nitroarginine. In organ bath experiments, electrical stimulation produced a tetrodotoxin-sensitive frequency-dependent contraction. The NK1 receptor antagonists, FK 888 (1-10 microM) and GR 82,334 (0.3-3 microM) markedly reduced but did not abolish the nonadrenergic noncholinergic response. The NK2 receptor antagonist, GR 94,800 (0.3-3 microM) was partly effective at 3 microM. The combined administration of FK 888 (10 microM) and GR 94,800 (3 microM) or GR 82,334 and GR 94,800 abolished the nonadrenergic noncholinergic contraction. The response to a prolonged period of stimulation (3 Hz for 5 min) was evenly depressed by FK 888 or GR 82,334, while GR 94,800 was more effective in inhibiting the late (87% inhibition) than the peak response (25% inhibition). In the presence of nifedipine (1 microM) a marked inhibition of the nonadrenergic noncholinergic contraction was observed and a time lag was evident between stimulus application and onset of contraction, which showed slow onset and offset kinetics. The nifedipine-resistant nonadrenergic noncholinergic contraction was unaffected by FK 888 or GR 82,334 but was suppressed by GR 94,800. Submaximally effective (1-3 nM) concentrations of substance P and neurokinin A produced distinct patterns of contraction: the response to substance P was fast and declined rapidly toward baseline; the response to neurokinin A was slow and sustained. In the presence of nifedipine, the response to substance P was greatly depressed and became slower in onset; nifedipine did not affect the contraction to neurokinin A but slowed its time-course. In sucrose gap experiments, either a short (10 Hz for 1 s) or a prolonged period of electrical stimulation (3 Hz for 3 min) evoked membrane depolarization, action potentials and contraction: in response to the "prolonged" stimulation, distinct phasic and tonic component of contraction were observed. Nifedipine abolished action potentials and the phasic contraction produced by a short period of stimulation, reduced by about 50% the maximal contraction developed during the prolonged stimulation without affecting the amplitude of the tonic response. In the presence of nifedipine, GR 82,334 (3 microM) blocked the membrane depolarization but did not affect contraction; GR 94,800 (0.1 microM) did not affect depolarization but abolished contraction.(ABSTRACT TRUNCATED AT 400 WORDS)

NK-1 receptor mediation of neurogenic plasma extravasation in rat skin.

1. Plasma extravasation was induced by electrical nerve stimulation and by perfusion of tachykinins over a vacuum-induced blister base on rat footpad. 2. Stimulation of the sciatic nerve (18 V, 15 Hz, 0.5 ms) for 20 min produced a significant increase in the protein content of the perfusate. The response in capsaicin pretreated rats was only 4% of the control response. This indicates that the electrically-induced plasma extravasation response was mediated by capsaicin-sensitive sensory fibres. 3. Exogenous perfusion of the mammalian tachykinins substance P, neurokinin A and neurokinin B and the non-mammalian tachykinins physalaemin, kassinin and eledoisin was used to determine the tachykinin receptor type mediating the plasma extravasation response. Dose-response curves of the tachykinins (10(-9) M-10(-4) M) gave a rank order of potency of substance P = physalaemin greater than eledoisin greater than or equal to kassinin greater than neurokinin B = neurokinin A. 4. In addition, specific agonists of neurokinin receptors were perfused. Perfusion of [Glp6, D-Pro9] SP6-11 and [Glp6, L-Pro9]SP6-11 demonstrated that the L-Pro isomer was much more potent than the D-Pro isomer. 5. The rank order of potency and the greater potency of [Glp6, L-Pro9]SP6-11 over its D-isomer indicate an NK-1 neurokinin receptor mediates plasma extravasation in rat footpad skin.

Contractile effects of substance P and other tachykinins on the mouse isolated distal colon.

1. Substance P (SP), physalaemin, eledoisin and kassinin induced concentration-related contractions of the longitudinal muscle of the mouse distal colon. The responses were not antagonized by atropine (1.5 x 10(-7) M), mepyramine (2.5 x 10(-7) M), methysergide (5 x 10(-7) M), timolol (10(-6) M), phentolamine (10(-6) M) or naloxone (4 x 10(-7) M). They were enhanced by tetrodotoxin (TTX, 1.5 x 10(-7) M). These observations indicate that the contractile responses to the tachykinins result from a direct activation of smooth muscle cells. 2. The contractile activity provoked by SP and physalaemin was inhibited by nifedipine (a Ca2+-entry blocker) and was abolished in Ca2+-free EGTA solution. Such data suggest that the myogenic effects of SP and physalaemin are mainly dependent on their ability to promote Ca2+ influx. 3. Eledoisin and kassinin evoked a contractile response in the absence of external Ca2+ and their myogenic activity was, to some extent, resistant to the inhibitory effect of nifedipine. This may indicate that an additional process, probably the release of an intracellularly bound Ca2+ store, participates in the mechanism by which eledoisin and kassinin contract the mouse distal colon. 4. After desensitization of the mouse distal colon to SP, the contractile activity provoked by SP or physalaemin was totally abolished whilst the responses evoked by eledoisin and kassinin were barely affected. These observations and other experimental findings indirectly support the assumption that the mouse distal colon could possess different tachykinin-binding sites.

Characterization of receptors mediating contraction induced by tachykinins in the guinea-pig isolated common bile duct.

1. We studied the effect of the natural tachykinins and of synthetic agonists selective for the tachykinin NK1, NK2 and NK3 receptors, on the motility of guinea-pig isolated common bile duct longitudinally-oriented smooth muscle. 2. All the tachykinins tested (both natural and synthetic) produced a concentration-dependent contractile response of the guinea-pig isolated common bile duct: these effects underwent a marked tachyphylaxis, especially the responses elicited by NK1 and NK3 receptor-selective agonists. 3. Among the natural tachykinins neurokinin B (EC50 = 3.2 nM; 95% c.l. = 2.0-5.1; n = 4) was the most potent, being about 40 and 25 fold more potent than substance P (EC50 = 121.6 nM; 95% c.l. = 94-157; P < 0.01; n = 4) and neurokinin A (EC50 = 83.4 nM; 95% c.l. = 62-112; P < 0.01; n = 4), respectively. Among the synthetic analogues the NK3 receptor-selective agonist senktide (EC50 = 1.1 nM; 95% c.l. = 0.7-1.8; n = 8) was the most potent, being about 120, 110 and 20 fold more potent than [Sar9]substance P sulfone (NK1 receptor-selective) (EC50 = 130.4 nM; 95% c.l. = 99-172; P < 0.01; n = 8), [beta Ala8]NKA (4-10) (NK2 receptor-selective) (EC50 = 120.1 nM; 95% c.l. = 95-151; P < 0.01; n = 8) and septide (NK1 receptor-selective) (EC50 = 22.6 nM; 95% c.l. = 18-28; P < 0.01; n = 8), respectively. All tachykinins (natural or synthetic receptor agonists) produced a similar Emax, averaging about 50% of that produced by KCl (80 mM). 4. Atropine (1 microM) did not affect the responses to either NK1 or NK2 receptor-selective agonists, whereas it reduced the Emax of senktide by about 50%, without affecting its potency (EC50). Tetrodotoxin (1 microM) totally blocked senktide-induced contractions, as did the combined pretreatment with atropine plus the tachykinin NK1 and NK2 receptor-selective antagonists GR 82334 and MEN 11420 (1 microM each), respectively. 5. GR 82334 (1 microM) blocked with apparent competitive kinetics septide- (apparent pKB = 7.46 +/- 0.10; n = 5) and [Sar9]substance P sulfone- (apparent pKB = 6.80 +/- 0.04; n = 4) induced contractions. MEN 11420 (30-300 nM), a novel potent NK2 receptor antagonist, potently antagonized [beta Ala8]NKA (4-10), with competitive kinetics (pKB = 8.25 +/- 0.08; n = 12: Schild plot slope = -0.90; 95% c.l. = -1.4; -0.35). The NK3 receptor-selective antagonist SR 142801 (30 nM) produced insurmountable antagonism of the senktide-induced contractions (Emax inhibited by 64%). None of the above antagonists, tested at the highest concentrations employed against tachykinins, affected the concentration-response curve to methacholine (0.1-300 microM). 6. We conclude that tachykinins produce contraction of the guinea-pig isolated common bile duct by stimulating NK1, NK2 and NK3 receptors. The responses obtained by activating NK1 and NK2 receptors are atropine-resistant. The contraction obtained by stimulating NK3 receptors is totally neurogenic, being mediated by the release of endogenous acetylcholine and tachykinins; the latter act, in turn, on postjunctional tachykinin NK1/NK2 receptors. The role of the NK3 receptor as prejunctional mediator of the excitatory transmission operated by tachykinins is discussed.

Contractile responses to substance P and related peptides of the isolated muscularis mucosae of the guinea-pig oesophagus.

The site of action of substance P and related tachykinins with respect to isotonic contractions was examined on the isolated muscularis mucosae attached to the submucous plexus of the guinea-pig oesophagus. Substance P (greater than 30 nM) produced a concentration-dependent contraction of the muscularis mucosae (EC50 1.9 +/- 0.5 microM, n = 10). The contractions were rapid in onset (2 min or less), sustained, reversible by washing and the preparation did not show tachyphylaxis. Eledoisin and physalaemin produced similar sustained contraction of the muscularis mucosae. The order of sensitivity was eledoisin greater than substance P greater than physalaemin. Contractions induced by 1 microM of each tachykinin were not significantly modified by incubation of the tissue with substance P or eledoisin (10 microM for 30 min). The contractile responses to tachykinins were unaffected by tetrodotoxin (0.3 microM), atropine (0.3 microM), phentolamine (1 microM), chlorpheniramine (1 microM), methysergide (1 microM), baclofen (100 microM) and verapamil (10 microM), but were abolished by the incubation of the tissue with calcium-free, EGTA (0.1 mM)-containing Tyrode solution. A substance P antagonist, [D-Pro2, D-Trp7,9]-substance P (greater than 0.1 microM), produced a transient contraction of the muscularis mucosae and the smooth muscle regained its original tone within 6 to 10 min. Contractions to the tachykinins were now inhibited by the antagonist (0.1-10 microM) in a concentration-dependent manner, the order of sensitivity being physalaemin greater than substance P = eledoisin. The cholinergically mediated electrically (0.1 Hz, 0.5 ms, supramaximal voltage)-induced twitch contractions of the muscularis mucosae were not significantly modified by substance P (0.01-0.3 microM). 7 The present results indicate that substance P and related tachykinins contract the isolated muscularis mucosae of the guinea-pig oesophagus by a direct action on the smooth muscle, probably by stimulating SP-E receptors.

Hyperalgesia produced by intrathecal substance P and related peptides: desensitization and cross desensitization.

The hyperalgesic effect of intrathecally administered substance P (SP), physalaemin, eledoisin and eledoisin-related peptide (ERP) was investigated in the rat tail flick test. Hyperalgesia produced by SP (2.5-15 micrograms, 1.9-11 nmol) was maximal 10-20 min after injection, lasted 30 min and was dose-related. The effect was mimicked by all of the peptides examined. The rank order of potency was physalaemin greater than SP greater than eledoisin greater than ERP. Desensitization to the hyperalgesic effect of SP was produced by three repeated intrathecal injections. Rats desensitized to SP no longer responded to physalaemin or ERP, indicating cross-desensitization. Phentolamine continued to produce hyperalgesia following such desensitization. The demonstration of a hyperalgesic effect for SP provides further support for a role for SP in nociceptive transmission. The receptor mediating this effect appears to be a SP-P subtype. Cross-desensitization between peptides suggests an action on the same receptor.

Neurokinin-induced changes in pial artery diameter in the anaesthetized guinea-pig.

1. The effects of selective neurokinin agents on pial artery diameter, measured with an on-line image analyser, have been studied in anaesthetized guinea-pigs in order to characterize the neurokinin receptors present on pial arteries. 2. Perivascular injection of either substance P (0.01-1 microM) or the selective NK1 receptor agonists, substance P methyl ester (SPOMe, 0.01-1 microM) and GR73632 (0.1 microM), increased pial artery diameter. 3. In contrast, the selective NK2 receptor agonist, GR64349 (1 microM), produced a small vasoconstriction while the NK3 receptor-selective agonist, senktide (1 microM) was inactive. 4. Co-administration of GR82334 (1 microM), a selective NK1 receptor antagonist, inhibited the vasodilatation produced by SPOMe (0.1 microM) but not that caused by calcitonin gene-related peptide (CGRP, 0.01 microM). 5. The results are consistent with an involvement of NK1 receptors in the neurokinin-induced increase in guinea-pig pial artery diameter.