Tachykinin NK1 but not NK2 receptors mediate non-cholinergic excitatory junction potentials in the circular muscle of guinea-pig colon

1. The effect of tachykinin NK1 and NK2 receptor antagonists on noncholinergic excitatory junction potentials (e.j.ps) evoked by electric field stimulation (EFS) in the circular muscle of the guinea-pig proximal colon was investigated by means of a sucrose-gap technique. 2. In the presence of 1 microM atropine, submaximal EFS (10 Hz, 20-30 V, 0.5 ms pulse width, 1 s train duration) evoked an inhibitory junction potential (i.j.p.) followed by e.j.p. with superimposed action potentials (APs) and contraction. Addition of either NG-nitro-L-arginine (L-NOARG, 0.1 mM) or apamin (0.1 microM) inhibited the evoked i.j.p. and the combined administration of the two agents almost abolished it. In the presence of both L-NOARG and apamin, an atropine-resistant e.j.p. was the only electrical response evoked by EFS in 50% of cases and a small i.j.p. (10% of original amplitude) followed by e.j.p. was evident in the remainder. 3. In the presence of L-NOARG and apamin, the tachykinin NK1 receptor antagonists, (+/-)-CP 96,345 and GR 82,334 (10 nM-3 microM) concentration-dependently inhibited the atropine-resistant e.j.p. and accompanying contraction evoked by EFS. EC50 values were: 0.77 microM (e.j.p. inhibition) and 0.22 microM (inhibition of contraction) for (+/-)-CP 96,345; 0.61 microM (e.j.p. inhibition) and 0.20 microM (inhibition of contraction) for GR 82,334. The tachykinin NK2 receptor antagonists, MEN 10,376 (up to 3 microM) and SR 48,968 (up to 1 microM) had no effect on the atropine-resistant e.j.p. MEN 10,376 (3 microM) but not SR 48,968 produced a slight inhibition of the evoked contraction. 4. (+/- )-CP 96,345 (3 microM) and GR 82,334 (3 microM) markedly reduced (81 and 89% inhibition, respectively)the atropine-resistant ej.p. in the absence of L-NOARG and apamin, without affecting the ij.p. MEN 10,376 (3 microM) and SR 48,968 (1 microM) had no significant effect on noncholinergic ij.p. and ej.p. evoked in the absence of apamin and L-NOARG.5. The electrical and mechanical responses to the NK, receptor agonist [Sar9]substance P (SP) sulfone were blocked by (+/-)-CP 96,345 (3 1M) or GR 82,334 (3 microM) which, at the same concentration, failed to affect the responses to the NK2 receptor agonist [PAla8] neurokinin A (NKA) (4-10). In contrast, MEN10,376 (3 microM) or SR 48,968 (1 microM) blocked the response to [beta Ala8]NKA(4-10) without affecting the response to [Sar9]SP sulfone.6. In the presence of L-NOARG and apamin, and in the absence of atropine, EFS of low pulse width(0.02-0.03 ms, other parameters as above) produced cholinergic ej.ps and contraction which were unaffected by GR 82,334 (3 microM). (+/-)-CP 96,345 (3 JAM) produced 24% reduction in the area of the atropine-sensitive ej.p. without affecting the peak amplitude of ej.p. or contraction.7. These findings demonstrate that the noncholinergic ej.ps and accompanying contraction of the circular muscle of the guinea-pig colon are produced through activation of intramural tachykininergic nerves and that the resultant smooth muscle response is almost entirely mediated through NK1 receptors.     

Characterization of tachykinin NK2 receptor on dog proximal colon. Antagonism by MEN 10,627 and SR 48,968

The nature of the tachykinin receptors involved in the contraction of the circular muscle of dog colon has been investigated. The following rank order of potency for agonists was obtained: [Sar9,Met(O2)11]substance P > or = neurokinin A > [beta-Ala8]neurokinin A-(4-10) > [MePhe7]neurokinin B. The efficacy of the tachykinin NK2 receptor agonists was significantly greater than that of the tachykinin NK1 receptor agonists and of carbachol. A concentration-dependent rightward shift of the motor response to neurokinin A (obtained in the presence of (+/-)-CP 96,345) was induced by peptide and non-peptide tachykinin NK2 receptor antagonists with this rank order: MEN 10,627 = SR 48,968 > L 659,877 > MEN 10,376 > MDL 28,564. MEN 10,627 and SR 48,968 affinities were similar to those measured in human tissues. In conclusion, the tachykinin NK2 receptor plays a predominant role in tachykinin-induced contraction of the canine colonic circular muscle and this tissue could be useful to predict the pharmacological actions of MEN 10,627 and SR 48,968 in human colon.     

Intercostal pedicle flap for thoracic oesophageal perforations

Since exogenously applied tachykinins (substance P and neurokinin A) prevent the neurogenic hyperaemia which is elicited by acid back-diffusion in the rat stomach, we investigated whether endogenous tachykinins would act in a similar manner. Acid back-diffusion, induced by perfusing the stomach with 15% ethanol in the presence of 0.05 M HCI, increased gastric mucosal blood flow (GMBF) by 60-100% as determined by hydrogen clearance in urethane-anaesthetized rats. This response remained unchanged after pretreatment with the tachykinin NK1 receptor antagonist SR 140,333 (300 nmol/kg) but tended to be enhanced by the NK2 receptor antagonist MEN 10,627 (200 nmol/kg). When given during ongoing acid back-diffusion, MEN 10,627 significantly enhanced the acid-evoked vasodilatation as compared with vehicle or SR 140,333. We conclude that endogenously released tachykinins, acting via NK2 receptors, limit the gastric hyperaemic response to acid.     

Inhibition of nitric oxide synthesis reveals non-cholinergic excitatory neurotransmission in the canine proximal colon

1. Neuromuscular transmission in the circular muscle of the canine proximal colon was examined, in the presence and absence of nitric oxide synthase inhibitors, by use of mechanical and intracellular microelectrode recording techniques. 2. Electrical field stimulation (EFS; 0.1-20 HZ) produced frequency-dependent contractions of circular muscle strips which reached a maximum at 15 Hz. These responses were enhanced by NG-monomethyl-L-arginine (L-NMMA; 300 microM) and reduced by atropine (1 microM). The effects of L-NMMA were reversed by L-arginine (3 mM). All responses to EFS were abolished by tetrodotoxin (1 microM). 3. In the presence of atropine, phentolamine and propranolol (all at 1 microM; 'non-adrenergic, non-cholingergic (NANC) conditions'), EFS evoked frequency-dependent inhibition of phasic contractions which reached a maximum at 5 Hz. At higher frequencies of EFS, inhibition diminished, and these responses were followed by post-stimulus excitation. 4. Under NANC conditions and in the presence of L-NG-nitroarginine methyl ester (L-NAME; 200 microM), EFS evoked contractions at frequencies of 5 Hz or greater. These contractions were reduced by co-incubation with L-arginine (2 mM) and abolished by tetrodotoxin (1 microM). 5. In the presence of atropine (1 microM), EFS (5-20 Hz) caused frequency-dependent inhibition of electrical slow waves. In the presence of L-NAME (100 microM) and atropine, the inhibitory response to EFS was abolished and an increase in slow wave duration was seen at stimulation frequencies greater than 5 Hz. The effects of EFS on slow wave duration were abolished by tetrodotoxin (1 microM). 6. Atropine-resistant contractions to EFS were enhanced by indomethacin (10 microM) and reduced or abolished by the non-selective NK1/NK2 tachykinin receptor antagonist D-Pro2, D-Trp7,9 SP, and by the selective NK2 receptor antagonist MEN 10,376 (10 microM).7. Exogenous tachykinins mimicked non-cholinergic excitatory electrical and mechanical responses. The rank order of potency for contraction was neurokinin A>neurokinin B>substance P, suggesting a predominance of the NK2 sub-type of tachykinin receptors on colonic smooth muscle cells. Low concentrations of neurokinin A also increased the amplitude and duration of electrical slow waves.8. These results suggest that: (i) in previous studies, non-cholinergic excitatory responses were masked by the simultaneous release of NO; (ii) non-cholinergic excitatory responses occur throughout the period of stimulation and are not manifest only as 'rebound' excitation; (iii) one or more tachykinins, possibly,acting via NK2 receptors, may mediate non-cholinergic excitatory responses.     

Excitatory motor and electrical effects produced by tachykinins in the human and guinea-pig isolated ureter and guinea-pig renal pelvis

1. In isolated tissue experiments, neurokinin A (NKA) produced concentration-dependent contraction of human and guinea-pig ureter (pD2 = 6.7 and 7.2, respectively); an effect greatly reduced (>80% inhibition) by the tachykinin NK2 receptor-selective antagonist MEN 11420 (0.1 microM). The tachykinin NK1 and NK3 receptor agonists septide and senktide, respectively, were ineffective. 2. Electrical field stimulation (EFS) of the guinea-pig isolated renal pelvis produced an inotropic response blocked by MEN 11420 (0.01-1 microM). In the same preparation MEN 11420 (0.1 microM) blocked (apparent pK(B) = 8.2) the potentiation of spontaneous motor activity produced by the NK2 receptor-selective agonist [betaAla8]NKA(4-10). 3. In sucrose-gap experiments, EFS evoked action potentials (APs) accompanied by phasic contractions of human and guinea-pig ureter, which were unaffected by tetrodotoxin or MEN 11420 (3 microM), but were blocked by nifedipine (1-10 microM). NKA (1-3 microM) produced a slow membrane depolarization with superimposed APs and a tonic contraction with superimposed phasic contractions. NKA prolonged the duration of EFS-evoked APs and potentiated the accompanying contractions. MEN 11420 completely prevented the responses to NKA in both the human and guinea-pig ureter. 4. Nifedipine (1-10 microM) suppressed the NKA-evoked APs and phasic contractions in both human and guinea-pig ureter, and slightly reduced the membrane depolarization induced by NKA. A tonic-type contraction of the human ureter in response to NKA persisted in the presence of nifedipine. 5. In conclusion, tachykinins produce smooth muscle excitation in both human and guinea-pig ureter by stimulating receptors of the NK2 type only. NK2 receptor activation depolarizes the membrane to trigger the firing of APs from latent pacemakers.     

Evidence that tachykinins are the main NANC excitatory neurotransmitters in the guinea-pig common bile duct

Application of electrical field stimulation (EFS; trains of 10 Hz, 0.25 ms pulse width, supramaximal voltage for 60 s) to the guinea-pig isolated common bile duct pretreated with atropine (1 microM), produced a slowly-developing contraction ('on' response) followed by a quick phasic 'off' contraction ('off peak' response) and a tonic response ('off late' response), averaging 16+/-2, 73+/-3 and 20+/-4% of the maximal contraction to KCl (80 mM), n=20 each, respectively. Tetrodotoxin (1 microM; 15 min before) abolished the overall response to EFS (n 8). Neither in vitro capsaicin pretreatment (10 microM for 15 min), nor guanethidine (3 microM, 60 min before) affected the excitatory response to EFS (n 5 each), showing that neither primary sensory neurons, nor sympathetic nerves were involved. Nomega-nitro-L-arginine (L-NOARG, 100 microM, 60 min before) or naloxone (10 microM, 30 min before) significantly enhanced the 'on' response (294+/-56 and 205+/-25% increase, respectively; n=6-8, P<0.01) to EFS. The combined administration of L-NOARG and naloxone produced additive enhancing effects (655+/-90% increase of the 'on' component, n = 6, P<0.05). The tachykinin NK2 receptor-selective antagonist MEN 11420 (1 microM) almost abolished both the 'on' and 'off late' responses (P<0.01: n=5 each) to EFS, and reduced the 'off-peak' contraction by 55+/-8% (n=5, P<0.01). The subsequent administration of the tachykinin NK1 receptor-selective antagonist GR 82334 (1 microM) and of the tachykinin NK3 receptor-selective antagonist SR 142801 (30 nM), in the presence of MEN 11420 (1 microM), did not produce any further inhibition of the response to EFS (P>0.05; n=5 each). At 3 microM, GR 82334 significantly reduced (by 68+/-9%, P<0.05, n=6) the 'on' response to EFS. The contractile 'off peak' response to EFS observed in the presence of both MEN 11420 and GR 82334 (3 microM each) was abolished (P<0.01; n=6) by the administration of the P2 purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM). PPADS (30 microM) selectively blocked (75+/-9 and 50+/-7% inhibition, n = 4 each) the contractile responses produced by 100 and 300 microM ATP. Tachykinin-containing nerve fibres were detected by using immunohistochemical techniques in all parts of the bile duct, being distributed to the muscle layer and lamina propria of mucosa. In the terminal part of the duct (ampulla) some labelled ganglion cells were observed. In conclusion, this study shows that in the guinea-pig terminal biliary tract tachykinins, released from intrinsic neuronal elements, are the main NANC excitatory neurotransmitters, which act by stimulating tachykinin NK2 (and possibly NK1) receptors. ATP is also involved as excitatory neurotransmitter. Nitric oxide and opioids act as inhibitory mediators/modulators in this preparation.     

Are inner or outer hair cells the source of summating potentials recorded from the round window?

Previous studies indicated that antidromic stimulation of capsaicin-sensitive vagal afferent fibers activated, via peripheral release of tachykinins, nonadrenergic, noncholinergic parasympathetic ganglion neurons that mediate relaxations of guinea pig trachealis. On the basis of the effects of selective agonists and inhibition with a nonselective receptor antagonist (SR 48968), we speculated that tachykinin-mediated activation of neurokinin3 (NK3) receptors might be involved. Using the recently developed NK3-selective receptor antagonist SR 142801, we further assessed the role of NK3 receptors in these relaxant responses. Relaxations of the guinea pig trachea elicited by antidromic stimulation of capsaicin-sensitive vagal afferent nerves were markedly inhibited by 0.3 microM SR 142801 and were abolished by a combination of SR 142801 and either of the NK1-selective receptor antagonists SR 140333 and CP 99994 (0.3 microM each). The NK3 receptor antagonist had similar effects on the relaxant responses elicited by capsaicin and substance P, but it had no effect on relaxations of the trachealis elicited by electrical field stimulation of the postganglionic nerves that innervate the trachealis or by stimulation of the preganglionic parasympathetic vagal nerves that innervate the trachea. These results and the observation that the ganglion neurons that mediate these responses are densely innervated by substance P-containing nerve fibers lead us conclude that stimulation of capsaicin-sensitive visceral afferent fibers activates, upon peripheral release of tachykinins, nonadrenergic, noncholinergic inhibitory neurons innervating guinea pig trachealis via activation of both NK3 and NK1 receptors.     

Stimulation of airway sensory nerves by cyclosporin A and FK506 in guinea-pig isolated bronchus

We have investigated the contractile property of cyclosporin A and FK506 in guinea-pig isolated bronchus. Cyclosporin A (10 microM) failed to significantly attenuate the excitatory non-adrenergic non-cholinergic (eNANC) and cholinergic contractile response (per cent methacholine Emax) induced by electrical field stimulation (EFS). In contrast, eNANC responses were significantly attenuated by both the neurokinin (NK)-1 and (NK)-2 receptor antagonists, N-acetyl-L-tryptophan 3,5-bis (trifluoromethyl)-benzyl and SR48968, respectively. Cyclosporin A and FK506 caused a concentration-dependent contraction in guinea-pig isolated bronchus, which was significantly attenuated by NK-1 and NK-2 receptor antagonists. The capsaicin receptor antagonist, capsazepine (10 microM) significantly reduced the contractile response to cyclosporin A and capsaicin, but not to FK506. The N-type calcium channel blocker, omega-Conotoxin (omegaCTX: 10 nM), significantly reduced the contractile response to FK506 and the eNANC response following EFS. In contrast, omega-CTX failed to significantly reduce the contractile potency to capsaicin or cyclosporin A. In bronchial preparations desensitized by repeated application of capsaicin (1 microM), the contractile responses to both cyclosporin A (100 microM) and FK506 (100 microM), were significantly reduced. In contrast, the contractile responses to substance P and neurokinin A (10 microM) were not altered. Furthermore, repeated application of cyclosporin A (100 microM) significantly inhibited the contractile response to capsaicin (1 microM). The findings from this study would indicate that cyclosporin A and FK506 mediate contraction of guinea-pig isolated bronchus secondary to the release of neuropeptides from airway sensory nerves. However, the release of sensory neuropeptides appears to be mediated via different mechanisms for cyclosporin A and FK506, the former by stimulation of the vanilloid receptor and the latter via opening of N-type calcium channels.     

Nutritional assessment of folate and cyanocobalamin status in a Spanish elderly group

The mammalian tachykinins, neurokinin A (NKA) and NKA(4-10), along with the tachykinin NK2 receptor-selective antagonist MEN 10,376, were compared to their C-terminal free acid derivatives, NKA-OH, NKA(4-10)-OH and MEN 10,456, respectively, on several in vitro bioassays for NK1, NK2 and NK3 tachykinin receptors. NKA-OH and NKA(4-10)-OH were much weaker agonists than NKA or NKA(4-10) in the endothelium-deprived rabbit pulmonary artery (endowed with NK2A receptors) and in the guinea pig isolated bronchus (endowed with NK2A and NK1 receptors), where they produced submaximal contractile responses, and were inactive in the hamster isolated trachea (endowed with NK2B receptors) and in the rat isolated portal vein (endowed with NK3 receptors). At NK1 receptors of the guinea pig isolated ileum, NKA-OH produced weak agonist responses, whereas NKA(4-10)-OH was ineffective. In sharp contrast, MEN 10,456, while maintaining the same antagonist potency of the parent compound MEN 10,376 in the rabbit pulmonary artery and hamster isolated trachea, developed a clear-cut agonist character in the rat isolated portal vein, guinea pig isolated ileum and guinea pig isolated bronchus. The agonist responses produced by MEN 10,456 (10 microM) were reduced by MEN 10,376 in the guinea pig isolated bronchus and by the NK1 receptor antagonist GR 82,334 in the guinea pig isolated ileum. These results, although indicating the importance of C-terminal amidation for the agonist activity of natural tachykinins, suggest that the C-terminal amide group may not be directly involved in stimulation of the tachykinin receptors, but could induce agonist activity through a conformation effect.     

Tachykinins and reflexly evoked atropine-resistant motility in the guinea pig colon in vivo

Distension of a balloon placed in the proximal colon of anesthetized, guanethidine- and naloxone-pretreated guinea pigs elicited a series of long-lasting regular phasic pressure waves which were suppressed by hexamethonium. Activity evoked by a low degree of balloon distension was largely, but not completely, suppressed by atropine. Further balloon distension in atropine-treated animals enabled us to study the effect of tachykinin receptor antagonists on the atropine-resistant and hexamethonium-sensitive response to distension. The selective tachykinin receptor antagonists, (+/-)-CP 96,345 for the NK-1 receptor and L 659,877, MEN 10,376 and SR 48,968 for the NK-2 receptor, inhibited with varying potency the atropine-resistant response to distension. These antagonists also blocked the contraction of the guinea pig colon produced by the i.v. administration of selective NK-1 and NK-2 receptor agonists. In vitro experiments, using mucosa-free circular muscle strips from the guinea pig colon, proved the existence of functional NK-1 and NK-2 receptors in this tissue. We conclude that both NK-1 and NK-2 receptors participate in the atropine-resistant reflex contractions produced by localized balloon distension of the guinea pig colon in vivo.     

Pharmacological characterization of tachykinin-induced intracellular calcium rise in a human NK2 receptor transfected cell line

We have examined the effect of various natural and synthetic tachykinins on the steady state Ca(++)-rise ([Ca++]i) in transfected chinese hamster ovary cells expressing recombinant human Neurokinin 2 (NK2) receptors. The rank order of potency with natural tachykinins was NeurokininA > Neurokinin B > Eledoisin > Physaelamin > substance P. The selective NK2 agonist, [beta-Ala8]NKA(4-10) was very potent, with an EC50 value of 4.83 x 10(-9) M whereas Senktide, MePhe7NKB and Sar9, (MetO2)11 substance P, selective NK3 and NK1 agonists, respectively, did not have any effect on [Ca++]i in hrNK2CHO cells, suggesting a selective and preferential recognition and activation of NK2 receptors in these cells. (+/-) SR 48968, a selective NK2 antagonist, abolished the beta-AlaNKA-induced [Ca++]i with an IC50 value of 0.7 nM. Two other peptidic NK2 antagonists, MEN 10376 and L-658977, were less active with IC50 values of 49 nM and 5.29 microM, respectively. In contrast, (+/-) CP-96,345 and (+/-)CP-99,994 and RP 67580, all selective NK1 antagonists, did not have any effect on the beta-AlaNKA-induced [Ca++]i in hrNK2CHO cells (+/-) SR 140333, a potent and selective NK1 antagonist, had a 35% inhibition under similar conditions. These data demonstrate a high selectivity and sensitivity to NK2 receptor mediated [Ca++]i in rhNK2R-CHO cells and may be of value as a rapid, selective test of drug action at the human NK2 receptors in vitro.     

The arterial blood supply of the human patella. Its clinical importance for the operating technique in vascularized knee joint transplantations

1 This study aimed to assess the effect of cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum calcium (Ca) pump, against contractile responses produced by selective tachykinin NK1 and NK2 receptor agonists, [Sar9]substance P (SP) sulfone and [beta Ala8]neurokinin A (NKA) (4-10), respectively, on the circular muscle of guinea-pig colon. All experiments were performed in the presence of atropine (1 microM) and indomethacin (10 microM). 2 In organ bath experiments, a submaximally equieffective concentration of the two agonists (10 nM) was selected: [Sar9]SP sulfone (10 nM) produced a biphasic contraction, the two amplitudes averaging 75 +/- 2 and 43 +/- 3% of the maximal response to KCl (80 mM) at 1 and 15 min from application of the agonist, respectively. CPA (3 microM for 60 min) slightly reduced the phasic response to [Sar9]SP sulfone (16 +/- 4% inhibition) and markedly suppressed the tonic component (89 +/- 3% inhibition). 3 The contraction produced by [beta Ala8]NKA (4-10) (10 nM) was more sustained than that induced by the NK1 receptor agonist: it averaged 69 +/- 5 and 73 +/- 4% of the response to KCl at 1 and 15 min from application of the agonist, respectively. CPA slightly and evenly depressed the response to [beta Ala8]NKA (4-10) (18 +/- 7 and 21 +/- 5% inhibition at 1 and 15 min). 4 In the presence of tachykinin NK1 and NK2 receptor antagonists (SR 140333 and MEN 10627, respectively, 1 microM each) and of L-nitroarginine (100 microM), KCl (40 mM) produced a distinct phasic and tonic contraction which was suppressed by 1 mM nifedipine. CPA (3 microM) did not affect the phasic contraction to KCl but abolished the tonic component of the response. 5 In the presence of 1 microM nifedipine, the response to [beta Ala8]NKA (4-10) was slightly depressed (32 +/- 6% inhibition) in its early component only, while the response to [Sar9]SP sulfone was abolished. CPA produced a slight inhibition (15 +/- 9 and 33 +/- 10% at 1 and 15 min, respectively) of the nifedipine-resistant response to [beta Ala8]NKA (4-10), an effect similar to that observed in the absence of nifedipine. Therefore, a large part of the response to [beta Ala8]NKA (4-10) persisted in the presence of both CPA and nifedipine. 6 In the sucrose gap, a prolonged superfusion with [Sar9]SP sulfone (0.1 microM for 5 min) produced sustained depolarization with superimposed spikes and contraction. CPA (3 microM) produced transient depolarization and contraction. In the presence of CPA, the initial responses (depolarization, spikes and contraction) to [Sar9]SP sulfone were unaffected but the sustained component of contraction was absent; the latter effect was accompanied by a suppression of spikes while the sustained depolarization was present. 7 We conclude that, during sustained depolarization produced by the NK1 receptor agonist, blockade of the sarcoplasmic reticulum Ca pump by CPA produces a faster Ca-dependent inactivation of Ca channels, thereby eliminating spikes and abolishing the tonic component of contraction. Ca mobilization/reuptake from a CPA-sensitive store seems to be of minor importance for regulating the NK2 receptor-mediated contractile responses.     

Distinct modifications by neurokinin1 (SR140333) and neurokinin2 (SR48968) tachykinin receptor antagonists of the N-methyl-D-aspartate-evoked release of acetylcholine in striosomes and matrix of the rat striatum

The effects of SR140333 and SR48968 (neurokinin1 and neurokinin2 tachykinin receptor antagonists, respectively) on the N-methyl-D-aspartate-evoked release of [3H]acetylcholine (previously formed from [3H]choline) were investigated in striosome-enriched areas and in the matrix of the rat striatum using an in vitro microsuperfusion method. In both striatal compartments, SR140333 and SR48968 did not modify the 50 microM N-methyl-D-aspartate-evoked release of [3H]acetylcholine. However, in low concentrations, both SR140333 (0.1 microM to 1 pM) and SR48968 (0.1 microM to 0.1 nM) markedly enhanced the 1 mM N-methyl-D-aspartate (+10 microM D-serine)-evoked release of [3H]acetylcholine in striosome-enriched areas. These responses were dopamine-dependent since they were not observed any more following the local blockade of D2 receptors by sulpiride or of dopamine synthesis by alpha-methyl-p-tyrosine. A dopamine-dependent disinhibitory effect (of lower amplitude) on the 1 mM N-methyl-D-aspartate (+10 microM D-serine)-evoked release of [3H]acetylcholine was also induced by SR48968 (0.1 microM to 0.1 nM) (but not by SR140333) in the matrix. In addition, in the matrix, as shown only in the presence of alpha-methyl-p-tyrosine, both SR140333 and SR48968 reduced the 1 mM N-methyl-D-aspartate (+10 microM D-serine)-evoked response and these non-dopamine-mediated inhibitory effects only occurred at the highest tested concentration (0.1 microM) of the antagonists. Indicating the specificity of these responses, the effects of SR140333 were reproduced by RP67580, another neurokinin1 receptor antagonist and, as expected from previous binding studies, corresponding SR140333 and SR48968 enantiomers were without effect. These results suggest that under potent stimulation of N-methyl-D-aspartate receptors, endogenously released substance P and neurokinin A (or related tachykinins) regulate differently the N-methyl-D-aspartate-evoked release of [3H]acetylcholine in striosomes and in the matrix. The inhibitory effects of these tachykinins on the evoked release of [3H]acetylcholine are mediated by dopamine. On the contrary, their facilitatory responses are only observed in the matrix under blockade of dopamine transmission.     

Effect of a non-peptide NK-2 tachykinin receptor antagonist on LH, FSH, and prolactin release by rat hemipituitaries in vitro

Tachykinins are present in the anterior pituitary gland and there is evidence that they may have a direct intrapituitary role influencing the secretion of some of the hormones released by this gland. In this investigation, we have studied the effect of the non-peptide NK-2 receptor antagonist SR 48,968 (Sanofi Recherche) on the basal release of LH, FSH, and prolactin by rat hemipituitaries incubated in vitro, and also on the response to GnRH. SR 48,968 significantly inhibited prolactin release into the medium. The highest doses of this compound stimulated the basal release of LH by hemipituitaries from castrated, castrated testosterone-treated, and ovariectomized estradiol-treated rats, but not from intact male rats. SR 48,968 significantly inhibited the release of LH in response to GnRH. Since some tachykinin receptor antagonists have been demonstrated to act also on calcium channels, studies with verapamil, a calcium channel antagonist, were also carried out for comparison. Verapamil inhibited prolactin release into the medium and decreased the LH response to GnRH. These results suggest that tachykinins that bind NK-2 receptors, may have an intrapituitary role stimulating the release of prolactin, and that they may also modulate the response of the gonadotrophs to GnRH. The fact that verapamil shares some of the actions exerted by NK-2 receptor antagonists on the pituitary glandm however, suggests the possibility that some of the effects of NK-2 receptor antagonists may be mediated through calcium channel antagonism. Therefore, the results observed with the use of some of these antagonists should be interpreted with great caution.     

Excitatory transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of cannabinoid CB1 receptors

1. The effect of cannabinoid drugs has been investigated on cholinergic and non-adrenergic non-cholinergic (NANC) contractile responses to the circular smooth muscle of guinea-pig ileum elicited by electrical field stimulation (EFS). 2. The cannabinoid receptor agonist WIN 55,212-2 (1-1000 nM) and the putative endogenous ligand anandamide (0.1-100 microM) both produced a concentration-dependent inhibition of the cholinergic (9-57% and 1-51% inhibition) and NANC (9 55% and 2-57% inhibition) contractile responses. WIN 55,212-2 and anandamide did not modify the contractions produced by exogenous acetylcholine or substance P. 3. Apamin (30 nM), a blocker of Ca2+-activated K+ channels, reduced the inhibitory effect of WIN 55,212-2 on cholinergic, but not NANC, contractile response. NG-nitro-L-arginine methyl ester (100 microM), an inhibitor of nitric oxide synthase, or naloxone (1 microM), an opioid receptors antagonist, did not modify the inhibitory effect of WIN 55,212-2 on both cholinergic and NANC contractions. 4. The inhibitory effects of WIN 55,212-2 and anandamide on both cholinergic and NANC contractile response was competitively antagonized by the cannabinoid CB1 receptor antagonist SR 141716A (10-1000 nM). 5. In absence of other drugs, SR 141716A (1-1000 nM) enhanced cholinergic (1-45% increase) and NANC (2-38% increase) contractile responses elicited by electrical stimulation, but did not modify the contractions produced by acetylcholine or substance P. 6. It is concluded that activation of prejunctional cannabinoid CB1 receptors produces inhibition of cholinergic and NANC excitatory responses in the guinea-pig circular muscle. The inhibition of cholinergic (but not NANC) transmission involves activation of apamin-sensitive K+ channels. In addition, an endogenous cannabinoid ligand could inhibit cholinergic and NANC transmission in the guinea-pig ileal circular muscle.     

Smoke-induced airway hyperresponsiveness to inhaled wood smoke in guinea pigs: tachykininergic and cholinergic mechanisms

The smoke-induced airway hyperresponsiveness (SIAHR) to inhaled wood smoke was investigated in anesthetized guinea pigs. Two smoke challenges (each 10 ml) separated by 30 min were delivered into the lungs by a respirator. In control animals, SIAHR was evidenced by an average bronchoconstrictive response (an increase in total lung resistance) to the second smoke challenge (SM2) that was approximately 4.3-fold greater than that to the first challenge (SM1). Pretreatment with CP-96,345 and SR-48,968 (neurokinin-1 and -2 receptor antagonists; each 1 mg/kg) in combination totally prevented this SIAHR, while pretreatment with CP-96,344 and SR48,965 (inactive enantiomers of CP-96,345 and SR-48,968, each 1 mg/kg) in combination failed to do so. Pretreatment with CP-96,345 (1 mg/kg), SR48,968 (1 mg/kg), or atropine (50 microg/kg) significantly alleviated this SIAHR. Pretreatment with phosphoramidon [an inhibitor of neutral endopeptidase (NEP); 2 mg/kg], which suppresses the degradation of tachykinins, induced an increase in airway reactivity that largely mimicked this SIAHR. The NEP activity measured in airway tissues excised 30 min after SM1 was significantly lower than that in air control value. These results suggest that 1) a prior wood smoke exposure induces an airway hyperresponsiveness to the subsequent wood smoke inhalation, 2) a tachykininergic mechanism involving both neurokinin-1 and -2 receptors is essential for, and a cholinergic mechanism is also involved in the development of this SIAHR, and 3) inactivation of airway NEP by wood smoke may contribute to this SIAHR.     

Acetylcholine and tachykinin receptor antagonists attenuate wood smoke-induced bronchoconstriction in guinea pigs

To study the mechanisms of wood smoke-induced bronchoconstriction, we measured total lung resistance (RL) and dynamic lung compliance (Cdyn) in anesthetized and mechanically ventilated guinea pigs. Airway exposure to various doses of wood smoke (lauan wood; 5, 10, and 15 breaths) resulted in a dose-dependent increase in RL and decrease in Cdyn. The smoke-induced changes in RL and Cdyn were significantly attenuated by pretreatment with atropine, CP-96,345 [(2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-aza bicyclo(2.2.2.)-octan-3-amine; a tachykinin NK1 receptor antagonist], and SR-48,968 [(S)-N-methyl-N(4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophen yl)-butyl)benzamide; a tachykinin NK2 receptor antagonist] in combination, atropine alone, and SR-48,968 alone, but were not significantly affected by pretreatment with the inactive enantiomers of CP-96,345 and SR-48,968, CP-96,345 alone, indomethacin (a cyclooxygenase inhibitor), and MK-571 [((3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl((3-dimethyl amino-3-oxo-propyl)thio)methyl)propanoic acid; a leukotriene D4 receptor antagonist]. The activity of airway neutral endopeptidase, a major enzyme for tachykinin degradation, was not significantly influenced by wood smoke during the development of bronchoconstriction. We conclude that: (1) both cholinergic mechanisms and endogenous tachykinins, but not cyclooxygenase products or leukotriene D4, play an important role in the acute bronchoconstriction induced by wood smoke, and (2) the contribution of tachykinins to this airway response is primarily mediated via the activation of tachykinin NK2 receptors, but is not associated with inactivation of the airway neutral endopeptidase.     

Expression of rat NK-2 (neurokinin A) receptor in E. coli

With the goal of obtaining sufficient functional protein for structural analysis, rat neurokinin-2 receptor was produced in Escherichia coli by linking it to the periplasmic maltose-binding protein. As a first step, we present a biochemical and pharmacological investigation of the recombinant receptor. Western-blots showed that the fusion protein was associated with the membranes. The agonist [4,5-3H-Leu9]neurokinin A and the NK-2 antagonist [3H]SR48,968 bound to the receptor in a highly specific manner. Saturation binding of the [3H]agonist demonstrated a single class of receptors (KD = 10.5 nM, Bmax = 2.5 pmol/mg protein). The [3H]antagonist bound with higher affinity to a larger receptor population (KD = 0.2 nM, Bmax = 7.2 pmol/mg protein). Competition of [3H]agonist binding with other agonists demonstrated a potency order of: neurokinin A > [Nle10]NKA(4-10) = [beta-Ala8]NKA(4-10) > substance P > senktide Against the [3H]antagonist, agonists were only partially inhibitory. Selective NK-2 antagonists inhibited binding of both [3H]ligands with an identical order of potency: SR48,968 > R396 > MEN10,376, which is consistent with NK-2 receptor pharmacology in rat tissue.     

Charge transfer of 0.5-, 1.5-, and 5-keV protons with atomic oxygen: Absolute differential and integral cross sections

We investigated the electrocortical (E.Co.G) correlates of visceral (topical capsaicin application or overdistension of the urinary bladder) and somatic (perineal pinching) painful stimulation in urethane-anesthetized rats and their modulation by intrathecal application of selective tachykinins receptors (NK 1 and NK 2) antagonists. Vesical overdistension or topical capsaicin on the bladder serosal surface produced an immediate and lasting E.Co.G. desynchronization resembling a cortical arousal. A second application of capsaicin was ineffective. Bladder contraction induced by topical acetylcholine did not alter E.Co.G. A desynchronized E.Co.G. was also induced by pinching of the perineal area of the rat. Intrathecal administration of lidocaine at lumbosacral level abolished the E.Co.G. desynchronization induced by both visceral and somatic noxious stimulation. On the other hand capsaicin-induced or over-distension (but not pinching-induced) E.Co.G. desynchronization disappeared in animals systemically pretreated with capsaicin or after intrathecal administration of NK 1 tachykinin receptor antagonists such as the peptide GR 82334 or the nonpeptide RP 67580, whereas the inactive enantiomer RP 68651 or the nonpeptide NK 2 antagonists SR 48968 were ineffective. In conclusion, the experimental model described herein, allowing a quantitative analysis of the E.Co.G. correlates of visceral and somatic noxious stimulation in urethane-anesthetized rats, provides evidence for a specific neural pathway carrying bladder-arising visceral (both mechanical and chemical) nociception that uses pelvic capsaicin-sensitive afferents projecting to NK 1 (but not NK 2) bearing spinal neurons and that ultimately leads to activation of cortical areas.