Effect of early cyclosporine levels on kidney allograft rejection

Experiments were performed on strips of mouse stomach and urinary bladder to characterize the receptors involved in the contractile responses of these tissues to neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and neuropeptide gamma (NP gamma). The neurokinin receptors were characterized by using assays with selective agonists as well as peptide and nonpeptide antagonists and by applying the two Schild criteria for receptor classification, namely, the order of potency of agonists and the apparent affinity of competitive antagonists. The mouse stomach contains primarily NK1 and NK2 functional sites and possibly some NK3 receptors, whereas the urinary bladder possesses only the NK2 receptor. The rank order of potency of agonists in the stomach is Ac[Arg6,Sar9,Met(O2)11]SP-(6-11) > NKA > SP > [beta-Ala8]NKA-(4-10) > NKB > [MePhe7]NKB. Among the selective agonists, Ac[Arg6,Sar9,Met(O2)11]SP-(6-11) is more active than SP and [Sar9,Met(O2)11]SP on the NK1 receptor, whereas the order of potency on the NK2 receptor is NKA > NP gamma > or = [beta-Ala8]NKA-(4-10) > [Nle10]NKA-(4-10). The order of potency of agonists in the bladder is NP gamma > NKA > [beta-Ala8]NKA-(4-10). The myotropic responses mediated by NK1 selective agonists are blocked by SR 140333 (pA2 8.57) and those mediated by the NK2 selective agonists are inhibited by SR 48968 (pA2 9.05). RP 67580 (pA2 8.41) is more active than CP 99994 (pA2 6.06) on the mouse NK1 receptor. The NK1 receptor of the mouse shows, therefore, a pharmacological profile similar to that of the NK1 receptor of the rat. Similarly, MEN 10627 (pA2 9.20) is more active than R 396 (pA2 6.21), suggesting that the mouse NK2 receptor is similar to that of the rabbit. The mouse NK2 receptor of the urinary bladder shows similarity with that of the stomach, but is less sensitive to [beta-Ala8]NKA-(4-10).     

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.     

SR 140333 prevents potentiation by citric acid of plasma exudation induced by histamine in airways

We here report a model of potentiation by citric acid of airway microvascular leakage induced by histamine and its modification by the tachykinin NK1 and NK2 receptor antagonists, SR 140333 ((S)1-{2-[3-(3,4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)p iperidin- 3-yl]ethyl}-phenyl-1-azoniabicyclo[2.2.2]octane, chloride) and SR 48968 (S)-N-methyl-N-[4-(4-acetyl-amino-4-phenylpiperidino)-2-(3,4- dichlorophenyl-butyl]benzamide. Guinea-pigs exposed to an acrosol of citric acid 0.4 M for 1 h developed 24 h later a hyperresponsiveness to histamine-induced microvascular leakage measured by Evans blue dye extravasation. SR 140333, but not SR 48968 (1 mg kg-1 given each once 30 min before citric acid exposure), prevented this potentiation. These results provide further evidence of the role of tachykinin and tachykinin NK1 receptor stimulation on airway hyperresponsiveness and its neurogenic inflammatory component.     

Expression of a retrovirally transduced gene under control of an internal housekeeping gene promoter does not persist due to methylation and is restored partially by 5-azacytidine treatment

BACKGROUND: Tachykinins, such as substance P, might be involved in the development of airway hyperresponsiveness and airway inflammation. OBJECTIVE: This study was designed to investigate the effects of the tachykinin NK1 receptor antagonist SR 140333 (Nolpitantium) and the NK2 receptor antagonist SR 48968 (Saredutant) on the activation of alveolar macrophages in the guinea-pig. METHODS: Guinea-pigs sensitized and challenged by ovalbumin administered by aerosol or naive guinea-pigs were exposed by aerosol to the neutral endopeptidase, phosphoramidon and, 15 min later, to substance P. Twenty-four hours later, bronchoalveolar lavages were performed and the cell composition of bronchoalveolar lavage fluids and the arachidonate release from alveolar macrophages stimulated in vitro with fMLP were evaluated. RESULTS: Antigen challenge in sensitized guinea-pigs induced an increase in the total number of cells and granulocytes in the bronchoalveolar lavage fluids that was not reduced by pre-treatment of guinea-pigs with a single dose of SR 140333 or SR 48968 (1 mg/kg). Substance P exposure in phosphoramidon-pretreated guinea-pigs did not induce an increase in the total number of cells. In contrast, antigen or substance P exposure induced a significant increase in the in vitro fMLP-induced arachidonate release from alveolar macrophages. Pre-treatment of the guinea pigs with SR 140333 or SR 48968 did not reduce the increase in arachidonate release from fMLP-stimulated alveolar macrophages from sensitized and challenged guinea-pigs. Pre-treatment of the animals by SR 140333 and SR 48968 reduced the enhanced arachidonate release induced by fMLP from substance P-exposed guinea-pigs. CONCLUSION: The present data demonstrate the importance of NK1- and NK2-receptor stimulation in the development of substance P-induced increased reactivity of alveolar macrophages.     

Renal effects of intracerebroventricularly injected tachykinins in the conscious saline-loaded rat: receptor characterization

1. The effects of intracerebroventricularly (i.c.v.) injected substance P (SP), neurokinin A (NKA) and [MePhe7]neurokinin B (NKB) were investigated on renal excretion of water, sodium and potassium in the conscious saline-loaded rat. The central effects of [MePhe7]NKB were characterized with selective tachykinin antagonists for NK1 (RP 67580), NK2 (SR 48968) and NK3 (R 820) receptors. 2. Whereas SP or NKA (65 or 650 pmol) failed to modify the renal responses, [MePhe7]NKB (65-6500 pmol) produced dose-dependent and long-lasting (30-45 min) decreases in renal excretion of water (maximal reduction at 65 pmol: from 66.14 +/- 7.62 to 21.07 +/- 3.79 microliters min-1), sodium (maximal reduction at 65 pmol: from 10.19 +/- 2.0 to 1.75 +/- 0.48 mumol min-1) and potassium (maximal reduction at 65 pmol: from 4.31 +/- 1.38 to 0.71 +/- 0.27 mumol min-1). While 650 pmol [MePhe7]NKB elevated urinary osmolality, neither 65 pmol nor 6.5 nmol [MePhe7]NKB altered this parameter. 3. Both the antidiuresis and antinatriuresis induced by [MePhe7]NKB (65 pmol) were significantly blocked by the prior i.c.v. injection of R 820 (1.3 nmol, 5 min earlier), although the potassium excretion was only partially reduced. However, R 820 did not affect the antidiuresis and antinatriuresis elicited by endothelin-1 (1 pmol, i.c.v.). On its own, R 820 decreased renal potassium excretion with no effect on urinary osmolality and renal excretion of water and sodium. The i.c.v. co-injection of RP 67580 and SR 48968 (6.5 nmol each, 5 min earlier) failed to modify the renal responses to [MePhe7]NKB in a similar study. 4. The central effects of [MePhe7]NKB (65 pmol) on renal excretion were blocked by the prior i.v. administration of a linear peptide vasopressin V2 receptor antagonist (50 micrograms kg-1, 5 min earlier). 5. These results suggest that the central NK3 receptor, probably located in the hypothalamus, is implicated in the renal control of water and electrolyte homeostasis through the release of vasopressin in the conscious saline-loaded rat.     

Potencies of agonists acting at tachykinin receptors in the oestrogen-primed rat uterus: effects of peptidase inhibitors

The uterotonic potencies of the naturally occurring mammalian tachykinins and the synthetic subtype-selective agonist analogues of these agents [Lys5,MeLeu9,Nlel0]neurokinin A-(4-10) and [Nle10]neurokinin A-(4-10) (tachykinin NK2 receptor-selective), [Sar9,Met(O2)11]substance P (tachykinin NK1 receptor-selective) and senktide (tachykinin NK3 receptor-selective) were determined using preparations from oestradiol-treated rats. The endopeptidase 24.11 inhibitor, N-[N-[1-(S)-carboxyl-3-phenylpropyl]-(S)-phenyl-alanyl-(S)-isoserine+ ++ (SCH 39370), potentiated responses to neurokinin A, neurokinin B and substance P, but not to [Lys5,MeLeu9,Nle10)]neurokinin A-(4-10) or senktide. [Nle10]neurokinin A-(4-10) effects were potentiated by SCH 39370 with amastatin and those to [Sar9,Met(O2)11]substance P were potentiated by SCH 39370 and captopril in combination. In the presence of optimal concentrations of peptidase inhibitors the relative order of agonist potency was: neurokinin A > substance P > neurokinin B for the naturally occurring mammalian tachykinins and [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) > [Nle10]neurokinin A-(4-10) > [Sar9,Met(O2)11]substance P > senktide for the synthetic tachykinin analogues. Thus, while a tachykinin NK2 receptor predominates in the oestrogen-primed uterus, a tachykinin NK1 receptor may also be present. The non-peptide tachykinin NK3 receptor antagonist, SR 142801, did not antagonise the effects of senktide suggesting that tachykinin NK3 receptors do not mediate its relatively minor effect on the uterus of the oestrogen-primed rat.     

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.     

A tachykinin NK3 receptor antagonist, SR 142801 (osanetant), prevents substance P-induced bronchial hyperreactivity in guinea-pigs

Aerosolized substance P (0.1 M, for 30 min) induced airway hyperresponsiveness in guinea-pigs 24 h after they were pre-treated with salbutamol (8.7 mM by aerosol for 10 min) and phosphoramidon (0.1 mM by aerosol for 10 min). This was displayed by an exaggerated response to the bronchoconstrictor effect of acetylcholine. A microvascular leakage hypersensitivity also occurred and was demonstrated by a potentiation of the plasma protein extravasation from bronchial vessels induced by histamine. The aim of this study was to investigate the effects of the non-peptide and potent tachykinin NK3 receptor antagonist, SR 142801 (osanetant), in comparison with those of the tachykinin NK1 and NK2 receptor antagonists, SR 140333 (nolpitantium) and SR 48968 (saredutant) respectively, on substance P. When given once at 1 mg/kg i.p. 45 min before exposure to substance P, SR 142801 prevented both hyperresponsiveness to acetylcholine and the potentiation of histamine-induced increase in microvascular permeability. SR 142801 did not exhibit any tachykinin NK1 or NK2 antagonistic activity in experiments on guinea-pig isolated airways, in vitro or in vivo. The results suggest that tachykinin NK3 receptors might be involved in these substance P-induced effects on airways.     

Characterization of the tachykinin receptors involved in spinal and supraspinal cardiovascular regulation

The pharmacological characterization of the tachykinin receptors involved in spinal and supraspinal cardiovascular regulation is reviewed in this report. In conscious rats, substance P (SP), neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK), and neuropeptide gamma (NP gamma) were injected either intrathecally (i.t.) or intracerebroventricularly (i.c.v.), and their effects were assessed on mean arterial blood pressure (MAP) and heart rate (HR). Moreover, selective antagonists for NK1 ((+/-)-CP-96045 and RP-67580), NK2 (SR-48968), and NK3 (R-486) receptors were tested against the agonists. I.t. tachykinins elicited dose-dependent increases in MAP and HR (NPK > NP gamma > SP > NKA > NKB). The cardiovascular response to i.t. SP, NPK, and NP gamma was significantly attenuated by the prior i.t. administration of (+/-)-CP-96345 and RP-67580 but not by SR-48968 and R-486. By the i.c.v. route, tachykinins also elicited pressor and tachycardiac responses dose dependently (NPK > NP gamma > SP > NKA > NKB). Senktide and [MePhe7]NKB, two NK3-selective agonists, were slightly more potent than NKB on both parameters. Whereas the cardiovascular response to NPK was largely blocked by (+/-)-CP-96345 and RP-67580, that to SP was reduced by 40-50%. This treatment had no effect on the cardiovascular response to NKA and [MePhe7]NKB. Conversely, SR-48968 reduced by 40-50% the NKA-induced cardiovascular changes without affecting the central mediated effects of NPK, SP, and [MePhe7]NKB. However, when coadministered, RP-67580 and SR-48968 abolished the effects to SP and NKA while leaving untouched those induced by [MePhe7]NKB. Finally, the central effects mediated by [MePhe7]NKB, senktide, and NKB were blocked by R-486. These findings suggest that the i.t. action of tachykinins on the rat cardiovascular system is mediated by a NK1 receptor in the spinal cord, while NK1, NK2, and NK3 receptors are likely involved in the supraspinal (hypothalamus) effects of these neuropeptides. It is also concluded that NPK is a pure and powerful NK1 agonist, in contrast to SP and NKA, which are not selective for NK1 and NK2 receptors, respectively.     

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.     

Airway responses to capsaicin in guinea pigs: role of NK-1 and NK-2 neurokinin receptors

The role of NK-1 and NK-2 receptors on the pulmonary response to capsaicin in guinea pigs was evaluated using intravenous infusion of selective nonpeptide antagonists of NK 1 (CP 96345, 300 nmol/kg, and SR 140333, 300 nmol/kg) and NK-2 (SR 48968, 100 nmol/kg) neurokinin receptors. Maximal values of pulmonary dynamic elastance (Edyn) and pulmonary resistance (RL) after capsaicin infusion were significantly lower in the presence of SR 48968 (p < .005). Morphometric analysis of lungs obtained by quick-freezing showed significant attenuation of airway contraction and peribronchiolar edema formation in the presence of NK-2 antagonist (p < .001). When compared to guinea pigs that received only capsaicin, animals that received SR 140333 or CP 96345 showed lower values of Edyn, RL, airway contraction, and peribronchiolar edema, but only the difference in Edyn values was significant. The combination of NK-1 and NK-2 antagonists was not more effective than NK-2 antagonist alone in attenuating capsaicin effects. The results suggest that airway effects of capsaicin are mainly mediated by activation of NK-2 receptors although NK-1 receptors may also play a role.     

Involvement of tachykinin NK1 and NK2 receptors in substance P-induced microvascular leakage hypersensitivity and airway hyperresponsiveness in guinea-pigs

Tachykinins, such as substance P, might be involved in the development of airway hyperresponsiveness (AHR) and airway inflammation. However, it is unknown which tachykinin receptors mediate these biological activities. The effects of two antagonists of tachykinin neurokinin-1 (NK1) and tachykinin neurokinin-2 (NK2) receptors, SR 140333 and SR 48968, respectively, were investigated on substance P (SP)-induced airway hyperresponsiveness and potentiation of the histamine-induced increase in microvascular leakage, in phosphoramidon-pretreated guinea-pigs. Guinea-pigs were pretreated with phosphoramidon (0.1 mM aerosol for 15 min) and exposed 15 min later to saline solution alone or to saline solution containing SP (0.1 mg.mL-1 for 30 min). Twenty four hours later, the animals were anaesthetized and prepared for the recording of the pulmonary inflation pressure (PIP) to acetylcholine or for the investigation of microvascular leakage to histamine. Pretreatment of the guinea-pigs with a single dose of SR 48968 (1 mg.kg-1, i.p.) 30 min before SP exposure, significantly prevented the development of AHR, whereas SR 140333 (1 mg.kg-1, i.p.) did not. In a second set of experiments, phosphoramidon-pretreated guinea-pigs exposed to SP presented a significant potentiation of the histamine-induced increase in microvascular leakage in pulmonary airways. When the guinea-pigs were pretreated with SR 140333, an inhibition of the increased microvascular leakage to histamine was observed. In contrast, no significant inhibitory activity was noted when the guinea-pigs were pretreated with SR 48968. The present data demonstrate the importance of tachykinin NK2 receptor stimulation in the development of airway hyperresponsiveness and that of tachykinin NK1 receptor stimulation in microvascular leakage hypersensitivity in phosphoramidon-pretreated and substance P-exposed guinea-pigs. The results also suggest a dissociation between the presence of microvascular leakage and the occurrence of airway hyperresponsiveness.     

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.     

Tachykinins may mediate capsaicin-induced, but not vagally induced motility in porcine antrum

Tachykinins are thought to be involved in extrinsic control of motility in the gastrointestinal tract. Using the isolated perfused porcine antrum with intact vagal innervation, we studied the effects of substance P, neurokinin A and capsaicin infusion, and electrical stimulation of the vagus nerves on antral motility without or with infusion of non-peptide antagonists for NK-1 receptors (CP96345) and NK-2 receptors (SR48968). Substance P and neurokinin A stimulated antral motility in a dose-dependent manner. The effect could be inhibited by atropine or a combination of the NK-1 and NK-2 receptor antagonists. Electrical stimulation of the vagus nerves and infusion of capsaicin (10(-5) M) stimulated antral motility. Vagally induced motility was not influenced by infusion of CP96345 and SR48968, whereas the effect of capsaicin was blocked. We conclude that tachykinins may be involved in regulation of antral motility through sensory nerves in the porcine antrum, but they do not seem to be involved in vagal regulation of antral motility.     

Heterogeneity of tachykinin receptors in the rabbit lung

The tachykinins, substance P (SP) and neurokinin A (NKA), are agonists for the NK(1) and NK(2) receptors, respectively. Tachykinins have various respiratory effects, including bronchoconstriction. This study characterizes tachykinin binding sites in the rabbit lung. We hypothesize that (2-[(125)I]iodohistidyl(1))Neurokinin A ([(125)I]NKA) interacts with NK1 and NK2 binding sites in the rabbit lung. The K d determined from saturation isotherms was 0.69 times/divided by 1.14 nM (geometric mean times/divided by SEM) and the B max was 4.15 + or - 0.22 femtomole/mg protein (arithmetic mean + or - SEM). Competitive inhibition studies with NKA, SP and various selective tachykinin agonists showed the rank order of potency; [beta-Ala(8)]-Neurokinin A 4-10 = SP > NKA > [Sar(9),Met(02)11]-Substance P. [beta-Ala(8)]-Neurokinin A 4-10, a selective NK(2) agonist, and SP inhibition of [(125)I]NKA binding were best described using a two-site model. Competitive inhibition studies using the selective nonpeptide NK(2) antagonist (SR 48968) and the selective nonpeptide NK(1) antagonist (CP 96,345) revealed Ki's of 5.5 nM and 8.1 nM, respectively. Our data therefore suggest that [(125)I]NKA binds to both the NK(1) and NK(2) receptors in the lung.     

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.     

Tachykinins mediate noncholinergic excitatory neural responses in the circular muscle of rat proximal colon

Using the sucrose-gap technique, we attempted to assess a role for tachykinins (TKs) in mediating noncholinergic excitatory junction potential (EJP) and contraction, in the circular muscle of rat proximal colon. Excitatory responses were evoked by submaximal electrical field stimulation (EFS) in the presence of atropine (1 microM), guanethidine (1 microM), indomethacin (10 microM), and N(omega)-nitro-L-arginine methyl ester (L-NAME) (100 microM). The NK1 receptor antagonist, SR 140,333 (up to 3 microM) or the NK2 receptor antagonists, SR 48,968 and MEN 10,627 (up to 5 microM) produced a partial inhibition of the excitatory responses to EFS. The co-administration of the selective NK1 and NK2 receptor antagonists produced additive effects on the responses to EFS. Selective NK1 receptor agonist, [Sar9, Met (O2)11]-substance P, induced depolarization and contraction, antagonized by SR 140,333, but not by NK2 receptor antagonists. NK2 receptor agonist, [betaAla8]-neurokinin A (4-10), also produced electrical and mechanical excitatory effects that were antagonized by SR 48,968 or MEN 10,627, but not by the NK1 receptor antagonist. Our results provide evidence that, in circular muscle of rat colon, endogenous tachykinins are the main excitatory transmitters for nonadrenergic, noncholinergic (NANC) excitation and their action is mediated by both NK1 and NK2 receptors.     

Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds

1. Ouabain, an inhibitor of Na+/K+ ATPase induces the release of acetylcholine from central and myenteric cholinergic neurones principally due to partial depolarization of the cell membrane. The effect of ouabain has been examined on neurogenic contractions in the guinea-pig ileum arising from either electrical field stimulation or from naloxone in morphine-exposed preparations. 2. Guinea-pig isolated ileum preparations were stimulated transmurally (0.1 Hz, 0.3 ms, 200 mA) to elicit contractions of the myenteric plexus-longitudinal smooth muscle. 3. Incubation with morphine (0.3 microM, 60 min) was followed by naloxone (1 microM) which produced withdrawal contractions in 16/26 preparations (median of 10.7 [2.2-40.0]% of a maximal contracture to KCl (60 mM)). 4. In parallel experiments, ouabain (1 microM) was added to the tissue before exposure to morphine (0.3 microM, 60 min). Naloxone (1 microM) subsequently displayed a withdrawal contraction in all 26/26 tissues (57.9 [30.5-151.7]% of a maximal contracture to KCl (60 mM). 5. Ouabain neither affected the concentration-dependent contractions of guinea-pig ileum produced by carbachol nor the inhibition of electrically-evoked contraction produced by morphine (0.3 microM). 6. The muscarinic antagonist atropine (0.1 microM) antagonized control naloxone withdrawal responses. The atropine resistant component, evident in ouabain-treated tissues, was blocked by SR140333((S)1-[2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenyla cetyl)piperidin-3-yl]ethyl]-4-phenyl-1-azoniabicyclo[2.2. 2]-octane, chloride), a substance P antagonist. 7. Clonidine (alpha2-adrenoceptor agonist) inhibited electrically-evoked contractions. Exposure to the alpha2-adrenoceptor antagonist RX811059 (2-(2-ethoxy-1,4-benzodioxan-2-yl)-2-imidazoline), resulted in a contracture which was not significantly enhanced by ouabain (1 microM). 8. Ouabain selectively potentiates the naloxone-induced withdrawal contraction following acute exposure to morphine the major components of which are mediated by both acetylcholine and substance P.     

Cardiovascular and behavioural effects of intracerebroventricularly administered tachykinin NK3 receptor antagonists in the conscious rat

1. In the conscious rat, three tachykinin NK3 receptor antagonists, namely SR142801 ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)pro pyl)-4-phenylpiperidin-4-yl)-N-methylacetamide), R820 (3-indolylcarbonyl-Hyp-Phg-N(Me)-Bzl) and R486 (H-Asp-Ser-Phe-Trp-beta-Ala-Leu-Met-NH2) were assessed against the intracerebroventricular (i.c.v.) effects induced by senktide, a selective NK3 receptor agonist, on mean arterial blood pressure (MAP), heart rate (HR) and motor behaviour. 2. Senktide (10-650 pmol per animal; i.c.v; n = 4-16) at the lowest dose caused a significant fall in MAP (-10 +/- 6 mmHg), while at the highest doses (100 and 650 pmol), senktide caused a rise in MAP (9 +/- 3 and 12 +/- 1 mmHg, respectively) when compared to vehicle. The intermediate doses (25 and 65 pmol) had no effect on MAP. The highest two doses caused a tachycardia of 62 +/- 15 and 88 +/- 8 beats min(-1), respectively. The dose of 65 pmol had a biphasic effect on HR, an initial bradycardia of 47 +/- 12 beats min(-1) followed by a tachycardia of 46 +/- 14 beats min(-1). The lowest doses caused either a rise of 52 +/- 10 beats min(-1) (25 pmol) or no effect (10 pmol) on HR. All doses of senktide caused similar increases in face washing, sniffing and wet dog shakes except at the dose of 100 pmol, when wet dog shakes were more than double those observed with the other doses. 3. The antagonist SR142801 (100 pmol -65 nmol per animal; i.c.v.; n = 6-8) caused increases in MAP at the highest two doses (6.5 and 65 nmol) while HR, dose-dependently, increased (23 +/- 6 to 118 +/- 26 beats min[-1]) and the onset dose-dependently decreased. The (R)-enantiomer, SR142806 (100 pmol - 65 nmol per animal; i.c.v.; n = 6-8) only caused rises in MAP (13 +/- 2 mmHg) and HR (69 +/- 11 beats min[-1]) at the highest dose. These drugs had no apparent effect on behaviour, except for the highest dose of SR142801 which increased sniffing. The antagonist R820 (650 pmol - 6.5 nmol per animal; i.c.v.; n = 6) had no effect on MAP or HR and only increased sniffing behaviour at 6.5 nmol. At 650 pmol (n = 6), R486 had no effect on any variable, but at 3.25 nmol, i.c.v. (n = 4) a delayed tachycardia and a significant increase in all behavioural variables were observed. 4. The cardiovascular responses induced by 6.5 nmol SR142801 and 25 pmol senktide were inhibited by R820 (6.5 nmol, 5 min earlier i.c.v.). In contrast, R820 failed to affect the central cardiovascular and behavioural responses induced by 10 pmol [Sar9, Met(O2)11]substance P, a NK1 receptor selective agonist. The senktide-induced behavioural changes were not inhibited by R820 (6.5 nmol, i.c.v.) while R486 (650 pmol, i.c.v.) blocked both the cardiovascular and behavioural responses to 25 pmol senktide. A mixture of antagonists for NK1 (RP67580; 6.5 nmol) and NK2 (SR48968; 6.5 nmol) receptors injected i.c.v. did not affect the cardiovascular response to SR142801. Cross-desensitization was shown between the central responses to SR142801 and senktide, but not between SR142801 and [Sar9, Met(O2)11]substance P. 5. The antagonists SR142801 and SR142806 (6.5-650 nmol kg(-1); n = 5-7), given i.v., did not evoke any cardiovascular or behavioural changes, except a delayed bradycardia for SR142806 (650 nmol kg[-1]), and also failed to inhibit the increase in MAP evoked by senktide (4 nmol kg(-1), i.v.). However, at the highest dose, both drugs slightly reduced the senktide-induced tachycardia. 6. Although the present data are consistent with the in vitro pharmacological bioassays and binding data, showing that SR142801 is a poor antagonist at rat peripheral NK3 receptors, they suggest that SR142801 has a partial agonist action at these receptors centrally. A separation of the cardiovascular and behavioural effects mediated by central NK3 receptor activation was achieved with SR142801 and R820 but not with R486. These results could be explained by the existence of NK3 receptor subtypes in the rat or by the differential activation and inhibition of the same receptor protein linked to the production of different second messengers. Differences in the pharmacokinetic or pharmacodynamic properties of the antagonists cannot be excluded at this time.