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.     

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.     

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.     

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 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.     

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.     

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.     

Capsaicin treatment induces muscarinic hyperreactivity in guinea pig trachea: a warning

Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a widely used tool for the depletion of neuropeptides from sensory C-fibres. Upon capsaicin treatment tachykinins are released, resulting in a variety of responses in the airways. We showed that after capsaicin (0.3 microM; 30 min) treatment of guinea pig tracheal smooth muscle preparations, the maximal contraction of the trachea after methacholine stimulation was strongly increased (capsaicin: 1.147 +/- 0.050 g vs. control: 0.717 +/- 0.047 g). This effect was completely nullified after pretreatment with capsazepine (2-[2-(4-chlorophenyl)ethyl-amino-thiocarbonyl]-7,8-dihydroxy-2,3, 4,5-tetrahydro-1H-2benzazepine; a vanilloid receptor antagonist) and YM38336 (a dual tachykinin NK1 and tachykinin NK2 receptor antagonist). Our results serve as a warning against using capsaicin as a putatively clean pharmacological tool to deplete the neuropeptides from pools on the C-fibres because we showed that capsaicin also strongly influences basal mechanisms in tracheal smooth muscle control.     

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.     

Forty-two years in cardiology

1. Incubation of proximal segments of the rat isolated duodenum with NG-nitro-L-arginine (L-NOARG; 3-100 microM) produced a concentration-dependent increase in both resting tone and the amplitude of the spontaneous contractions. These effects were attenuated by concurrent incubation with L-arginine (1 mM) but not D-arginine (1 mM). 2. These changes in resting tone and motility induced by L-NOARG (30 microM) were substantially reduced by concurrent incubation with tetrodotoxin (1 microM) or hexamethonium (10 microM), implicating the involvement of a local neuronal response. 3. The L-NOARG-induced increase in duodenal motility was not, however, inhibited by atropine (1 microM), guanethidine (6.4 microM) phentolamine (1 microM), or indomethacin (10 microM), indicating a non-cholinergic, non-adrenergic and non-prostanoid-mediated contractile response. 4. The NK1/NK2 tachykinin receptor antagonist, (D-Pro2, D-Trp7.9 substance P, 1-10 microM), and the NK2-receptor antagonists, MEN 10,207 and MEN 10,376 (1-5 microM), concentration-dependently reduced the effect of L-NOARG (30 microM) on spontaneous duodenal motility. 5. The resting tone and amplitude of the spontaneous contractions was likewise increased by incubation with NG-monomethyl-L-arginine (L-NMMA; 100-1000 microM). However, incubation with L-NMMA (100 microM) attenuated the actions of more potent L-NOARG (30 microM) on resting motility. 6. Administration of E.coli endotoxin (3 mg kg-1, i.v.) to the rat 5 h prior to tissue removal, at a time of known induction of NO synthase, reduced the amplitude of spontaneous contractions of the isolated duodenum, an effect inhibited by pretreatment of the rats with dexamethasone (1 mg kg-1) 2 h prior to endotoxin challenge. 7. These findings indicate a role of endogenous NO in the modulation of spontaneous tone and motility in the rat duodenum. Induction of NO synthase may result in a reduction in spontaneous motility of the tissue. By contrast, inhibition of constitutive NO biosynthesis unmasks a contractile response that is neuronally mediated and involves tachykinin NK2 receptors.     

Nociceptin inhibits non-adrenergic non-cholinergic contraction in guinea-pig airway

1. Electrical field stimulation (EFS) of guinea-pig isolated main bronchi induced a non-adrenergic non-cholinergic (NANC) contractile response. Nociceptin (0.01-1 microm) significantly inhibited the contractile response to EFS (P<0.01), but not to capsaicin (P>0.05). 2. The mu-, delta- and kappa-opioid receptor antagonists, naloxone (0.3 microM), naltrindole (3 microM) and norbinaltorphimine (1 microm), respectively, did not significantly affect the inhibitory effect of nociceptin (0.03 microM; P>0.05). 3. The novel nociceptin antagonist, [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 (0.03-1 microM); the sigma ligands, carbetapentane (30 microM), 3-phenylpiperidine (30-100 microM) and (+)-cyclazocine (10-100 microM) significantly reversed the inhibitory effect of nociceptin (0.03 microM, P<0.05). In contrast, rimcazole, did not significantly reverse the inhibitory effect of nociceptin (0.03 microM) at any concentration tested (P>0.05). 4. EFS of guinea-pig bronchial preparations significantly increased SP-LI release above basal SP-LI (P<0.05). In the presence of nociceptin (1 microM), EFS induced a significant increase in SP-LI release above basal SP-LI release (P<0.05). Nociceptin caused a 59+11% (n=5) inhibition of EFS-induced release of SP-LI. 5. Nociceptin reduces the release of sensory neuropeptides induced by EFS, but not capsaicin, from guinea-pig airways. These experiments provide further evidence for a role for nociceptin in regulating the release of sensory neuropeptides in response to EFS.     

Activation of the cloned human NK3 receptor in Chinese Hamster Ovary cells characterized by the cellular acidification response using the Cytosensor microphysiometer

1. The aim of the present study was to validate the Cytosensor microphysiometer, a novel system that measures the extracellular acidification rate as a reliable index of the integrated functional response to receptor activation, as a method for studying NK3 receptor pharmacology, and then to use this system to assess the functional activity of novel compounds at this receptor. 2. The selective NK3 agonist senktide caused reproducible, concentration-related increases in acidification ratein CHO-NK3 cells, with a pEC50 value of 8.72+/-0.11 (n=15). [Beta-Ala8]NKA(4-10), the selective NK2 agonist, elicited a much weaker response (pEC50=6.68+/-0.08, n=4), while the NK1-selective agonist substance P methylester only caused a very weak response at concentrations > or =3 microM (n=2). The rank order of potency for the endogenous tachykinins NKB>NKA>substance P (n=3) confirmed the response was mediated by the NK3 receptor. Moreover, the actual potencies obtained were consistent with affinities measured in radioligand binding studies. 3. The novel compounds PD156319-121 (0.3-1 microM), PD161182 (10-300 nM), PD168001 (10-100 nM) and PD168073 (10-100 nM) all acted as surmountable antagonists of the senktide-induced acidification response, with pA2 values of 7.49, 8.67, 9.17 and 9.25 respectively (n=3-5). In comparison the known NK3 antagonist SR142801 (10-100 nM) had a pA2 value of 8.83 (n=8) for the interaction with senktide. Again, these values are consistent with the radioligand binding data. 4. Amiloride (1 mM) inhibited the senktide-induced acidification response by 68.3+/-3.3 (n=4), indicating that the Na+/H+ antiporter plays an important role in this response, and this is consistent with the importance of this antiporter in other acidification responses. 5. Inhibition of protein kinase C with staurosporine (0.1 microM), or depletion of the intracellular Ca2+ stores with thapsigargin (1 microM), both resulted in a reduction in the maximum response to senktide (63.3+/-1.7 and 68.9+/-3.2% respectively, n=3-5), and co-application of these inhibitors abolished the response (n=3). This strongly suggested that the NK3 receptor was coupling via phospholipase C (PLC), as would be expected, although this could not be confirmed by the use of the putative PLC/PLA2 inhibitor U73122. 6. In conclusion, we have demonstrated the utility of the Cytosensor in the characterization of functional responses to agonists, and assessment of the affinities of antagonists in CHO cells expressing the human NK3, and have shown that our series of novel compounds are non-peptide NK3 antagonists of high affinity, as exemplified by PD168073.     

The ETA antagonist CI-1020 inhibits hypoxic pulmonary vasoconstriction in small isolated rat pulmonary arteries

Hypoxic pulmonary vasoconstriction (HPV) of rat pulmonary arteries in vitro occurs in four phases. Initial vasodilation (phase 1), is followed by transient contraction (phase 2), further vasodilation (phase 3) and finally a second sustained contraction (phase 4). We have investigated the role of ET-1 in HPV using the ETA receptor antagonist CI-1020. Small rat pulmonary arteries (SPA, n=32, diameter=454+/-22 microM) were mounted in a wire myograph. Two contractions to 80 microM KCl ensured response reproducibility and relaxation to 10 microM acetyl choline following constriction with 100 microM prostaglandin F2alpha (PGF2alpha) to indicate endothelial integrity. A control hypoxic response was produced following priming with 5 microM PGF2alpha. Vessels (n=8) were then exposed to either vehicle or CI-1020 (1, 10 or 100 microM) for 30 min in the dark before re-exposure to PGF2alpha and hypoxia. Responses were standardized as a percentage of contraction to 80 mM KCl. Vehicle caused an increase in phase 2 of HPV of +2.51+/-4.20% (expressed as difference between pre- and post-drug values). CI-1020 (1, 10 and 100 microM) caused a significant reduction in phase 2 of HPV of -9. 76+/-1.40%, -9.23+/-2.30% and -7.96+/-1.70%, respectively (P<0.05). These results suggest that phase 2 of HPV in rat SPA is attributed, in part, to the action of ET-1 at the ETA receptor.     

Correct blue-light regulation of pea Lhcb genes in an Arabidopsis background

The phasic contraction of the isolated guinea pig vas deferens induced by adenosine 5'-triphosphate (ATP) 1mM was significantly augmented by acidification of bathing solution induced by administration of hydrochloric acid (HCL) 10mM (pH = 6.87 +/- 0.015 (n = 5); mean +/- S.E.), while the tonic contraction induced by norepinephrine (NE) 10microM was significantly depressed by HCl 10mM. The contractile response to ATP 1mM was markedly potentiated in the presence of NE 10 microM. The potentiated contractile response to ATP 1mM in the presence of NE 10microM was significantly augmented by HCl 1mM or 10mM. The potentiating ratio of the contraction induced by ATP 1mM in the presence of NE 10microM to that induced by ATP 1mM alone was almost unaffected by the administration of HCl. Electrical field stimulation (EFS) produced a biphasic contractile response of the isolated guinea pig vas deferens; viz. the first rapid phasic contractile response and the second slow maintained tonic contractile response. The phasic contractile response to EFS was significantly augmented by HCl 10mM. These results may indicate that acidification of the medium potentiates the neurochemical transmission between the nerve terminals and the smooth muscle of vas deferens via sensitization of P2X (presumably P2X1 or P2X2) receptors existing in the smooth muscle.     

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.     

Effect of mycophenolic acid on TNF alpha-induced expression of cell adhesion molecules in human venous endothelial cells in vitro

1. The characteristic features of the endothelium-mediated regulation of the electrical and mechanical activity of the smooth muscle cells of cerebral arteries were studied by measuring membrane potential and isometric force in endothelium-intact and -denuded strips taken from the rabbit middle cerebral artery (MCA). 2. In endothelium-intact strips, histamine (His, 3-10 microM) and high K+ (20-80 mM) concentration-dependently produced a transient contraction followed by a sustained contraction. Noradrenaline (10 microM), 5-hydroxytryptamine (10 microM) and 9,11-epithio-11, 12-methano-thromboxane A2 (10 nM) each produced only a small contraction (less than 5% of the maximum K+-induced contraction). 3. N(G)-nitro-L-arginine (L-NOARG, 100 microM), but not indomethacin (10 microM), greatly enhanced the phasic and the tonic contractions induced by His (1-10 microM) in endothelium-intact, but not in endothelium-denuded strips, suggesting that spontaneous or basal release of nitric oxide (NO) from endothelial cells potently attenuates the His-induced contractions. Acetylcholine (ACh, 0.3-3 microM) caused concentration-dependent relaxation (maximum relaxation by 89.7 +/- 7.5%, n=4, P<0.05) when applied to endothelium-intact strips precontracted with His. L-NOARG had little effect on this ACh-induced relaxation (n=4; P<0.05). Apamin (0.1 microM), but not glibenclamide (3 microM), abolished the relaxation induced by ACh (0.3-3 microM) in L-NOARG-treated strips (n=4, P<0.05). 4. In endothelium-intact tissues, His (3 microM) depolarized the smooth muscle membrane potential (by 4.4 +/- 1.8 mV, n = 12, P < 0.05) whereas ACh (3 microM) caused membrane hyperpolarization (-20.9 +/- 3.0 mV, n = 25, P< 0.05). The ACh-induced membrane hypepolarization persisted after application of L-NOARG (-23.5 +/- 5.9 mV, n=8, P<0.05) or glibenclamide (-20.6 +/- 5.4 mV, n=5, P<0.05) but was greatly diminished by apamin (reduced to - 5.8 +/- 3.2 mV, n = 3, P< 0.05). 5. Sodium nitroprusside (0.1-10 microM) did not hyperpolarize the smooth muscle cell membrane potential (0.2 +/- 0.3 mV, n=4, P>0.05) but it greatly attenuated the His-induced contraction in endothelium-denuded strips (n-4, P<0.05). 6. These results suggest that, under the present experimental conditions: (i) spontaneous or basal release of NO from endothelial cells exerts a significant negative effect on agonist-induced contractions in rabbit MCA, and (ii) ACh primarily activates the release of endothelium-derived hyperpolarizing factor (EDHF) in rabbit MCA.     

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.     

Nitrergic modulation of cholinergic responses in the opossum lower oesophageal sphincter

1. Electrical field stimulation (EFS) of the superfused lower oesophageal sphincter from opossum (Monodelphis domestica) elicited biphasic responses. The first phase (relaxation) was strictly dependent on the duration of the EFS. The second phase (contraction) started following termination of the EFS (< or = 15 Hz). EFS at frequencies above 15 Hz led only to contraction, which started immediately upon initiation of the stimulation. 2. In the presence of NG-nitro-L-arginine (L-NOARG; 0.1-300 microM), the relaxation phase was abolished and the contractile response started with the initiation of EFS (at all frequencies) and was greater in magnitude. The contractile response to EFS was completely blocked with scopolamine (10 microM). 3. Exogenous acetylcholine (1-100 microM) elicited concentration-dependent contractions of the sphincter in the presence of botulinum toxin. These contractions were abolished when EFS was applied during administration of acetylcholine. This inhibitory effect of EFS was completely reversed when the tissue was treated with L-NOARG (100 microM). 4. These results suggest that the cholinergic response in the opossum lower oesophageal sphincter is under nitrergic control.     

Depolarization-dependent effect of flavonoids in rat uterine smooth muscle contraction elicited by CaCl2

1. The effects of the flavonoids genistein (3-60 microM), kaempferol (3-60 microM) and quercetin (1-100 microM) on KCl (60 mM)-induced tonic contraction in rat uterus and their modifications with the inhibitor of cAMP-dependent protein kinases (TPCK, 3 microM), the inhibitor of ornithine decarboxylase [alpha-difluoromethyl ornithine (DFMO), 10 mM] and the polyamine spermine (1 mM) have been assayed. The effects of the three flavonoids were also studied on the contraction elicited by CaCl2 (30 microM to 10 mM) on rat uterus incubated in medium lacking calcium and supplemented with 33, 60 or 90 mM of KCl. For comparison, the effects of the calcium channel blockers nifedipine and verapamil and the activator of adenylyl cyclase forskolin were assayed on contractions induced by KCl and CaCl2. 2. Genistein (IC50: 20.2 +/- 1.0 microM, n = 11), kaempferol (IC50: 10.1 +/- 0.8 microM, n = 8) and quercetin (IC50: 13.2 +/- 0.5 microM, n = 8) relaxed the tonic contraction induced by KCl (60 mM) in a concentration-dependent way. Verapamil (IC50: 70.1 +/- 5.8 nM, n = 7), nifedipine (IC50: 8.4 +/- 0.7 nM, n = 6) and forskolin (IC50: 0.62 +/- 0.08 microM, n = 14) also relaxed the KCl-induced contraction. TPCK (3 microM) significantly antagonized the effect of quercetin, kaempferol and forskolin (P < 0.01) but did not modify the effect of genistein. 3. Spermine (1 mM) increased the effects of genistein and verapamil and antagonized the effect of quercetin but did not modify those of kaempferol and forskolin. DFMO (10 mM) did not modify the effect of quercetin but increased that of genistein and antagonized those of kaempferol and forskolin. The addition of spermine (1 mM) plus DFMO (10 mM) antagonized the effect of quercetin. Spermine counteracted the effect of DFMO on forskolin but not on genistein. 4. KCl (33, 60 or 90 mM) did not produce contraction in calcium-free solution, but CaCl2 (30 microM to 10 mM) induced concentration-dependent contraction after depolarizing with KCl. The EC50 values for CaCl2 were: 0.74 +/- 0.08 (n = 12), 0.34 +/- 0.03 (n = 14) and 0.48 +/- 0.02 (n = 12) mM in a medium with 33, 60 or 90 mM of KCl, respectively. 5. Genistein (20 microM), kaempferol (10 microM), quercetin (15 microM), verapamil (70 nM), nifedipine (10 nM) and forskolin (0.5 microM) inhibited the concentration-response curve to CaCl2 in medium supplemented with 33, 60 or 90 mM of KCl. The effect of kaempferol was independent of the concentration of KCl in the incubation medium. However, the inhibitory effect of genistein on CaCl2-induced contraction was inversely related to the concentration of KCl in the medium. On the contrary, the effect of quercetin was directly related to the concentration of KCl in the medium. 6. The antagonism of verapamil, nifedipine and forskolin on CaCl2-induced contraction seems to be related to the degree of depolarization because increasing the KCl in the medium counteracted their effects. 7. Our results suggest that (1) cAMP contributes to the relaxant effects of quercetin and kaempferol on KCl (60 mM)-induced tonic contraction; (2) polyamines are involved in the relaxant effects of forskolin and kaempferol on KCl-induced tonic contraction but not on CaCl2-induced contraction in the depolarized uterus, and (3) the flavonoids assayed also possess a calcium antagonist action but show a different behavior toward the calcium channel blockers and the cAMP enhancer forskolin.     

Endothelin-1 affects capsaicin-evoked release of neuropeptides from rat vas deferens

Capsaicin-sensitive neurones release a number of neuropeptides, such as substance P, neurokinin A, somatostatin and calcitonin gene-related peptide (CGRP), which exert a number of effects on smooth muscle tissues. Endothelin-1 was thought to potentiate the capsaicin-evoked release of neuropeptides from sensory neurones of the rat. We have investigated the neuromodulatory effects of endothelin-1 on capsaicin-induced release of neurotransmitters from rat vas deferens. Capsaicin and human alpha calcitonin gene-related peptide (human alphaCGRP) reduced the rat vas deferens twitch responses induced by electrical field stimulation. Human beta calcitonin gene-related peptide-(8-37) [human betaCGRP-(8-37)] (1 microM), a selective alphaCGRP receptor antagonist, antagonized the inhibitory effects of both drugs. Endothelin-1 concentration dependently evoked an increase in basal tone of the musculature and potentiated the amplitude of the electrically stimulated responses, blocking inhibitory effects of capsaicin but not of human alphaCGRP. Moreover, endothelin-1 did not markedly change the inhibitory effects of papaverine (0.1-100 microM) or isoprenaline (1 nM-100 microM) on responses to electrical field stimulation. FR 139317 [(N,N-hexamethylene) carbamoyl-Leu-D-Trp(N-Me)-D-2-Pya], a selective endothelin ET(A) receptor antagonist, administered 30 min before endothelin-1 restored the capsaicin effects whereas BQ 788 [Dmpc-gamma-MeLeu-D-Trp-(1-methoxycarbonyl)-D-Nle], a selective endothelin ET(B) receptor antagonist, was completely ineffective. The endothelin-1-induced block of the capsaicin effect was resistant to tetrodotoxin (1 microM) and 30-min pre-treatment with MEN 10.627 (cyclo[(Met-Asp-Trp-Phe-Dap-Leu) cyclo (2beta-5beta)]), a selective tachykinin NK2 receptor antagonist, did not abolish the endothelin-1 effect on the inhibitory response to capsaicin. These results suggest that endothelin-1 selectively inhibits the capsaicin-induced release of neurotransmitters from rat vas deferens and these effects are mediated via endothelin ET(A) receptors but not by tachykinin release.