Treatment of acquired haemophilia with recombinant activated FVII: a critical appraisal.

Acquired haemophilia is a rare bleeding disorder usually caused by the spontaneous formation of inhibitory antibodies to coagulation FVIII. The disease occurs most commonly in the elderly, and although acquired haemophilia may be associated with a variety of underlying conditions, up to 50% of reported cases are idiopathic. Treatment options have traditionally involved human FVIII or FIX replacement therapy (if the inhibitor titre allows), porcine FVIII or the use of activated pro-thrombin complex concentrates. Recombinant activated coagulation FVII (rFVIIa) was available on an emergency and compassionate use basis from 1988 to 1999 at sites in Europe and North America. It has been registered in Europe for use in treating acquired haemophilia since 1996 and has recently been licensed for this indication in the United States. By directly activating FX on the surface of activated platelets at the site of injury (thereby bypassing FVIII and FIX), rFVIIa can circumvent the actions of inhibitory antibodies present in acquired haemophilia patients. This paper provides an overview of experiences with rFVIIa for the treatment of acquired haemophilia from the NovoSeven compassionate and emergency use programmes (1989-1999), the Hemophilia and Thrombosis Research Society Registry, and independent published reports from January 1999 to September 2005. rFVIIa has been reported to provide safe and effective haemostasis as a first line therapy in patients of all ages for a variety of surgical and non-surgical bleeding situations.

Cyclosporin for severe childhood atopic dermatitis: short course versus continuous therapy.

Cyclosporin (CyA) has been shown to be highly effective and well tolerated in the short-term treatment of severe childhood atopic dermatitis; however, there is limited experience in its longer-term use. The aim of this study was to compare multiple short courses of CyA with continuous therapy for 1 year, with respect to efficacy, safety, tolerability and quality of life. Children aged 2-16 years, with a diagnosis of severe atopic dermatitis refractory to topical steroid therapy, were randomly assigned to receive short course therapy (multiple courses of 12 weeks) or continuous therapy. The starting dose and maximum dose for all patients was 5 mg/kg per day. Disease activity was monitored using the Six Area Six Sign Atopic Dermatitis score and the 'Rule of Nines' area score. Pruritus, sleep disturbance and irritability were measured using visual analogue scales, and topical therapy was monitored. Safety measurements included monitoring of serum creatinine, blood pressure and adverse events. Forty patients were included in the efficacy analysis, 21 of whom were randomized to the short course group (of whom six were withdrawn) and 19 to the continuous group (of whom five were withdrawn). Significant improvements were seen in all efficacy parameters at every time-point. There were no significant differences between groups, although the improvement was more consistent in the continuous arm. In the short course arm, 7 out of 21 patients could be managed by at least two short courses. The remaining 14 patients includes 12 who could not be controlled by at least two short courses, one patient who failed to return after week 12 and another patient who was withdrawn at week 4 due to an adverse event. Quality of life improved for both the children and their families. Tolerability was considered good or very good in at least 80% of the patients at week 12 and at the end of the study. No clinically significant change was seen in mean serum creatinine and no change was seen in mean blood pressure in either group. CyA is effective in controlling severe atopic dermatitis in children over a 1-year period and is well tolerated. More consistent control is achieved with continuous treatment; however, short course therapy was adequate for some patients, indicating that treatment should be tailored to the individual patient's needs. Short course treatment may produce prolonged remission in some cases and reduce the cumulative exposure to the drug.

Treatment of psoriasis with intermittent short course cyclosporin (Neoral). A multicentre study.

A 1 year, prospective multicentre study was performed to investigate the efficacy and safety of intermittent treatment with cyclosporin in psoriasis vulgaris. Subjects received cyclosporin (Neoral) 5 mg/kg per day until achieving 90% reduction in area affected, or for a maximum of 12 weeks. Those failing to demonstrate a satisfactory response were withdrawn. When further treatment was required, cyclosporin was recommenced. This cycle was repeated up to three times. Psoriasis activity was recorded using the area affected and sign scores for erythema, scaling and infiltration. Overall assessments of response and tolerability were recorded. Forty-one subjects, mean age 36, mean PASI 12.8, entered the first treatment period. Thirty-three received a second period of treatment and 16 a third. Eighteen failed to complete the study as planned: five were withdrawn due to adverse events, four due to treatment failure and nine due to protocol violations. At the end of each treatment period, significant improvements were seen in all efficacy parameters. Overall response was graded as 'considerable improvement' or 'minimal or no symptoms', by over 80% of subjects and investigators. Median intervals to relapse for subjects remaining in the study were 72 days (range 28-329) and 53 days (range 14-141) after periods 1 and 2, respectively. There were significant increases in mean serum creatinine and blood pressure during each treatment period. However, there were no significant differences in either parameter between baseline and the final follow-up visit. At the end of each treatment period, overall tolerability of the treatment was considered 'good' or 'very good' by over 80% of subjects and investigators.

Phospholipase A2 and protein kinase C contribute to myofilament sensitization to 5-HT in the rabbit mesenteric artery.

1. Calcium (Ca2+, 0.1-100 microM) stimulated concentration-dependent contractions in small strips from the rabbit mesenteric artery in which the smooth muscle cells had been permeabilized with Staphylococcus aureus alpha-toxin. 2. 5-Hydroxytryptamine (5-HT) and phenylephrine, each in the presence of 10 microM guanosine 5'-triphosphate (GTP), concentration-dependently stimulated additional contractions in strips sub-maximally contracted by the presence of a buffered concentration of calcium (0.3 microM). All the additional contraction was abolished with the selective inhibitor of protein kinase C, Ro 31-8220 (10 microM). 3. Quinacrine (10-50 microM), an inhibitor of phospholipase A2, selectively inhibited the sensitization to 5-HT, but did not alter the sensitization to either phenylephrine or GTP. 4. Myofilament sensitization to calcium was mimicked by exogenous arachidonic acid (300 microM, in the presence of indomethacin, miconazole and BW755c) and the stable analogue of arachidonic acid, 5,8,11,14-eicosatetrayonic acid (ETYA, 100 microM), and in both cases did not require the additional presence of GTP. Ro 31-8220, but not quinacrine, reduced the sensitization to arachidonic acid by around 30%. 5. These results indicate that G protein-linked myofilament sensitization to calcium in the mesenteric artery that follows the activation of 5-HT receptors, but not alpha 1-receptors, involves phospholipase A2. The sensitization stimulated by each of these different receptors, and a component of the response to arachidonic acid, also appears to involve the activation of protein kinase C.

Characterisation of the endothelin receptor mediating contraction of human pulmonary artery using BQ123 and Ro 46-2005.

We have characterised the endothelin receptor mediating contraction of human isolated pulmonary artery. Endothelin-1 induced a concentration-dependent contraction of human endothelium-denuded pulmonary artery (EC50 5.6 nM). In contrast, endothelin-3 produced only a small contraction (approximately 12% of maximum endothelin-1 response) at the highest concentration tested (1 microM). The ETB receptor-selective agonist, sarafotoxin S6c (0.1 nM to 1 microM) did not cause contraction of human pulmonary artery. Pretreatment of human pulmonary artery with BQ123 (1-10 microM), an ETA receptor-selective blocking drug, resulted in a concentration-dependent, surmountable antagonism of endothelin-1-induced contractions (apparent pKB 6.6-7.0). Schild analyses yielded a shallow slope (0.58), which was significantly less than unity and, consequently, the calculated pA2 (8.1) was greater than the individual pKB values. Pretreatment of human pulmonary artery with Ro 46-2005 (30 microM), a non-peptide. non-selective endothelin receptor-blocking drug, resulted in a surmountable antagonism of endothelin-1-induced contractions (apparent pKB 5.5). In conclusion, endothelin-1-induced contraction of human pulmonary artery appears to be mediated predominantly via ETA receptors, although the shallow Schild slope observed with BQ123 indicates possible receptor heterogeneity.

Characterization of three non-peptide endothelin receptor ligands using human cloned ETA and ETB receptors.

1. A number of putative endothelin (ET) receptor ligands were synthesized with a view to assessing their relative affinity for human recombinant ET receptors. 2. Human (h) and endothelin ETA and ETB receptor open reading frames were cloned by reverse transcription-polymerase chain reaction into the mammalian expression vector pcDNA1 and stable cell lines were created by transfection of Chinese hamster ovary cells. 3. Scatchard analyses of saturation isotherms for the specific binding of [125I]-endothelin-1 ([125I]-ET-1) to membranes, prepared from Chinese hamster ovary cells transfected with hETA or hETB receptors, yielded values for equilibrium dissociation constants (Kd) of 20.5 +/- 1.8 pM and 25.5 +/- 5.5 pM, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogeneous, non-interacting receptor populations. 4. Pharmacological characterization of the transfected hETA and hETB receptors was undertaken by measuring the relative abilities of ETA and ETB receptor-selective peptide ligands to inhibit binding of [125I]ET-1. For interaction with hETA receptors, the relative order of potency was ET-1 > ET-3 = FR139317 = BQ123 >[Ala1,3,11,15]-ET-1 = sarafotoxin S6c (S6c). In contrast, the relative order of potency, at hETB receptors, was ET-1 = ET-3 = [Ala1,3,11,15]-ET-1 = S6c >> FR139317 = BQ123. 5. The novel non-peptide ligands, Ro 46-2005, SB 209670 and BMS 182874, were found to inhibit [125I]-ET-1 binding to human recombinant ETA and ETB receptors. At hETA receptors, the calculated pIC50 values were 6.7 (Ro 46-2005), 8.7 (SB 209670) and 5.8 (BMS 182874), while at hETB receptors, the corresponding pIC50 values were 6.8, 7.5 and <5, respectively.6. In conclusion, we have characterized the pharmacology of human cloned ETA and ETB receptors and used these in membrane binding assays to determine the affinity and selectivity of three structurally diverse non-peptide ET receptor ligands. SB 209670 is, to date, the highest affinity non-peptide ligand to be described for ET receptors. As such, it may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of endothelins.

Human saphenous vein contains both endothelin ETA and ETB contractile receptors.

We have investigated the endothelin receptor subtypes mediating contraction in isolated preparations of human saphenous vein. Endothelin-1 (EC50: 17.8 nM), endothelin-3 (EC50: 82.3 nM) and the endothelin ETB receptor-selective agonists, [Ala1,3,11,15]endothelin-1 (EC50: 63 nM) and sarafotoxin S6c (EC50: 0.75 nM) all produced concentration-dependent contractions of human saphenous vein, although [Ala1,3,11,15]endothelin-1 and sarafotoxin S6c only produced a contraction in approximately 50% of the preparations tested. The endothelin ETA receptor antagonist, BQ123 (D-Val,Leu,D-Trp,D-Asp,Pro; 10 microM), antagonized endothelin-1-induced contractions with an estimated potency (pKB approximately 6.0) which was an order of magnitude lower than reported previously for non-human isolated vascular tissues from other species (pA2 values approximately 7.0). These data suggest that both endothelin ETA and endothelin ETB receptors can mediate vascular smooth muscle contraction in human saphenous vein.

The effect of endothelins on nitric oxide and prostacyclin production from human umbilical vein, porcine aorta and bovine carotid artery endothelial cells in culture.

1. This study has investigated the effects of the endothelin isopeptides, endothelin-1 (ET-1), ET-2 and ET-3 on the production of the endothelium-derived relaxing factors, nitric oxide (NO) and prostacyclin (PGI2) from primary cultures of endothelial cells obtained from human umbilical vein (HUVECS), porcine aorta (PAECS) and bovine carotid artery (BCAECS). 2. NO generation was assessed indirectly by measuring production of cyclic GMP and PGI2 formation was measured by radioimmunoassay of 6-keto PGF1 alpha. 3. In HUVECS, histamine (1 microM) increased cyclic GMP and 6-keto PGF1 alpha production by 12.6 +/- 2.0 and 4.9 +/- 0.7 fold respectively over the corresponding basal values. Haemoglobin (10 microM) and the NO synthase inhibitor NG-monomethyl-L-arginine (10 microM) significantly inhibited the increase in cyclic GMP formation in response to histamine but had no effect on 6-keto PGF1 alpha production. In contrast to histamine, the endothelin isopeptides (ET-1, ET-2 and ET-3; 0.01-1000 nM) produced no significant change in either cyclic GMP or 6-keto PGF1 alpha production in HUVECS. 4. In a separate series of experiments, ET-3 (0.01-1000 nM) also failed to produce any significant change in cyclic GMP or 6-keto PGF1 alpha production from primary cultures of PAECS and BCAECS. In contrast, bradykinin (0.1 microM) and sodium nitroprusside (1 mM) were used as positive control agents and increased cyclic GMP production in these cells. 5. In conclusion, the endothelin isopeptides do not release NO and PGI2 from primary cultures of HUVECS, PAECS and BCAECS. This suggests that endothelin receptors are either absent from these cells or are not coupled to NO or PGI2 production.

Investigations into factors determining the duration of action of the beta 2-adrenoceptor agonist, salmeterol.

1. This study has explored the mechanism underlying the long duration of action of the beta 2-adrenoceptor agonist, salmeterol. 2. Salmeterol, salbutamol and isoprenaline caused a concentration-related inhibition of electrically-induced contractile responses of the guinea-pig superfused trachea preparation. The effects of both isoprenaline and salbutamol were rapid in onset and rapidly reversed upon removal of the agonist. In contrast, the effects of salmeterol were slower in onset and could not be reversed by superfusion of the tissue with agonist-free Krebs solution even for periods of up to 10 h. 3. The effects of salmeterol were, however, readily reversed by a number of beta-adrenoceptor blocking drugs, as was the effect of a continuous infusion of isoprenaline. Upon removal of the antagonist, however, the effects of salmeterol and of the isoprenaline infusion were reasserted at a rate which was inversely related to the lipophilicity of a beta-adrenoceptor blocking drugs. 4. Salmeterol inhibited the binding of [125I]-(-)-iodopindolol (100 pM) to rat lung membranes (pIC50 7.1), with isoprenaline (pIC50 6.2) and salbutamol (pIC50 5.1) having lower potencies. The inhibition of binding by salmeterol was apparently non-competitive, whereas that produced by salbutamol and isoprenaline was competitive in nature. 5. Isoprenaline and salbutamol rapidly dissociated from their binding sites, whereas in marked contrast, the binding of salmeterol showed no dissociation for periods of up to 1 h. 6. These data are consistent with the mechanism in which salmeterol binds adjacent to the active site of the beta 2-adrenoceptor, such that the drug cannot be washed out of the tissue, yet can interact with and activate the receptor. This latter property is susceptible to antagonism by beta-adrenoceptor blocking drugs but is reassured when the antagonists are removed.

Human placental membranes contain predominantly ETB receptors.

The endothelin (ET) receptors on human placental membranes (HPMs), coupled to fluomicrospheres, have been characterized by examining the binding of 125I-ET-1 in a scintillation proximity assay (SPA). Specific binding of 125I-ET-1 was potently inhibited by ET-1 (IC50: 80 pM), ET-3 (IC50: 170 pM), and the ETB receptor-selective agonists sarafotoxin S6c (S6c; IC50: 210 pM) and alanine1,3,11,15-ET-1 (4-Ala-ET-1; IC50: 3.56 nM). In contrast, the ETA receptor-selective antagonist BQ123 (D-Val-Leu-D-Trp-D-Asp-Pro) only weakly (28% at 10 microM) inhibited 125I-ET-1 binding. In addition, the inhibition curves for ET-3 and 4-Ala-ET-1 were shallow, with slopes less than unity, indicating binding-site heterogeneity. In keeping with this finding, the presence of a small population of ETA receptors was confirmed by the ability of BQ123 (1 microM) to reduce the maximum binding capacity of the HPMs for 125I-ET-1 by approximately 17%, without affecting the affinity for the radioligand. In conclusion, these results suggest that the HPM-SPA system contains predominantly (approximately 80%) ETB receptors, with a small ETA receptor population. These findings should be taken into account when this assay system is used to identify novel endothelin receptor ligands.

Endothelin ETA and ETB receptors mediate vascular smooth-muscle contraction.

Endothelin (ET) ETA receptors on vascular smooth muscle are believed to mediate the vasoconstrictor effects of ET isopeptides, and ETB receptors on the endothelium are thought to mediate the vasodilator effects. This study has investigated the receptors mediating endothelin-induced contraction of isolated ring preparations of rat thoracic aorta (RTA) and rabbit carotid artery (RCA), pulmonary artery (RPA), and jugular vein (RJV). In RTA and RCA, ET-1 (EC50 4.5 and 5.2 nM, respectively) was 82- and 108-fold, respectively, more potent than ET-3, whereas the ETB receptor-selective agonists sarafotoxin S6c (S6c) and Ala1,3,11,15-ET-1 (4-Ala-ET-1) were without effect up to > or = 1 microM. In contrast, in RPA and RJV, ET-1 (EC50 3.1 and 0.7 nM, respectively) and ET-3 (EC50 4.4 and 0.9 nM, respectively) were equipotent, and 4-Ala-ET-1 (EC50 10.7 and 2.1, respectively) and S6c (EC50 0.4 and 0.1 nM, respectively) were potent contractile agonists. The ETA receptor antagonist BQ123 (D-Val-Leu-D-Trp-D-Asp-Pro) competitively antagonized the effects of ET-1 in RTA and RCA (pA2 values 6.9 +/- 0.1 and 6.8 +/- 0.2, respectively) but did not antagonize (at 10 microM) contractions to ET-1, ET-3, or 4-Ala-ET-1 in RPA and RJV. In conclusion, contraction of vascular smooth muscle by endothelins can be mediated by both ETA and ETB receptors.

The pharmacology of salmeterol.

Salmeterol was developed to provide prolonged bronchodilatation to control nocturnal symptoms and improve maintenance therapy in asthmatic patients. Salmeterol is > 10,000 times more lipophilic than salbutamol and has greater affinity for the beta 2-adrenoceptor. Membrane binding is non-competitive and dissociation is slow so that its effects last for many hours. Despite this, salmeterol does not accumulate in tissues. Its mechanism of action can be explained by binding to a specific exo-site domain of the beta 2-receptor protein to produce continuous stimulation of the active site of the receptor, which gives salmeterol a profile of pharmacological activity unlike that of other beta 2-agonists. Due to its potent and prolonged activation of beta 2-adrenoceptors in airway smooth muscle cells, endothelial cells, mast cells and epithelial cells, salmeterol induces prolonged bronchodilatation, reduced vascular permeability, inhibition of inflammatory mediators, stimulation of ciliary function and modulation of ion and water transport across the bronchial mucosa.

Endothelin ETA and ETB receptors mediate vascular smooth muscle contraction.

1. We have investigated the receptors mediating endothelin-induced contraction of rabbit isolated jugular vein (RJV) and rat isolated thoracic aorta (RTA). 2. Endothelin-1 (ET-1) and endothelin-3 (ET-3) contracted RJV preparations with similar potency (EC50 values approximately 1 nM), whereas, ET-1 (EC50:4.5 nM) was approximately 80 fold more potent than ET-3 in contracting RTA. In addition, the ETB receptor-selective agonist [Ala1,3,11,15]ET-1 contracted RJV (EC50:2.1 nM) but not RTA. 3. The ETA receptor antagonist, BQ123, competitively antagonized (pA2 6.93) the contraction of RTA produced by ET-1, but had no effect (at 10 microM) on the contractile effects of either ET-1, ET-3 or [Ala1,3,11,15]ET-1 in RJV. 4. These data suggest that both ETA and ETB receptors can mediate vascular smooth muscle contraction.

Studies on the mechanism of 5-HT1 receptor-induced smooth muscle contraction in dog saphenous vein.

1. We have investigated the mechanism of smooth muscle contraction evoked by activation of 5-HT1-like receptors in dog isolated saphenous vein. 2. In the presence of the 5-HT2 receptor antagonist, ritanserin (0.1 microM), concentration-effect curves (10 nM-300 microM) for 5-hydroxytryptamine (5-HT)-induced smooth muscle contraction were biphasic. This could be attributed to a direct action on 5-HT1-like receptors at low concentrations of 5-HT (10 nM-10 microM) and an indirect (through the release of noradrenaline from sympathetic neurones) activation of postjunctional alpha-adrenoceptors at higher 5-HT concentrations. In contrast, concentration-effect curves (100 nM-100 microM) for sumatriptan-induced contractions were not biphasic, and were due solely to activation of 5-HT1-like receptors. 3. Smooth muscle contractions evoked either by low concentrations of 5-HT or by sumatriptan were abolished by removal of extracellular calcium and were markedly inhibited, but not abolished, by the calcium channel blocker, verapamil (1-30 microM). In contrast, contractions evoked by high concentrations of 5-HT were markedly less sensitive to removal of extracellular calcium or to verapamil. 4. 5-HT and sumatriptan also inhibited (to a maximum of about 50%) prostaglandin E2 (PGE2, 5 microM)-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation. This effect was mimicked by the alpha 2-adrenoceptor agonist, azepexole (B-HT933) but not by the alpha 1-adrenoceptor agonist, methoxamine.5. In contrast to mediation of smooth muscle contraction, the 5-HT1-like receptor-mediated inhibition of PGE2-stimulated cyclic AMP formation evoked by 5-HT or sumatriptan was not attenuated by removal of extracellular calcium or by verapamil (1 microM).6. A directly-acting inhibitor of adenylyl cyclase, 2',3'-dideoxyadenosine (1 mM) inhibited PGE2-stimulated cyclic AMP formation but did not produce smooth muscle contraction.7. These results suggest that contractile responses of dog isolated saphenous vein arising through activation of 5-HT1-like receptors are associated with both an influx of extracellular calcium ions (to a large extent via voltage-dependent channels) and an inhibition of adenylyl cyclase. However, although these two responses are coupled to the same receptor, they appear to be independent.

Characterization of the 5-HT receptor mediating endothelium-dependent relaxation in porcine vena cava.

1. 5-Hydroxytryptamine (5-HT) relaxes rings of neonatal porcine isolated vena cava by both an endothelium-dependent and an endothelium-independent mechanism. The receptor mediating the latter response has been shown to be a 5-HT1-like receptor (positively coupled to adenylyl cyclase) located on the vascular smooth muscle. The features of the endothelium-dependent response to 5-HT in this preparation are now described. 2. In ring preparations contracted with the stable thromboxane-A2-mimetic, U-46619 (10 nM), and in the presence of the 5-HT2 receptor antagonist ketanserin (1 microM), low concentrations of 5-HT (1-100 nM) evoked an endothelium-dependent, rapid, 'spike-like' relaxation. Higher concentrations of 5-HT (0.1-10 microM) elicited a more sustained, but endothelium-independent relaxation. 3. Relaxation induced by low concentrations (1-100 nM) of 5-HT was abolished by endothelium removal, and was markedly (but not totally) inhibited by the guanylate cyclase inhibitor, methylene blue (10 microM) or by the inhibitor of endothelium-derived nitric oxide (NO) synthesis, L-NG-monomethylarginine (L-NMMA; 100-500 microM). 4. The endothelium-dependent response to 5-HT was mimicked by alpha-methyl-5-HT, 5-methoxytryptamine, tryptamine and 2-methyl-5-HT, but not by sumatriptan or 8-hydroxy-di-n-propylaminotetralin (8-OH-DPAT) at concentrations up to 10 microM. In contrast, relaxation evoked by 5-carboxamidotryptamine (5-CT) was endothelium-independent. 5. The endothelium-dependent relaxation induced by 5-HT or alpha-methyl-5-HT was antagonized by methysergide, methiothepin, cyproheptadine and metergoline, but not by ketanserin, spiperone, ondansetron, verapamil, cyanopindolol, mesulergine, ICS 205-930, or indomethacin. 6. These results suggest that the endothelium-dependent relaxation of porcine vena cava induced by 5-HT is largely mediated by the release of NO (although other endothelium-derived relaxing factors may also be involved) and that 5-HT is acting at a receptor which is not '5-HT1-like', 5-HT2, 5-HT3 or 5-HT4 and is not comparable to recognised 5-HT receptor ligand binding sites. The characteristics of this receptor are discussed in relation to the endothelial 5-HT receptor types in other blood vessels.

Sumatriptan (GR43175) inhibits cyclic-AMP accumulation in dog isolated saphenous vein.

Sumatriptan (GR43175) contracts rings of dog isolated saphenous vein by an action at 5-HT1-like receptors. We have now examined the effects of sumatriptan on prostaglandin E2(PGE2)-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in this tissue. Sumatriptan and 5-hydroxytryptamine (5-HT) produced a concentration-dependent inhibition of PGE2-stimulated cyclic AMP accumulation (EC50 values of 250 nM and 80 nM respectively), responses that were mimicked by 5-carboxamidotryptamine but not by U-46619 or methoxamine. The response to sumatriptan (1 microM) was antagonised by methiothepin (1 microM), but not by metergoline (0.1 microM), spiperone (1 microM) or ondansetron (GR38032, 1 microM). These results suggest that 5-HT1-like receptors which mediate contraction of the dog isolated saphenous vein are negatively coupled to adenylate cyclase in this preparation.

5-HT1D binding sites in porcine brain can be sub-divided by GR43175.

We have examined the binding of 5-carboxamidotryptamine (5-CT) and GR43175 (3-(2-dimethylamino)ethyl-N-methyl-1H-indole-5-methane sulphonamide) to 5-HT1D sites labelled with [3H]-5-hydroxytryptamine [( 3H]-5-HT) in neonatal porcine caudate membranes. In competition studies, 5-CT produced shallow inhibition curves (Ki 138 nM, slope 0.31), indicating binding site heterogeneity, while GR43175 interacted with a single population of binding sites (Ki 251 nM, slope 0.98), producing a maximum of only 52% inhibition of [3H]-5-HT binding compared to 100% for 5-HT or 5-CT. In the presence of excess GR43175 (10 microM), 5-CT produced a monophasic inhibition curve with a Ki value of 800 nM for the remaining sites (slope 0.89). These preliminary data suggest that under the conditions employed, GR43175, and to a lesser extent 5-CT, may discriminate between two sub-populations of 5-HT1D binding sites in porcine brain.

Further characterization of the 5-HT receptor mediating vascular relaxation and elevation of cyclic AMP in porcine isolated vena cava.

1. 5-Hydroxytryptamine (5-HT) and 5-carboxamidotryptamine (5-CT) produce both smooth muscle relaxation and elevation of tissue adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in isolated rings of neonatal porcine vena cava. We now present studies attempting to characterize in more detail the 5-HT receptor mediating these responses. 2. Both 5-HT and 5-CT relaxed porcine isolated vena cava rings (EC50 values 200 nM and 4 nM respectively) and elevated tissue cyclic AMP levels (EC50 values 1500 nM and 16 nM respectively). For both responses 5-CT was approximately 50-100 fold more potent than 5-HT. 3. Both 5-CT-induced smooth muscle relaxation and cyclic AMP elevation were potently and specifically antagonized to a similar extent by methiothepin, methysergide and spiperone. 4. At concentrations up to 1 microM, 8-hydroxy-2-(di-n-propylamino) tetralin, buspirone, ipsapirone, n,n-dipropyl-5-CT, cyanopindolol, RU24969, ketanserin, GR38032 and GR43175 were devoid of both agonist and antagonist activity for both responses. 5. These findings suggest that the same 5-HT1-like receptor mediates both smooth muscle relaxation and elevation of cyclic AMP. This receptor is unlike any known 5-HT1 ligand binding site or adenylate cyclase-coupled 5-HT receptor in brain tissues.