Saxena, P.R.

Migraine headache is not associated with cerebral or meningeal vasodilatationa 3T magnetic resonance angiography study (Article)

2009-06-01T00:00:00Z

Current and prospective pharmacological targets in relation to antimigraine action (Article)

2008-10-01T00:00:00Z

Migraine is a recurrent incapacitating neurovascular disorder characterized by unilateral and throbbing headaches associated with photophobia, phonophobia, nausea, and vomiting. Current specific drugs used in the acute treatment of migraine interact with vascular receptors, a fact that has raised concerns about their cardiovascular safety. In the past, α-adrenoceptor agonists (ergotamine, dihydroergotamine, isometheptene) were used. The last two decades have witnessed the advent of 5-HT1B/1D receptor agonists (sumatriptan and second-generation triptans), which have a well-established efficacy in the acute treatment of migraine. Moreover, current prophylactic treatments of migraine include 5-HT2 receptor antagonists, Ca2+ channel blockers, and β-adrenoceptor antagonists. Despite the progress in migraine research and in view of its complex etiology, this disease still remains underdiagnosed, and available therapies are underused. In this review, we have discussed pharmacological targets in migraine, with special emphasis on compounds acting on 5-HT (5-HT1-7), adrenergic (α1, α2, and β), calcitonin gene-related peptide (CGRP 1 and CGRP2), adenosine (A1, A2, and A3), glutamate (NMDA, AMPA, kainate, and metabotropic), dopamine, endothelin, and female hormone (estrogen and progesterone) receptors. In addition, we have considered some other targets, including gamma-aminobutyric acid, angiotensin, bradykinin, histamine, and ionotropic receptors, in relation to antimigraine therapy. Finally, the cardiovascular safety of current and prospective antimigraine therapies is touched upon.

The effect of maternal ketanserin treatment on foetal 5-HT receptor function in umbilical cord artery of pre-eclamptic patients (Article)

2007-11-01T00:00:00Z

Background: Maternal treatment with the 5-HT2Areceptor antagonist ketanserin (KT) in pre-eclamptic patients is associated with a high placental transmission of KT, resulting in pharmacologically active levels of KT in the umbilical cord artery (UCA) and the neonate. Prolonged exposure to a 5-HT receptor antagonist may influence the functionality of foetal 5-HT receptors and compromise foetal development. Objective: To study whether exposure to KT influences the characteristics of foetal 5-HT receptors, functional studies were performed on 5-HT2Aand 5-HT1B/1Dreceptors in UCA from pre-eclamptic patients treated with KT. Methods: UCAs were obtained, immediately after delivery, from pre-eclamptic patients (n = 7), treated antenatally with intravenous KT. Pre-eclamptic patients (n = 13), not treated with KT (non-KT), were included as a control group. Segments of UCA were prepared and mounted in tissue baths and isometric force changes were determined. Cumulative concentration response curves to 5-HT and to the 5-HT1B/1Dreceptor agonist sumatriptan were constructed in the absence or presence of the 5-HT2Areceptor antagonist KT or the 5-HT1B/1Dreceptor antagonist GR125743, respectively. Results: All UCA segments showed contractile responses to both 5-HT and sumatriptan, and the concentration response curves showed a rightward shift with increasing concentrations of KT and GR125743, respectively, indicating the presence of functional 5-HT2Aand 5-HT1B/1Dreceptors in the foetal tissue. No significant differences were found in maximum response (Emax)(expressed in percent of response on 100 mM KCl) or potency (pEC50) of 5-HT in both groups (Emax= 141 ± 7.7%, pEC50= 7.67 ± 0.26 in KT-treated group and Emax= 162 ± 12.6%, pEC50= 7.69 ± 0.14 in non-KT treated group, respectively). No significant differences were found in the potency of the antagonist KT in both study groups (pKb= 7.65 ± 0.31 in KT group and 7.46 ± 0.17 in non-KT group, respectively). Similarly, with sumatriptan, no significant differences were found between KT-treated patients and non-KT treated patients (Emax= 142 ± 16.2 and 140 ± 14.7%, respectively, pEC50= 6.17 ± 0.37 and 6.41 ± 0.28 respectively, pKbof GR125743 = 7.83 ± 0.48 and 8.43 ± 0.29, respectively). Conclusion: Foetal exposure to KT in pre-eclamptic patients does not seem to influence the functional characteristics of 5-HT2Aand 5-HT1B/1Dreceptors in the UCA. Copyright

Effects of female sex hormones on responses to CGRP, acetylcholine, and 5-HT in rat isolated arteries (Article)

2007-04-01T00:00:00Z

Background. - Female sex hormones are implicated in the modulation of reactivity of a wide range of blood vessels under physiological as well as pathological conditions. Migraine, a neurovascular syndrome, is 3 times more prevalent in women during their reproductive period than in men. Objective. - This study sets out to investigate the effects of the female sex steroids, 17β-estradiol and progesterone (separately and in combination) on vasoactive responses to calcitonin gene-related peptide (CGRP), acetylcholine, and 5-hydroxytryptamine (5-HT) in rat isolated mesenteric, caudal, and basilar arteries. Methods. - Female Sprague-Dawley rats were ovariectomized (Day 0) and 7 days later subcutaneously implanted with pellets releasing over a 21-day period 17β-estradiol (0.25 mg), progesterone (50 mg), their combination, or placebo. On days 25-28, the animals were killed, arteries isolated and mounted in Mulvany myographs, and cumulative concentration response curves to CGRP, acetylcholine, and 5-HT were constructed. Results. - The relaxant responses to CGRP were significantly potentiated in mesenteric and caudal arteries from rats treated with 17β-estradiol as compared to the placebo-treated rats. Acetylcholine-induced relaxations were potentiated in the caudal artery from rats treated with the combination of 17β-estradiol and progesterone, as compared to that from placebo-treated rats. The 5-HT-induced contractions in the 3 arteries were not significantly different in efficacy or potency. Conclusion. - Our results show that 17β-estradiol potentiates CGRP-induced relaxations in the mesenteric and caudal arteries, while the combination treatment enhances acetylcholine-induced relaxations in the caudal artery. Although these in vitro experiments have been carried out in rats and a direct extrapolation to migraine in humans is not possible, our results may provide a new avenue to study the effects of sex steroids on vascular reactivity.

Pharmacological characterisation of capsaicin-induced relaxations in human and porcine isolated arteries (Article)

2007-03-01T00:00:00Z

Capsaicin, a pungent constituent from red chilli peppers, activates sensory nerve fibres via transient receptor potential vanilloid receptors type 1 (TRPV1) to release neuropeptides like calcitonin gene-related peptide (CGRP) and substance P. Capsaicin-sensitive nerves are widely distributed in human and porcine vasculature. In this study, we examined the mechanism of capsaicin-induced relaxations, with special emphasis on the role of CGRP, using various pharmacological tools. Segments of human and porcine proximal and distal coronary arteries, as well as cranial arteries, were mounted in organ baths. Concentration response curves to capsaicin were constructed in the absence or presence of the CGRP receptor antagonist olcegepant (BIBN4096BS, 1 μM), the neurokinin NK1receptor antagonist L-733060 (0.5 μM), the voltage-sensitive calcium channel blocker ruthenium red (100 μM), the TRPV1 receptor antagonist capsazepine (5 μM), the nitric oxide synthetase inhibitor Nω-nitro-l-arginine methyl ester HCl (l-NAME; 100 μM), the gap junction blocker 18α-glycyrrhetinic acid (10 μM), as well as the RhoA kinase inhibitor Y-27632 (1 μM). Further, we also used the K+channel inhibitors 4-aminopyridine (1 mM), charybdotoxin (0.5 μM)+apamin (0.1 μM) and iberiotoxin (0.5 μM)+apamin (0.1 μM). The role of the endothelium was assessed by endothelial denudation in distal coronary artery segments. In distal coronary artery segments, we also measured levels of cyclic adenosine monophosphate (cAMP) after exposure to capsaicin, and in human segments, we also assessed the amount of CGRP released in the organ bath fluid after exposure to capsaicin. Capsaicin evoked concentration-dependent relaxant responses in precontracted arteries, but none of the above-mentioned inhibitors did affect these relaxations. There was no increase in the cAMP levels after exposure to capsaicin, unlike after (exogenously administered) α-CGRP. Interestingly, there were significant increases in CGRP levels after exposure to vehicle (ethanol) as well as capsaicin, although this did not induce relaxant responses. In conclusion, the capsaicin-induced relaxations of the human and porcine distal coronary arteries are not mediated by CGRP, NK1, NO, vanilloid receptors, voltage-sensitive calcium channels, K+channels or cAMP-mediated mechanisms. Therefore, these relaxant responses to capsaicin are likely to be attributed to a non-specific, CGRP-independent mechanism.

Rat carotid artery responses to α-adrenergic receptor agonists and 5-HT after ovariectomy and hormone replacement (Article)

2007-02-01T00:00:00Z

Objective. - To compare the contractile responses to α-adrenergic receptor agonists and 5-HT in the rat carotid artery after ovariectomy and subsequent hormone replacement with 17β-estradiol, progesterone, or the combination of 17β-estradiol and progesterone. Background. - The prevalence of migraine is higher in women than in men, and changes in 17β-estradiol levels seem to affect the frequency of attacks in female migraineurs. However, the underlying mechanisms are not yet completely understood. Methods. - After 1 week of acclimatization (Day 0), female Sprague-Dawley rats were either sham-operated or bilateral ovariectomized. One week later (Day 7), the ovariectomized rats were subcutaneously implanted with a pellet releasing over a 21-day period either placebo, 0.25 mg 17β-estradiol, 50 mg progesterone, or the combination of the 2 hormones. Blood samples were collected on Days 0, 7, and 21 to measure plasma norepinephrine and epinephrine. On days 25 to 28, the animals were killed to isolate carotid artery and mount its segments in Mulvany myographs. Cumulative concentration response curves to α-adrenergic receptor agonists and 5-HT were constructed in the absence or presence of suitable antagonists. Results. - The potency of norepinephrine in ovariectomized rats was significantly reduced in animals treated with progesterone as compared to those with placebo. In placebo-treated ovariectomized animals there was a noticeable response mediated by α2-adrenoceptors, in contrast to that in sham-operated or ovariectomized rats treated with 17β-estradiol and progesterone, either alone or in combination. The plasma levels of norepinephrine and epinephrine were not significantly affected by either ovariectomy or the subsequent hormone replacement. The potency of 5-HT was significantly reduced in animals having circulating sex hormones as compared to that in placebo-treated ovariectomized animals. Conclusion. - Taken together, our results indicate that circulating progesterone and/or 17β-estradiol may reduce the contraction of the rat carotid artery in response to norepinephrine and 5-HT. This effect of female sex hormones might be one of the factors through which these hormones aggravate migraine in women.

Female sex hormones and rat dural vasodilatation to CGRP, periarterial electrical stimulation and capsaicin (Article)

2007-02-01T00:00:00Z

Background. - The prevalence of migraine is 2 to 3-fold higher in females than in males, and it is intricately related to the levels of female sex hormones. These hormones may regulate the synthesis and receptor expression of calcitonin gene-related peptide (CGRP), which mediates neurogenic dural vasodilatation and is implicated in migraine pathogenesis. Objective. - To investigate the effects of the female sex steroids, 17β-estradiol and progesterone, separately and in combination, on dural vasodilatation induced by αCGRP, periarterial electrical stimulation and capsaicin in ovariectomized rats, using intravital microscopy. Methods. - Sprague-Dawley rats were ovariectomized and, 7 days later, subcutaneously implanted with 21-day release pellets of 17β-estradiol, progesterone, their combination or placebo. On day 19 to 21, the animals were anesthetized, overlying bone thinned to visualize the middle meningeal artery and vasodilator responses to αCGRP (10 to 3000 ng kg-1), periarterial electrical stimulation (25 to 125 μA) and capsaicin (0.3 to 18 μg kg-1) elicited. Results. - There were no significant differences in the vasodilator potency or efficacy of αCGRP or capsaicin in the different groups studied. In contrast, the vasodilator response to electrical stimulation was significantly higher in rats treated with 17β-estradiol (Emax:157 ± 19%) as compared to those observed after placebo treatment (Emax:93 ± 11%). Conclusion. - Our results show that, in contrast to CGRP- or capsaicin-induced dural vasodilatation, 17β-estradiol enhanced neurogenic vasodilatation, suggesting increased CGRP release from perivascular nerves. This may be one of the mechanisms through which 17β-estradiol exacerbates migraine in women.

Potential role of female sex hormones in the pathophysiology of migraine (Article)

2007-02-01T00:00:00Z

Clinical evidence indicates that female sex steroids may contribute to the high prevalence of migraine in women, as well as changes in the frequency or severity of migraine attacks that are in tandem with various reproductive milestones in women's life. While female sex steroids do not seem to be involved in the pathogenesis of migraine per se, they may modulate several mediators and/or receptor systems via both genomic and non-genomic mechanisms; these actions may be perpetuated at the central nervous system, as well as at the peripheral (neuro)vascular level. For example, female sex steroids have been shown to enhance: (i) neuronal excitability by elevating Ca2+and decreasing Mg2+concentrations, an action that may occur with other mechanisms triggering migraine; (ii) the synthesis and release of nitric oxide (NO) and neuropeptides, such as calcitonin gene-related peptide CGRP, a mechanism that reinforces vasodilatation and activates trigeminal sensory afferents with a subsequent stimulation of pain centres; and (iii) the function of receptors mediating vasodilatation, while the responses of receptors inducing vasoconstriction are attenuated. The serotonergic, adrenergic and γ-aminobutyric acid (GABA)-ergic systems are also modulated by sex steroids, albeit to a varying degree and with potentially contrasting effects on migraine outcome. Taken together, female sex steroids seem to be involved in an array of components implicated in migraine pathogenesis. Future studies will further delineate the extent and the clinical relevance of each of these mechanisms, and will thus expand the knowledge on the femininity of migraine.

Angiotensin II type 2 receptor-mediated vasodilation in human coronary microarteries. (Article)

2004-05-18T00:00:00Z

BACKGROUND: Angiotensin (Ang) II type 2 (AT2) receptor stimulation results in coronary vasodilation in the rat heart. In contrast, AT2 receptor-mediated vasodilation could not be observed in large human coronary arteries. We studied Ang II-induced vasodilation of human coronary microarteries (HCMAs). METHODS AND RESULTS: HCMAs (diameter, 160 to 500 microm) were obtained from 49 heart valve donors (age, 3 to 65 years). Ang II constricted HCMAs, mounted in Mulvany myographs, in a concentration-dependent manner (pEC50, 8.6+/-0.2; maximal effect [E(max)], 79+/-13% of the contraction to 100 mmol/L K+). The Ang II type 1 receptor antagonist irbesartan prevented this vasoconstriction, whereas the AT2 receptor antagonist PD123319 increased E(max) to 97+/-14% (P<0.05). The increase in E(max) was larger in older donors (correlation DeltaE(max) versus age, r=0.47, P<0.05). The PD123319-induced potentiation was not observed in the presence of the NO synthase inhibitor L-NAME, the bradykinin type 2 (B2) receptor antagonist Hoe140, or after removal of the endothelium. Ang II relaxed U46619-preconstricted HCMAs in the presence of irbesartan by maximally 49+/-16%, and PD123319 prevented this relaxation. Finally, radioligand binding studies and reverse transcription-polymerase chain reaction confirmed the expression of AT2 receptors in HCMAs. CONCLUSIONS: AT2 receptor-mediated vasodilation in the human heart appears to be limited to coronary microarteries and is mediated by B2 receptors and NO. Most likely, AT2 receptors are located on endothelial cells, and their contribution increases with age.

Bradykinin-induced relaxation of coronary microarteries: S-nitrosothiols as EDHF? (Article)

2004-05-01T00:00:00Z

1. To investigate whether S-nitrosothiols, in addition to NO, mediate bradykinin-induced vasorelaxation, porcine coronary microarteries (PCMAs) were mounted in myographs. 2. Following preconstriction, concentration-response curves (CRCs) were constructed to bradykinin, the NO donors S-nitroso-N-penicillamine (SNAP) and diethylamine NONOate (DEA-NONOate) and the S-nitrosothiols L-S-nitrosocysteine (L-SNC) and D-SNC. All agonists relaxed PCMAs. L-SNC was approximately 5-fold more potent than D-SNC. 3. The guanylyl cyclase inhibitor ODQ and the NO scavenger hydroxocobalamin induced a larger shift of the bradykinin CRC than the NO synthase inhibitor L-NAME, although all three inhibitors equally suppressed bradykinin-induced cGMP responses. 4. Complete blockade of bradykinin-induced relaxation was obtained with L-NAME in the presence of the large- and intermediate-conductance Ca(2+)-activated K(+)-channel (BK(Ca), IK(Ca)) blocker charybdotoxin and the small-conductance Ca(2+)-activated K(+)-channel (SK(Ca)) channel blocker apamin, but not in the presence of L-NAME, apamin and the BK(Ca) channel blocker iberiotoxin. 5. Inhibitors of cytochrome P450 epoxygenase, cyclooxygenase, voltage-dependent K(+) channels and ATP-sensitive K(+) channels did not affect bradykinin-induced relaxation. 6. SNAP-, DEA-NONOate- and D-SNC-induced relaxations were mediated entirely by the NO-guanylyl cyclase pathway. L-SNC-induced relaxations were partially blocked by charybdotoxin+apamin, but not by iberiotoxin+apamin, and this blockade was abolished following endothelium removal. ODQ, but not hydroxocobalamin, prevented L-SNC-induced increases in cGMP, and both drugs shifted the L-SNC CRC 5-10-fold to the right. 7. L-SNC hyperpolarized intact and endothelium-denuded coronary arteries. 8. Our results support the concept that bradykinin-induced relaxation is mediated via de novo synthesized NO and a non-NO, endothelium-derived hyperpolarizing factor (EDHF). S-nitrosothiols, via stereoselective activation of endothelial IK(Ca) and SK(Ca) channels, and through direct effects on smooth muscle cells, may function as an EDHF in porcine coronary microarteries.

Mediators of bradykinin-induced vasorelaxation in human coronary microarteries. (Article)

2004-02-01T00:00:00Z

To investigate the mediators of bradykinin-induced vasorelaxation in human coronary microarteries (HCMAs), HCMAs (diameter approximately 300 microm) obtained from 42 heart valve donors (20 men and 22 women; age range, 3 to 65 years; mean age, 46 years) were mounted in Mulvany myographs. In the presence of the cyclooxygenase inhibitor indomethacin, bradykinin relaxed preconstricted HCMAs in a concentration-dependent manner. N(G)-nitro-L-arginine methyl ester and ODQ (inhibitors of nitric oxide [NO] synthase and guanylyl cyclase, respectively) and the NO scavenger hydroxocobalamin, either alone or in combination, shifted the bradykinin concentration-response curve to the right. Removal of H2O2 (with catalase), inhibition of cytochrome P450 epoxygenase (with sulfaphenazole or clotrimazole) or gap junctions (with 18alpha-glycyrrhetinic acid or carbenoxolone), and blockade of large- (BK(Ca)) and small- (SK(Ca)) conductance Ca2+-dependent K+ channels (with iberiotoxin and apamin), either alone or in addition to hydroxocobalamin, did not affect bradykinin. In contrast, complete blockade of bradykinin-induced relaxation was obtained when we combined the nonselective BK(Ca) and intermediate-conductance (IK(Ca)) Ca2+-dependent K+ channel blocker charybdotoxin and apamin with hydroxocobalamin. Charybdotoxin plus apamin alone were without effect. Inhibition of inwardly rectifying K+ channels (K(IR)) and Na+/K+-ATPase (with BaCl2 and ouabain, respectively) shifted the bradykinin concentration-response curve 10-fold to the right but did not exert an additional effect in the presence of hydroxocobalamin. In conclusion, bradykinin-induced relaxation in HCMAs depends on (1) the activation of guanylyl cyclase, K(IR), and Na(+)/K(+)-ATPase by NO and (2) IK(Ca) and SK(Ca) channels. The latter are activated by a factor other than NO. This factor is not a cytochrome P450 epoxygenase product or H2O2, nor does it depend on gap junctions or BK(Ca) channels.

Effects of the CGRP receptor antagonist BIBN4096BS on capsaicin-induced carotid haemodynamic changes in anaesthetised pigs. (Article)

2003-09-01T00:00:00Z

1. Calcitonin gene-related peptide (CGRP), a potent vasodilator released from capsaicin-sensitive trigeminal sensory nerves, seems to be involved in the pathogenesis of migraine. Hence, CGRP receptor antagonists may serve as a novel treatment for migraine. This study was therefore designed to investigate the effects of BIBN4096BS (100, 300 and 1000 microg kg-1, i.v.), a potent and selective CGRP receptor antagonist, on capsaicin-induced carotid haemodynamic changes in anaesthetised pigs. Both vagosympathetic trunks were cut and phenylephrine was infused into the carotid artery (i.c.) to support carotid vascular tone. 2. Infusions of capsaicin (0.3, 1, 3 and 10 microg kg-1 min-1, i.c.) did not alter the heart rate, but dose-dependently increased the mean arterial blood pressure. This moderate hypertensive effect was not modified by BIBN4096BS. 3. Capsaicin infusion (10 microg kg-1 min-1, i.c.) increased total carotid, arteriovenous anastomotic and tissue blood flows and conductances as well as carotid pulsations, but decreased the difference between arterial and jugular venous oxygen saturations. These responses to capsaicin were dose-dependently blocked by BIBN4096BS. 4. Capsaicin infusion (10 microg kg-1 min-1, i.c.) more than doubled the jugular venous plasma concentration of CGRP. This effect was not blocked, but rather increased, by BIBN4096BS. 5. The above results show that BIBN4096BS behaves as a potent antagonist of capsaicin-induced carotid haemodynamic changes that are mediated via the release of CGRP. Therefore, this compound may prove effective in the treatment of migraine.

ACE-versus chymase-dependent angiotensin II generation in human coronary arteries: a matter of efficiency? (Article)

2003-01-01T00:00:00Z

OBJECTIVE: The objective of this study was to investigate ACE- and chymase-dependent angiotensin I-to-II conversion in human coronary arteries (HCAs). METHODS AND RESULTS: HCA rings were mounted in organ baths, and concentration-response curves to angiotensin II, angiotensin I, and the chymase-specific substrate Pro(11)-D-Ala(12)-angiotensin I (PA-angiotensin I) were constructed. All angiotensins displayed similar efficacy. For a given vasoconstriction, bath (but not interstitial) angiotensin II during angiotensin I and PA-angiotensin I was lower than during angiotensin II, indicating that interstitial (and not bath) angiotensin II determines vasoconstriction. PA-angiotensin I increased interstitial angiotensin II less efficiently than angiotensin I. Separate inhibition of ACE (with captopril) and chymase (with C41 or chymostatin) shifted the angiotensin I concentration-response curve approximately 5-fold to the right, whereas a 10-fold shift occurred during combined ACE and chymase inhibition. Chymostatin, but not captopril and/or C41, reduced bath angiotensin II and abolished PA-Ang I-induced vasoconstriction. Perfused HCA segments, exposed luminally or adventitially to angiotensin I, released angiotensin II into the luminal and adventitial fluid, respectively, and this release was blocked by chymostatin. CONCLUSIONS: Both ACE and chymase contribute to the generation of functionally active angiotensin II in HCAs. However, because angiotensin II loss in the organ bath is chymase-dependent, ACE-mediated conversion occurs more efficiently (ie, closer to AT(1) receptors) than chymase-mediated conversion.

Prorenin-induced myocyte proliferation: no role for intracellular angiotensin II (Article)

2002-03-04T00:00:00Z

Cardiomyocytes bind, internalize, and activate prorenin, the inactive precursor of renin, via a mannose 6-phosphate receptor (M6PR)--dependent mechanism. M6PRs couple directly to G-proteins. To investigate whether prorenin binding to cardiomyocytes elicits a response, and if so, whether this response depends on angiotensin (Ang) II, we incubated neonatal rat cardiomyocytes with 2 nmol/L prorenin and/or 150 nmol/L angiotensinogen, with or without 10 mmol/L M6P, 1 micromol/L eprosartan, or 1 micromol/L PD123319 to block M6P and AT(1) and AT(2) receptors, respectively. Protein and DNA synthesis were studied by quantifying [(3)H]-leucine and [(3)H]-thymidine incorporation. For comparison, studies with 100 nmol/L Ang II were also performed. Neither prorenin alone, nor angiotensinogen alone, affected protein or DNA synthesis. Prorenin plus angiotensinogen increased [(3)H]-leucine incorporation (+21 +/- 5%, mean +/- SEM, P<0.01), [(3)H]-thymidine incorporation (+29 +/- 6%, P<0.01), and total cellular protein (+14 +/- 3%, P<0.01), whereas Ang II increased DNA synthesis only (+34 +/- 7%, P<0.01). Eprosartan, but not PD123319 or M6P, blocked the effects of prorenin plus angiotensinogen as well as the effects of Ang II. Medium Ang II levels during prorenin and angiotensinogen incubation were <1 nmol/L. In conclusion, prorenin binding to M6PRs on cardiomyocytes per se does not result in enhanced protein or DNA synthesis. However, through Ang II generation, prorenin is capable of inducing myocyte hypertrophy and proliferation. Because this generation occurs independently of M6PRs, it most likely depends on the catalytic activity of intact prorenin in the medium (because of temporal prosegment unfolding) rather than its intracellular activation. Taken together, our results do not support the concept of Ang II generation in cardiomyocytes following intracellular prorenin activation.

Enhanced expression of fibroblast growth factors and receptor FGFR-1 during vascular remodeling in chronic obstructive pulmonary disease (Article)

2002-01-01T00:00:00Z

Important characteristics of chronic obstructive pulmonary disease (COPD) include airway and vascular remodeling, the molecular mechanisms of which are poorly understood. We assessed the role of fibroblast growth factors (FGF) in pulmonary vascular remodeling by examining the expression pattern of FGF-1, FGF-2, and the FGF receptor (FGFR-1) in peripheral area of lung tissues from patients with COPD (FEV(1) < or = 75%; n = 15) and without COPD (FEV(1) > or = 85%; n = 13). Immunohistochemical staining results were evaluated by digital video image analysis as well as by manual scoring. FGF-1 and FGFR-1 were detected in vascular smooth muscle (VSM), airway smooth muscle, and airway epithelial cells. FGF-2 was localized in the cytoplasm of airway epithelium and in the nuclei of airway smooth muscle, VSM, and endothelial cells. In COPD cases, an unequivocal increase in FGF-2 expression was observed in VSM (3-fold, P = 0.001) and endothelium (2-fold, P = 0.007) of small pulmonary vessels with a luminal diameter under 200 micro m. In addition, FGFR-1 levels were elevated in the intima (1.5-fold, P = 0.05). VSM cells of large (> 200 micro m) pulmonary vessels showed increased staining for FGF-1 (1.6-fold, P < 0.03) and FGFR-1 (1.4-fold, P < 0.04) in COPD. Pulmonary vascular remodeling, assessed as the ratio of alpha-smooth muscle actin staining and vascular wall area with the lumen diameter, was increased in large vessels of patients with COPD (P = 0.007) and was inversely correlated with FEV(1) values (P < 0.007). Our results suggest an autocrine role of the FGF-FGFR-1 system in the pathogenesis of COPD-associated vascular remodeling.

Vasoconstriction is determined by interstitial rather than circulating angiotensin II. (Article)

2002-01-01T00:00:00Z

1. We investigated why angiotensin (Ang) I and II induce vasoconstriction with similar potencies, although Ang I-II conversion is limited. 2. Construction of concentration-response curves to Ang I and II in porcine femoral arteries, in the absence or presence of the AT(1) or AT(2) receptor antagonists irbesartan and PD123319, revealed that the approximately 2 fold difference in potency between Ang I and II was not due to stimulation of different AT receptor populations by exogenous and locally generated Ang II. 3. Measurement of Ang I and II and their metabolites at the time of vasoconstriction confirmed that, at equimolar application of Ang I and II, bath fluid Ang II during Ang I was approximately 18 times lower than during Ang II and that Ang II was by far the most important metabolite of Ang I. Tissue Ang II was 2.9+/-1.5% and 12.2+/-2.4% of the corresponding Ang I and II bath fluid levels, and was not affected by irbesartan or PD123319, suggesting that it was located extracellularly. 4. Since approximately 15% of tissue weight consists of interstitial fluid, it can be calculated that interstitial Ang II levels during Ang II resemble bath fluid Ang II levels, whereas during Ang I they are 8.8 - 27 fold higher. Consequently at equimolar application of Ang I and II, the interstitial Ang II levels differ only 2 - 4 fold. 5. Interstitial, rather than circulating Ang II determines vasoconstriction. Arterial Ang I, resulting in high interstitial Ang II levels via its local conversion by ACE, may be of greater physiological importance than arterial Ang II.

Cardiomyocytes bind and activate native human prorenin : role of soluble mannose 6-phosphate receptors (Article)

2001-01-01T00:00:00Z

Cardiomyocytes bind, internalize, and activate recombinant human prorenin through mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptors. To investigate whether this also applies to native human prorenin, neonatal rat myocytes were incubated for 4 hours at 37 degrees C with various prorenin-containing human body fluids. Uptake and activation by M6P/IGFII receptors were observed for plasma prorenin from subjects with renal artery stenosis and/or hypertension and for follicular fluid prorenin. The total amount of cellular renin and prorenin (expressed as percentage of the levels of renin and prorenin in the medium) after 4 hours of incubation was 4 to 10 times lower than after incubation with recombinant human prorenin. Although plasma contains alkaline phosphatases capable of inactivating the M6P label as well as soluble M6P/IGFII receptors that block prorenin binding in a competitive manner and proteins (eg, insulin, IGFII) that increase the number of cell-surface M6P/IGFII receptors, these factors were not responsible for the modest uptake of native human prorenin. Uptake did not occur during incubation of myocytes with plasma prorenin from anephric subjects or with amniotic fluid prorenin, and this was not due to the presence of excessively high levels of M6P/IGFII receptors and/or phosphatase activity in these fluids. In conclusion, myocytes are capable of binding, internalizing, and activating native human prorenin of renal and ovarian origin through M6P/IGFII receptors. Differences in prorenin glycosylation and/or phosphorylation as well as the concentration of soluble M6P/IGFII receptors and growth factors affecting cell-surface M6P/IGFII receptor density determine the amount of prorenin entering the heart and thus cardiac angiotensin II production.

High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin (Article)

2001-01-01T00:00:00Z

Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.

Bradykinin potentiation by angiotensin-(1-7) and ACE inhibitors correlates with ACE C- and N-domain blockade (Article)

2001-01-01T00:00:00Z

ACE inhibitors block B(2) receptor desensitization, thereby potentiating bradykinin beyond blocking its hydrolysis. Angiotensin (Ang)-(1-7) also acts as an ACE inhibitor and, in addition, may stimulate bradykinin release via angiotensin II type 2 receptors. In this study we compared the bradykinin-potentiating effects of Ang-(1-7), quinaprilat, and captopril. Porcine coronary arteries, obtained from 32 pigs, were mounted in organ baths, preconstricted with prostaglandin F(2alpha), and exposed to quinaprilat, captopril, Ang-(1-7), and/or bradykinin. Bradykinin induced complete relaxation (pEC(50)=8.11+/-0.07, mean+/-SEM), whereas quinaprilat, captopril, and Ang-(1-7) alone were without effect. Quinaprilat shifted the bradykinin curve to the left in a biphasic manner: a 5-fold shift at concentrations that specifically block the C-domain (0.1 to 1 nmol/L) and a 10-fold shift at concentrations that block both domains. Captopril and Ang-(1-7) monophasically shifted the bradykinin curve to the left, by a factor of 10 and 5, respectively. A 5-fold shift was also observed when Ang-(1-7) was combined with 0.1 nmol/L quinaprilat. Repeated exposure of porcine coronary arteries to 0.1 micromol/L bradykinin induced B(2) receptor desensitization. The addition of 10 micromol/L quinaprilat or Ang-(1-7) to the bath, at a time when bradykinin alone was no longer able to induce relaxation, fully restored the relaxant effects of bradykinin. Angiotensin II type 1 or 2 receptor blockade did not affect any of the observed effects of Ang-(1-7). In conclusion, Ang-(1-7), like quinaprilat and captopril, potentiates bradykinin by acting as an ACE inhibitor. Bradykinin potentiation is maximal when both the ACE C- and N-terminal domains are inhibited. The inhibitory effects of Ang-(1-7) are limited to the ACE C-domain, raising the possibility that Ang-(1-7) synergistically increases the blood pressure-lowering effects of N-domain-specific ACE inhibitors.

AT(2) receptor-mediated vasodilation in the heart: effect of myocardial infarction (Article)

2001-01-01T00:00:00Z

To investigate the functional consequences of postinfarct cardiac angiotensin (ANG) type 2 (AT(2)) receptor upregulation, rats underwent coronary artery ligation or sham operation and were infused with ANG II 3-4 wk later, when scar formation is complete. ANG II increased mean arterial pressure (MAP) more modestly in infarcted animals than in sham animals. The AT(1) receptor antagonist irbesartan, but not the AT(2) receptor antagonist PD123319, decreased MAP and antagonized the ANG II-mediated systemic hemodynamic effects. Myocardial (MVC) but not renal vascular conductance (RVC) was diminished in infarcted versus sham rats. ANG II did not affect MVC and reduced RVC in all rats. MVC was unaffected by irbesartan and PD123319 in all animals. However, with PD123319, ANG II reduced MVC in sham but not infarcted animals, and, with irbesartan, ANG II increased MVC in infarcted but not sham animals. Irbesartan increased RVC and antagonized the ANG II-mediated renal effects in all animals. RVC, at baseline or with ANG II, was not affected by PD123319 in infarcted and sham animals. In conclusion, coronary but not renal AT(2) receptor stimulation results in vasodilation, and this effect is enhanced in infarcted rats.

L-NAME-resistant bradykinin-induced relaxation in porcine coronary arteries is NO-dependent: effect of ACE inhibition. (Article)

2000-01-01T00:00:00Z

1. NO synthase (NOS)inhibitors partially block bradykinin (BK)-mediated vasorelaxation. Here we investigated whether this is due to incomplete NOS inhibition and/or NO release from storage sites. We also studied the mechanism behind ACE inhibitor-mediated BK potentiation. 2. Porcine coronary arteries (PCAs) were mounted in organ baths, preconstricted, and exposed to BK or the ACE-resistant BK analogue Hyp(3)-Tyr(Me)(8)-BK (HT-BK) with or without the NOS inhibitor L-NAME (100 microM), the NO scavenger hydroxocobalamin (200 microM), the Ca(2+)-dependent K(+)-channel blockers charybdotoxin+apamin (both 100 nM), or the ACE inhibitor quinaprilat (10 microM). 3. BK and HT-BK dose-dependently relaxed preconstricted vessels (pEC(50) 8.0+/-0.1 and 8.5+/-0.2, respectively). pEC(50)'s were approximately 10 fold higher with quinaprilat, and approximately 10 fold lower with L-NAME or charybdotoxin+apamin. Complete blockade was obtained with hydroxocobalamin or L-NAME+ charybdotoxin+apamin. 4. Repeated exposure to 100 nM BK or HT-BK, to deplete NO storage sites, produced progressively smaller vasorelaxant responses. With L-NAME, the decrease in response occurred much more rapidly. L-Arginine (10 mM) reversed the effect of L-NAME. 5. Adding quinaprilat to the bath following repeated exposure (with or without L-NAME), at the time BK and HT-BK no longer induced relaxation, fully restored vasorelaxation, while quinaprilat alone had no effect. Quinaprilat also relaxed vessels that, due to pretreatment with hydroxocobalamin or L-NAME+charybdotoxin+apamin, previously had not responded to BK. 6. In conclusion, L-NAME-resistant BK-induced relaxation in PCAs depends on NO from storage sites, and is mediated via stimulation of guanylyl cyclase and/or Ca(2+)-dependent K(+)-channels. ACE inhibitors potentiate BK independent of their effect on BK metabolism.

Molecular cloning, sequence analysis and pharmacological properties of the porcine 5-HT(1D) receptor. (Article)

2000-01-01T00:00:00Z

A cDNA encoding the full-length 5-HT(1D) receptor derived from porcine cerebral cortex was amplified, cloned and sequenced, using guinea-pig 5-HT(1D) receptor coding sequence oligonucleotide primers in reverse transcription-polymerase chain reaction (RT - PCR). The 5' and 3' ends of the porcine 5-HT(1D) receptor cDNA were verified by inverse PCR. Sequence analysis of porcine 5-HT(1D) receptor cDNA revealed an open reading frame of 1134 nucleotides encoding a polypeptide of 377 amino acids having 92% homology with the human 5-HT(1D) receptor and 88 - 90% homology with other species homologues. The porcine 5-HT(1D) receptor cDNA was further subcloned into a mammalian expression vector pcDNA3 and expressed in monkey Cos-7 cells. Radioligand binding assays using either [(3)H]-5-CT or [(3)H]-GR125743 on Cos-7 cell membranes showed that pK(i) values of 14 serotonin ligands were highly correlated with those obtained with the human 5-HT(1D) receptor. Nonetheless, a selective antagonist at the human 5-HT(1D) receptor, BRL15572, only poorly recognized the porcine homologue. Using membranes from cells co-expressing the porcine 5-HT(1D) receptor and rat G(alphail)Cys(351) Ile protein, it was shown that 5-HT and zolmitriptan increased, while ketanserin decreased basal [(35)S]-GTPgammaS binding. The potency of zolmitriptan in the [(35)S]-GTPgammaS binding assay (pEC(50): 8. 46+/-0.08) agreed with its affinity in displacing the radioligands [(3)H]-5-CT and [(3)H]-GR125743 (pK(i): 8.38+/-0.15 and 8.67+/-0.08, respectively). In conclusion, we have established the cDNA sequence and pharmacology of the cloned porcine 5-HT(1D) receptor. This information would be useful in exploring the role of divergent amino acid residues in the receptor-ligand interaction as well as the role of 5-HT(1D) receptor in pathophysiological processes relevant for novel drug discovery in diseases such as migraine.

Tumor necrosis factor-alpha enhances mRNA expression and secretion of interleukin-6 in cultured human airway smooth muscle cells (Article)

2000-01-01T00:00:00Z

Airway smooth muscle (ASM) is considered to be an end-target cell for the effects of mediators released during airway wall inflammation. Several reports suggest that activated ASM may be capable of generating various proinflammatory cytokines. We investigated the effects of tumor necrosis factor (TNF)-alpha, a potent proinflammatory cytokine, on cultured human ASM cells by examining the expression and release of the cytokine interleukin (IL)-6, cell proliferation, and the expression pattern of c-fos and c-jun, two nuclear proto-oncogenes constituting the activator protein-1 transcription factor. Growth-arrested cell monolayers were stimulated with human recombinant TNF-alpha in a concentration- and time-dependent manner. TNF-alpha stimulated the expression of IL-6 messenger RNA (mRNA), which was detected after 15 min, reaching a maximum at 1 h. IL-6 protein was readily detected in ASM cell-conditioned medium after 2 h of TNF-alpha stimulation. Protein levels increased in a time- and concentration-dependent manner. Release of IL-6 elicited by TNF-alpha was significantly inhibited by dexamethasone, cycloheximide, and nordihydroguaiaretic acid (NDGA). TNF-alpha did not alter DNA biosynthesis up to 48 h or cell numbers up to 120 h. Northern blot analysis of proto-oncogene expression revealed that c-fos and c-jun mRNA levels were elevated after 30 min of TNF-alpha incubation with maximum levels at 1 h and 45 min, respectively. Expression of c-fos mRNA was downregulated by NDGA. Four hours of TNF-alpha treatment resulted in translocation of c-jun immunofluorescence from the cytoplasm to the nucleus in human ASM cells. Our results suggest that despite the lack of a mitogenic response to TNF-alpha, upregulation of primary response genes in human ASM cells may account for the induction of proinflammatory cytokines, such as IL-6, in human airways.

Localization and production of angiotensin II in the isolated perfused rat heart (Article)

1998-01-01T00:00:00Z

We used a modification of the isolated perfused rat heart, in which coronary effluent and interstitial transudate were separately collected, to investigate the localization and production of angiotensin II (Ang II) in the heart. During combined renin (0.7 to 1.5 pmol Ang I/mL per minute) and angiotensinogen (6 to 12 pmol/mL) perfusion (4 to 8 mL/min) for 60 minutes (n=3), the steady-state levels of Ang II in interstitial transudate in two consecutive 10-minute periods were 4.3+/-1.5 and 3.6+/-1.5 fmol/mL compared with 1.1+/-0.4 and 1.1+/-0.6 fmol/mL in coronary effluent (mean+/-half range). During perfusion with Ang II (n=5), steady-state Ang II in interstitial transudate was 32+/-19% of arterial Ang II compared with 65+/-16% in coronary effluent (mean+/-SD, P<.02). During perfusion with Ang I (n=5), Ang II in interstitial transudate was 5.1+/-0.6% of arterial Ang I compared with 2.2+/-0.3% in coronary effluent (P<.05). The tissue concentration of Ang II in the combined renin/angiotensinogen perfusions (per gram) was as high as the concentration in interstitial transudate (per milliliter). Addition of losartan (10(-6) mol/L) to the renin/angiotensinogen perfusion (n=3) had no significant effect on the tissue level of Ang II, whereas losartan in the perfusions with Ang I (n=5) or Ang II (n=5) decreased tissue Ang II to undetectably low levels. The results indicate that the heart is capable of producing Ang II and that this can lead to higher levels in tissue than in blood plasma. Cardiac Ang II does not appear to be restricted to the extracellular fluid. This is in part due to AT1-receptor-mediated cellular uptake of extracellular Ang II, but our results also raise the possibility of intracellular Ang II production.

Angiotensin II induces hypertrophy of human airway smooth muscle cells: expression of transcription factors and transforming growth factor-beta1 (Article)

1998-01-01T00:00:00Z

Increased smooth muscle mass due to hyperplasia and hypertrophy of airway smooth muscle (ASM) cells is a common feature in asthma. Angiotensin II (Ang II), a potent vasoconstrictor and mitogen for a wide variety of cells, has recently been implicated in bronchoconstriction in asthmatics. However, a possible mitogenic role as well as underlying molecular mechanisms of this octapeptide in human ASM cells are not yet known. We studied the effects of Ang II on ASM cell proliferation and growth and on the expression of three transcription factors, egr-1, c-fos, and c-jun, as well as a cytokine, transforming growth factor-beta1 (TGF-beta1). Human ASM cells were isolated by enzymatic digestion of bronchial smooth muscle obtained from lung resection tissue. Confluent cells were growth-arrested and subsequently incubated with Ang II (100 nM) for different time periods and processed for the measurement of cell growth and gene expression. Ang II significantly induced DNA and protein synthesis in human ASM cells at 8 h, resulting in a net increase in the accumulation of protein over DNA (i.e., cellular hypertrophy) at 16 h of incubation. Cell counts and MTT-reduction assay, however, showed no increase in cell number as a result of Ang II stimulation. Ang II stimulated the expression of egr-1 and c-fos as early as 15 min, reaching maximum levels at 45 min, whereas the expression of c-jun peaked at 2 h of Ang II exposure. Furthermore, steady-state mRNA levels of TGF-beta1 were upregulated by Ang II after 4 h and reached peak levels at 16 h of incubation. Secretion of biologically active TGF-beta1 from human ASM cells was significantly (P <= 0.02) enhanced by Ang II incubation after 8 h, which remained elevated until 24 h. Our results suggest that the Ang II-induced transient early expression of transcription factors may regulate autocrine genes like TGF-beta1, of which the subsequent late upregulation could contribute to cellular hypertrophy during, for example, airway remodeling in asthma.

Coronary side-effect potential of current and prospective antimigraine drugs (Article)

1998-01-01T00:00:00Z

BACKGROUND: The antimigraine drugs ergotamine and sumatriptan may cause angina-like symptoms, possibly resulting from coronary artery constriction. We compared the coronary vasoconstrictor potential of a number of current and prospective antimigraine drugs (ergotamine, dihydroergotamine, methysergide and its metabolite methylergometrine, sumatriptan, naratriptan, zolmitriptan, rizatriptan, avitriptan). METHODS AND RESULTS: Concentration-response curves to the antimigraine drugs were constructed in human isolated coronary artery segments to obtain the maximum contractile response (Emax) and the concentration eliciting 50% of Emax (EC50). The EC50 values were related to maximum plasma concentrations (Cmax) reported in patients, obtaining Cmax/EC50 ratios as an index of coronary vasoconstriction occurring in the clinical setting. Furthermore, we studied the duration of contractile responses after washout of the acutely acting antimigraine drugs to assess their disappearance from the receptor biophase. Compared with sumatriptan, all drugs were more potent (lower EC50 values) in contracting the coronary artery but had similar efficacies (Emax <25% of K+-induced contraction). The Cmax of avitriptan was 7- to 11-fold higher than its EC50 value, whereas those of the other drugs were <40% of their respective EC50 values. The contractile responses to ergotamine and dihydroergotamine persisted even after repeated washings, but those to the other drugs declined rapidly after washing. CONCLUSIONS: All current and prospective antimigraine drugs contract the human coronary artery in vitro, but in view of low efficacy, these drugs are unlikely to cause myocardial ischemia at therapeutic plasma concentrations in healthy subjects. In patients with coronary artery disease, however, these drugs must remain contraindicated. The sustained contraction by ergotamine and dihydroergotamine seems to be an important disadvantage compared with sumatriptan-like drugs.

Nature of 5-HT1-like receptors mediating depressor responses in vagosympathectomized rats; close resemblance to the cloned 5-ht7 receptor (Article)

1997-07-14T00:00:00Z

It has been suggested that the late hypotensive response to serotonin (5-hydroxytryptamine; 5-HT) in vagosympathectomized rats is mediated by '5-HT1-like' receptors since this effect is mimicked by 5-carboxamidotryptamine (5-CT), is not modified by cyproheptadine, ketanserin or MDL 72222, but it is blocked by met hysergide. The present study was set out to reanalyze this suggestion in terms of the classification schemes proposed in 1994 and 1996 by the NC-IUPHAR subcommittee on the classification and nomenclature of 5-HT receptors. I.v. bolus injections of 5-CT (0.01-0.3 μg · kg-1), 5-HT (1-30 μg · kg-1) and 5-methoxytryptamine (5-MeO-T; 1-30 μg · kg-1) produced dose-dependent hypotensive responses with a rank order of agonist potency: 5-CT >> 5-HT ≤ 5-methoxytryptamine with sumatriptan (30-1000 μg · kg-1) inactive. The depressor responses to 5-HT and 5-CT were not attenuated by i.v. GR127935 (300-3000 μg · kg-1) or equivalent volumes of saline. In contrast, lisuride, methiothepin, mesulergine, metergoline and clozapine dose-dependently antagonized the responses to 5-HT and 5-CT; the rank order of apparent pA2values against 5-HT and 5-CT, respectively, was: lisuride (7.7; 7.8) > methiothepin (6.8; 7.0) ≤ mesulergine (6.4; 6.6) > clozapine (5.7; 5.8); metergoline displayed variable potencies (5.6; 6.4). Except for lisuride, which also affected isoprenaline-induced hypotension, the antagonism by the other drugs was selective. Based upon the above rank order of agonist potency, the blockade by a series of drugs showing high affinity for the cloned 5-ht7receptor and the lack of blockade by GR127935, our results indicate that the 5-HT receptor mediating hypotension in vagosympathectomized rats is operationally similar to other putative 5-ht7receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine coronary and external carotid arteries and guinea-pig ileum as well as feline tachycardia).

5-Hydroxytryptamine-induced contractions of the human isolated saphenous vein: involvement of 5-HT2 and 5HT1D-like receptors and a comparison with grafted veins (Article)

1992-01-01T00:00:00Z

The receptors mediating the contractile effect of 5-hydroxytryptamine (5-HT) on the human isolated saphenous vein, obtained from 42 patients undergoing coronary bypass surgery, have been further characterized using a number of 5-HT-related drugs. The rank order of agonist potency was 5-carboxamidotryptamine (5-CT) approximately 5-HT greater than methysergide approximately sumatriptan approximately alpha-methyl-5-HT approximately 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indolesuccinate (RU 24969) approximately 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) greater than 2-methyl-5-HT greater than 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT). Flesinoxan was inactive as an agonist. Ketanserin (1 mumol/l) hardly affected sumatriptan-induced contractions but it caused a rightward shift of the upper part of the concentration-response curve of 5-HT and 5-CT. The same concentration of ketanserin caused a parallel rightward shift of the concentration-response curves of alpha-methyl-5-HT and DOI with pKB values of 7.1 and 7.1, respectively. The responses to sumatriptan were antagonized by methiothepin (0.1 mumol/l), metergoline (0.1 and 1 mumol/l), rauwolscine (1 mumol/l) and cyanopindolol (1 mumol/l); the calculated pKB values were 7.3, 6.9, 7.3, 6.7 and 6.5, respectively. Contractions to 5-HT were antagonized by methysergide (1 mumol/l), methiothepin (0.1 mumol/l; pKB = 7.1), ICS 205-930 (1 mumol/l; pKB = 5.9) and flesinoxan (30 mumol/l; pKB = 5.3). Remarkably, the contractions elicited by 2-methyl-5-HT were not attenuated by ICS 205-930, but were antagonized by methiothepin (0.1 mumol/l) and, more markedly, by ketanserin (1 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)