Differential distribution of 5HT1D- and 5HT1B-immunoreactivity within the human trigemino-cerebrovascular system: implications for the discovery of new antimigraine drugs

Sumatriptan, a 5HT_1B/1D-receptor agonist, is clinically effective as an antimigraine agent. Its therapeutic action may result partly from vasoconstriction of excessively dilated cranial blood vessels (a 5HT_1B-receptor mediated response). The antimigraine activity of sumatriptan may also result from inhibition of the release of vasoactive neuropeptides from trigeminal sensory fibres within the meninges. The identity of the 5HT_1B/1D-receptor subtype mediating this effect is unknown. Using 5HT_1D- and 5HT_1B-receptor-specific antibodies we have demonstrated a differential distribution of these receptor subtypes within the human trigemino-cerebrovascular system. Only 5HT_1B-receptor protein was detected on dural arteries. In contrast, only 5HT_1D-receptor protein was detected on trigeminal sensory neurones including peripheral and central projections to dural blood vessels and to the medulla. Within the medulla 5HT_1D-receptor protein was confined to discrete areas associated with the trigeminal sensory system. These findings have important implications for the design of new antimigraine drugs.     

Differential effects of the 5HT1B/1D receptor agonist naratriptan on trigeminal versus spinal nociceptive responses

In vivo electrophysiological assays in anesthetized rats have been used to compare the effects of the 5HT_1B/1D receptor agonist, naratriptan, on central trigeminal nociceptive processing from dural and cutaneous inputs with its effects on nociceptive processing in the spinal cord. Naratriptan inhibited responses of single trigeminal neurons, to noxious electrical and mechanical stimulation of the dura and face, dose dependently by a maximum of 67±3% and 70±18%, respectively, at 3 mg kg⁻¹ i.v. In contrast, naratriptan did not affect spinal dorsal horn neuronal responses to noxious mechanical stimulation of the hind-paw. These findings suggest that 5HT_1B/1D receptors have differential effects on nociceptive processing in the trigeminal versus spinal dorsal horns and provide a potential explanation for the lack of general analgesic effects of brain penetrant 5HT_1B/1D agonist antimigraine drugs.     

Central 5HT1A-receptor binding in normotensive and spontaneously hypertensive rats

Central 5HT1A-receptors have been found with a high density in brainstem and are involved in cardiovascular control. The high-affinity binding of [3H]-8-OH-DPAT, a specific 5HT1A-receptor ligand, was measured in medulla oblongata of normotensive and spontaneously hypertensive rats (SHR). The maximal number of [3H]-8-OH-DPAT binding site (Bmax) was significantly increased in SHR when compared to normotensive rats. Whether central 5HT1A-receptors are involved in the etiology of hypertension or are influenced by other neurochemical events remains to be investigated.     

The antagonist properties of S 11978 on 5HT3 receptors in N1E-115 neuroblastoma cells

Summary— The effects of the novel antagonist S 11978 (Endo-7-[(8-methyl-8-azabicyclo[3,2,1]-3-octyl)oxycarbonyl] benzo[b] thiophene) on 5HT₃ receptors were examined in N1E-115 mouse neuroblastoma x rat glioma hybrid cells, with radioligand binding and whole cell patch clamp techniques. The 5HT₃ receptor ligand [³H] quipazine was displaced by ICS 205–930, GR 38032F and S 11978 with K_I values of 2.25 nM, 36.5 nM and 1.75 nM respectively. Electrophysiological studies showed that S 11978 is a potent 5HT₃ antagonist: IC₅₀ values for inhibition of 5HT-induced inward current by ICS 205–930, GR 38032F and S 11978 were 0.22 nM, 0.63 nM and 0.43 nM respectively at a holding potential of-65 mV. It is concluded that S 11978 is a potent, high affinity 5HT₃ receptor antagonist.     

5HT2 receptors in cerebral cortex of migraineurs studied using PET and 18F-fluorosetoperone

Since the brain 5HT₂ receptors might be implicated in migraine pathogenesis, we have used positron emission tomography and ¹⁸F-fluorosetoperone, a 5HT₂ specific radioligand, to investigate in vivo the cortical 5HT₂ receptors in migraine subjects. Nine migraineurs who had either migraine with and without aura ( n = 5) or only migraine without aura ( n = 4) were studied between attacks. Twelve unmedicated healthy subjects of similar mean age were used as controls. Brain radioactivity was measured after ¹⁸F-setoperone IV injection for 90 min. A decrease of the regional specific distribution volumes (SDV) of the ligand was observed both in migraineurs and in controls. The age adjusted group means of SDV did not differ between patients and controls for the whole and for the right or left frontal, temporal, parietal and occipital cortex. These results suggest that cortical 5HT₂ receptors may be unaltered between attacks in migraine sufferers.     

Migraine association and linkage analyses of the human 5-hydroxytryptamine (5HT2A) receptor gene

5-Hydroxytryptamine (5HT), commonly known as serotonin, which predominantly serves as an inhibitory neurotransmitter in the brain, has long been implicated in migraine pathophysiology. This study tested an Mspl polymorphism in the human 5HT_2A receptor gene ( HTR2A ) and a closely linked microsatellite marker (D13S126), for linkage and association with common migraine. In the association analyses, no significant differences were found between the migraine and control populations for both the Mspl polymorphism and the D13S126 microsatellite marker. The linkage studies involving three families comprising 36 affected members were analysed using both parametric (FASTLINK) and non-parametric (MFLINK and APM) techniques. Significant close linkage was indicated between the Mspl polymorphism and the D13S126 microsatellite marker at a recombination fraction (θ) of zero (lod score=7.15). Linkage results for the Mspl polymorphism were not very informative in the three families, producing maximum and minimum lod scores of only 0.35 and 0.39 at recombination fractions (θ) of 0.2 and 0.00, respectively. However, linkage analysis between the D13S126 marker and migraine indicated significant non-linkage (lod2) up to a recombination fraction (θ) of 0.028. Results from this study exclude the HTR2A gene, which has been localized to chromosome 13q14-q21, for involvement with common migraine.     

Influence of a selective 5HT1-receptor agonist GR43175 on platelet responsiveness

The possible interaction of sumatriptan, a selective 5HT₁-receptor agonist, with platelet responsiveness has been investigated. Stimulation of platelet rich plasma with sumatriptan (1–100 μM) did not induce shape change, aggregation or modification of intraplatelet cytosolic calcium levels. Total inhibition of aggregation induced by 20 μM 5HT was observed in platelets preincubated for 20 min with 100 μM sumatriptan. In the same model, platelet stimulation with 4 μM adenosine 5'-diphosphate (ADP), concentration known to induce an irreversible single-phase curve, determined a decrease of aggregatory response. Concentrations from I μM to 50 μM of sumatriptan did not influence the aggregatory response induced by 5HT and ADP. These effects appear not to be determined by modifications of platelet calcium homeostasis. The possibility to modulate platelet responsiveness by sumatriptan offers a further approach for evaluating the probable link between platelet behaviour and pathophysiology of migraine.     

5-HYDROXYTRYPTAMINE STIMULATES TWO DISTINCT ADENYLATE CYCLASE ACTIVITIES IN RAT BRAIN: HIGH-AFFINITY ACTIVATION IS RELATED TO A 5-HT1 SUBTYPE DIFFERENT FROM 5-HT1A, 5-HT1B, AND 5-HT1C

5-HT binding sites of the 5-HT1 type are heterogeneous and appear to comprise several subtypes (5-HT1A, 5-HT1B and 5-HT1C); their physiological role is as yet unclear. The stimulation of adenylate cyclase induced by 5-HT has been investigated in membrane fractions prepared from rat brain cortex. Enzymatic activity was determined by measuring cAMP production with an HPLC technique. It was shown that 5-HT stimulates adenylate cyclase activity with 2 activation constants (Kact): one shows a high apparent affinity (Kact = 0.8 nM) and the other a lower apparent affinity (Kact = 0.30 microM). The latter activity, induced by micromolar concentrations of 5-HT, was inhibited by spiperone at concentrations that block 5-HT1A binding. 5-Methoxytryptamine, bufotenin, and LSD also had a stimulatory biphasic effect on adenylate cyclase activity, whereas trifluoromethylphenylpiperazine, 5-carboxyamidotryptamine, 8-hydroxy-(2-di-n-propylamino)tetralin, RU 24969 had a monophasic effect. Enzyme activation by drugs acting in the micromolar range was inhibited by spiperone (1 microM), suggesting a link between this activation and 5-HT1A sites. On the other hand, the high-affinity activation of the enzyme induced by 5-HT, 5-methoxytryptamine, bufotenin, LSD, and the activation induced by TFMPP were not inhibited by spiperone (1 microM), by propranolol (3 microM), or by mesulergine (0.1 microM), which selectively block 5-HT1A, 5-HT1B, and 5-HT1C sites. Inhibition was produced by dihydroergotamine, methysergide, cinanserin, and mianserin, but not by naloxone, phenoxybenzamine, and phentolamine. Therefore, these activations seem related to 5-HT1 receptors but not to 5-HT1A, 5-HT1B, or 5-HT1C sites. Accordingly, binding of [3H]5-HT to 5-HT1-like sites was examined in the presence of spiperone (1 microM) and propranolol (3 microM); in these conditions, a high-affinity site (KD = 3.4 nM) was indeed revealed. The relative potencies of a series of drugs that stimulate or inhibit the activation of the adenylate cyclase with a high affinity and their ability to inhibit this binding of [3H]5-HT showed a positive correlation, strongly suggesting a direct relation between this recognition site for 5-HT and the production of a second messenger (cAMP). Moreover, this potential receptor is shown to be heterogeneously distributed within the brain, and was localized postsynaptically at serotonergic synapses.     

Pharmacology of the Selective 5-HT1B/1D Agonist Frovatriptan

Objective.—To determine the pharmacological profile of frovatriptan.
Background.—Frovatriptan is a new 5-HT_1B/1D agonist developed for the treatment of migraine.
Methods.—Pharmacological studies were performed using in vitro and in vivo techniques.
Results.—Radioligand-binding studies showed that frovatriptan has a high affinity for 5-HT_1B and 5-HT_1D receptors, and moderate affinity for 5-HT_1A, 5-HT_1F, and 5-HT₇ receptors. In vitro, frovatriptan acts as a potent full agonist at human cloned 5-HT_1B and 5-HT_1D receptors, and as a moderately potent full agonist at 5-HT₇ receptors. Studies of frovatriptan in isolated human arteries demonstrated a lower threshold for constriction of cerebral than coronary vasculature and a bell-shaped dose-response curve was apparent in the coronary arteries. In anesthetized dogs, frovatriptan administration produced no measurable effect on cardiac function or on blood pressure. Frovatriptan had no effects on coronary blood flow following transient coronary artery occlusion, whereas sumatriptan produced a prolonged and significant decrease in coronary blood flow.
Conclusion.—The pharmacology of frovatriptan suggests that it should be an effective agent for the acute treatment of migraine, with a low potential for undesirable peripheral effects.     

MCL1 and MCL6 Compared to V1 and V6 in Distinguishing Aberrant Supraventricular from Ventricular Ectopic Beats

Use of V1 and V6 has been suggested for distinguishing aberrant supraventricular from ventricular ectopy. For two decades, "modified" leads MCL1 and MCL6 have been widely used as V1 and V6 substitutes for bedside monitoring, but their use has never been validated. To determine the value of MCL1 and MCL6, 81 morphologically distinct wide QRS ectopic beats were recorded from 46 patients during cardiac electrophysiological study. As determined by the His-bundle electrogram, 31 of the ectopics were aberrant supraventricular, 50 were ventricular. A new criterion, measurement of QRS onset to the predominant peak or nadir of the complex, was valuable in diagnosing wide complexes in MCL6 and V6. An interval of 50 msec or less predicted aberrant supraventricular ectopy; an interval of 70 msec or more predicted ventricular ectopy. There was agreement between the modified and conventional precordial leads regarding which QRS patterns were useful in distinguishing aberrant supraventricular from ventricular ectopy. A greater proportion of wide complexes in MCL1 and V1 exhibited patterns useful in making the diagnosis compared to MCL6 and V6. Using well-established criteria, the proportion of correct diagnoses that was made from individual leads was: MCL1 = 86%, V1 = 85%, MCL6 = 72%, V6 = 67%. The bedside leads (MCL1 and MCL6) were not statistically different in diagnostic accuracy from their conventional lead counterparts (V1 and V6); however, MCL1 and V1 were superior to MCL6 and V6. When the new criterion was added to make the diagnosis from MCL6 and V6, no difference in diagnostic accuracy was present between the four leads.     

5-HT1B Receptor Polymorphism and Clinical Response to Sumatriptan

The 5-HT₁ receptor agonist, sumatriptan, is highly effective in the treatment of migraine. Some patients, however, do not respond or experience recurrence of the headache. In addition, some patients report chest symptoms after sumatriptan. We investigated whether these different responses could be attributed to genetic diversity of the 5-HT_1B receptor, which most likely mediates the therapeutic action and the coronary side effects of sumatriptan. Allele frequencies of two polymorphisms in the 5-HT_1B receptor gene ( G861C and T-261G ) were investigated in migraine patients with consistently good response to sumatriptan (n=14), with no response (n=12), with recurrence of the headache (n=12), with chest symptoms (n=13), and in patients without chest symptoms (n=27). Allele frequencies (G:0.74; C:0.26 at nt 861 and T:0.39; G:0.61 at nt -261) did not differ between patient groups, indicating that genetic diversity of the 5-HT_1B receptor does not seem to be involved in the different clinical responses to sumatriptan.     

Exclusion of 5-HT2A and 5-HT2C Receptor Genes as Candidate Genes for Migraine

Several lines of investigation suggest that the serotonergic system may be involved in the pathogenesis of migraine. In particular, drugs which block 5-HT₂ receptor subtypes appear to be effective migraine prophylactic agents. Therefore, chromosomal DNA regions overlapping the 5-HT_2A (13q14-q22) and 5-HT_2c (Xq22-25) receptor loci were analyzed for possible linkage to the clinical diagnosis of migraine. No evidence for linkage to either chromosomal region was found, although a small subset of migrainous families showed positive likelihood of odds (LOD) scores. However, a homogeneity (HOMOG) analysis provided no statistical evidence for locus heterogeneity. The coding region of the 5-HT_2A and 5-HT_2c receptor genes was also analyzed in migraine patients and unaffected controls using polmerase chain reaction and direct sequencing. No mutations were found in the deduced amino acid sequence of either receptor in the sample of migraineurs tested. These results indicate that DNA-based mutations in the 5-HT_2A and 5-HT_2c receptors are not generally involved in the pathogenesis of migraine.     

Radioimmunoassay of Prostaglandins Fα, E1 and E2 in Human Plasma

Abstract. Antibodies against prostaglandins (PG)F₂α, E₁ and E₂ were obtained in rabbits immunized with respectively PG F₂α, PG E₁ and PG E₂ conjugated to bovine serum albumin by carbodiimide. A radioimmunoassay capable of measuring 7 pg of PG Fα, 2 pg of PG E₂ and 14 pg of PG Ej in human peripheral plasma is described. Plasma samples (pH 3, citric acid) are extracted with cyclohexane: ethyl acetate, 1:1 and then chromatographed on silicic acid columns to separate the prostaglandins into three fractions: fraction I, PG A, PG B and some unknown immunoraactive compounds; fraction II, PG E and fraction III PG Fα. The recovery is 80 %± 6. 2. Mean plasma levels iu adults of PG Fa and PG E, expressed in pg/ml: -PG Fα 12 ± 2. 8 (n = 25 men), 8 ± 2. 3 (n = 18 women, follicular phase), 7 ± 1. 4 (n = 18 women, luteal phase). -PG E₁ 40. 5 + 7. 6 (n = 13 men), 38 + 17. 1 (n = 10 women). -PG E₂ 4. 5 ± 1 (n = 12 adult subjects).
The major characteristics of the method described herein are the following: - a large volume of plasma has to be processed (10 ml or more for PG Fa and PG E₁, 5 ml or more for PG E₂). - a chromatographic step is necessary to separate the different prostaglandins which makes it possible to circumvent problems of immunological cross reactivity and interference with unknown immunoreactive compounds. - great care has been taken in collection of blood samples, especially to insure complete removal of blood cells namely platelets.     

Anti-A1 Lectins from Crotalaria

Anti-A₁ lectins useful for blood typing have been extracted from seeds of Crotalaria vitellina , a tropical seashore leguminous bush.