Identification and quantification of the quality markers and anti-migraine active components in Chuanxiong Rhizoma and Cyperi Rhizoma herbal pair based on chemometric analysis between chemical constituents and pharmacological effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Chuanxiong Rhizoma and Cyperi Rhizoma (CRCR), an ancient and classic herbal pair, has been used in herbal medicines for treating migraine, but its effective components are not clear. AIM OF THE STUDY: The present study aimed to identify and quantify the quality markers and anti-migraine active components in CRCR based on chemometric analysis between chemical constituents and pharmacological effects. MATERIALS AND METHODS: The HPLC fingerprints of eight batches of CRCR samples were obtained, and their characteristic common peaks were identified by HPLC-ESI-Q-TOF-MS/MS. The therapeutic effects of eight batches of CRCR samples on nitroglycerin-induced migraine rats were evaluated by migraine-related neurotransmitters and neuropeptides. Similarity analysis, hierarchical cluster analysis and principal component analysis were applied to screen the quality markers. Artificial neural network and partial least squares regression models were used to screen the anti-migraine compounds by correlating the chemical constituents in HPLC fingerprints and pharmacological indicators. RESULTS: Eighteen characteristic common peaks were found in the HPLC fingerprints, including eleven known compounds and seven unknown compounds. Ferulic acid (FA), senkyunolide I (SI), senkyunolide A (SA), 3-n-butylphthalide (NBP), Z-ligustilide (LIG), Z-3-butylidenephthalide (BDPH), nookatone (NKT), levistilide A (LA), α-cyperone (CYP) and other five unknown compounds (P1, P2, P7, P8 and P9) were identified as quality markers. SA, NBP, LIG, NKT, CYP and other three unknown compounds (P1, P4 and P9) can be considered as anti-migraine prototype compounds. The quality markers and anti-migraine active components were further quantified in CRCR extract, rat serum and cerebral cortex by UPLC-MS/MS, which gives a clue to track the dynamic changes of the contents of the main constituents. CONCLUSIONS: Our study explored the anti-migraine material basis, and could lay a foundation for the improvement of the quality control of CRCR in practice.

The effect of intravenous administration of liposomal curcumin in addition to sumatriptan treatment in an experimental migraine model in rats.

BACKGROUND: Curcumin has antioxidative properties that could be useful in various diseases due to its ability to act on multiple targets of various cellular pathways. We aimed to assess the efficacy of liposomal curcumin compared with curcumin solution, when in addition to sumatriptan (ST) treatment, in an experimental migraine model induced with nitroglycerin (NTG) in rats. METHODS: Seven groups of 9 rats each were investigated: control group without migraine (1 mL saline solution intraperitoneal injection [ip]), control group with induced migraine, NTG+ST group (ST), NTG+ST+curcumin1 (CC1) group - 1 mg/100 g body weight (bw), NTG+ST+CC2 - 2 mg/100 g bw, NTG+ST+liposomal curcumin1 (lCC1) group - 1 mg/100 g bw, and NTG+ST+lCC2 (lCC2) group - 2 mg/100 g bw. NTG and ST were administered as 1 mL ip NTG | 1 mg/100 g bw and 1 mL ip ST | 1 mg/100 g bw, respectively. Plasma total oxidative stress (TOS), malondialdehyde (MDA), nitric oxide (NOx), thiol levels, as well as total antioxidative capacity (TAC) were assessed. The nociception process was assessed by counting the number of flinches and shakes after the formalin test. RESULTS: The plasma TOS, MDA, and NOx levels, as oxidative stress parameters, were significantly decreased in the curcumin-treated groups, especially where curcumin was in liposomal form. The thiol and TAC were also improved by the curcumin treatment, with the best results obtained for the liposomal curcumin. The closest number of flinches and shakes to the control group was obtained for the group treated with liposomal curcumin at a dose of 2 mg/100 g bw. CONCLUSION: Liposomal curcumin in a dose of 2 mg/100 g bw when in addition to ST treatment could be an optimum therapeutic strategy for migraine attacks and could represent a base for future clinical research and application.

The in vitro pharmacology and non-clinical cardiovascular safety studies of a novel 5-HT4 receptor agonist, DSP-6952.

The pharmacological activity of DSP-6952, a novel compound was investigated, compared to that of clinically efficacious gastrointestinal (GI) prokinetic 5-hydroxytryptamine4 (5-HT4) receptor agonists. DSP-6952 had a strong affinity of Ki = 51.9 nM for 5-HT4(b) receptor, and produced contraction in the isolated guinea pig colon with EC50 of 271.6 nM and low intrinsic activity of 57%, similar to tegaserod and mosapride. In the development of the 5-HT4 receptor agonists, cardiovascular risk was deliberately evaluated, because some related prokinetics were reported to cause with cardiovascular adverse events, such as ventricular arrhythmias or ischemia. DSP-6952 showed minimal effects up to 100 µM in human ether-a-go-go-related gene (hERG) channels or guinea pig cardiomyocytes. In telemetered conscious monkeys, DSP-6952 did not affect blood pressure or any electrocardiogram (ECG) up to 180 mg/kg, p.o.; however, DSP-6952 transiently increased heart rate, as well as in anesthetized dogs. The positive chronotropic effects of DSP-6952 were completely antagonized by a 5-HT4 receptor antagonist, and another 5-HT4 receptor agonist, TD-5108 also increased heart rate. These effects are considered a class effect seen in clinically developing and marketed 5-HT4 receptor agonists, and have not been regarded as a critical issue in clinical use. DSP-6952 did not induce contraction in the rabbit coronary artery up to 100 µM, which differed from tegaserod or sumatriptan. These results show that DSP-6952 does not have cardiac ischemic risk via coronary vasoconstriction. In conclusion, DSP-6952 is a promising GI prokinetic compound with partial 5-HT4 receptor agonistic activity as well as a favorable cardiovascular safety profile.

Triptan-induced disruption of trigemino-cortical connectivity.

OBJECTIVE: The 5-HT1B/D agonists (triptans) are specific headache medications that have no effect on pain as such. Although they are routinely used in the treatment of acute migraine attacks, the underlying mechanisms of action are still a matter of debate. METHODS: Forty-three healthy participants underwent fMRI while receiving trigemino-nociceptive stimulation and control stimuli in a standardized fMRI paradigm. Using a crossover, double-blind, placebo-controlled design, 21 participants (10 women, mean age 26.9, range 20-37 years) received sumatriptan and 22 participants (11 women, mean age 25.5, range 22-32 years) received acetylsalicylic acid (ASA). Administration of medication and saline was randomized between participants of each group resulting in half of the participants receiving saline and the other half receiving the respective medication during the first fMRI data acquisition. RESULTS: While mean pain intensity ratings did not differ significantly between control and medication nor between medications, we found a significant blood oxygen level-dependent signal increase in the trigeminal nuclei and the thalamus after sumatriptan treatment compared with placebo or ASA. In addition, we specifically looked for the pharmacologic modulation of functional coupling between trigeminal nuclei and higher brain areas, i.e., trigemino-cortical pathways, and found a strong coupling during the saline condition, which was altered by sumatriptan but not after ASA administration. CONCLUSION: These data suggest that a specific functional inhibition of trigemino-cortical projections is one of the reasons that triptans, unlike pain killers, act highly specifically on headache and migraine but not pain as such.

Effects of DA-9701, a novel prokinetic agent, on gastric accommodation in conscious dogs.

BACKGROUND AND AIM: DA-9701, a novel prokinetic agent formulated with Pharbitis Semen and Corydalis Tuber, has strong prokinetic effects, and enhances gastric compliance in conscious dogs. In this study, the effects of DA-9701 on gastric accommodation were studied in conscious dogs. METHODS: Beagle dogs with an implanted gastric cannula in the stomach were used in this study. After an overnight fast, the dogs received DA-9701 orally, or served as a positive control that received sumatriptan or a negative control before ingestion of a meal. The basal and postprandial gastric volumes were monitored at a constant operating pressure using an electronic barostat. To investigate the long-lasting effects on increased postprandial gastric volume, the area under the volume versus time curve (AUC) was calculated. RESULTS: DA-9701 significantly increased the basal gastric volume compared to the negative controls (P < 0.05); the effects were comparable to sumatriptan. DA-9701 and sumatriptan significantly increased gastric accommodation compared to the negative control (P < 0.05). In the negative control, the gastric volume reached the maximal volume 40 min after the meal, and then gradually decreased. However, with DA-9701, the increased gastric volume remained significantly elevated for 60 min postprandially (P < 0.05). DA-9701 significantly increased the value of AUC compared to the negative control; this was observed during both the early and late postprandial phases (P < 0.05). CONCLUSIONS: A novel prokinetic agent, DA-9701, improved gastric accommodation by increasing the postprandial gastric volume; these effects persisted for 60 min after a meal.

[The pharmacological mechanism of gastrodin on calcitonin gene-related peptide of cultured rat trigeminal ganglion].

The Chinese herbal medicine Tianma (Gastrodia elata) has been used for treating and preventing primary headache over thousands of years, but the exact pharmacological mechanism of the main bioactive ingredient gastrodin remains unclear. In present study, the effects of gastrodin on calcitonin gene-related peptide (CGRP) and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) expression were observed in rat trigeminal ganglion (TG) after in vitro organ culture to explore the underlying intracellular mechanism of gastrodin on primary vascular-associated headache. CGRP-immunoreactivity (CGRP-ir) positive neurons count, positive area, mean optical density and integrated optical density by means of immunohistochemistry stain were compared at different concentrations of gastrodin, which was separately co-incubated with DMEM in SD rat TG for 24 hours. Only at 5 or 10 mmol L(-1) concentration, gastrodin demonstrated significantly concentration-dependent reduction of CGRP-ir (+) expression and its action closed to 1.2 mmol L(-1) sumatriptan succinate. While at 2.5, 20, and 40 mmol L(-1) concentration, gastrodin did not show remarkable effects on CGRP-ir (+) expression. The optimal concentration of gastrodin (5 and 10 mmol L(-1)) similarly inhibited CGRP-mRNA expression level separately compared with 1.2 mmol L(-1) sumatriptan succinate and 10 micromol L(-1) flunarizine hydrochloride, which was quantitatively analyzed by real-time PCR (RT-PCR). pERK1/2 level was examined by Western blotting after co-cultured with optimal concentration of gastrodin and effective specific ERK1/2 pathway inhibitors PD98059, U0126. The result indicated that gastrodin significantly reduced pERK1/2 protein actions similarly to ERK1/2 pathway specific blockade. It suggests ERK1/2 signaling transduction pathway may be involved in gastrodin intracellular mechanism. This study indicates gastrodin (5 and 10 mmol L(-1)) can remarkably reduce CGRP-ir (+) neuron, CGRP-mRNA and pERK1/2 expression level in cultured rat TG, with its actions similar to the effective concentration of sumatriptan succinate, flunarizine hydrochloride and specific ERK1/2 pathway blocker. The intracellular signaling transduction ERK1/2 pathway may be involved in the gastrodin reducing CGRP up-regulation in rat TG after organ culture.

The central analgesia induced by antimigraine drugs is independent from Gi proteins: superiority of a fixed combination of indomethacin, prochlorperazine and caffeine, compared to sumatriptan, in an in vivo model.

A hypofunctionality of Gi proteins has been found in migraine patients. The fixed combination of indomethacin, prochlorperazine and caffeine (Indoprocaf) is a drug of well-established use in the acute treatment of migraine and tension-type headache. The aim of this study was to investigate if Indoprocaf was able to exert its central antinociceptive action when Gi proteins activity is abolished by pertussis toxin (PTX), compared to its single active ingredients and to sumatriptan. The mice model of abdominal constriction test induced by an i.p. injection of a 0.6% solution of acetic acid was used. The study showed that Indoprocaf (a fixed combination of indomethacin 1 mg/kg, prochlorperazine 1 mg/kg and caffeine 3 mg/kg, s.c.) and sumatriptan (20 mg/kg, s.c.) exert their central antinociceptive action independently from the Gi proteins. In addition, the antinociceptive efficacy of Indoprocaf in this study was statistically superior to that of sumatriptan. This study also showed that the single active ingredients of Indoprocaf, indomethacin (1 mg/kg, s.c.), prochlorperazine (1 mg/kg, s.c.) and caffeine (3 mg/kg, s.c.), were able to exert their central antinociceptive action independently from the Gi proteins. However, Indoprocaf at analgesic doses was able to abolish almost completely the abdominal constrictions, with a statistically higher efficacy compared to the single active ingredients, showing an important synergic effect of Indoprocaf. This synergic effect was evident not only when Gi proteins activity was abolished by PTX, but also under control condition, when Gi proteins were active. This study suggests that the central antinociceptive action induced by antimigraine drugs is independent from Gi proteins.

Goshuyuto, a traditional Japanese medicine for migraine, inhibits platelet aggregation in guinea-pig whole blood.

The effects of goshuyuto and chotosan, traditional Japanese medicines, on collagen-induced platelet aggregation were examined using guinea-pig blood. Goshuyuto at the concentration of 1,000 mug/mL inhibited collagen-induced platelet hyper-aggregation to the same degree as aspirin at the concentration of 100 mumol/L, but chotosan did not. Goshuyuto is composed of four medicinal herbs. Of them, aqueous extracts of Evodiae Fructus and Zingiberis Rhizoma inhibited platelet aggregation, but aqueous extracts of Zizyphi Fructus and Ginseng Radix did not. Two components of Zingiberis Rhizoma, 6-shogaol and 6-gingerol, also inhibited platelet aggregation. These results suggest that Evodiae Fructus and Zingiberis Rhizoma may play important roles in the anti-aggregation effects of goshuyuto and that 6-shogaol and 6-gingerol are among the active ingredients. Therefore, goshuyuto may ameliorate migraine by preventing the hyper-aggregation of platelets in migraine with aura.

Validation of a rat behavioral avoidance model from a drug delivery perspective.

Conventional taste-masking strategies are used to overcome the bitter taste perception of pharmaceuticals by coating the drug particles and/or adding flavoring agents. However, for certain product categories such as rapid dissolve sublingual tablets, taste-masking is challenging. Programs exploring such formulation strategies in the LO-CS phase or post CS phase possess very little toxicological information available in order to conduct human taste panel studies. The potential of a bitter taste perception can present a significant business risk. The objective of the study was to validate a rat behavioral avoidance model that identifies bitter-tasting compounds. Most classic bitter substances elicit a response in the micromolar concentration range while most drugs elicit a response in the millimolar range, hence a validation exercise was conducted to examine if the existing biological model was sensitive enough to identify known bitter tasting drugs as such. Five compounds: ergotamine tartrate, fluoxetine, sucrose, sumatriptan and povidone were chosen to represent a spectrum of compounds. The bitter tasting compounds were identified as such in the model. Based on these results, the assay may serve as a useful surrogate test to identify compounds that may have bitter taste issues.

Evaluation of the anti-emetic potential of anti-migraine drugs to prevent resiniferatoxin-induced emesis in Suncus murinus (house musk shrew).

Activation of vanilloid receptors has commonly been used to facilitate neurogenic inflammation and plasma exudation to model components of the pathogenesis of migraine; however, these studies have been performed mainly in species lacking the emetic reflex. In the present studies, therefore, we used Suncus murinus, a species of insectivore capable of emesis, to investigate if the vanilloid receptor agonist resiniferatoxin is capable of modeling the emesis associated with migraine. Resiniferatoxin (100 nmol/kg, s.c.) induced an emetic response that was antagonized significantly (P<0.05) by ruthenium red (1-3 micromol), (2R-trans)-4-[1-[3,5-bis(trifluromethyl)benzoyl]-2-(phenylmethyl)-4-piperidinyl]-N-(2,6-dimethylphenyl)-1-acetamide (S)-hydroxybutanedioate (R116301; 10-100 micromol/kg), and scopolamine (1 micromol/kg), but not by dihydroergotamine (0.3-3 micromol/kg), sumatriptan (1-10 micromol/kg), methysergide (1-10 micromol/kg), tropanyl 3,5-dichlorobenzoate (MDL72222; 3-30 micromol/kg), ondansetron (0.3-3 micromol/kg), metoclopramide (3-30 micromol/kg), domperidone (3-30 micromol/kg), diphenhydramine (1-10 micromol/kg), or indomethacin (3-30 micromol/kg). The failure of a wide range of representative anti-migraine drugs to reduce retching and vomiting limits the use of this model to identify/investigate novel treatments for the emesis (and nausea) associated with migraine attacks in humans. However, the results provide further evidence for the involvement of a novel vanilloid receptor in resiniferatoxin-induced emesis and implicate both tachykinins and acetylcholine in the pathway(s) activated by resiniferatoxin in S. murinus.

Involvement of 5-HT1B receptors in triptan-induced contractile responses in guinea-pig isolated iliac artery.

Using a series of triptans we characterized in vitro the 5-hydroxytryptamine (5-HT) receptor that mediates the contraction in guinea-pig iliac arteries moderately precontracted by prostaglandin F2alpha (PGF2alpha). Additionally, we investigated by reverse-transcriptase polymerase chain reaction (RT-PCR) which triptan-sensitive receptor is present in this tissue. Frovatriptan, zolmitriptan, rizatriptan, naratriptan, sumatriptan, and almotriptan contracted guinea-pig iliac arteries with pD2 values of 7.52+/-0.04, 6.72+/-0.03, 6.38+/-0.06, 6.22+/-0.05, 5.86+/-0.05 and 5.26+/-0.04 respectively. For comparison, the pD2 values for 5-HT and 5-carboxamidotryptamine (5-CT) were 7.52+/-0.02 and 7.55+/-0.03 respectively. In contrast to all other triptans tested, the concentration-response curve for eletriptan was biphasic (first phase: 0.01-3 microM, pD2 approximately 6.6; second phase: > or = 10 microM). Contractions to 5-HT, 5-CT, frovatriptan, zolmitriptan, rizatriptan, naratriptan, sumatriptan, almotriptan, and eletriptan (first phase) were antagonized by the 5-HT1B/1D receptor antagonist GR127935 (10 nM) and the 5-HT1B receptor antagonist SB216641 (10 nM). RT-PCR studies in guinea-pig iliac arteries showed a strong signal for the 5-HT1B receptor while expression of 5-HT1D and 5-HT1F receptors was not detected in any sample. The present results demonstrate that triptan-induced contraction in guinea-pig iliac arteries is mediated by the 5-HT1B receptor. The guinea-pig iliac artery may be used as a convenient in vitro model to study the (cardio)vascular side-effect potential of anti-migraine drugs of the triptan family.

Nitroglycerin-induced nNOS increase in rat trigeminal nucleus caudalis is inhibited by systemic administration of lysine acetylsalicylate but not of sumatriptan.

Systemic administration of nitroglycerin (NTG), a nitric oxide (NO) donor, in migraineurs triggers after several hours an attack of which the precise mechanisms are unknown. We found previously in rats that nitroglycerin (10 mg/kg s.c.) is able to increase significantly after 4 h the number of neuronal nitric oxide synthase (nNOS)-immunoreactive neurones in the cervical part of trigeminal nucleus caudalis. In the present experiments, we demonstrate that the 5-HT1B/D agonist sumatriptan (0.6 mg/kg s.c.) does not alter this phenomenon when given before NTG. By contrast, pretreatment with lysine acetylsalicylate (50 mg/kg i.m.) attenuates the NTG-induced nNOS expression in the superficial laminae of trigeminal nucleus caudalis. These findings suggest that effect of NTG on nNOS at a high dosage may involve the cycloxygenase pathway and that activation of the peripheral 5-HT1B/D receptors is not able to modify this effect. These data could help to better understand the role of NO in the pathogenesis of headaches and the action of antimigraine drugs.

Preclinical studies characterizing the anti-migraine and cardiovascular effects of the selective 5-HT1D receptor agonist PNU-142633.

The present study describes the preclinical pharmacology of a highly selective 5-HT1D receptor agonist PNU-142633. PNU-142633 binds with a Ki of 6 nm at the human 5-HT1D receptor and a Ki of> 18 000 nm at the human 5-HT1B receptor. The intrinsic activity of PNU-142633 at the human 5-HT1D receptor was determined to be 70% that of 5-HT in a cytosensor cell-based assay compared with 84% for that of sumatriptan. PNU-142633 was equally effective as sumatriptan and a half-log more potent than sumatriptan in preventing plasma protein extravasation induced by electrical stimulation of the trigeminal ganglion. Like sumatriptan, PNU-142633 reduced the increase in cat nucleus trigeminal caudalis blood flow elicited by electrical stimulation of the trigeminal ganglion compared with the vehicle control. The direct vasoconstrictor potential of PNU-142633 was evaluated in vascular beds. Sumatriptan increased vascular resistance in carotid, meningeal and coronary arteries while PNU-142633 failed to alter resistance in these vascular beds. These data are discussed in relation to the clinical findings of PNU-142633 in a phase II acute migraine study.

The potential anti-migraine compound SB-220453 does not contract human isolated blood vessels or myocardium; a comparison with sumatriptan.

The mechanistically novel benzopyran derivative SB-220453, which is undergoing clinical evaluation in migraine, exhibits a high affinity for a selective, but not yet characterized, binding site in the human brain. It inhibits nitric oxide release and cerebral vasodilatation following cortical spreading depression as well as carotid vasodilatation induced by trigeminal nerve stimulation in the cat. The aim of our study was to investigate the contractile properties of SB-220453 on a number of human isolated blood vessels (coronary artery, saphenous vein and middle meningeal artery) as well as atrial and ventricular cardiac trabeculae. While sumatriptan induced marked contractions in three blood vessels investigated, SB-220453 was devoid of any effect. In atrial and ventricular cardiac trabeculae, neither SB-220453 nor sumatriptan displayed a positive or negative inotropic effect. Since SB-220453 did not contract the middle meningeal artery, we conclude that potential anti-migraine effects are not mediated via a direct cerebral vasoconstriction. The lack of activity of SB-220453 in coronary artery, saphenous vein and cardiac trabeculae demonstrates that the compound is unlikely to cause adverse cardiac side-effects.

Functional profile of almotriptan in animal models predictive of antimigraine activity.

Almotriptan is a new 5-HT(1B/1D) receptor agonist whose clinical efficacy for the treatment of migraine attacks has been demonstrated in Phase III clinical trials. We now compare the functional profile of almotriptan (assessed using animal models) with that of sumatriptan. Almotriptan selectively increased carotid vascular resistance in anaesthetised cats after intravenous or intraduodenal administration (ED(100)=11 microg/kg, i.v.; ED(50)=339 microg/kg, i. d.) and in anaesthetised beagle dogs following intravenous administration (ED(50)=116 microg/kg). A study in anaesthetised cats also demonstrated that almotriptan acts by selectively increasing the resistance of the carotid arteriovenous anastomoses without adversely affecting brain irrigation. In addition, almotriptan inhibited meningeal extravasation produced by electrical stimulation of the trigeminal ganglion in anaesthetised guinea pigs in the dose range of 0.3-3 mg/kg, i.v. In conclusion, almotriptan is both a selective constrictor affecting intracranial blood vessels and an inhibitor of neurogenically evoked plasma protein extravasation of the dura mater.

Human 5-HT1B receptor stimulated inositol phospholipid hydrolysis in CHO cells: synergy with Gq-coupled receptors.

We have previously reported that the transfected Gi/Go protein-coupled human adenosine A1 receptor (expressed at 200 fmol/mg of protein) and the endogenous 5-HT1B receptor (not detectable using radioligand binding) suppress forskolin-stimulated cyclic AMP accumulation and stimulate increases in [Ca2+]i in Chinese hamster ovary cells (CHO). In addition, co-activation of the adenosine A1 receptor (but not the 5-HT1B receptor) potentiates the hydrolysis of inositol phospholipids elicited by receptors coupled to Gq-proteins (Dickenson and Hill, 1996. Eur. J. Pharmacol. 320, 141-151). In order to establish whether this difference in ability to modulate Gq-coupled receptor responses is a consequence of low 5-HT1B receptor density, we have stably transfected CHO-KI cells with the human 5-HT1Dbeta cDNA (the human homologue of the rodent 5-HT1B receptor). We initially isolated a clonal cell line (designated CHO5-HT1B cells) displaying moderate specific [3H]5-HT binding (pKd of 8.17+/-0.07 and a Bmax of 140 fmol/mg protein). In CHO5-HT1B cells, the selective human 5-HT1B/1D receptor agonist sumatriptan produced a concentration-dependent inhibition of forskolin-stimulated cyclic AMP accumulation (pEC50=7.92+/-0.04). Sumatriptan also elicited a moderate and pertussis toxin-sensitive increase in [3H]inositol phosphate formation in CHO-5HT1B cells (pEC50=6.51+/-0.05). Finally, sumatriptan synergistically enhanced P2U purinoceptor stimulated [3H]inositol phosphate accumulation through a pertussis toxin-sensitive mechanism. These findings clearly show the significance of 5-HT1B receptor expression level in determining whether 5-HT1B receptor activation can modulate the accumulation of [3H]inositol phosphates elicited by a Gq-protein coupled receptor. The observation that 5-HT1B receptor activation can potentiate Gq-coupled receptor stimulated second messenger responses may have an important physiological role in the regulation of vascular smooth muscle contraction.

Regulation of serotonin release in the frontal cortex and ventral hippocampus of homozygous mice lacking 5-HT1B receptors: in vivo microdialysis studies.

To assess the involvement of the serotonin receptor subtype 5-HT1B as terminal autoreceptor regulating 5-HT release in mice, we compared basal values and potassium-evoked changes of extracellular 5-HT levels obtained by in vivo microdialysis in two serotoninergic terminal projection areas of conscious wild-type mice with those measured in homozygous mutant mice lacking the gene encoding the 5-HT1B receptor. In the frontal cortex and ventral hippocampus, basal and K+-evoked 5-HT release did not differ between the two strains of mice studied. The infusion via reverse microdialysis of the selective 5-HT1B receptor agonist CP-93,129 (500 nM) decreased significantly K+-evoked 5-HT release in the frontal cortex (by -44%) and ventral hippocampus (by -32%) of wild-type mice but had no effect in mutants. In a similar manner, the mixed 5-HT1B-5-HT1D receptor agonist sumatriptan (800 nM) decreased significantly K+-evoked 5-HT release in the frontal cortex (by -46%) of wild-type mice but had no effect in mutants. These results demonstrated that 5-HT1B knockout mice are not as sensitive to full (CP-93,129) and mixed (sumatriptan) 5-HT1B receptor agonists as are wild-type mice. These data provide in vivo evidence that, in mice, 5-HT1B, but not 5-HT1D, autoreceptors inhibit 5-HT release at nerve terminals located in the frontal cortex and ventral hippocampus.