Stimulation of Neurite Outgrowth in Cerebrocortical Neurons by Sodium Channel Activator Brevetoxin-2 Requires Both N-Methyl-D-aspartate Receptor 2B (GluN2B) and p21 Protein (Cdc42/Rac)-Activated Kinase 1 (PAK1).

N-methyl-D-aspartate (NMDA) receptors play a critical role in activity-dependent dendritic arborization, spinogenesis, and synapse formation by stimulating calcium-dependent signaling pathways. Previously, we have shown that brevetoxin 2 (PbTx-2), a voltage-gated sodium channel (VGSC) activator, produces a concentration-dependent increase in intracellular sodium [Na+]I and increases NMDA receptor (NMDAR) open probabilities and NMDA-induced calcium (Ca2+) influxes. The objective of this study is to elucidate the downstream signaling mechanisms by which the sodium channel activator PbTx-2 influences neuronal morphology in murine cerebrocortical neurons. PbTx-2 and NMDA triggered distinct Ca2+-influx pathways, both of which involved the NMDA receptor 2B (GluN2B). PbTx-2-induced neurite outgrowth in day in vitro 1 (DIV-1) neurons required the small Rho GTPase Rac1 and was inhibited by both a PAK1 inhibitor and a PAK1 siRNA. PbTx-2 exposure increased the phosphorylation of PAK1 at Thr-212. At DIV-5, PbTx-2 induced increases in dendritic protrusion density, p-cofilin levels, and F-actin throughout the dendritic arbor and soma. Moreover, PbTx-2 increased miniature excitatory post-synaptic currents (mEPSCs). These data suggest that the stimulation of neurite outgrowth, spinogenesis, and synapse formation produced by PbTx-2 are mediated by GluN2B and PAK1 signaling.

Antillatoxin-Stimulated Neurite Outgrowth Involves the Brain-Derived Neurotrophic Factor (BDNF) - Tropomyosin Related Kinase B (TrkB) Signaling Pathway.

Voltage-gated sodium channel (VGSC) activators promote neurite outgrowth by augmenting intracellular Na+ concentration ([Na+]i) and upregulating N-methyl-d-aspartate receptor (NMDAR) function. NMDAR activation stimulates calcium (Ca2+) influx and increases brain-derived neurotrophic factor (BDNF) release and activation of tropomyosin receptor kinase B (TrkB) signaling. The BDNF-TrkB pathway has been implicated in activity-dependent neuronal development. We have previously shown that antillatoxin (ATX), a novel lipopeptide isolated from the cyanobacterium Moorea producens, is a VGSC activator that produces an elevation of [Na+]i. Here we address the effect of ATX on the synthesis and release of BDNF and determine the signaling mechanisms by which ATX enhances neurite outgrowth in immature cerebrocortical neurons. ATX treatment produced a concentration-dependent release of BDNF. Acute treatment with ATX also resulted in increased synthesis of BDNF. ATX stimulation of neurite outgrowth was prevented by pretreatment with a TrkB inhibitor or transfection with a dominant-negative Trk-B. The ATX activation of TrkB and Akt was blocked by both a NMDAR antagonist (MK-801) and a VGSC blocker (tetrodotoxin). These results suggest that VGSC activators such as the structurally novel ATX may represent a new pharmacological strategy to promote neuronal plasticity through a NMDAR-BDNF-TrkB-dependent mechanism.

Unanticipated Characteristics of a Selective, Potent Neuromedin-U Receptor 2 Agonist.

Neuromedin-U (NMU) mediates several physiological functions via its two cognate receptors, NMUR1 and NMUR2. Disentangling the individual roles of each receptor has largely been undertaken through the use of transgenic mice bearing a deletion in one of the two receptors or by testing native molecules (NMU or its truncated version NMU-8) in a tissue-specific manner, in effect, taking advantage of the distinct receptor expression profiles. These strategies have proved quite useful despite the inherent limitations of overlapping receptor roles and potential compensatory influences of germline gene deletion. With these considerations in mind, the availability of potent, selective NMU compounds with appropriate pharmacokinetic profiles would advance the capabilities of investigators undertaking such efforts. Here, we evaluate a recently reported NMUR2-selective peptide (compound 17) for its in vitro potency (mouse and human), binding affinity, murine pharmacokinetic properties, and in vivo effects. Despite being designed as an NMUR2 agonist, our results show compound 17 unexpectedly binds but does not have functional activity on NMUR1, thereby acting as an R1 antagonist while simultaneously being a potent NMUR2 agonist. Furthermore, evaluation of compound 17 across all known and orphan G-protein-coupled receptors demonstrates multiple receptor partners beyond NMUR2/R1 binding. These properties need to be appreciated for accurate interpretation of results generated using this molecule and may limit the broader ability of this particular entity in disentangling the physiological role of NMU receptor biology.

Evaluation of VGF peptides as potential anti-obesity candidates in pre-clinical animal models.

VGF is a peptide precursor expressed in neuroendocrine cells that is suggested to play a role in the regulation of energy homeostasis. VGF is proteolytically cleaved to yield multiple bioactive peptides. However, the specific actions of VGF-derived peptides on energy homeostasis remain unclear. The aim of the present work was to investigate the role of VGF-derived peptides in energy homeostasis and explore the pharmacological actions of VGF-derived peptides on body weight in preclinical animal models. VGF-derived peptides (NERP-1, NERP-2, PGH-NH2, PGH-OH, NERP-4, TLQP-21, TLQP-30, TLQP-62, HHPD-41, AQEE-30, and LQEQ-19) were synthesized and screened for their ability to affect neuronal activity in vitro on hypothalamic brain slices and modulate food intake and energy expenditure after acute central administration in vivo. In addition, the effects of NERP-1, NERP-2, PGH-NH2, TLQP-21, TLQP-62, and HHPD-41 on energy homeostasis were studied after chronic central infusion. NERP-1, PGH-NH2, HHPD-41, and TLQP-62 increased the functional activity of hypothalamic neuronal networks. However, none of the peptides altered energy homeostasis after either acute or chronic ICV administration. The present data do not support the potential use of the tested VGF-derived peptides as novel anti-obesity drug candidates.

A Comparison of the Ability of Leu8- and Pro8-Oxytocin to Regulate Intracellular Ca2+ and Ca2+-Activated K+ Channels at Human and Marmoset Oxytocin Receptors.

The neurohypophyseal hormone oxytocin (OT) regulates biologic functions in both peripheral tissues and the central nervous system. In the central nervous system, OT influences social processes, including peer relationships, maternal-infant bonding, and affiliative social relationships. In mammals, the nonapeptide OT structure is highly conserved with leucine in the eighth position (Leu8-OT). In marmosets (Callithrix), a nonsynonymous nucleotide substitution in the OXT gene codes for proline in the eighth residue position (Pro8-OT). OT binds to its cognate G protein-coupled receptor (OTR) and exerts diverse effects, including stimulation (Gs) or inhibition (Gi/o) of adenylyl cyclase, stimulation of potassium channel currents (Gi), and activation of phospholipase C (Gq). Chinese hamster ovary cells expressing marmoset or human oxytocin receptors (mOTRs or hOTRs, respectively) were used to characterize OT signaling. At the mOTR, Pro8-OT was more efficacious than Leu8-OT in measures of Gq activation, with both peptides displaying subnanomolar potencies. At the hOTR, neither the potency nor efficacy of Pro8-OT and Leu8-OT differed with respect to Gq signaling. In both mOTR- and hOTR-expressing cells, Leu8-OT was more potent and modestly more efficacious than Pro8-OT in inducing hyperpolarization. In mOTR cells, Leu8-OT-induced hyperpolarization was modestly inhibited by pretreatment with pertussis toxin (PTX), consistent with a minor role for Gi/o activation; however, the Pro8-OT response in mOTR and hOTR cells was PTX insensitive. These findings are consistent with membrane hyperpolarization being largely mediated by a Gq signaling mechanism leading to Ca2+-dependent activation of K+ channels. Evaluation of the influence of apamin, charybdotoxin, paxilline, and TRAM-34 demonstrated involvement of both intermediate and large conductance Ca2+-activated K+ channels.

Upregulation of RGS2: a new mechanism for pirfenidone amelioration of pulmonary fibrosis.

BACKGROUND: Pirfenidone was recently approved for treatment of idiopathic pulmonary fibrosis. However, the therapeutic dose of pirfenidone is very high, causing side effects that limit its doses and therapeutic effectiveness. Understanding the molecular mechanisms of action of pirfenidone could improve its safety and efficacy. Because activated fibroblasts are critical effector cells associated with the progression of fibrosis, this study investigated the genes that change expression rapidly in response to pirfenidone treatment of pulmonary fibroblasts and explored their contributions to the anti-fibrotic effects of pirfenidone. METHODS: We used the GeneChip microarray to screen for genes that were rapidly up-regulated upon exposure of human lung fibroblast cells to pirfenidone, with confirmation for specific genes by real-time PCR and western blots. Biochemical and functional analyses were used to establish their anti-fibrotic effects in cellular and animal models of pulmonary fibrosis. RESULTS: We identified Regulator of G-protein Signaling 2 (RGS2) as an early pirfenidone-induced gene. Treatment with pirfenidone significantly increased RGS2 mRNA and protein expression in both a human fetal lung fibroblast cell line and primary pulmonary fibroblasts isolated from patients without or with idiopathic pulmonary fibrosis. Pirfenidone treatment or direct overexpression of recombinant RGS2 in human lung fibroblasts inhibited the profibrotic effects of thrombin, whereas loss of RGS2 exacerbated bleomycin-induced pulmonary fibrosis and mortality in mice. Pirfenidone treatment reduced bleomycin-induced pulmonary fibrosis in wild-type but not RGS2 knockout mice. CONCLUSIONS: Endogenous RGS2 exhibits anti-fibrotic functions. Upregulated RGS2 contributes significantly to the anti-fibrotic effects of pirfenidone.

Synthesis and biological evaluation of palmyrolide A macrocycles as sodium channel blockers towards neuroprotection.

Palmyrolide A is a neuroprotective macrolide isolated by Gerwick and coworkers in 2010. This natural product is known to suppress neuronal spontaneous calcium ion oscillations through its voltage-gated sodium channel blocking ability which is of significant interest in CNS drug discovery. Herein, we give a detailed account on total synthesis of (+)-palmyrolide A and synthesis of a focused library of macrocycles around the scaffold, followed by their biological evaluation. Use of the chiral pool approach, Zhu's oxidative homologation, access to unnatural cis-palmyrolide A, preparation of 18 new analogues and identification of macrolides with improved sodium channel blocking activity are the important features of the present paper. As a measure of potency as voltage-gated sodium channel blockers, all the synthesized analogues were profiled for their ability to inhibit the veratridine-stimulated Na(+) influx in murine primary neuronal cultures. Four macrocycles were found to be more potent or comparable to that of the natural product (-)-palmyrolide A. The most potent compound from this series 20 was structurally simplified and readily accessible in good quantities for further biological profiling.

Expanding the Described Metabolome of the Marine Cyanobacterium Moorea producens JHB through Orthogonal Natural Products Workflows.

Moorea producens JHB, a Jamaican strain of tropical filamentous marine cyanobacteria, has been extensively studied by traditional natural products techniques. These previous bioassay and structure guided isolations led to the discovery of two exciting classes of natural products, hectochlorin (1) and jamaicamides A (2) and B (3). In the current study, mass spectrometry-based 'molecular networking' was used to visualize the metabolome of Moorea producens JHB, and both guided and enhanced the isolation workflow, revealing additional metabolites in these compound classes. Further, we developed additional insight into the metabolic capabilities of this strain by genome sequencing analysis, which subsequently led to the isolation of a compound unrelated to the jamaicamide and hectochlorin families. Another approach involved stimulation of the biosynthesis of a minor jamaicamide metabolite by cultivation in modified media, and provided insights about the underlying biosynthetic machinery as well as preliminary structure-activity information within this structure class. This study demonstrated that these orthogonal approaches are complementary and enrich secondary metabolomic coverage even in an extensively studied bacterial strain.

Detailed analysis of (-)-palmyrolide a and some synthetic derivatives as voltage-gated sodium channel antagonists.

A small library of synthetic (-)-palmyrolide A diastereomers, analogues, and acyclic precursors have been examined with respect to their interaction with voltage-gated sodium channels (VGSCs). Toward this goal, the ability of (-)-palmyrolide A and analogues to antagonize veratridine-stimulated Na(+) influx in primary cultures of mouse cerebrocortical neurons was assessed. We found that synthetic (-)-palmyrolide A and its enantiomer functioned as VGSC antagonists to block veratridine-induced sodium influx. A detailed NMR and computational analysis of four diastereomers revealed that none had the same combination of shape and electrostatic potential as exhibited by natural (-)-palmyrolide A. These data indicate that the relative configuration about the tert-butyl and methyl substituents appears to be a prerequisite for biological function. Additional testing revealed that the enamide double bond was not necessary for blocking veratridine-induced sodium influx, whereas the acyclic analogues and other macrolide diastereomers tested were inactive as inhibitors of VGSCs, suggesting that the intact macrolide was required.

Gambierol inhibition of voltage-gated potassium channels augments spontaneous Ca2+ oscillations in cerebrocortical neurons.

Gambierol is a marine polycyclic ether toxin produced by the marine dinoflagellate Gambierdiscus toxicus and is a member of the ciguatoxin toxin family. Gambierol has been demonstrated to be either a low-efficacy partial agonist/antagonist of voltage-gated sodium channels or a potent blocker of voltage-gated potassium channels (Kvs). Here we examined the influence of gambierol on intact cerebrocortical neurons. We found that gambierol produced both a concentration-dependent augmentation of spontaneous Ca(2+) oscillations, and an inhibition of Kv channel function with similar potencies. In addition, an array of selective as well as universal Kv channel inhibitors mimicked gambierol in augmenting spontaneous Ca(2+) oscillations in cerebrocortical neurons. These data are consistent with a gambierol blockade of Kv channels underlying the observed increase in spontaneous Ca(2+) oscillation frequency. We also found that gambierol produced a robust stimulation of phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). Gambierol-stimulated ERK1/2 activation was dependent on both inotropic [N-methyl-d-aspartate (NMDA)] and type I metabotropic glutamate receptors (mGluRs) inasmuch as MK-801 [NMDA receptor inhibitor; (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate], S-(4)-CGP [S-(4)-carboxyphenylglycine], and MTEP [type I mGluR inhibitors; 3-((2-methyl-4-thiazolyl)ethynyl) pyridine] attenuated the response. In addition, 2-aminoethoxydiphenylborane, an inositol 1,4,5-trisphosphate receptor inhibitor, and U73122 (1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), a phospholipase C inhibitor, both suppressed gambierol-induced ERK1/2 activation, further confirming the role of type I mGluR-mediated signaling in the observed ERK1/2 activation. Finally, we found that gambierol produced a concentration-dependent stimulation of neurite outgrowth that was mimicked by 4-aminopyridine, a universal potassium channel inhibitor. Considered together, these data demonstrate that gambierol alters both Ca(2+) signaling and neurite outgrowth in cerebrocortical neurons as a consequence of blockade of Kv channels.

Novel C-1 substituted cocaine analogs unlike cocaine or benztropine.

Despite a wealth of information on cocaine-like compounds, there is no information on cocaine analogs with substitutions at C-1. Here, we report on (R)-(-)-cocaine analogs with various C-1 substituents: methyl (2), ethyl (3), n-propyl (4), n-pentyl (5), and phenyl (6). Analog 2 was equipotent to cocaine as an inhibitor of the dopamine transporter (DAT), whereas 3 and 6 were 3- and 10-fold more potent, respectively. None of the analogs, however, stimulated mouse locomotor activity, in contrast to cocaine. Pharmacokinetic assays showed compound 2 occupied mouse brain rapidly, as cocaine itself; moreover, 2 and 6 were behaviorally active in mice in the forced-swim test model of depression and the conditioned place preference test. Analog 2 was a weaker inhibitor of voltage-dependent Na+ channels than cocaine, although 6 was more potent than cocaine, highlighting the need to assay future C-1 analogs for this activity. Receptorome screening indicated few significant binding targets other than the monoamine transporters. Benztropine-like "atypical" DAT inhibitors are known to display reduced cocaine-like locomotor stimulation, presumably by their propensity to interact with an inward-facing transporter conformation. However, 2 and 6, like cocaine, but unlike benztropine, exhibited preferential interaction with an outward-facing conformation upon docking in our DAT homology model. In summary, C-1 cocaine analogs are not cocaine-like in that they are not stimulatory in vivo. However, they are not benztropine-like in binding mechanism and seem to interact with the DAT similarly to cocaine. The present data warrant further consideration of these novel cocaine analogs for antidepressant or cocaine substitution potential.

Mixed lineage kinase 3 modulates ß-catenin signaling in cancer cells.

Expression of ß-catenin is strictly regulated in normal cells via the glycogen synthase kinase 3ß (GSK3ß)- adenomatous polyposis coli-axin-mediated degradation pathway. Mechanisms leading to inactivation of this pathway (example: activation of Wnt/ß-catenin signaling or mutations of members of the degradation complex) can result in ß-catenin stabilization and activation of ß-catenin/T-cell factor (TCF) signaling. ß-Catenin-mediated cellular events are diverse and complex. A better understanding of the cellular signaling networks that control ß-catenin pathway is important for designing effective therapeutic strategies targeting this axis. To gain more insight, we focused on determining any possible cross-talk between ß-catenin and mixed lineage kinase 3 (MLK3), a MAPK kinase kinase member. Our studies indicated that MLK3 can induce ß-catenin expression via post-translational stabilization in various cancer cells, including prostate cancer. This function of MLK3 was dependent on its kinase activity. MLK3 can interact with ß-catenin and phosphorylate it in vitro. Overexpression of GSK3ß-WT or the S9A mutant was unable to antagonize MLK3-induced stabilization, suggesting this to be independent of GSK3ß pathway. Surprisingly, despite stabilizing ß-catenin, MLK3 inhibited TCF transcriptional activity in the presence of both WT and S37A ß-catenin. These resulted in reduced expression of ß-catenin/TCF downstream targets Survivin and myc. Immunoprecipitation studies indicated that MLK3 did not decrease ß-catenin/TCF interaction but promoted interaction between ß-catenin and KLF4, a known repressor of ß-catenin/TCF transcriptional activity. In addition, co-expression of MLK3 and ß-catenin resulted in significant G(2)/M arrest. These studies provide a novel insight toward the regulation of ß-catenin pathway, which can be targeted to control cancer cell proliferation, particularly those with aberrant activation of ß-catenin signaling.

Estrogen suppresses MLK3-mediated apoptosis sensitivity in ER+ breast cancer cells.

Little knowledge exists about the mechanisms by which estrogen can impede chemotherapy-induced cell death of breast cancer cells. 17beta-Estradiol (E(2)) hinders cytotoxic drug-induced cell death in estrogen receptor-positive (ER(+)) breast cancer cells. We noted that the activity of the proapoptotic mixed lineage kinase 3 (MLK3) kinase was relatively higher in estrogen receptor-negative (ER(-)) breast tumors, suggesting that E(2) might inhibit MLK3 activity. The kinase activities of MLK3 and its downstream target, c-Jun NH(2)-terminal kinase, were rapidly inhibited by E(2) in ER(+) but not in ER(-) cells. Specific knockdown of AKT1/2 prevented MLK3 inhibition by E(2), indicating that AKT mediated this event. Furthermore, MLK3 inhibition by E(2) involved phosphorylation of MLK3 Ser(674) by AKT, attenuating the proapoptotic function of MLK3. We found that a pan-MLK inhibitor (CEP-11004) limited Taxol-induced cell death and that E(2) accentuated this limitation. Taken together, our findings indicate that E(2) inhibits the proapoptotic function of MLK3 as a mechanism to limit cytotoxic drug-induced death of ER(+) breast cancer cells.

Novel cell death signaling pathways in neurotoxicity models of dopaminergic degeneration: relevance to oxidative stress and neuroinflammation in Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative movement disorder characterized by extensive degeneration of dopaminergic neurons in the nigrostriatal system. Neurochemical and neuropathological analyses clearly indicate that oxidative stress, mitochondrial dysfunction, neuroinflammation and impairment of the ubiquitin-proteasome system (UPS) are major mechanisms of dopaminergic degeneration. Evidence from experimental models and postmortem PD brain tissues demonstrates that apoptotic cell death is the common final pathway responsible for selective and irreversible loss of nigral dopaminergic neurons. Epidemiological studies imply both environmental neurotoxicants and genetic predisposition are risk factors for PD, though the cellular mechanisms underlying selective dopaminergic degeneration remain unclear. Recent progress in signal transduction research is beginning to unravel the complex mechanisms governing dopaminergic degeneration. During the 12th International Neurotoxicology meeting, discussion at one symposium focused on several key signaling pathways of dopaminergic degeneration. This review summarizes two novel signaling pathways of nigral dopaminergic degeneration that have been elucidated using neurotoxicity models of PD. Dr. Anumantha Kanthasamy described a cell death pathway involving the novel protein kinase C delta isoform (PKCdelta) in oxidative stress-induced apoptotic cell death in experimental models of PD. Dr. Ajay Rana presented his recent work on the role of mixed lineage kinase-3 (MLK3) in neuroinflammatory processes in neurotoxic cell death. Collectively, PKCdelta and MLK3 signaling pathways provide new understanding of neurodegenerative processes in PD, and further exploration of these pathways may translate into effective neuroprotective drugs for the treatment of PD.

TRAF2-MLK3 interaction is essential for TNF-alpha-induced MLK3 activation.

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-alpha (TNF-alpha) and specifically activates c-Jun N-terminal kinase (JNK) on TNF-alpha stimulation. The mechanism by which TNF-alpha activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-alpha treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-alpha treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-alpha in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-alpha-induced activation of MLK3 and its downstream target, JNK.

Current and prospective pharmacological targets in relation to antimigraine action.

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, alpha-adrenoceptor agonists (ergotamine, dihydroergotamine, isometheptene) were used. The last two decades have witnessed the advent of 5-HT(1B/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-HT(2) receptor antagonists, Ca(2+) channel blockers, and beta-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-HT(1-7)), adrenergic (alpha(1), alpha(2,) and beta), calcitonin gene-related peptide (CGRP(1) and CGRP(2)), adenosine (A(1), A(2), and A(3)), 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 phe-124-Cys and A-161T variants of the human 5-HT1B receptor gene are not major determinants of the clinical response to sumatriptan.

BACKGROUND: The 5-HT(1B/1D) receptor agonist sumatriptan is highly effective in the treatment of migraine. However, some patients do not respond to sumatriptan or experience recurrence of the headache after initial relief. In addition, some patients report chest symptoms after the use of sumatriptan. OBJECTIVE: To assess whether 2 genetic variants (F124C changing a phenylalanine for a cysteine and polymorphism A/T at nucleotide position -161 in the 5' regulatory region) of the 5-HT(1B) receptor play a major role in the therapeutic response to sumatriptan. The 5-HT(1B) receptor most likely mediates the therapeutic action and coronary side effects of sumatriptan, and both F124C and A-161T have relevant functional consequences on either the affinity of sumatriptan to bind to the 5-HT(1B) receptor or on receptor expression level itself, respectively. METHOD: Genomic DNA of a relatively small but very well-characterized set of 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 patients without chest symptoms (n = 27) was available for the genetic analyses and screened for the F124C variant and the A-161T polymorphism in the human 5-HT(1B) receptor gene. RESULTS: F124C was not detected in any of the patients studied. In addition, we did not observe drastic changes in allele frequencies of the A-161T polymorphism that might hint to a causal relation with the therapeutic effect of sumatriptan. CONCLUSION: We have not obtained any evidence that variants F124C and A-161T of the 5-HT(1B) receptor are major determinants in the clinical response to sumatriptan.

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

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, 17beta-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 17beta-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 17beta-estradiol as compared to the placebo-treated rats. Acetylcholine-induced relaxations were potentiated in the caudal artery from rats treated with the combination of 17beta-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 17beta-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.

A61603-induced contractions of the porcine meningeal artery are mediated by alpha1- and alpha2-adrenoceptors.

It has recently been shown that A61603 (N-[5-(4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7,8-tetrahydro-naphthalen-1-yl]methane sulphonamide), a potent alpha(1A)-adrenoceptor agonist, decreased carotid artery conductance in anaesthetized pigs by a novel non-adrenergic mechanism. In this study, we set out to pharmacologically characterize A61603-induced contractions of the porcine isolated meningeal artery. While the maximum contractile responses of the artery were similar, A61603 (E(max): 183 +/- 23% of 100 mM KCl; pEC(50): 7.25 +/- 0.18) was more potent than noradrenaline (E(max): 156 +/- 16%; pEC(50): 5.75 +/- 0.17) or phenylephrine (E(max): 163 +/- 20%; pEC(50): 5.63 +/- 0.02). Prazosin (pA(2): 9.36 +/- 0.23) and, to a lesser extent, rauwolscine (pK(b): 6.36 +/- 0.38) and yohimbine (pK(b): 7.30 +/- 0.15) antagonised the contractions to A61603. The 5-HT(1B) (GR127935; N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)[1,1-biphenyl]-4-carboxamide) and 5-HT(2) (ritanserin) receptor antagonists failed to affect the responses to A61603, but methiothepin, which, in addition, has a high affinity for alpha-adrenoceptors, proved an effective antagonist. The A61603-induced responses were suppressed by the cAMP stimulator forskolin, but not by the protein kinase C inhibitor chelerythrine. Our results suggest that the contraction of porcine isolated meningeal artery by A61603 is mediated via mainly alpha(1)-(probably alpha(1A)) and, to a lesser extent, alpha(2)-adrenoceptors, involving the adenylyl cyclase, but not the diacylglycerol pathway.

Female sex hormones and rat dural vasodilatation to CGRP, periarterial electrical stimulation and capsaicin.

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, 17beta-estradiol and progesterone, separately and in combination, on dural vasodilatation induced by alphaCGRP, 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 17beta-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 alphaCGRP (10 to 3000 ng kg(-1)), periarterial electrical stimulation (25 to 125 microA) and capsaicin (0.3 to 18 microg kg(-1)) elicited. RESULTS: There were no significant differences in the vasodilator potency or efficacy of alphaCGRP or capsaicin in the different groups studied. In contrast, the vasodilator response to electrical stimulation was significantly higher in rats treated with 17beta-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, 17beta-estradiol enhanced neurogenic vasodilatation, suggesting increased CGRP release from perivascular nerves. This may be one of the mechanisms through which 17beta-estradiol exacerbates migraine in women.