Amino acid sequences of helospectins, new members of the glucagon superfamily, found in Gila monster venom

The amino acid sequences of two closely related peptides from Gila monster (Heloderma suspectum) venom are reported. Helospectin I is a 38-residue peptide, His-Ser-Asp-Ala-Thr-Phe-Thr-Ala-Glu-Tyr-Ser-Lys-Leu-Leu-Ala-Lys-Leu-Ala- Leu-Gln - Lys-Tyr-Leu-Glu-Ser-Ile-Leu-Gly-Ser-Ser-Thr-Ser-Pro-Arg-Pro-Pro-Ser-Ser, and helospectin II is a 37-residue peptide identical to helospectin I except that it lacks serine 38. Helospectins are pancreatic secretagogues with structures and bioactivities similar to vasoactive intestinal peptide and other members of the glucagon superfamily. The relative significance of helospectin-I and helospectin-II is presently unknown. Comparison of the 28 residues of vasoactive intestinal peptide with residues 1-28 of helospectin shows that identical amino acids occur in 15 positions. Since members of the glucagon superfamily have similar structures but different biological actions, it is possible that helospectin is more closely related to a mammalian peptide awaiting discovery.     

Helospectin I and II evoke vasodilation in the intact peripheral microcirculation

Helospectin I and II, two closely related mammalian neuropeptides of the secretin/glucagons/vasoactive intestinal peptide (VIP) superfamily of peptides, are co-localized with VIP in nerve fibers surrounding vascular smooth muscle. However, the role if any, VIP receptors play in transducing the vasorelaxant effects of helospectin I and II in the intact peripheral microcirculation is uncertain. The purpose of this study was to determine whether helospectin I and II elicit vasodilation in the intact peripheral microcirculation and, if so, whether this response is mediated, in part, by VIP or pituitary adenylate cyclase activating peptide (PACAP) receptor engagement, and through local elaboration of cyclooxygenase products of arachidonic acid metabolism. Using intravital microscopy, we found that suffusion of helospectin I and II (each, 1.0 nmol) evoked potent vasodilation and of similar magnitude in the intact hamster cheek pouch microcirculation (P < 0.05). Suffusion of 0.1 nmol helospectin I and II had no significant effects on arteriolar diameter. Pretreatment with VIP(10-28), a VPAC1/VPAC2 receptor antagonist, or PACAP(6-38), a PAC1/VPAC2 receptor antagonist, had no significant effects on helospectin I- and II-induced responses. In addition, pretreatment with indomethacin had no significant effects on helospectin I- and II-induced vasodilation. Collectively, these data indicate that helospectin I and II evoke potent vasodilation in the intact peripheral microcirculation that is not transduced by VIP or PACAP receptors nor through cyclooxygenase products of arachidonic acid metabolism.     

Helospectin-like immunoreactivity in the esophagus

Helospectin I and II are two non-amidated, VIP-like peptides, isolated from the salivary gland venom of the lizard Heloderma horridum. The lower esophagus of cat, sheep and man was analyzed for helospectin-like immunoreactivity.

Immunocytochemistry revealed helospectin-immunoreactive nerve fibers in the muscle layers, submucosa and mucosa of all species studied. In myenteric ganglia helospectin-immunoreactive nerve fibers and nerve cell bodies could be seen. Double immunostaining for helospectin and vasoactive intestinal peptide (VIP) revealed their coexistence in nerve fibers and cell bodies throughout the lower esophagus of all species tested. Double immunostaining for helospectin and neuropeptide Y revealed their coexistence in nerve fibres surrounding vascular and non-vascular smooth muscle. In the cat and sheep (but not in man) a subpopulation of the helospectin/VIP-containing fibers stored, in addition, substance P.

The helospectin-immunoreactive material in the esophagus probably constitutes a novel neuropeptide. The distribution of the VIP/helospectin-immunoreactive neurons and fibers indicates their possible involvement in the regulation of motor and secretory activities.     

Epac as a novel effector of airway smooth muscle relaxation

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)‐elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP‐mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre‐contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine‐induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine‐induced RhoA activation, measured by both stress fibre formation and pull‐down assay whereas the same Epac activation prevented methacholine‐induced Rac1 inhibition measured by pull‐down assay. Epac‐driven inhibition of both methacholine‐induced muscle contraction by Toxin B‐1470, and MLC phosphorylation by the Rac1‐inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac‐mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre‐contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.     

Targeting the restricted α-subunit repertoire of airway smooth muscle GABAA receptors augments airway smooth muscle relaxation

The prevalence of asthma has taken on pandemic proportions. Since this disease predisposes patients to severe acute airway constriction, novel mechanisms capable of promoting airway smooth muscle relaxation would be clinically valuable. We have recently demonstrated that activation of endogenous airway smooth muscle GABA(A) receptors potentiates β-adrenoceptor-mediated relaxation, and molecular analysis of airway smooth muscle reveals that the α-subunit component of these GABA(A) receptors is limited to the α(4)- and α(5)-subunits. We questioned whether ligands with selective affinity for these GABA(A) receptors could promote relaxation of airway smooth muscle. RT-PCR analysis of GABA(A) receptor subunits was performed on RNA isolated by laser capture microdissection from human and guinea pig airway smooth muscle. Membrane potential and chloride-mediated current were measured in response to GABA(A) subunit-selective agonists in cultured human airway smooth muscle cells. Functional relaxation of precontracted guinea pig tracheal rings was assessed in the absence and presence of the α(4)-subunit-selective GABA(A) receptor agonists: gaboxadol, taurine, and a novel 8-methoxy imidazobenzodiazepine (CM-D-45). Only messenger RNA encoding the α(4)- and α(5)-GABA(A) receptor subunits was identified in RNA isolated by laser capture dissection from guinea pig and human airway smooth muscle tissues. Activation of airway smooth muscle GABA(A) receptors with agonists selective for these subunits resulted in appropriate membrane potential changes and chloride currents and promoted relaxation of airway smooth muscle. In conclusion, selective subunit targeting of endogenous airway smooth muscle-specific GABA(A) receptors may represent a novel therapeutic option for patients in severe bronchospasm.     

Activation of endogenous GABAA channels on airway smooth muscle potentiates isoproterenol-mediated relaxation

Reactive airway disease predisposes patients to episodes of acute smooth muscle mediated bronchoconstriction. We have for the first time recently demonstrated the expression and function of endogenous ionotropic GABA(A) channels on airway smooth muscle cells. We questioned whether endogenous GABA(A) channels on airway smooth muscle could augment beta-agonist-mediated relaxation. Guinea pig tracheal rings or human bronchial airway smooth muscles were equilibrated in organ baths with continuous digital tension recordings. After pretreatment with or without the selective GABA(A) antagonist gabazine (100 muM), airway muscle was contracted with acetylcholine or beta-ala neurokinin A, followed by relaxation induced by cumulatively increasing concentrations of isoproterenol (1 nM to 1 muM) in the absence or presence of the selective GABA(A) agonist muscimol (10-100 muM). In separate experiments, guinea pig tracheal rings were pretreated with the large conductance K(Ca) channel blocker iberiotoxin (100 nM) after an EC(50) contraction with acetylcholine but before cumulatively increasing concentrations of isoproterenol (1 nM to 1 uM) in the absence or presence of muscimol (100 uM). GABA(A) activation potentiated the relaxant effects of isoproterenol after an acetylcholine or tachykinin-induced contraction in guinea pig tracheal rings or an acetylcholine-induced contraction in human endobronchial smooth muscle. This muscimol-induced potentiation of relaxation was abolished by gabazine pretreatment but persisted after blockade of the maxi K(Ca) channel. Selective activation of endogenous GABA(A) receptors significantly augments beta-agonist-mediated relaxation of guinea pig and human airway smooth muscle, which may have important therapeutic implications for patients in severe bronchospasm.     

Unlike VIP, the VIP-related peptides PACAP, helodermin and helospectin suppress electrically evoked contractions of rat vas deferens

We have compared the effects of vasoactive intestinal peptide (VIP) and of the VIP-related peptides pituitary adenylate cyclase activating peptide (PACAP) 1–27 and 1–38, helodermin, helospectin I and helospectin II, on the electrically evoked twitches in the isolated vas deferens of the rat. While VIP was virtually without effect, PACAP 1–38 suppressed the electrically evoked twitches effectively and in a concentration-dependent manner (pIC₅₀ value 7.5). The naturally occurring N-terminal fragment PACAP 1–27 was less effective than PACAP 1–38 (I_max values 37.2% suppression compared to 76.5%) and less potent. The C-terminal fragment PACAP 16–38 was virtually inactive. Also helodermin and helospectin I+II suppressed the electrically evoked twitches effectively and in a concentration-dependent manner (pIC₅₀ values 6.9; 7.2; 6.8, respectively). The three peptides produced similar maximum reduction of the twitches (74–80%).

The findings suggest that PACAP, helodermin and helospectin suppress the electrically evoked contractions in the rat vas deferens via receptors distinct from VIP receptors.     

Airway smooth muscle relaxation induced by 5-HT(2A) receptors: role of Na(+)/K(+)-ATPase pump and Ca(2+)-activated K(+) channels

AIMS: Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. MAIN METHODS: Using organ baths, patch clamp, and intracellular Ca(2+) measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. KEY FINDINGS: A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 muM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 muM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT(1A), 5-HT(1B)/5-HT(1D) and 5-HT(2B) receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 muM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K(+) currents (I(K(Ca))), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. SIGNIFICANCE: During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na(+)/K(+)-ATPase pump, and increasing activity of the high-conductance Ca(2+)-activated K(+) channels, probably via 5-HT(2A) receptors.     

Hyperoxia impairs airway relaxation in immature rats via a cAMP-mediated mechanism.

Hyperoxic exposure enhances airway reactivity in newborn animals, possibly due to altered relaxation. We sought to define the role of prostaglandinand nitric oxide-mediated mechanisms in impaired airway relaxation induced by hyperoxic stress. We exposed 7-day-old rat pups to either room air or hyperoxia (>95% O2) for 7 days to assess airway relaxation and cAMP and cGMP production after electrical field stimulation (EFS). EFS-induced relaxation of preconstricted trachea was diminished in hyperoxic vs. normoxic animals (P < 0.05). Indomethacin (a cyclooxygenase inhibitor) reduced EFS-induced airway relaxation in tracheae from normoxic (P < 0.05), but not hyperoxic, rat pups; however, in the presence of NG-nitro-L-arginine methyl ester (a nitric oxide synthase inhibitor) EFS-induced airway relaxation was similarly decreased in tracheae from both normoxic and hyperoxic animals. After EFS, the increase from baseline in the production of cAMP was significantly higher in tracheae from normoxic than hyperoxic rat pups, and this was accompanied by greater prostaglandin E2 release only in the normoxic group. cGMP production after EFS stimulation did not differ between normoxic and hyperoxic groups. We conclude that hyperoxia impairs airway relaxation in immature animals via a mechanism primarily involving the prostaglandin-cAMP signaling pathway with an impairment of prostaglandin E2 release and cAMP accumulation.     

G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M(2) muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene (GRK5(-/-)) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5(-/-) and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from beta(2)-adrenergic receptor activation were diminished in GRK5(-/-) mice. These data suggest that M(2) muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5(-/-) mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.     

Nitric oxide induces airway smooth muscle cell relaxation by decreasing the frequency of agonist-induced Ca2+ oscillations

Nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, but the underlying mechanism is not well understood. Consequently, we investigated the effects of NO on airway SMC contraction, Ca²⁺ signaling, and Ca²⁺ sensitivity in mouse lung slices with phase-contrast and confocal microscopy. Airways that were contracted in response to the agonist 5-hydroxytryptamine (5-HT) transiently relaxed in response to the NO donor, NOC-5. This NO-induced relaxation was enhanced by zaprinast or vardenafil, two selective inhibitors of cGMP-specific phosphodiesterase-5, but blocked by ODQ, an inhibitor of soluble guanylyl cyclase, and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Simultaneous measurements of airway caliber and SMC [Ca²⁺]_i revealed that airway contraction induced by 5-HT correlated with the occurrence of Ca²⁺ oscillations in the airway SMCs. Airway relaxation induced by NOC-5 was accompanied by a decrease in the frequency of these Ca²⁺ oscillations. The cGMP analogues and selective PKG activators 8Br-cGMP and 8pCPT-cGMP also induced airway relaxation and decreased the frequency of the Ca²⁺ oscillations. NOC-5 inhibited the increase of [Ca²⁺]_i and contraction induced by the photolytic release of inositol 1,4,5-trisphosphate (IP₃) in airway SMCs. The effect of NO on the Ca²⁺ sensitivity of the airway SMCs was examined in lung slices permeabilized to Ca²⁺ by treatment with caffeine and ryanodine. Neither NOC-5 nor 8pCPT-cGMP induced relaxation in agonist-contracted Ca²⁺-permeabilized airways. Consequently, we conclude that NO, acting via the cGMP–PKG pathway, induced airway SMC relaxation by predominately inhibiting the release of Ca²⁺ via the IP₃ receptor to decrease the frequency of agonist-induced Ca²⁺ oscillations.     

Purification and structure of exendin-3, a new pancreatic secretagogue isolated from Heloderma horridum venom.

An amino-terminal histidyl structure (His1) is characteristic of most peptides in the glucagon superfamily. An assay for His1 peptides performed by amino-terminal amino acid sequencing was used to screen venom from the Gila monster lizard, Heloderma horridum. Two His1 peptides were identified: helospectin and a new His1 peptide that has been named exendin-3 to indicate that it is the third peptide to be found in an exocrine secretion of Heloderma lizards which has endocrine activity, the first two being helospectin (exendin-1) and helodermin (exendin-2). In the lot of H. horridum venom tested, exendin-3 was 5-10-fold more abundant in molar concentration than helospectin. The structure of exendin-3 was analyzed by amino acid sequencing and mass spectrometry. Exendin-3 is a 39-amino acid peptide with a mass of 4200. It contains a carboxyl-terminal amide and has a strong homology with secretin at its amino-terminal 12 amino acids. The complete structure of exendin-3 is His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala- Val-Arg - Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser- amide. It is 32 and 26% homologous with helospectin and helodermin, respectively. It has greatest homology with glucagon (48%) and human glucagon-like peptide-1 (50%). Exendin-3 (3 microM) stimulated increases in cellular cAMP and amylase release from dispersed guinea pig pancreatic acini.     

Immunolocalization of a Na-K-2Cl cotransporter in human tracheobronchial smooth muscle.

Inhibition of the Na-K-2Cl (NKCC) cotransporter by loop diuretics is associated with airway relaxation, but there has been no direct evidence for the expression of this protein in airway smooth muscle. Thus we hypothesized that a NKCC cotransporter is present and functional in airway smooth muscle cells. Monoclonal and polyclonal antibodies were used first to demonstrate the presence of a NKCC cotransporter protein in isolated human fetal trachea and normal human bronchial smooth muscle cells (BSMC) by Western blotting. The cotransporter protein was then localized by immunohistochemical staining to airway smooth muscle cells in culture and in situ. The localization was confirmed by indirect immunofluorescence and laser confocal microscopy in the BSMC. Cotransporter function in BSMC was also confirmed in vitro by bumetanide-mediated inhibition of rubidium uptake. Our present findings thus document the presence of a functional NKCC cotransporter in human airway smooth muscle, providing a basis for defining the role of this ion cotransporter in airway smooth muscle function.     

Effects of alpha calcitonin gene-related peptide in human bronchial smooth muscle and pulmonary artery

Although airway and pulmonary vessel tone are regulated predominantly by cholinergic and adrenergic impulses, biologically active peptides such as calcitonin gene-related peptide (CGRP) may significantly influence human smooth muscle tone in normal and pathophysiological states. In the present study, the expression of CGRP and its receptor CGRPR-1 and the biological effect of the peptide were investigated in human airways and pulmonary arteries. Immunohistochemistry revealed the presence of CGRP in human airway nerves and neuro-epithelial cells, whereas the receptor was found in epithelial cells and smooth muscle myocytes of the bronchi and in pulmonary artery endothelium. On precontracted bronchi (3-4 mm in diameter) alpha-CGRP (0.01-10 nM) caused a concentration-dependent contraction on epithelium-denuded bronchi, whereas no significant effect was recorded in bronchi with intact epithelium. In pulmonary arteries (2-6 mm in diameter), alpha-CGRP caused a concentration-dependent relaxation of endothelium intact and denuded vessels. Pre-treatment with indomethacin, but not with l-NAME, prevented the relaxation induced by alpha-CGRP in pulmonary arteries suggesting that prostaglandins but not nitric oxide (NO) are involved in the intracellular signal transduction pathway. The effects induced by alpha-CGRP in bronchi and vessels were prevented by application of the antagonist CGRP((8-37)). In summary, the present studies examined the biological function of CGRP in human airways and demonstrated a constrictory effect of CGRP only in epithelium-denuded airway smooth muscle indicating an alteration of CGRP airway effects in respiratory tract pathological states with damaged epithelium such as chronic obstructive pulmonary disease or bronchial asthma.     

Homologous and heterologous regulation of the helodermin/vasoactive-intestinal-peptide response in the murine radiation leukemia-virus-induced lymphoma cell line BL/VL3

1. Functional vasoactive intestinal peptide (VIP)/helodermin receptors and beta 2-adrenoceptors coexist in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus (see preceding paper in this journal). 2. Short-term (5-30 min) exposures of BL/VL3 cells to VIP or isoproterenol induced both homologous and heterologous desensitization. The potency of VIP and isoproterenol to desensitize was similar to their potency to occupy receptors and activate adenylate cyclase. 3. Long-term (16-h) exposure of BL/VL3 cells to VIP induced homologous down regulation only, whereas isoproterenol induced both homologous and heterologous down regulation. The potency of VIP, peptide histidine isoleucinamide, helodermin, helospectin, and [D-Phe2]VIP on the one hand, and of isoproterenol on the other hand, to decrease homologous responses was comparable to their potency for receptor occupancy and adenylate cyclase activation.     

Peptidergic innervation of the human clitoris.

In the present study, the distributions of neuropeptides in the normal human clitoris and in a clitoris from an adrenogenital syndrome (AGS) was demonstrated by immunohistochemistry (IHC). Immunohistochemical screening detected a complex network of nerve fibers containing vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), neuropeptide tyrosine (neuropeptide Y), C-flanking peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP) and substance P immunoreactivities. Special attention was given to the VIP-related peptide helospectin, that has been detected in neuronal elements in the clitoris. No visible differences between the localization and distribution of peptidergic nerve fibers of normal and hypertrophic clitoris from AGS have been observed. Co-localization studies showed the co-existence of VIP, PHM and partly helospectin and neuropeptide Y with CPON within nerve fibers in the cavernous tissue and substance P and CGRP co-expression in nerve fibers especially underneath and within the glans clitoris.     

Modeling the airway epithelium in allergic asthma: Interleukin-13-induced effects in differentiated murine tracheal epithelial cells

Summary Mucous cells of the airway epithelium play a crucial role in the pathogenesis of human inflammatory airway diseases. Therefore, it is of importance to complement in vivo studies that use murine models of allergic asthma with in vitro mechanistic studies that use murine airway epithelial cells, including mucus-containing cells. In this study, we report the development and characterization of an in vitro culture system for primary murine tracheal epithelial (MTE) cells comprising ciliated cells and a substantial number of mucous cells. The increase in mucous cell number over that observed in the native murine airway, or in previously described murine cultures, creates a culture intermediate between the in vivo murine airway epithelium and in vitro cultures of human airway epithelial cells. To establish the usefulness of this culture system for the study of epithelial effects during inflammatory airway diseases, the cells were exposed to interleukin (IL)-13, a central inflammatory mediator in allergic asthma. The IL-13 induced two characteristic epithelial effects, proliferation and modulation of MUC5AC gene expression. There was a concentration dependence of these events, wherein high concentrations of IL-13 (10 ng/ml) induced proliferation, whereas lower concentrations (1 ng/ml) increased MUC5AC mRNA (where mRNA is messenger RNA). Interestingly, these effects occurred in an inverse manner, with the high concentration of IL-13 also provoking a significant decrease in MUC5AC gene expression. Thus, MTE cells cultured in this manner may provide an important link between experimental findings from animal models of allergic asthma and their application to human disease.     

Relaxing effects of 5-oxo-ETE on human bronchi involve BK Ca channel activation

The present study investigated the ability of 5-oxo-EicosaTetraEnoic acid (5-oxo-ETE) for modulating airway smooth muscle (ASM) tone in human bronchi. 5-Oxo-ETE induced a concentration-dependent relaxing effect on human bronchi pre-contracted with methacholine (MCh) and arachidonic acid (AA). This relaxing response was highly sensitive to Iberiotoxin (IbTx), a large conducting Ca(2+)-activated K(+) channel (BK(Ca)) inhibitor. Furthermore, microelectrode measurements revealed that 5-oxo-ETE (0.1-10 microM) hyperpolarizes the membrane potential of human bronchial ASM cells. These hyperpolarizing effects were also inhibited in the presence of 10nM IbTx. Lastly, 5-oxo-ETE was shown to directly activate reconstituted BK(Ca) channels derived from human airway smooth muscles. In summary, the 5-oxo-ETE eicosanoid activates a specific K(+) conductance, involved in membrane hyperpolarization, which in turn reduces Ca(2+) entry and facilitates relaxation of smooth muscle cells.     

Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction

Bitter taste receptors (TAS2Rs) on the tongue probably evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants that, when activated, lead to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased intracellular calcium ([Ca2(+)](i)) in ASM in a Gβγ-, phospholipase Cβ (PLCβ)- and inositol trisphosphate (IP?) receptor-dependent manner, which would be expected to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM and dilation of airways that was threefold greater than that elicited by β-adrenergic receptor agonists. The relaxation induced by TAS2Rs is associated with a localized [Ca2(+)](i) response at the cell membrane, which opens large-conductance Ca2(+)-activated K(+) (BK(Ca)) channels, leading to ASM membrane hyperpolarization. Inhaled bitter tastants decreased airway obstruction in a mouse model of asthma. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants.