Patent Application
20150202212
The invention generally relates to methods and compositions for identifying novel therapeutic agents, and related methods of use thereof. More particularly, the invention relates to novel methods for identifying agents, e.g., bitter tastants, which are useful in treating smooth muscle disorders, pharmaceutical compositions comprising such agents and related methods of use.
Airway obstructive diseases, such as asthma and chronic obstructive pulmonary disease (COPD), have become increasingly prevalent, currently affecting more than 300 million people worldwide. Asthma is an obstructive lung disease where the bronchial tubes (airways) are extra sensitive and, when inflamed, can cause muscles around the airways to tighten making the airways narrower. Asthma is usually triggered by dust, pollen and other allergens, upper respiratory tract infections, etc. COPD, also known as chronic obstructive lung disease, is the occurrence of chronic bronchitis or emphysema, a pair of commonly co-existing diseases of the lungs in which the airways become narrowed, which limits air flow to and from the lungs and causes shortness of breath.
Dysfunction of airway smooth muscle (ASM) cells in the respiratory tree plays a pivotal role in promoting progression of airway obstructive diseases and in contributing to their symptoms. (Grainge, et al. 2011 New Engl J Med 364:2006-2015; Hershenson, et al. 2008 Annual Rev Pathol: Mechanisms of Disease 3:523-555; Tliba, et al. 2009 Annual Rev Physiol 71:509-535.) With their ability to contract and relax, smooth muscle cells regulate the diameter and length of conducting airways, controlling dead space and resistance to airflow. Excessive contraction of smooth muscle cells can be life-threatening as they can cause the airway to fully close.
There is an unmet clinical need for new and effective treatments for smooth muscle disorders: (1) their prevalence has almost doubled worldwide in the last few decades, (2) many asthmatics and COPD patients do not respond well to current bronchodilators, and (3) no major breakthrough in bronchodilator development has been achieved since the discovery of specific 132 adrenergic receptor agonists almost fifty years ago.
Bronchodilators have been used to treat asthmatic attacks and to manage COPD. (Fanta 2009 New Engl J Med 360:1002-1014; Han, et al. 2011 Proc of Am Thoracic Soc 8:356-362.) Existing bronchodilators, however, have undesirable side effects and are not sufficiently effective for severe asthmatics and many COPD patients. Understanding the mechanisms regulating ASM holds the promise of developing more effective and safe bronchodilators.
Bitter tastants represent a new class of compounds with potential as potent bronchodilators. Bitter taste, the most sensitive of the five basic tastes, is key to animal and human survival since it help them avoid harmful toxins and noxious substances. Deshpande et al. recently found that cultured ASM cells express G-protein coupled bitter taste receptors (TAS2Rs), a class of proteins long thought to be expressed only in the specialized epithelial cells in the taste buds of the tongue. (Deshpande, et al. 2010 Nat Med 16:1299-1304; Chandrashekar, et al. 2000 Cell 100:703-711; Ruiz-Avila, et al. 1995 Nature 376:80-85; Wong, et al. 1996 Nature 381:796-800; Zhang, et al. 2003 Cell 112:293-301.) Bitter tastants with diverse chemical structures have been shown to cause greater ASM relaxation in vitro than β2 adrenergic agonists, the most commonly used bronchodilators to treat asthma and COPD. (Deshpande, et al. 2010 Nat Med 16:1299-1304; Zhang, et al. 2012 Nat Med 18:648-650.) Moreover, these compounds can effectively relieve in vivo asthmatic airway obstruction than 132 adrenergic agonists in a mouse model of asthma, making them highly attractive bronchondilators for asthma and COPD.
Bitter tastant-induced bronchodilation was unexpected, because these agents appeared to increase intracellular Ca2+ concentration ([Ca2+]i) to a level comparable to that produced by potent bronchoconstrictors, which should have led to smooth muscle contraction. (Deshpande, et al. 2010 Nat Med 16:1299-1304; Somlyo, et al. 1994 Nature 372:231-236.) To reconcile this apparent paradox, it was proposed that bitter tastants activate the canonical bitter taste signaling pathway (i.e., TAS2R-gustducin-phospholipase Cβ(PLCβ)-inositol 1,4,5-triphosphate receptor (IP3R)) to increase focal Ca2+ release from endoplasmic reticulum, which then activate large-conductance Ca2+-activated K+ (BK) channels thereby hyperpolarizing the membrane. (Deshpande, et al. 2010 Nat Med 16:1299-1304.) It was, however, subsequently demonstrated through patch-clamp recordings that bitter tastants do not activate BK channels but rather inhibit them. (Zhang, et al. 2012 Nat Med 18:648-650.) Moreover, three different BK channel blockers did not affect the bronchodilation induced by bitter tastants.
The apparent conundrum of putative [Ca2+]I elevation leading to relaxation may be attributed to the fact that Ca2+ responses to bitter tastants were assessed in cultured human ASM cells, while the contractile responses to them were investigated in freshly dissected ASM tissues. (Deshpande, et al. 2010 Nat Med 16:1299-1304.) It is well known that cultured smooth muscle cell lines alter their phenotype, i.e., losing their ability to contract and relax. (Chamley-Campbell, et al. 1979 Physiol Rev 59:1-61; Hall, et al. 1995 Am J Physiol 268:L1-11.) It is likely their Ca2+ response is also modified. To understand bitter tastant-induced bronchodilation, it is necessary to study the contraction and the underlying signaling in freshly isolated ASM tissues and cells.
Thus, in addition to an ongoing need for agents, such as bitter tastants, that are therapeutically effective in treating ASM-related diseases, an urgent need remains for novel methodologies for screening and testing compounds, such as bitter tastants.
The invention provides a novel methodology for identifying agents that are useful as therapeutic agents for smooth muscle disorders. The invention also provides pharmaceutical compositions, and methods thereof, useful in preventing, treating or managing smooth muscle disorders.
In one aspect, the invention generally relates to a method for identifying a candidate compound for treating or preventing a smooth muscle disorder. The method includes: (1) contacting a test compound with a cell of a smooth muscle tissue or organ; and (2) measuring the intracellular Ca2+ concentration before and after contacting the test compound, whereby a decrease of 30% or greater after contacting the test compound is indicative of the activity of the test compound. In certain preferred embodiments, the method further includes, after contacting a test compound: measuring the cell length before and after contacting with the test compound, wherein an increase of 20% or greater is indicative of the activity of the test compound. In certain preferred embodiments, the test compounds are bitter tastants.
In another aspect, the invention generally relates to a method for treating or preventing a smooth muscle disorder in a mammal, including human. The method includes administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound having the structural formula of (I):
or a pharmaceutically acceptable salt, ester or prodrug thereof, wherein each of R1, R2, R3, R4, R5 and R6 is independently selected from hydrogen, OH, alkyl, alkoxy, and halogen; n is an integer from 0 to about 4.
In yet another aspect, the invention generally relates to a pharmaceutical composition for treating or preventing a smooth muscle disorder in a mammal, including human, comprising a therapeutically effective amount of a pharmaceutical composition comprising a compound having the structural formula of (I):
or a pharmaceutically acceptable salt, ester or prodrug thereof, wherein each of R1, R2, R3, R4, R5 and R6 is independently selected from hydrogen, OH, alkyl, alkoxy, and halogen; n is an integer from 0 to about 4.
The present invention provides novel methodologies for screening and testing compounds, such as bitter tastants. The unique approach disclosed herein is based on a better understanding of the underlining mechanisms regulating ASM. The invention also provides pharmaceutical compositions and methods of use of certain bitter tastants that are therapeutically effective in treating ASM-related diseases.
Smooth muscles express bitter taste receptors, the activation of which induces profound changes in the contractility of smooth muscle. Bitter taste receptors are believed to be targets for treating diseases or disorders in smooth muscle. Bitter compounds represent a good starting point for developing therapeutics that relax airway smooth muscle more effective than β2 agonists, the commonly used bronchidilators for airway obstructive diseases. Some bitter compounds may be capable of relaxing bronchoconstrictor pre-contracted airways that are resistant to β2 agonist treatment. A unique and effective screening methodology has been developed and disclosed herein that is promised to change the paradigm of research and development on smooth muscle disorders.
As disclosed first herein, bitter tastants activate the canonical bitter taste signaling cascade, slightly increasing global the intracellular Ca2+ concentration ([Ca2+]i) in resting cells, but not to a level sufficient to cause contraction. However, bitter tastants reverse the increase in [Ca2+]i evoked by bronchoconstrictors, leading to bronchodilation. This reversal is mediated by the suppression of L-type voltage-dependent Ca2+ channels (VDCCs) in a gustducin βγ subunit-dependent, yet PLCβ- and IP3R-independent manner. Hence, it is believed that TAS2R activation in ASM stimulates two opposing Ca2+ signaling pathways, both mediated by Gβγ subunits, which increases [Ca2+]i at rest but blocks activated L-type VDCCs reversing the contraction they cause. Therefore, bitter tastants can generate different and opposing Ca2+ signals depending upon the cellular environment.
The present invention revealed two major differences in Ca2+ signaling compared to a prior study by Deshpande et al. (Deshpande, et al. 2010 Nat Med 16:1299-1304.) First, Desphande et al. reported that bitter tastant increased [Ca2+]i to a level comparable to bronchoconstictors. In freshly isolated ASM, we found that bitter tastants only modestly increased [Ca2+]i to a level much lower than that produced by bronchconstrictors. Second, Deshpande et al. reported that bitter tastants generate local Ca2+ events. In freshly isolated ASM, in contrast, we found that bitter tastants did not increase local Ca2+ releases such as Ca2+ puffs and Ca2+ sparks. A reason for these discrepancies may be because Despande et al.'s studies were conducted in cultured ASM cell lines, as oppose to freshly isolated ASM, which display a different phenotype by altering the expression of receptors, ion channels and contractile proteins. (Chamley-Campbell, et al. 1979 Physiol Rev 59:1-61; Hall, et al. 1995 Am J Physiol 268:L1-11.) Additionally, we found that bitter tastants do not activate BK channels. Thus, the evidence establish that bitter tastant-induced bronchodilation is highly unlikely to result from the generation of local Ca2+ events, which in turn activate BK channel and hyperpolarize the membrane as proposed previously. (Deshpande, et al. 2010 Nat Med 16:1299-1304.)
By simultaneously measuring [Ca2+]i and cell shortening, we found that bitter tastant's ability to reverse the increase in [Ca2+]i caused by bronconstrictors is the underlying signal producing the bronchodilation. The conclusion that [Ca2+]i is the critical signal governing ASM contractility was supported by at least three lines of evidence. First, in the presence of bronchoconstrictors, bitter tastants lowered [Ca2+]i while at the same time relaxing the precontracted cells. This response was found to be reversible. Second, clamping intracellular [Ca2+]i to levels produced by the bronchoconstrictors (low μM) prevented bitter tastants from relaxing airways. Third, enhancing and blocking Ca2+ influx via L-type Ca2+ channels oppositely regulated the relaxation mediated by bitter tastants.
Gustducin Gβγ inhibits L-type VDCCs to cause bronchodilation, highlighting the importance of these channels in mediating bronchoconstriction and their potential as a target for bronchodilators. Indeed, L-type VDCCs are expressed in ASM cells and their activation causes these cells to fully contract. (Du, et al. 2006 J Biol Chem 281:30143-30151; Kotlikoff 1988 Am J Physiol 254:C793-801; Liu, et al. 2006 Am J Physiol Lung Cell Mol Physiol 291:L281-288; Zhuge, et al. 2010 J Biol Chem. 285:2203-2210.) Activation of these channels is a key mechanism underlying bronchoconstrictor-induced contraction. (Gosens, et al. 2006 Respiratory Res 7:73; Hirota, et al. 2003 British J Anaesthesia 90:671-675; Kajita, et al. 1993 Am J Physiol 264:L496-503; Liu, et al. 2006 Am J Physiol Lung Cell Mol Physiol 291:L281-288.) Moreover, antagonists of L-type VDCCs are effective in relieving airway spasm in animal models of asthma and in at least a subset of asthmatic patients. (Ahmed, et al. 1988 J Allergy & Clinical Immunol 81:133-144; Barnes, et al. 1981 Thorax. 36:726-730; Harman, et al. 1987 Am Rev Respir Dis 136:1179-1182; Patel, et al. 1985 Eur J Respir Dis. 67:269-271.) Although L-type VDCCs in smooth muscle can be modulated by a variety of means including phosphorylation and Ca2+, this is the first demonstration that a Gβγ can inhibit L-type VDCCs in smooth muscle. (Gui, et al. 2006 J Biol Chem 281:14015-14025; Le Blanc, et al. 2004 Circulation Res 95:300-307; Liao, et al. 2005 Cardiovascular Res 68:197-203; Thakali, et al. 2010 Circulation Res 106:739-747; Zhong, et al. 2001 J Physiol 531:105-115.) Given that Gβγ can directly inhibit K+ channels and N type Ca2+ channels in several cell types (Herlitze, et al. 1996 Nature 380:258-262; Ikeda 1996 Nature 380:255-258; Reuveny, et al. 1994 Nature 370:143-146; Wickman, et al. 1994 Nature 368:255-257), it is likely that Gβγ acts on L-type VDCCs in a similar manner.
The opposing Ca2+ signals mediated by Gβγ upon activation of G-protein coupled bitter taste receptors (TAS2Rs) revealed in this study are unique. It is expected that gustducin Gβγ activates PLCβ to generate IP3 and release Ca2+ from endo/sarcoplasmic reticulum to raise [Ca2+]i in ASM cells. But, unexpectedly, gustducin Gβγ also suppresses Ca2+ signaling mediated by Mch, which largely activates M3 muscarinic acetylcholine receptor, a Gq family receptor. In general, Gβγ from the Gi/Go family (to which TAS2Rs belong) tends to potentiate, rather than, inhibit the Ca2+ responses caused by the Gq family. (Cheng, et al. 2002 Biochem J 364:33-39; Samways, et al. 2003 Biochem J 375:713-720.) It remains to be determined whether the inhibition of Ca2+ signaling by TAS2R activation is Gβγ isoform specific. Since Gβγ also mediates the ASM contractions induced by activation of M2 muscarinic acetylcholine receptors and γ-aminobutyric acid-B receptors, our present findings suggested that Gβγ reversal of the rise in [Ca2+]I caused by bronchoconstrictors is isoform specific, and is likely via Gβ3γ13 dimers which are released upon activation of TAS2Rs. (Mizuta, et al. 2011 Am J Respiratory Cell and Mol Biol 45:1232-1238; Nino, et al. 2012 PLoS ONE 7:e32078; Huang, et al. 1999 Nat Neurosci 2:1055-1062.)
Investigation was directed at how bitter tastants affected both [Ca2+]i and ASM contraction in freshly isolated airway cells and tissue from mouse and human. Fluo-3 was used to assess the effect of bitter tastants on [Ca2+]i. Chloroquine and denatonium, two substances commonly used to study bitter taste signaling, were used as bitter tastants.
It is worth mentioning that virtually all of the studies of bitter taste signaling in taste buds and extraoral tissues have focused on the responses mediated by bitter tastants alone. (e.g., Chandrashekar, et al. 2000 Cell 100:703-711; Ruiz-Avila, et al. 1995 Nature 376:80-85; Wong, et al. 1996 Nature 381:796-800; Zhang, et al. 2003 Cell 112:293-301; Janssen, et al. 2011 Proc of Nat Academy of Sci 108:2094-2099; Shah, et al. 2009 Science 325:1131-1134; Tizzano, et al. 2010 Proc of Nat Academy of Sci 107:3210-3215.) The opposing Ca2+ signaling mediated by Gβγ as disclosed herein may operate in these systems when they are stimulated by a combination of bitter tastants and other activators.
Bitter tastants induce a stronger bronchodilation in both in vitro and in vivo asthmatic mouse models than do β2 agonists, the most commonly used bronchodilators for treating asthma and COPD. Therefore, these compounds are promising candidates to be developed as a new class of bronchodilators. The findings in the present study provide the cellular and molecular rationale for this line of inquiry. Searching for these bitter tastants is of clinical significance because the current bronchodilators are insufficient for treating severe asthma and many COPD patients.
TAS2Rs had long been thought to function only in specialized epithelial cells in the taste buds of the tongue. However, studies in recent years have demonstrated that activation of TAS2Rs can generate different biological responses in a variety of extraoral tissues. Bitter tastants were found to cause airway smooth muscle relaxation in vitro in normal mice and human lung specimens, and in vivo in asthmatic mice. This relaxation is greater than that produced by β2 adrenergic agonists, the most commonly used bronchodilators for symptomatic relief in asthma and chronic obstructive pulmonary disease.
To uncover the underlining mechanism, freshly isolated airway tissue and airway smooth muscle cells from mice and humans have been studied using a combination of Ca2+ imaging, patch-clamp recording, single cell shortening/tissue contraction assay, and pharmacology. The results showed that activation of TAS2Rs in airway smooth muscle releases the G-protein gustducin βγ. Surprisingly, gustducin βγ, on the one hand, mediates an modest elevation in intracellular Ca2+ concentration ([Ca2+]i) in resting cells and, on the other hand, reverses the rise in [Ca2+]i seen in cells treated with bronchoconstrictors (e.g. Gq-coupled receptor agonists), meant to simulate asthma, by suppressing L-type voltage-dependent Ca2+ channels, thereby producing relaxation.
Disclosed first herein is that Gβγ mediates opposing Ca2+ signaling mechanisms, which uncovers a new form of signaling that integrates two major cellular signaling systems (i.e., G-protein coupled receptor and Ca2+) since Gβγ from the Gi/Go family usually potentiates, rather than, inhibits the responses by the Gq family. This mechanism likely operates in many types of smooth muscle—a tissue essential for virtually all the internal hollow organs in animals and human, and involved in an array of diseases or disorders such as hypertension and overactive bladder.
The present invention provides (1) a cellular and molecular basis of a new form of bronchodilation, bitter tastant-induced bronchodialtion, and (2) a molecular explanation for a new class of bronchodilators potentially better than β2 adrenergic agonists. More importantly from a drug development perspective, the invention reveals a Ca2+ effect that is large enough to be well suited for screening and identifying potent bronchodilators from among the many thousands of available bitter tastants. A critical step in identifying highly potent bitter tastants is developing reliable and highly effective screening methodologies. The better understanding of Ca2+ dynamics in response to bitter tastants enables a methodology that is promised to accelerate screening and identification of potent bronchodilators from a new class of compounds. Measurements of [Ca2+]i (and optionally in conjunction with measurement of cell shortening) as disclosed herein provide a robust and quantitative approach that represents a powerful new paradigm for identifying bronchodilators from among the many bitter tastants available. Furthermore, the invention provides promising candidates for treating asthma and COPD.
Thus, in one aspect, the invention generally relates to a method for identifying a candidate compound for treating or preventing a smooth muscle disorder. The method includes: (1) contacting a test compound with a cell of a smooth muscle tissue or organ; and (2) measuring the intracellular Ca2+ concentration before and after contacting the test compound, whereby a decrease of 30% or greater after contacting the test compound is indicative of the activity of the test compound.
In certain preferred embodiments, the intracellular Ca2+ concentration decreased 40% or greater after contacting the test compound is indicative of the activity of the test compound. In certain preferred embodiments, the intracellular Ca2+ concentration decreased 50% or greater after contacting the test compound is indicative of the activity of the test compound. In certain preferred embodiments, the intracellular Ca2+ concentration decreased 60% or greater after contacting the test compound is indicative of the activity of the test compound. In certain preferred embodiments, the intracellular Ca2+ concentration decreased 70% or greater after contacting the test compound is indicative of the activity of the test compound. In certain preferred embodiments, the intracellular Ca2+ concentration decreased 80% or greater after contacting the test compound is indicative of the activity of the test compound. In certain preferred embodiments, the intracellular Ca2+ concentration decreased 90% or greater after contacting the test compound is indicative of the activity of the test compound.
In certain preferred embodiments, the method further includes, after contacting a test compound: measuring the cell length before and after contacting with the test compound, wherein an increase of 20% or greater is indicative of the activity of the test compound. In certain preferred embodiments, the method further includes, after contacting a test compound: measuring the cell length before and after contacting with the test compound, wherein an increase of 25% or greater is indicative of the activity of the test compound. In certain preferred embodiments, the method further includes, after contacting a test compound: measuring the cell length before and after contacting with the test compound, wherein an increase of 30% or greater is indicative of the activity of the test compound. In certain preferred embodiments, the method further includes, after contacting a test compound: measuring the cell length before and after contacting with the test compound, wherein an increase of 35% or greater is indicative of the activity of the test compound. In certain preferred embodiments, the method further includes, after contacting a test compound: measuring the cell length before and after contacting with the test compound, wherein an increase of 40% or greater is indicative of the activity of the test compound.
In certain preferred embodiments, the test compounds are selected from bitter tastants.
In certain preferred embodiments, the smooth muscle tissue or organ is part of the respiratory tract. In certain preferred embodiments, the smooth muscle tissue or organ is part of a blood vessel. In certain preferred embodiments, the smooth muscle tissue or organ is part of the gastrointestinal tract. In certain preferred embodiments, the smooth muscle tissue or organ is part of the urinary tract. In certain preferred embodiments, the smooth muscle tissue or organ is part of internal anal sphincter. In certain preferred embodiments, the smooth muscle tissue or organ is part of pulmonary artery.
The invention is also directed at compounds identified, via the disclosed methods, to have activity in treating or preventing a smooth muscle disorder.
In another aspect, the invention generally relates to a method for treating or preventing a smooth muscle disorder in a mammal, including human. The method includes administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a compound having the structural formula of (I):
or a pharmaceutically acceptable salt, ester or prodrug thereof, wherein each of R1, R2, R3, R4, R5 and R6 is independently selected from hydrogen, OH, alkyl, alkoxy, and halogen; n is an integer from 0 to about 4 (e.g., 0, 1, 2, 3, 4).
In yet another aspect, the invention generally relates to a pharmaceutical composition for treating or preventing a smooth muscle disorder in a mammal, including human, comprising a therapeutically effective amount of a pharmaceutical composition comprising a compound having the structural formula of (I):
or a pharmaceutically acceptable salt, ester or prodrug thereof, wherein each of R1, R2, R3, R4, R5 and R6 is independently selected from hydrogen, OH, alkyl, alkoxy, and halogen; n is an integer from 0 to about 4 (e.g., 0, 1, 2, 3, 4).
In certain preferred embodiments, the compound has the structural formula:
In certain preferred embodiments, the smooth muscle disorder is an airway obstructive disease. In certain preferred embodiments, the airway obstructive disease is asthma. In certain preferred embodiments, the airway obstructive disease is chronic obstructive pulmonary disease (COPD). In certain preferred embodiments, the smooth muscle disorder is anal sphincter disorder. In certain preferred embodiments, the smooth muscle disorder is urethral obstruction. In certain preferred embodiments, the smooth muscle disorder is associated with cystic fibrosis. In certain preferred embodiments, the smooth muscle disorder is associated with fecal incontinence and constipation. In certain preferred embodiments, the smooth muscle disorder is associated with pulmonary hypertension.
In certain preferred embodiments, R1 is hydrogen. In certain preferred embodiments, R1 is OH. In certain preferred embodiments, R2 is hydrogen. In certain preferred embodiments, R2 is OH.
In certain preferred embodiments, the compound is:
In certain preferred embodiments, the compound is:
It should be noted that, although the disclosed screening methods are especially suited for bitter compounds, they can be adopted to effectively screen non-bitter compounds. Additionally, bitter compounds may be of a variety of chemical structures and may be naturally occurring or synthetic. Exemplary bitter compounds include those found at: http://en.wikipedia.org/wiki/Category:Bitter_compounds (accessed on Jul. 19, 2012) and derivatives and analogs thereof. For example:
Aloin
Alpha acid
Amarogentin
Andrographolide
Bitrex
Brucine
Caffeine
Denatonium
Eugenin
Hesperidin
Humulone
Isohumulone
Kaempferol 3-O-rutinoside
6-Methoxymellein
Naringin
Papaverine
Phenylthiocarbamide
Propylthiouracil
Exemplary pharmaceutical compositions of the invention include: lung aerosol compositions to prevent, treat or manage asthma, chronic obstructive pulmonary disease, cystic fibrosis; topical preparations to prevent, treat or manage fecal incontinence, haemorrhoids and anal fissure; compositions suitable for oral administration to prevent, treat or manage fecal incontinence and constipation; compositions suitable for oral administration or injection to prevent, treat or manage pulmonary hypertension; compositions suitable for oral administration or injection to prevent, treat or manage urethral obstruction.
Ca2+ response to bitter tastants in resting cells was examined. In contrast to the marked increase in global [Ca2+]i reported in resting cultured human ASM cells (Deshpande, et al. 2010 Nat Med 16:1299-1304), we observed, in resting native ASM from mouse, that chloroquine (0.1 mM) only modestly raised global [Ca2+]i (and to a level much lower than when cells contracted after application of Mch at 0.01 μM-100 μM) (
To examine whether this modest increase in [Ca2+]i is sufficient to trigger contraction, smooth muscle force formation in mouse airways was measured. As shown in
Mouse ASM cells exhibit spontaneous Ca2+ sparks resulting from the opening of ryanodine receptors in the sarcoplasmic reticulum (Zhuge, et al. 2010 J Biol Chem 285:2203-2210). To test whether bitter tastants generate local Ca2+ events as proposed by others (Deshpande, et al. 2010 Nat Med 16:1299-1304), ASM cells were stimulated with chloroquine (10 μM, a concentration around EC50) for 2 mins and measured Ca2+ sparks. Off 40 chloroquine-stimulated cells, 27 cells generated a global [Ca2+]i increase that precluded an accurate estimate of Ca2+ sparks. In the remaining 13 cells without a detectable global rise in [Ca2]i, chloroquine inhibited the spark frequency but had no effect on the amplitude (Frequency (Hz): 2.13±0.24 in control and 1.62±0.21 with chloroquine (n=13, P<0.05, paired student's t-test); Amplitude (ΔF/F0 at the brightest location): 20.6±1.69 in control and 18.1±1.3 with chloroquine (n=13, P>0.05, paired student's t-test)). To test whether spontaneous Ca2+ sparks mask the effect of bitter tastants on other forms of local Ca2+ releases, such as Ca2+ puffs due to the opening of IP3Rs (Smith, et al. 2009 Proceedings the Nat Academy of Sci 106:6404-6409), the Ca2+ responses to chloroquine in ASM cells pretreated with 100 μM ryanodine was examined. In these cells, prior to chloroquine application, no spontaneous sparks were observed (n=14). Chloroquine (10 μM) increased global [Ca2+] by 12±4% (ΔF/F0 at its brightest location) in 9 cells, and failed to cause any detectable Ca2+ increase in 5 cells. There were no detectable local Ca2+ events produced in any of the 14 cells. These results indicate that chloroquine at 10 μM does not increase local Ca2+ events (either Ca2+ puffs or Ca2+ sparks).
Next examined was the cause of the modest global [Ca2+]i rise by bitter tastants. Since in taste cells, bitter tastants bind to TAS2R to activate the pertussis toxin (PTX) sensitive G-protein gustducin, which in turn induces a PLCβ2 and IP3 signaling cascade (Hoon, et al. 1995 Biochem J 309 (Pt 2):629-636; Spielman, et al. 1996 Am J Physiol 270:C926-931), it was studied whether bitter tastants activate this TAS2R signaling pathway. In native ASM cells, PTX (1 μg/ml, and 6-8 hr pretreatment), reduced the chloroquine-induced increase in global [Ca2+]i to 21.1±8.6% of the control cells (n=20;
Bitter Tastant-Induced Bronchodilation is Due to Reversal of the Rise in Global [Ca2+]i Caused by Bronchoconstrictors
Bitter tastants at μM levels can modestly increase [Ca2+]i in resting cells, but this raises a conundrum as they also can fully relax airways precontracted by bronchoconstrictors. (Deshpande, et al. 2010 Nat Med 16:1299-1304; Zhang, et al. 2012 Nat Med 18:648-650.) In light of the fact that an increase in [Ca2+]i is the primary signal for contraction in all smooth muscle, we explored how bitter tastants affect [Ca2+]i evoked by bronchoconstrictors. To better quantify these effects, we measured ASM Ca2+ response and cell shortening at the same time. The cells were stimulated with methacholine (Mch), a stable analogue of acetylcholine that is the major neurotransmitter in parasympathetic nerves. As expected, Mch (100 μM) rapidly increased [Ca2+]i as fluo-3 fluorescence increased by 162±26% (ΔF/F0), and concurrently caused cell shortening by 49±8% (n=21;
The inverse relationship between changes in [Ca2+]I and the resulting cell length (i.e., lowering [Ca2+]I results in cell lengthening) in response to bitter tastants indicates that bitter tastants reduce [Ca2+]i, leading to bronchodilation. If this is the case, one would expect that bitter tastant-induced bronchodilation could be prevented if [Ca2+]i was clamped to a physiologically high level. To test this possibility, we used staphylococcal α-toxin (16,000 μ/ml) to make the ASM membrane permeable to ions such that the intracellular [Ca2+]i could be controlled at will. A major advantage of using this toxin is that it does not damage the cells; thus signaling processes such as the G-protein-coupled receptor mediated signaling remain intact. (Kitazawa, et al. 1989 J Biol Chem 264:5339-5342.) As shown in
Mch activates both the M3 muscarinic acetylcholine receptor (M3R)-Gq-PLCβ-IP3 pathway and the M2 muscarinic acetylcholine receptor (M2R)-Gi/o pathway to raise [Ca2+]i by releasing Ca2+ from internal stores and inducing Ca2+ influx from the extracellular space (Gosens, et al. 2006 Respiratory Research 7:73; Hirota, et al. 2003 British J Anaesthesia 90:671-675; Kajita, et al. 1993 Am J of Physiology 264:L496-503; Liu, et al. 2006 Am J Physiol Lung Cell Mol Physiol 291:L281-288). It has been suggested that Ca2+ release from the internal stores contributes to the early phase of Mch-induced contraction, and Ca2+ influx via L-type voltage-dependent Ca2+ channels (VDCCs) is largely required to sustain elevated [Ca2+]i and for contraction. Indeed, the sustained contraction by Mch in mouse ASM is largely dependent on Ca2+ influx (
To directly examine the inhibitory role of bitter tastants on L-type VDCCs, we studied the effect of bitter tastants on KCl-induced increases in [Ca2+]i and contraction, and on L-type VDCC currents using patch clamp recording. KCl is a desirable bronchoconstrictor for this line of experiments because most likely it does not involve complex signaling processes (as does Mch). To test this, we compared the contraction and [Ca2+]i response to KCl in the presence of extracellular Ca2+ and in its absence. In Ca2+ containing medium, KCl (60 mM) induced a prominent increase in [Ca2+]i (
Give the action of KCl as revealed in
To address the signaling basis underlying bitter tastant inhibition of L-type VDCCs, we studied the impact of perturbing TAS2R signaling on bitter tastant-induced reversal of the [Ca2+]i increase in response to KCl. Pretreatment with PTX at 1 μg/ml for 6-8 hours prevented chloroquine-induced reversal of the KCl-induced increase in [Ca2+]i as did gallein (20 μM) and Anti-βγ, a Gβγ blocking peptide (1 μM) (
When administered alone to ASM cells at rest, bitter tastants activate the canonical TAS2R signaling pathway to modestly raise [Ca2+]i (
Bitter Tastants Reverse Contractile Agonist-Induced Increase in [Ca2+]i in Isolated Smooth Muscle Cells from Mouse Internal Anal Sphincter
As shown in
Tests showed that bitter compounds including two bile acids (e.g., DCA and CDCA shown below) can fully relax smooth muscle from airways, internal anal sphincter, pulmonary artery, and urethra from mouse, and smooth muscle from airways and pulmonary artery from human (
Results also showed that some bitter compounds relax airway smooth muscle, yet contract internal anal sphincter smooth muscle (
Based on the molecular mechanisms proposed herein by which bitter tastants relax smooth muscle, a method was developed for screening agents for smooth muscle relaxants. Results showed that bitter tastants reverse bronchoconstrictor-induced increase in intracellular Ca concentration ([Ca2+]i) is the underlying signal for their relaxation. This phenomon can be demonstrated robustly and quantatively by simulateous measurement of [Ca2+]i and cell length change at single cell level. The screening method disclosed herein is well suited for identifying relaxants from among the many thousands of available bitter tastants.
Experimental protocols for animal research were approved by the Institutional Animal Care and Use Committees at the University of Massachusetts Medical School (protocol A-1473 to R.Z).
C57/BL6 mice from 7 to 12 weeks of age were anesthetized with intraperitoneally injected pentobarbitone (50 mg kg−1), and the trachea and mainstem bronchi were quickly removed and placed in a pre-chilled dissociation solution consisting of (in mM): 135 NaCl, 6 KCl, 5 MgCl2, 0.1 CaCl2, 0.2 EDTA, 10 HEPES, and 10 Glucose (pH 7.3). The tracheas and bronchi were dissected free from the surface of the connective tissue. The tissue was incubated in the dissociation medium containing papain 30 unit/ml, 1 mM DTT, and 0.5 mg/ml BSA, at 35° C. for 30 min, and then transferred to a dissociation medium containing 3 unit/ml collagenase F and 0.5 mg/mL BSA, and incubated at 35° C. for another 15 min to produce isolated ASM cells. Finally, the tissue was agitated with a fire polished wide-bore glass pipette to release the cells.
C57/BL6 mice at 7-12 weeks of age were sacrificed and the entire respiratory trees were rapidly removed and immersed in Krebs physiologic solution containing (in mM) 118.07 NaCl, 4.69 KCl, 2.52 CaCl2, 1.16 MgSO4, 1.01 NaH2PO4, 25 NaHCO3, and 11.10 glucose. Trachea and bronchi were isolated and cut into rings (4 mm in length). The rings were mounted on a wire myograph chamber (Danish Myo Technology, Aarhus, Denmark), and a PowerLab recording device (AD Instruments) was used to record isometric tension. The ring preparations with zero tension were immersed in 5 ml of Krebs physiologic solution, bubbled with 95% 02 and 5% CO2 at 37° C. The basal tones were set at the level of approximately 2 mN. The order and treatment time of agonists and antagonists are indicated in the figure captions.
Human lung tissue was obtained (with informed consent) from patients undergoing surgery (lobectomy) for lung cancer at the Department of Surgery and the Department of Pathology at the Univ. of Massachusetts Memorial Med Ctr (Worcester, Mass.). The tumors were identified as nonsmall cell carcinoma (adenocarcinoma, squamous cell carcinoma). Intrapulmonary airways were dissected out and cleaned free of the connective tissues. These airways were either cut into the rings (4 mM in length) for force measurements the same as for mouse airway tissues, or digested with the same enzymes, dissociation medium and isolation procedure as for single mouse ASM cells. The experimental protocols on human tissues were approved by the Committee for Protection of Human Subjects in Research at the University of Massachusetts Medical School (Protocol 13590 to R.Z).
Fluorescence images using fluo-3 as a calcium indicator were obtained using a custom-built wide-field digital imaging system. The camera was interfaced to a custom made inverted microscope, and the cells were imaged using either a 20× Nikon 1.3 NA for global [Ca2+] measurement or a 60× Nikon 1.4 NA oil for Ca2+ spark measurement. The 488 nm line of an Argon Ion laser provided fluorescence excitation, with a shutter to control exposure duration, and emission of the Ca2+ indicator was monitored at wavelengths >500 nm. The images were acquired at the speed of either 1 Hz for global [Ca2+] measurement or 50 Hz for Ca2+ spark measurement. Subsequent image processing and analysis was performed off line using a custom-designed software package, running on a Linux/PC workstation. [Ca2+]i was represented as ΔF/F0*100 with F calculated by integrating fluo-3 over entire cells for global [Ca2+], or just the value at the brightest pixel (i.e., epicenter pixel) for Ca2+ sparks.
Membrane currents were recorded with an EPC10 HEKA amplifier under perforated whole-cell patch recording configuration. The extracellular solution contained (in mM): NaCl 126, Tetraethylammonium Cl 10, BaCl2 2.2, MgCl2 1, Hepes 10, and glucose 5.6; pH adjusted to 7.4 with NaOH. The pipette solution contained (in mM): CsCl 139, MgCl2 1, Hepes 10, MgATP 3, Na2ATP 0.5; pH adjusted to 7.3 with KOH; amphotericin B was freshly made and added to the pipette solution at a final concentration of 200 μg/ml. Whole-cell Ba2+ currents were evoked by step depolarization with 300 ms duration every 10 s from a holding potential of −70 mV at a 10 mV increment. Currents were leak corrected using a P/4 protocol.
Myocytes were placed into a recording chamber superfused with the bath solution for patch clamp experiments at room temperature. Cells loaded with Fluo-3 were imaged using a custom-built wide-field digital imaging system and their lengths were determined using custom software to manually trace down the center of the cell.
RT-PCR to Detect mRNA
The connective tissues in trachea and mainstem bronchi were carefully removed and the ASM were then quickly frozen in dry ice. The total RNA of the ASM was isolated with the TRIzol™ (Invitrogen) method following the manufacturer's guidelines; and cDNA was synthesized using extracted RNA with an Omniscript Reverse Transcription Kit (Qiagen). The specific primers, synthesized by Invitrogen, are listed in Table 1. β-actin was used as a positive control and the absence of DNA as a negative control, and the PCR reaction was carried out in a PCR mastercycler.
Reagents and their Application
All chemicals, except fluo-3 (Invitrogen Co, San Diego, Calif., USA), gallein (Tocris Bioscience, Bristol, United Kingdom), and Anti-βγ blocking peptide (AnaSpec, Fremont, Calif., USA), were purchased from Sigma-Aldrich Co. (St. Louis, Mo., USA). For single cell studies, agonists and antagonists were applied locally to cells via a picospritzer at a constant pressure, so that the duration of its action and concentration could be controlled easily.
Unless stated otherwise, data are reported as mean±s.e.m and n means numbers of cells or trachea and mainstem bronchi. Statistical analysis of differences was made with Student's paired or unpaired t-test and the significance level was set at p<0.05.
In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference, unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed.
References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.
The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples and the references to the scientific and patent literature included herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.
This application claims the benefit of priority from U.S. Provisional Application Ser. No. 61/677,674, filed on Jul. 31, 2012, the entire content of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US13/51543 | 7/22/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61677674 | Jul 2012 | US |
