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

Deshpande DA, Wang WC, McIlmoyle EL, Robinett KS, Schillinger RM, An SS, Sham JS, Liggett SB - Nat. Med. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
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 (IP₃) 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.

Show MeSH

Related in: MedlinePlus

Saccharin preferentially triggers localized [Ca2+]i responses in ASM cells. (a,c) Sequential confocal images of Fluo-3 loaded cells shows activation of localized [Ca2+]i increases in the cell periphery upon exposure of ASM cells to 0.3 mM saccharin, and a generalized increase in [Ca2+]i with exposure to 1.0 µM histamine. The images are Fluo-3 fluorescence after background subtraction and baseline normalized (F/F0) with intensity encoded by pseudo-color. The arrows highlight local [Ca2+]i “hot-spots”. (b,d) Local [Ca2+]i transients measured in regions of interest (ROI). Saccharin activated a rapid rise of Ca2+ in the peripheral end (ROI 1), but a smaller and gradual increase of [Ca2+]i in the central regions (ROI 2,3) of the cell. The histamine response (ROI 4–7) was asynchronous and was observed throughout the cells. (e) Confocal linescan imaging shows spatially and temporally resolved local [Ca2+]i events activated by saccharin in a peripheral site. The scan line (white dashed line) was placed within 1 µm parallel to the cell membrane at one end of an elongated ASM cell as shown in the left panel. Arrows indicate several local [Ca2+]i events that occur prior to the more defined increase within the isolated region. The bottom panel is the spatially averaged normalized fluorescence signal (F/F0) generated from the linescan. Results are from single experiments representative of five performed.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3066567&req=5

Figure 4: Saccharin preferentially triggers localized [Ca2+]i responses in ASM cells. (a,c) Sequential confocal images of Fluo-3 loaded cells shows activation of localized [Ca2+]i increases in the cell periphery upon exposure of ASM cells to 0.3 mM saccharin, and a generalized increase in [Ca2+]i with exposure to 1.0 µM histamine. The images are Fluo-3 fluorescence after background subtraction and baseline normalized (F/F0) with intensity encoded by pseudo-color. The arrows highlight local [Ca2+]i “hot-spots”. (b,d) Local [Ca2+]i transients measured in regions of interest (ROI). Saccharin activated a rapid rise of Ca2+ in the peripheral end (ROI 1), but a smaller and gradual increase of [Ca2+]i in the central regions (ROI 2,3) of the cell. The histamine response (ROI 4–7) was asynchronous and was observed throughout the cells. (e) Confocal linescan imaging shows spatially and temporally resolved local [Ca2+]i events activated by saccharin in a peripheral site. The scan line (white dashed line) was placed within 1 µm parallel to the cell membrane at one end of an elongated ASM cell as shown in the left panel. Arrows indicate several local [Ca2+]i events that occur prior to the more defined increase within the isolated region. The bottom panel is the spatially averaged normalized fluorescence signal (F/F0) generated from the linescan. Results are from single experiments representative of five performed.

Mentions: To further ascertain the mechanism by which bitter taste receptors evoke ASM relaxation, we used magnetic twisting cytometry15 to measure dynamic changes in stiffness of isolated human ASM cells (Fig. 3), thus removing any potential confoundment from unrecognized mechanisms present in intact tissue. In these experiments magnetic particles attached to the cell by a peptide linker provide a highly quantitative measurement of single-cell stiffness, with isoproterenol and histamine exposure resulting in the expected relaxation and contraction from baseline, respectively (Fig. 3a). Chloroquine and saccharin exposure (Fig. 3a) resulted in ASM relaxation at this single cell level, confirming that these bitter tastants act directly on smooth muscle cells, which is consistent with our findings in the coordinated relaxation response of intact airways. The relaxation response to saccharin was not blocked by the PKA inhibitor H89 (Fig. 3b) confirming results from cAMP and VASP phosphorylation measurements. Inhibition of PLC by U73122 eliminated the saccharin-promoted relaxation of isolated ASM (Fig. 3b). In light of our findings with PLC inhibition (as well as βγ inhibitors and IP3 receptor antagonists) on bitter tastant-promoted increases in [Ca2+]i (Fig. 1d), these results indicated that the relaxation response of these receptors in ASM is triggered by, or requires, intracellular Ca2+ release. Consistent with this concept, the chloroquine EC50 values for [Ca2+]i release in cultured ASM cells (70 ± 10 µM, Fig. 1a) and in the relaxation of intact airways (93 ± 4.3 µM, Fig. 2a) are virtually identical. The dependence of relaxation on SR Ca2+ release was further supported by results of studies with the SR Ca2+ re-uptake inhibitor thapsigargin, which depletes the SR of [Ca2+]i. Thapsigargin pre-incubation blocked chloroquine and other bitter tastant-mediated [Ca2+]i stimulation in ASM cells (Supplementary Fig. 7), and also chloroquine-mediated relaxation of intact airway rings (Supplementary Fig. 8). We considered a potential mechanism for Ca2+-mediated relaxation in ASM was via hyperpolarization due to stimulation of the large conductance Ca2+-activated K+-channel (BKCa). These channels are known to be expressed on human ASM16 (which we confirmed; Supplementary Fig. 1) and have been reported to regulate airway tone17. To test whether the observed relaxation is due to bitter tastant-triggered Ca2+ activation of BKCa, human ASM cells were pretreated with carrier, a Ca2+-dependent K+ channel antagonist charybdotoxin, or the specific BKCa channel antagonist iberiotoxin. As shown in Fig. 3b, both pretreatments ablated saccharin-mediated ASM relaxation as assessed in isolated cells. Similar results were found with chloroquine (data not shown). And finally, pretreatment with iberiotoxin also attenuated chloroquine-promoted relaxation in the isolated mouse airway (Fig. 3c). The relaxation of ASM from BKCa activation would be expected to be from membrane hyperpolarization18. ASM cells were loaded with a membrane potential-sensitive fluorescent dye19, and as shown in Fig. 3d, exposure to KCl and histamine resulted in the expected membrane depolarization. With exposure to the bitter tastants chloroquine and saccharin, membrane hyperpolarization was readily observed. Furthermore, bitter tastant-promoted membrane hyperpolarization was completely inhibited by iberiotoxin (Fig. 3e). Thus the highly efficacious bronchodilator response from ASM bitter taste receptors appears to be due to [Ca2+]i-dependent activation of BKCa, which is distinct from histamine promoted increases in [Ca2+]i which causes contraction. This suggested that the intracellular distribution of the Ca2+ responses to histamine and bitter tastants are different, and indeed, it is established that a high concentration of localized [Ca2+]i is associated with BKCa activation20. To define the characteristics of saccharin-promoted [Ca2+]i increases in ASM cells, real-time confocal imaging was performed in Fluo-3 loaded cells (Fig. 4a,c). As shown, localized [Ca2+]i signals were detected at the slender ends and sarcolemmal regions of ASM. This response was rapid (e.g., observed within 2.5 sec in the cross-sectional studies, Fig. 4a) and the magnitude was greater than at the central region of the myocytes (Fig. 4b). When using the line-scan mode at regions within 1 µm and parallel to the cell membrane of ASM cells, spatially and temporally discernible [Ca2+]i events were detected very early after the application of saccharin, prior to the subsequent sustained localized rise in [Ca2+]i (Fig. 4e). These results confirm the notion that saccharin promotes localized [Ca2+]i signals in ASM cells. In contrast, the response to histamine in ASM cells caused a rapid rise in [Ca2+]i throughout the cell (Fig. 4c,d), without the localized features observed with saccharin.


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

Deshpande DA, Wang WC, McIlmoyle EL, Robinett KS, Schillinger RM, An SS, Sham JS, Liggett SB - Nat. Med. (2010)

Saccharin preferentially triggers localized [Ca2+]i responses in ASM cells. (a,c) Sequential confocal images of Fluo-3 loaded cells shows activation of localized [Ca2+]i increases in the cell periphery upon exposure of ASM cells to 0.3 mM saccharin, and a generalized increase in [Ca2+]i with exposure to 1.0 µM histamine. The images are Fluo-3 fluorescence after background subtraction and baseline normalized (F/F0) with intensity encoded by pseudo-color. The arrows highlight local [Ca2+]i “hot-spots”. (b,d) Local [Ca2+]i transients measured in regions of interest (ROI). Saccharin activated a rapid rise of Ca2+ in the peripheral end (ROI 1), but a smaller and gradual increase of [Ca2+]i in the central regions (ROI 2,3) of the cell. The histamine response (ROI 4–7) was asynchronous and was observed throughout the cells. (e) Confocal linescan imaging shows spatially and temporally resolved local [Ca2+]i events activated by saccharin in a peripheral site. The scan line (white dashed line) was placed within 1 µm parallel to the cell membrane at one end of an elongated ASM cell as shown in the left panel. Arrows indicate several local [Ca2+]i events that occur prior to the more defined increase within the isolated region. The bottom panel is the spatially averaged normalized fluorescence signal (F/F0) generated from the linescan. Results are from single experiments representative of five performed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3066567&req=5

Figure 4: Saccharin preferentially triggers localized [Ca2+]i responses in ASM cells. (a,c) Sequential confocal images of Fluo-3 loaded cells shows activation of localized [Ca2+]i increases in the cell periphery upon exposure of ASM cells to 0.3 mM saccharin, and a generalized increase in [Ca2+]i with exposure to 1.0 µM histamine. The images are Fluo-3 fluorescence after background subtraction and baseline normalized (F/F0) with intensity encoded by pseudo-color. The arrows highlight local [Ca2+]i “hot-spots”. (b,d) Local [Ca2+]i transients measured in regions of interest (ROI). Saccharin activated a rapid rise of Ca2+ in the peripheral end (ROI 1), but a smaller and gradual increase of [Ca2+]i in the central regions (ROI 2,3) of the cell. The histamine response (ROI 4–7) was asynchronous and was observed throughout the cells. (e) Confocal linescan imaging shows spatially and temporally resolved local [Ca2+]i events activated by saccharin in a peripheral site. The scan line (white dashed line) was placed within 1 µm parallel to the cell membrane at one end of an elongated ASM cell as shown in the left panel. Arrows indicate several local [Ca2+]i events that occur prior to the more defined increase within the isolated region. The bottom panel is the spatially averaged normalized fluorescence signal (F/F0) generated from the linescan. Results are from single experiments representative of five performed.
Mentions: To further ascertain the mechanism by which bitter taste receptors evoke ASM relaxation, we used magnetic twisting cytometry15 to measure dynamic changes in stiffness of isolated human ASM cells (Fig. 3), thus removing any potential confoundment from unrecognized mechanisms present in intact tissue. In these experiments magnetic particles attached to the cell by a peptide linker provide a highly quantitative measurement of single-cell stiffness, with isoproterenol and histamine exposure resulting in the expected relaxation and contraction from baseline, respectively (Fig. 3a). Chloroquine and saccharin exposure (Fig. 3a) resulted in ASM relaxation at this single cell level, confirming that these bitter tastants act directly on smooth muscle cells, which is consistent with our findings in the coordinated relaxation response of intact airways. The relaxation response to saccharin was not blocked by the PKA inhibitor H89 (Fig. 3b) confirming results from cAMP and VASP phosphorylation measurements. Inhibition of PLC by U73122 eliminated the saccharin-promoted relaxation of isolated ASM (Fig. 3b). In light of our findings with PLC inhibition (as well as βγ inhibitors and IP3 receptor antagonists) on bitter tastant-promoted increases in [Ca2+]i (Fig. 1d), these results indicated that the relaxation response of these receptors in ASM is triggered by, or requires, intracellular Ca2+ release. Consistent with this concept, the chloroquine EC50 values for [Ca2+]i release in cultured ASM cells (70 ± 10 µM, Fig. 1a) and in the relaxation of intact airways (93 ± 4.3 µM, Fig. 2a) are virtually identical. The dependence of relaxation on SR Ca2+ release was further supported by results of studies with the SR Ca2+ re-uptake inhibitor thapsigargin, which depletes the SR of [Ca2+]i. Thapsigargin pre-incubation blocked chloroquine and other bitter tastant-mediated [Ca2+]i stimulation in ASM cells (Supplementary Fig. 7), and also chloroquine-mediated relaxation of intact airway rings (Supplementary Fig. 8). We considered a potential mechanism for Ca2+-mediated relaxation in ASM was via hyperpolarization due to stimulation of the large conductance Ca2+-activated K+-channel (BKCa). These channels are known to be expressed on human ASM16 (which we confirmed; Supplementary Fig. 1) and have been reported to regulate airway tone17. To test whether the observed relaxation is due to bitter tastant-triggered Ca2+ activation of BKCa, human ASM cells were pretreated with carrier, a Ca2+-dependent K+ channel antagonist charybdotoxin, or the specific BKCa channel antagonist iberiotoxin. As shown in Fig. 3b, both pretreatments ablated saccharin-mediated ASM relaxation as assessed in isolated cells. Similar results were found with chloroquine (data not shown). And finally, pretreatment with iberiotoxin also attenuated chloroquine-promoted relaxation in the isolated mouse airway (Fig. 3c). The relaxation of ASM from BKCa activation would be expected to be from membrane hyperpolarization18. ASM cells were loaded with a membrane potential-sensitive fluorescent dye19, and as shown in Fig. 3d, exposure to KCl and histamine resulted in the expected membrane depolarization. With exposure to the bitter tastants chloroquine and saccharin, membrane hyperpolarization was readily observed. Furthermore, bitter tastant-promoted membrane hyperpolarization was completely inhibited by iberiotoxin (Fig. 3e). Thus the highly efficacious bronchodilator response from ASM bitter taste receptors appears to be due to [Ca2+]i-dependent activation of BKCa, which is distinct from histamine promoted increases in [Ca2+]i which causes contraction. This suggested that the intracellular distribution of the Ca2+ responses to histamine and bitter tastants are different, and indeed, it is established that a high concentration of localized [Ca2+]i is associated with BKCa activation20. To define the characteristics of saccharin-promoted [Ca2+]i increases in ASM cells, real-time confocal imaging was performed in Fluo-3 loaded cells (Fig. 4a,c). As shown, localized [Ca2+]i signals were detected at the slender ends and sarcolemmal regions of ASM. This response was rapid (e.g., observed within 2.5 sec in the cross-sectional studies, Fig. 4a) and the magnitude was greater than at the central region of the myocytes (Fig. 4b). When using the line-scan mode at regions within 1 µm and parallel to the cell membrane of ASM cells, spatially and temporally discernible [Ca2+]i events were detected very early after the application of saccharin, prior to the subsequent sustained localized rise in [Ca2+]i (Fig. 4e). These results confirm the notion that saccharin promotes localized [Ca2+]i signals in ASM cells. In contrast, the response to histamine in ASM cells caused a rapid rise in [Ca2+]i throughout the cell (Fig. 4c,d), without the localized features observed with saccharin.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA.

ABSTRACT
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 (IP₃) 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.

Show MeSH
Related in: MedlinePlus