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Swelling-activated Ca2+ channels trigger Ca2+ signals in Merkel cells.

Haeberle H, Bryan LA, Vadakkan TJ, Dickinson ME, Lumpkin EA - PLoS ONE (2008)

Bottom Line: Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results.Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs.We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Graduate Program, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute to touch reception in the Merkel cell-neurite complex.

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Merkel cells swell in hypotonic solutions. Shape changes were monitored with plasmalemma-bound fluorescent microspheres using confocal microscopy.(A) A three-dimensional plot of fluorescent microspheres (black spots) coating the surfaces of a representative Merkel cell and the coverslip. The surface of the Merkel cell was reconstructed from the location of the micropheres (topographic scalebar: navy blue denotes the coverslip surface; dark red = 6 µm above the coverslip). Areas of low bead density are keratinocytes, which are poorly bound by the beads. (B) Plot of cell volume versus time for the cell shown in (A). The 20% hypotonic solution was administered at t = 0. Double slash marks denote a transient focal-plane change during the solution change. (C) Projections of confocal z-series of cells membranes stained with fluorescent sphingolipids. Processes 1–8 µm in length jutted from Merkel cells' surfaces (arrowheads). By comparison, a keratinocyte showed a smooth cell surface (upper right).
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pone-0001750-g002: Merkel cells swell in hypotonic solutions. Shape changes were monitored with plasmalemma-bound fluorescent microspheres using confocal microscopy.(A) A three-dimensional plot of fluorescent microspheres (black spots) coating the surfaces of a representative Merkel cell and the coverslip. The surface of the Merkel cell was reconstructed from the location of the micropheres (topographic scalebar: navy blue denotes the coverslip surface; dark red = 6 µm above the coverslip). Areas of low bead density are keratinocytes, which are poorly bound by the beads. (B) Plot of cell volume versus time for the cell shown in (A). The 20% hypotonic solution was administered at t = 0. Double slash marks denote a transient focal-plane change during the solution change. (C) Projections of confocal z-series of cells membranes stained with fluorescent sphingolipids. Processes 1–8 µm in length jutted from Merkel cells' surfaces (arrowheads). By comparison, a keratinocyte showed a smooth cell surface (upper right).

Mentions: Hypotonic solutions induce cell swelling that activates stretch-sensitive channels in bacteria [28], so we asked whether similar hypotonic-induced swelling occurred in Merkel cells. To ascertain if hypotonic solutions altered Merkel-cell volume, we monitored cell shape in three dimensions with fluorescent microspheres attached to the plasmalemma of Merkel cells while perfusing cells with a 20% hypotonic bath solution. Microspheres settled onto Merkel cells and the surrounding coverslip within 30 min of bath application and remained tightly coupled during solution changes (Fig. 2A, movie S1). Microspheres were imaged with high-speed confocal microscopy, and their positions were used to model the location of Merkel-cell surfaces in relation to the coverslip. By integrating the volume between reconstructed cell surfaces and the coverslip, we estimated that Merkel cells' volume in isotonic Ringer's solution was 334±39 µm3 (mean±SD, N = 8). To determine if Merkel cells swelled in response to a hypotonic stimulus, we imaged Merkel cells in time series while perfusing with 20% hypotonic Ringer's solution (Fig. 2B). In this condition, Merkel-cell volume was significantly higher (358±43 µm3, mean±SD, N = 8, p<0.001, paired Student's t test), which represents an average volume increase of 7.3±2.9%. Merkel cells began swelling within 7 s of the onset of hypotonic perfusion, which was the temporal resolution of the three-dimensional imaging. Merkel cells remained enlarged during 120 s of hypotonic stimulation and relaxed to their original volume after perfusion of isotonic Ringer's solution. One Merkel cell subjected to prolonged hypotonic stimulation showed volume decreases in ∼300 s.


Swelling-activated Ca2+ channels trigger Ca2+ signals in Merkel cells.

Haeberle H, Bryan LA, Vadakkan TJ, Dickinson ME, Lumpkin EA - PLoS ONE (2008)

Merkel cells swell in hypotonic solutions. Shape changes were monitored with plasmalemma-bound fluorescent microspheres using confocal microscopy.(A) A three-dimensional plot of fluorescent microspheres (black spots) coating the surfaces of a representative Merkel cell and the coverslip. The surface of the Merkel cell was reconstructed from the location of the micropheres (topographic scalebar: navy blue denotes the coverslip surface; dark red = 6 µm above the coverslip). Areas of low bead density are keratinocytes, which are poorly bound by the beads. (B) Plot of cell volume versus time for the cell shown in (A). The 20% hypotonic solution was administered at t = 0. Double slash marks denote a transient focal-plane change during the solution change. (C) Projections of confocal z-series of cells membranes stained with fluorescent sphingolipids. Processes 1–8 µm in length jutted from Merkel cells' surfaces (arrowheads). By comparison, a keratinocyte showed a smooth cell surface (upper right).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2365925&req=5

pone-0001750-g002: Merkel cells swell in hypotonic solutions. Shape changes were monitored with plasmalemma-bound fluorescent microspheres using confocal microscopy.(A) A three-dimensional plot of fluorescent microspheres (black spots) coating the surfaces of a representative Merkel cell and the coverslip. The surface of the Merkel cell was reconstructed from the location of the micropheres (topographic scalebar: navy blue denotes the coverslip surface; dark red = 6 µm above the coverslip). Areas of low bead density are keratinocytes, which are poorly bound by the beads. (B) Plot of cell volume versus time for the cell shown in (A). The 20% hypotonic solution was administered at t = 0. Double slash marks denote a transient focal-plane change during the solution change. (C) Projections of confocal z-series of cells membranes stained with fluorescent sphingolipids. Processes 1–8 µm in length jutted from Merkel cells' surfaces (arrowheads). By comparison, a keratinocyte showed a smooth cell surface (upper right).
Mentions: Hypotonic solutions induce cell swelling that activates stretch-sensitive channels in bacteria [28], so we asked whether similar hypotonic-induced swelling occurred in Merkel cells. To ascertain if hypotonic solutions altered Merkel-cell volume, we monitored cell shape in three dimensions with fluorescent microspheres attached to the plasmalemma of Merkel cells while perfusing cells with a 20% hypotonic bath solution. Microspheres settled onto Merkel cells and the surrounding coverslip within 30 min of bath application and remained tightly coupled during solution changes (Fig. 2A, movie S1). Microspheres were imaged with high-speed confocal microscopy, and their positions were used to model the location of Merkel-cell surfaces in relation to the coverslip. By integrating the volume between reconstructed cell surfaces and the coverslip, we estimated that Merkel cells' volume in isotonic Ringer's solution was 334±39 µm3 (mean±SD, N = 8). To determine if Merkel cells swelled in response to a hypotonic stimulus, we imaged Merkel cells in time series while perfusing with 20% hypotonic Ringer's solution (Fig. 2B). In this condition, Merkel-cell volume was significantly higher (358±43 µm3, mean±SD, N = 8, p<0.001, paired Student's t test), which represents an average volume increase of 7.3±2.9%. Merkel cells began swelling within 7 s of the onset of hypotonic perfusion, which was the temporal resolution of the three-dimensional imaging. Merkel cells remained enlarged during 120 s of hypotonic stimulation and relaxed to their original volume after perfusion of isotonic Ringer's solution. One Merkel cell subjected to prolonged hypotonic stimulation showed volume decreases in ∼300 s.

Bottom Line: Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results.Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs.We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Graduate Program, University of California San Francisco, San Francisco, California, United States of America.

ABSTRACT
Merkel cell-neurite complexes are highly sensitive touch receptors comprising epidermal Merkel cells and sensory afferents. Based on morphological and molecular studies, Merkel cells are proposed to be mechanosensory cells that signal afferents via neurotransmission; however, functional studies testing this hypothesis in intact skin have produced conflicting results. To test this model in a simplified system, we asked whether purified Merkel cells are directly activated by mechanical stimulation. Cell shape was manipulated with anisotonic solution changes and responses were monitored by Ca2+ imaging with fura-2. We found that hypotonic-induced cell swelling, but not hypertonic solutions, triggered cytoplasmic Ca2+ transients. Several lines of evidence indicate that these signals arise from swelling-activated Ca2+-permeable ion channels. First, transients were reversibly abolished by chelating extracellular Ca2+, demonstrating a requirement for Ca2+ influx across the plasma membrane. Second, Ca2+ transients were initially observed near the plasma membrane in cytoplasmic processes. Third, voltage-activated Ca2+ channel (VACC) antagonists reduced transients by half, suggesting that swelling-activated channels depolarize plasma membranes to activate VACCs. Finally, emptying internal Ca2+ stores attenuated transients by 80%, suggesting Ca2+ release from stores augments swelling-activated Ca2+ signals. To identify candidate mechanotransduction channels, we used RT-PCR to amplify ion-channel transcripts whose pharmacological profiles matched those of hypotonic-evoked Ca2+ signals in Merkel cells. We found 11 amplicons, including PKD1, PKD2, and TRPC1, channels previously implicated in mechanotransduction in other cells. Collectively, these results directly demonstrate that Merkel cells are activated by hypotonic-evoked swelling, identify cellular signaling mechanisms that mediate these responses, and support the hypothesis that Merkel cells contribute to touch reception in the Merkel cell-neurite complex.

Show MeSH
Related in: MedlinePlus