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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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GFP+ Merkel cells but not keratinocytes show cytoplasmic [Ca2+] increases in response to hypotonic solutions.(A) An epiflourescence micrograph shows GFP+ Merkel cells after two days in culture (scale bar: 20 µm). (B, C) Pseudocolor images of fura-2 fluorescence ratios (F340/F380) of cells just before perfusion (B) with a 20% hypotonic solution (232 mmol·kg−1), and 26 s after perfusion onset (C). Pseudocolor scale bar represents F340/F380 (range 0.3–3.0). (D) Plot of [Ca2+] in a keratinocyte (denoted by arrowhead in C, black) and a Merkel cell (arrow in C, green). (E) Plot of cytoplasmic [Ca2+] versus time for three representative Merkel cells perfused with bath solutions of decreasing osmolality. (F) Quantification of the proportion of total Merkel cells (N = 104) that responded to hypotonic solutions (see Methods, 10%: 263 mmol·kg−1; 20%: 232 mmol·kg−1: 30%: 203 mmol·kg−1).
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pone-0001750-g001: GFP+ Merkel cells but not keratinocytes show cytoplasmic [Ca2+] increases in response to hypotonic solutions.(A) An epiflourescence micrograph shows GFP+ Merkel cells after two days in culture (scale bar: 20 µm). (B, C) Pseudocolor images of fura-2 fluorescence ratios (F340/F380) of cells just before perfusion (B) with a 20% hypotonic solution (232 mmol·kg−1), and 26 s after perfusion onset (C). Pseudocolor scale bar represents F340/F380 (range 0.3–3.0). (D) Plot of [Ca2+] in a keratinocyte (denoted by arrowhead in C, black) and a Merkel cell (arrow in C, green). (E) Plot of cytoplasmic [Ca2+] versus time for three representative Merkel cells perfused with bath solutions of decreasing osmolality. (F) Quantification of the proportion of total Merkel cells (N = 104) that responded to hypotonic solutions (see Methods, 10%: 263 mmol·kg−1; 20%: 232 mmol·kg−1: 30%: 203 mmol·kg−1).

Mentions: In cells lacking cell walls, hypotonic extracellular solutions cause water flux across the cell membrane to induce cell swelling. If Merkel cells express ion channels activated by membrane tension, we reasoned that cell swelling might increase membrane tension to open such channels. The resulting membrane depolarization could then activate VACCs to allow Ca2+ influx. To determine if Merkel cells respond to changes in osmolality, we monitored intracellular Ca2+ with the ratiometric, fluorescent indicator fura-2. In epidermal-cell suspensions, Merkel cells represent ≈0.2% of dissociated cells. Using FACS we enriched GFP+ Merkel cells to approximately 85% percent: the remaining 15% consisted predominately of GFP-negative keratinocytes. Cells were subjected to Ringer's solutions of varying osmolality. Most Merkel cells showed an increase in free [Ca2+] in response to 20% hypotonic stimuli (65±3% cells, N = 19 experiments, 10–33 cells/experiment, Fig. 1A–C). In responding Merkel cells, the peak cytoplasmic Ca2+ transients ranged from threshold to 4 µM above resting Ca2+ levels (0.51±0.06 µM, mean±SEM, N = 19 experiments, 47–90% of cells responded per experiment). By contrast, Merkel cells showed no changes in cytoplasmic [Ca2+] in 30% hypertonic solutions (N = 35 cells). Keratinocytes showed no change in free [Ca2+] to osmotic strength changes (N = 30 cells, Fig. 1A–C, arrow head). Our results indicate that Merkel cells, but not keratinocytes, respond to hypotonic solutions with increased cytoplasmic [Ca2+].


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

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

GFP+ Merkel cells but not keratinocytes show cytoplasmic [Ca2+] increases in response to hypotonic solutions.(A) An epiflourescence micrograph shows GFP+ Merkel cells after two days in culture (scale bar: 20 µm). (B, C) Pseudocolor images of fura-2 fluorescence ratios (F340/F380) of cells just before perfusion (B) with a 20% hypotonic solution (232 mmol·kg−1), and 26 s after perfusion onset (C). Pseudocolor scale bar represents F340/F380 (range 0.3–3.0). (D) Plot of [Ca2+] in a keratinocyte (denoted by arrowhead in C, black) and a Merkel cell (arrow in C, green). (E) Plot of cytoplasmic [Ca2+] versus time for three representative Merkel cells perfused with bath solutions of decreasing osmolality. (F) Quantification of the proportion of total Merkel cells (N = 104) that responded to hypotonic solutions (see Methods, 10%: 263 mmol·kg−1; 20%: 232 mmol·kg−1: 30%: 203 mmol·kg−1).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2365925&req=5

pone-0001750-g001: GFP+ Merkel cells but not keratinocytes show cytoplasmic [Ca2+] increases in response to hypotonic solutions.(A) An epiflourescence micrograph shows GFP+ Merkel cells after two days in culture (scale bar: 20 µm). (B, C) Pseudocolor images of fura-2 fluorescence ratios (F340/F380) of cells just before perfusion (B) with a 20% hypotonic solution (232 mmol·kg−1), and 26 s after perfusion onset (C). Pseudocolor scale bar represents F340/F380 (range 0.3–3.0). (D) Plot of [Ca2+] in a keratinocyte (denoted by arrowhead in C, black) and a Merkel cell (arrow in C, green). (E) Plot of cytoplasmic [Ca2+] versus time for three representative Merkel cells perfused with bath solutions of decreasing osmolality. (F) Quantification of the proportion of total Merkel cells (N = 104) that responded to hypotonic solutions (see Methods, 10%: 263 mmol·kg−1; 20%: 232 mmol·kg−1: 30%: 203 mmol·kg−1).
Mentions: In cells lacking cell walls, hypotonic extracellular solutions cause water flux across the cell membrane to induce cell swelling. If Merkel cells express ion channels activated by membrane tension, we reasoned that cell swelling might increase membrane tension to open such channels. The resulting membrane depolarization could then activate VACCs to allow Ca2+ influx. To determine if Merkel cells respond to changes in osmolality, we monitored intracellular Ca2+ with the ratiometric, fluorescent indicator fura-2. In epidermal-cell suspensions, Merkel cells represent ≈0.2% of dissociated cells. Using FACS we enriched GFP+ Merkel cells to approximately 85% percent: the remaining 15% consisted predominately of GFP-negative keratinocytes. Cells were subjected to Ringer's solutions of varying osmolality. Most Merkel cells showed an increase in free [Ca2+] in response to 20% hypotonic stimuli (65±3% cells, N = 19 experiments, 10–33 cells/experiment, Fig. 1A–C). In responding Merkel cells, the peak cytoplasmic Ca2+ transients ranged from threshold to 4 µM above resting Ca2+ levels (0.51±0.06 µM, mean±SEM, N = 19 experiments, 47–90% of cells responded per experiment). By contrast, Merkel cells showed no changes in cytoplasmic [Ca2+] in 30% hypertonic solutions (N = 35 cells). Keratinocytes showed no change in free [Ca2+] to osmotic strength changes (N = 30 cells, Fig. 1A–C, arrow head). Our results indicate that Merkel cells, but not keratinocytes, respond to hypotonic solutions with increased cytoplasmic [Ca2+].

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