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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 display increased [Ca2+] near the plasmalemma in response to hypotonic stimuli.(A, B) Pseudocolor images of fura-2 fluorescence ratios in a Merkel cell before (A) and 16 s after (B) perfusion onset with a 20% hypotonic solution (232 mmol·kg−1). Pseudocolor scale bar shows F340/F380 and applies to (A–C). (C) Pseudocolor kymograph of the line shown in (A) and (B). Each pixel along the ordinate corresponds to a point on the line shown in (A) and (B). Pixels near the top and bottom of the kymograph show F340/F380 near the plasmalemma, whereas pixels in the middle correspond to the interior of the process. Time proceeds along the abscissa. The time points of (A) and (B) are indicated by arrows pointing to the kymograph in (C). Black lines denote the beginning and end of hypotonic perfusion. (D) Plot of [Ca2+] versus time for three positions along the cell process. Colored lines indicate the average of the corresponding colored box in (C). Red and green traces signify regions near the plasmalemma, whereas blue represents a region deeper in the cytoplasm.
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pone-0001750-g003: Merkel cells display increased [Ca2+] near the plasmalemma in response to hypotonic stimuli.(A, B) Pseudocolor images of fura-2 fluorescence ratios in a Merkel cell before (A) and 16 s after (B) perfusion onset with a 20% hypotonic solution (232 mmol·kg−1). Pseudocolor scale bar shows F340/F380 and applies to (A–C). (C) Pseudocolor kymograph of the line shown in (A) and (B). Each pixel along the ordinate corresponds to a point on the line shown in (A) and (B). Pixels near the top and bottom of the kymograph show F340/F380 near the plasmalemma, whereas pixels in the middle correspond to the interior of the process. Time proceeds along the abscissa. The time points of (A) and (B) are indicated by arrows pointing to the kymograph in (C). Black lines denote the beginning and end of hypotonic perfusion. (D) Plot of [Ca2+] versus time for three positions along the cell process. Colored lines indicate the average of the corresponding colored box in (C). Red and green traces signify regions near the plasmalemma, whereas blue represents a region deeper in the cytoplasm.

Mentions: Mechanosensitive ion channels are often located within specialized cellular processes that are thought to leverage forces to the channels. Hair cells have mechanoelectrical transduction channels near the tips of modified microvilli called stereocilia [29], and kidney cells detect fluid flow with mechanosensitive channels located in cilia [30]. Similarly, Merkel cells in vivo have actin-filled processes that penetrate overlying keratinocytes [5], [31]. To determine if Merkel cells extend cytoplasmic processes in vitro, we stained Merkel cells with fluorescent sphingolipids to visualize membrane morphology (Fig. 2C). We found that most cultured Merkel cells have processes arranged in a branch-like pattern, with numerous fine protrusions (1–8 µm in length) at the terminals of large processes (2–15 µm in length). Although the larger cytoplasmic processes were visible in fura-2 fluorescence images, fine protrusions were not (Figs. 1, 3).


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 display increased [Ca2+] near the plasmalemma in response to hypotonic stimuli.(A, B) Pseudocolor images of fura-2 fluorescence ratios in a Merkel cell before (A) and 16 s after (B) perfusion onset with a 20% hypotonic solution (232 mmol·kg−1). Pseudocolor scale bar shows F340/F380 and applies to (A–C). (C) Pseudocolor kymograph of the line shown in (A) and (B). Each pixel along the ordinate corresponds to a point on the line shown in (A) and (B). Pixels near the top and bottom of the kymograph show F340/F380 near the plasmalemma, whereas pixels in the middle correspond to the interior of the process. Time proceeds along the abscissa. The time points of (A) and (B) are indicated by arrows pointing to the kymograph in (C). Black lines denote the beginning and end of hypotonic perfusion. (D) Plot of [Ca2+] versus time for three positions along the cell process. Colored lines indicate the average of the corresponding colored box in (C). Red and green traces signify regions near the plasmalemma, whereas blue represents a region deeper in the cytoplasm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2365925&req=5

pone-0001750-g003: Merkel cells display increased [Ca2+] near the plasmalemma in response to hypotonic stimuli.(A, B) Pseudocolor images of fura-2 fluorescence ratios in a Merkel cell before (A) and 16 s after (B) perfusion onset with a 20% hypotonic solution (232 mmol·kg−1). Pseudocolor scale bar shows F340/F380 and applies to (A–C). (C) Pseudocolor kymograph of the line shown in (A) and (B). Each pixel along the ordinate corresponds to a point on the line shown in (A) and (B). Pixels near the top and bottom of the kymograph show F340/F380 near the plasmalemma, whereas pixels in the middle correspond to the interior of the process. Time proceeds along the abscissa. The time points of (A) and (B) are indicated by arrows pointing to the kymograph in (C). Black lines denote the beginning and end of hypotonic perfusion. (D) Plot of [Ca2+] versus time for three positions along the cell process. Colored lines indicate the average of the corresponding colored box in (C). Red and green traces signify regions near the plasmalemma, whereas blue represents a region deeper in the cytoplasm.
Mentions: Mechanosensitive ion channels are often located within specialized cellular processes that are thought to leverage forces to the channels. Hair cells have mechanoelectrical transduction channels near the tips of modified microvilli called stereocilia [29], and kidney cells detect fluid flow with mechanosensitive channels located in cilia [30]. Similarly, Merkel cells in vivo have actin-filled processes that penetrate overlying keratinocytes [5], [31]. To determine if Merkel cells extend cytoplasmic processes in vitro, we stained Merkel cells with fluorescent sphingolipids to visualize membrane morphology (Fig. 2C). We found that most cultured Merkel cells have processes arranged in a branch-like pattern, with numerous fine protrusions (1–8 µm in length) at the terminals of large processes (2–15 µm in length). Although the larger cytoplasmic processes were visible in fura-2 fluorescence images, fine protrusions were not (Figs. 1, 3).

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