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P2Y-mediated Ca²⁺ response is spatiotemporally graded and synchronized in sensory neurons: a two-photon photolysis study

SHINSUKE NAKAYAMA, TOSHIKAZU YAMASHITA^*, MASATO KONISHI^,1, HIROMI KAZAMA^* and SHINICHIRO KOKUBUN^*

^* Advanced Medical Research Center, School of Medicine, Nihon University, Tokyo;
Department of Cell Physiology, Nagoya University Graduate School of Medicine, Nagoya; and
Department of Physiology, Tokyo Medical University, Tokyo, Japan

¹Correspondence: Department of Physiology, Tokyo Medical University, Tokyo 160-8402, Japan. E-mail: mkonishi{at}tokyo-med.ac.jp

SPECIFIC AIMS

ATP is considered an initiator and modulator of noxious pain sensation. Recent studies have suggested that metabotropic and ionotropic purinoceptors play important roles. Intracellular Ca²⁺ ions trigger multiple cellular processes in DRG neurons, resulting in plastic changes of nociception through several potential mechanisms. We used the two-photon photolysis (TPP) technique to apply ATP locally and transiently, thus mimicking ATP release upon cell damage or exocytosis. Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in DRG neurons were simultaneously monitored using a confocal microscope system. Experiments were performed to assess possible mechanisms underlying spatiotemporal modulations of pain sensation via purinoceptors.

PRINCIPAL FINDINGS

1. Two-photon photolysis of caged ATP induces a [Ca²⁺]_i rise in DRG neurons
In cultured DRG neurons 9–18 µm in diameter, we applied ATP by use of the TPP of caged ATP and employed a confocal microscope system to collect [Ca²⁺]_i images sequentially. (Unless otherwise stated, "application of ATP" or "photolysis" represents use of this technique.)

Figure 1 A shows an example of a [Ca²⁺]_i response to application of ATP for 40 ms. Two cells are observed in transmission (Fig. 1A, a ) and [Ca²⁺]_i images (Fig. 1A, b-e ). The left graph and the [Ca²⁺]_i image (Fig. 1A, c ) clearly indicate that application of ATP for a short duration raised [Ca²⁺]_i in the lower DRG (red line) but did not in the other cell (blue line). Photolysis for a longer duration (100 ms) raised [Ca²⁺]_i in both DRG neurons (Fig. 1B ). In the present experiments, we therefore used short durations of the photolysis (40 ms) for local application of ATP and longer durations (100 ms) to apply ATP to multiple DRG neurons.

View larger version (19K): [in this window] [in a new window]   Figure 1. Effects of changing the TPP duration on [Ca2+]i in a pair of DRG neurons. A) A spot near the lower cell was illuminated with the Tsunami laser beam (pulse mode) for 40 ms. a) A transmission image with the TPP spot (red) and three ROIs (orange, green, and blue squares) used to estimate of [Ca2+]i are shown. [Ca2+]i images (b–e) were acquired just before and 4830, 8820, and 18900 ms after TPP, respectively. Right graph shows the time courses of the [Ca2+]i changes in the three regions of interest (ROIs) (a). Color of the ROI in panel a corresponds to that of the line in the right graph. The left graph (A) shows the time courses of changes in the mean [Ca2+]i of the soma of the two DRG neurons. Red and blue lines are for lower and upper neurons, respectively. The time of TPP is indicated by a red arrow. B) Records obtained by 100 ms TPP. a) A transmission image; b–g) [Ca2+]i images acquired just before and 1470, 2100, 3990, 9540, and 39480 ms after TPP. The graphs show time course of changes in the mean [Ca2+]i of the two neurons. Early changes in [Ca2+]i are expanded in the left graph.

In some DRG neurons with long neurites, photolysis of caged ATP for a short duration near a neurite (but >10 µm away from the soma) induced [Ca²⁺]_i rises only in the neurite; those for a long duration caused [Ca²⁺]_i rises in both soma and neurites.

2. [Ca²⁺]_i increases with a considerable latency in the majority of DRG neurons
When ATP was applied locally for 10–40 ms, the average times to peak amplitude of [Ca²⁺]_i (t_PCa) and to half-maximal amplitude (t_0.5PCa) were 3217 ± 2933 and 2216 ± 1463 ms, respectively (n=10). When longer durations of ATP application were used, t_PCa and t_0.5PCa were 2267 ± 889 and 1228 ± 610 ms, respectively (n=10). Latency of the ATP-induced [Ca²⁺]_i rise was significantly shortened by increasing the photolysis (ATP application) duration. The prolongation of ATP application duration also increased the magnitude of the [Ca²⁺]_i rise (F_peak/F₀: 1.84±0.30 to 2.46±0.77, n=10).

A small population of DRG neurons responded immediately to ATP application with no appreciable latency. Occasionally, we observed a [Ca²⁺]_i oscillation-like response with multiple phases after a TPP of caged ATP.

3. Intracellular Ca²⁺ stores are responsible for ATP-induced [Ca²⁺]_i rise
Application of ATP (100 ms) raised [Ca²⁺]_i in the absence of extracellular Ca²⁺. Similar ATP-induced [Ca²⁺]_i rises were observed, even when the extracellular K⁺ concentration was increased to 120 mM (in the absence of Ca²⁺). These results do not support that voltage-dependent mechanisms (e.g., facilitation of IP3 production during depolarization) play a major role.

4. [Ca²⁺]_i rise is caused via P2Y purinoceptors
TPP-induced [Ca²⁺]_i rises in the absence of extracellular Ca²⁺ suggest an involvement of P2Y purinoceptors. We examined the effect of suramin, an antagonist to P2Y and P2X purinoceptors. After the control [Ca²⁺]_i was observed under a Ca²⁺-free condition, 100 µM suramin was administered. This treatment completely suppressed the ATP-induced [Ca²⁺]_i rise.

The effect of ,ß-methylene ATP, a selective P2X purinoceptor-desensitizing agonist, was examined. Applications of desensitizing concentrations of ,ß-methylene ATP at 1–10 µM had little effect on the ATP-induced [Ca²⁺]_i rise.

5. Ryanodine as well as IP3 receptors are involved
Effects of PLC inhibitors were examined. Addition of either NCDC (10 µM) or U73122 (1 µM) to the extracellular medium significantly suppressed the ATP-induced [Ca²⁺]_i rise. The inhibitory effects of PLC inhibitors implied involvement of IP3. The effect of a membrane-permeable blocker for IP3 receptors was then examined in the absence of extracellular Ca²⁺. Application of ATP failed to evoke [Ca²⁺]_i transient in the presence of 10 µM 2-APB.

Ryanodine receptors also occur in the endoplasmic reticulum of DRG neurons. This family of intracellular Ca²⁺ release channels may contribute in the ATP-induced [Ca²⁺]_i rise via a Ca²⁺-induced Ca²⁺ release mechanism. The effect of ryanodine was therefore examined. Application of ATP (200 ms) failed to evoke a transient rise in [Ca²⁺]_i after application of ryanodine (10 µM), suggesting a possible co-contribution of ryanodine receptors in addition to IP3 receptors.

6. Local and transient application of ATP increases [Ca²⁺]_i in a graded and synchronized manner
To study the time course of [Ca²⁺]_i change with higher resolution, we monitored [Ca²⁺]_i in x-t scan mode. We found that [Ca²⁺]_i increased with the same time course in different regions of cytoplasm in DRG neurons even when ATP was applied by TPP for a short duration (local application).

Figure 2 shows the effect of changing the duration of ATP application in the presence of 10 µM Cd²⁺. Measurement of [Ca²⁺]_i in line scan mode revealed that prolongation of the photolysis duration reduced the latency and potentiated the magnitude of [Ca²⁺]_i rise. When [Ca²⁺]_i was averaged in the region indicated by the bar in Fig. 2A (Fig. 2B ), t_PCa was 5046, 3894, and 2853 ms for 20, 50, and 150 ms application of ATP, respectively; t_0.5PCa was 3416, 2590, and 1501 ms, respectively. The magnitude of the ATP-induced [Ca²⁺]_i rise (F_peak/F₀) increased from 1.41 to 2.00 and 2.85 by increasing the duration from 20 to 50 and 150 ms, respectively) (Fig. 2B , left).

View larger version (31K): [in this window] [in a new window]   Figure 2. Line scan measurements of [Ca2+]i obtained with TPPs of various durations. The TPP site and a scanned line are indicated in the transmission image. Each row shows 8 consecutive line scan images (F/F0, 512x290 pixels, pixel size 0.078 µmx1.1 ms). TPP duration was 20, 50, or 150 ms for upper, middle, and bottom rows, respectively. The timing of TPPs is indicated by red bars. For each series of images, 4 preceding frames were averaged to yield F0 of each pixel; F/F0 values were calculated pixel by pixel. Graphs in panel B show changes in [Ca2+]i induced by TPPs of the 3 different durations. [Ca2+]i was averaged in the region indicated by a bar in the left of the first line scan image of each row. The color of the bar in panel A corresponds to that of the graph line in panel B: orange, green and blue lines are for 20, 50, and 150 ms TPPs, respectively. In Ba, F/F0 is plotted against time by smoothing with adjacent 100 data points; in Bb, data points are further normalized by the peak amplitude of each series of data. Gaps correspond to intervals between each x-t image acquisition.

CONCLUSIONS AND SIGNIFICANCE

Since living cells contain ATP in millimolar concentrations, exocytosis and/or rupture of the plasma membrane upon nociception would temporarily produce localized high concentration of ATP in the vicinity of and during the event. Application of ATP by TPP seems to mimic such a transient ATP release from a point source in biological systems (Fig. 3 A). In the present study we have demonstrated that application of ATP using TPP enables us to discriminate rises in [Ca²⁺]_i in individual DRG cell bodies (Fig. 1) . It was also possible to elevate [Ca²⁺]_i in a part of a neurite on its own.

View larger version (51K): [in this window] [in a new window]   Figure 3. Summary schemas. A) Two-photon photolysis of caged ATP mimics ATP release upon exocytosis and/or rupture of the plasma membrane. B) Graded and synchronized nature of [Ca2+]i rise may have an important contribution to modulating pain sensation via cross-excitation in sensory ganglia. Strong stimuli would enlarge the area of cross-excitation and shorten the latency. This mechanism may be operating in many biological systems that express metabotropic purinoceptors other than sensory neurons.

The DRG neurons used in the present study (9–18 µm diameter) are categorized as small cells that are responsible for noxious stimuli. These cells express vanilloid receptors, and ATP is a plausible factor in modulating as well as initiating noxious stimuli. The extracellular ATP-dependent modulation observed in the present study likely is not limited in peripheral nociception, but is considered to be involved in a wide range of physiological functions. For instance, ATP is released from the bladder urothelium in response to pressure/stretch, consequently modulating urinary bladder functions via the afferent nerve activity. It has recently been shown that ATP is released within sensory ganglia. This mechanism seems likely to play a crucial role in functional cross-excitation between neighboring somata of sensory neurons without a synaptic contact (Fig. 3B ). Along with P2X receptor-mediated process, the P2Y receptor-mediated [Ca²⁺]_i rise observed in the present study is thought to contribute to the cross-excitation mechanism. Due to the graded nature of the [Ca²⁺]_i rise via P2Y receptors, the width of cross-excitation would be determined by the amount of ATP released from the somata of affected sensory neurons (perhaps reflecting the degree of stimuli).

In conclusion, transient and local applications of ATP by TPP revealed spatiotemporal features of the [Ca²⁺]_i response. Using this technique, we provide evidence that local [Ca²⁺]_i rises could be elicited via P2Y purinoceptors. In the majority of DRG neurons, the [Ca²⁺]_i rise occurred after a considerable latency. The time courses of the rising phase were essentially the same over the whole soma, once [Ca²⁺]_i had started to rise. The latency and magnitude of the [Ca²⁺]_i rise were graded by the duration of ATP application. This graded and synchronized nature of [Ca²⁺]_i rise in the sensory neurons possibly makes an important contribution to modulation of pain sensation in intact animals, especially for cross-excitation in DRG. According to the spatiotemporal variation in the [Ca²⁺]_i rise, DRG neuron activity would be modulated variably to express pain sensation. The present study indicated involvement of both IP3 and ryanodine receptors and intracellular Ca²⁺ release channels.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1933fje;

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