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Hydrogen sulfide induces calcium waves in astrocytes¹

YASUO NAGAI^*, MAMIKO TSUGANE^*,, JUN-ICHIRO OKA and HIDEO KIMURA^*,2

^* National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan; and
Faculty of Pharmaceutical Science, Tokyo University of Science, Noda, Chiba, Japan

²Correspondence: National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan. E-mail: kimura{at}ncnp.go.jp

SPECIFIC AIM

Hydrogen sulfide (H₂S), a toxic gas, is found in the brain (50–160 µM). H₂S modifies hippocampal long-term potentiation (LTP) by enhancing the activity of NMDA receptors and may function as a neuromodulator in the brain. We determined that H₂S increases intracellular Ca²⁺ and induces Ca²⁺ waves in primary cultures of astrocytes as well as hippocampal slices. We examined for a role of H₂S in Ca²⁺ waves induced in astrocytes.

PRINCIPAL FINDINGS

1. H₂S increases intracellular Ca²⁺ and induces Ca²⁺ waves in astrocytes
The observation that H₂S enhances the induction of hippocampal LTP suggests that H₂S may modulate some aspects of synaptic activity. Although H₂S enhances NMDA receptor-mediated responses to glutamate in neurons, the effects of H₂S in the absence of glutamate on brain cells are not well understood. To investigate the effect of H₂S alone, we measured changes in intracellular Ca²⁺ in primary cultures of rat brain cells enriched for neurons or glia using a Ca²⁺ imaging system with Calcium Green-1 as a Ca²⁺-sensitive fluorescent dye. Focal application of H₂S increased intracellular concentrations of Ca²⁺ in GFAP-positive astrocytes (Fig. 1 A, B). Forty seconds after application of H₂S, intracellular concentrations of Ca²⁺ began to increase and propagated to neighboring astrocytes (Fig. 1B ). The focus of astrocytes with high intracellular concentrations of Ca²⁺ moved to the neighboring astrocytes and continued propagating to make other foci (Fig. 1B ). Propagation of Ca²⁺ waves lasted 10 min after the cessation of the NaHS application. Although H₂S enhances the responses of neurons to NMDA, significant calcium responses to H₂S alone were not observed in neurons.

View larger version (88K): [in this window] [in a new window]   Figure 1. H2S increases intracellular Ca2+ and induces Ca2+ waves in astrocytes. A) GFAP-positive cultures of astrocytes. The left panel shows 12-day-old-cultures of astrocytes prepared from embryonic day 17 rat hippocampi. The same cultures were stained with an antibody against GFAP (right panel). B) Calcium imaging of responses of astrocytes to focally applied NaHS. A glass micropipette filled with 160 µM NaHS was positioned immediately above cell surfaces without touching them (first panel). The application of NaHS was started at time 0 and lasted for 40 s. Indicated times are minutes and seconds after the initial application. Note that basal salt solution (BSS) flows from left to right, but Ca2+ waves propagate even to the left of the spot applied NaHS, ruling out the possibility that the diffusion of NaHS is responsible for propagation. Only cells attached or closely located propagate Ca2+ waves. C) Increase in the intracellular Ca2+ and Ca2+ waves induced by NaHS.

There is a difference in the time course of the increase in intracellular Ca²⁺ between the astrocytes exposed directly to H₂S and those activated by the propagated Ca²⁺ waves. Intracellular Ca²⁺ in the astrocytes exposed to H₂S sharply increased and gradually decayed whereas the propagated Ca²⁺ waves showed oscillations with a faster decay (Fig. 1C ). These observations suggest that the initial increase in the intracellular Ca²⁺ induced by H₂S may be regulated by a mechanism different from the propagated Ca²⁺ waves.

To examine whether the responses to H₂S observed in cultures of astrocytes also occur in intact brain tissue, responses to H₂S were investigated in hippocampal slices. The bath application of 200 µM NaHS induces increases in intracellular concentrations of Ca²⁺ in hippocampal slices, suggesting the responsiveness of cells to H₂S is similar in brain slices and cultures of astrocytes.

2. Responses to H₂S require extra- and intracellular Ca²⁺ stores
Whether the increase in intracellular Ca²⁺ induced by H₂S is dependent on extracellular Ca²⁺ was examined by measuring the effect of NaHS in a Ca²⁺-free medium. The increase in intracellular Ca²⁺ induced by NaHS was greatly suppressed in the Ca²⁺-free medium. The influx of ⁴⁵Ca²⁺ was measured in cultures of glial cells: 200 µM NaHS increases the influx of Ca²⁺ similar to that caused by ionomycin, a Ca²⁺ ionophore.

The dependency of responses to H₂S on intracellular Ca²⁺ stores was examined by applying 1 µM thapsigargin for 90 min to deplete intracellular Ca²⁺ stores. Responses to NaHS were suppressed but much less than those to ATP or glutamate. These observations indicate that H₂S increases intracellular concentrations of Ca²⁺ largely by inducing Ca²⁺ influx and, to a lesser extent, through release from intracellular Ca²⁺ stores.

3. Responses to H₂S are suppressed by La³⁺ and Gd³⁺
Since H₂S increases intracellular Ca²⁺, H₂S may activate a channel or a receptor associated with a channel that is permeable to Ca²⁺. To assay the involvement of Ca²⁺ channels in response to H₂S, the effect of Ca²⁺ channel blockers was examined. Trivalent cations La³⁺ and Gd³⁺, known blockers of Ca²⁺ channels, potently suppress responses to NaHS and NaHS induced Ca²⁺ uptake. Although inhibition was less potent than La³⁺ and Gd³⁺, ruthenium red, a blocker of ryanodine receptors that inhibits voltage-gated Ca²⁺ channels, suppressed responses to NaHS.

4. Involvement of H₂S in Ca²⁺ waves in astrocytes induced by neuronal excitation
Interactions between neurons and glia may modulate synaptic transmission. To examine whether neuronal excitation induces Ca²⁺ waves, responses of astrocytes were investigated in mixed cultures of neurons and astrocytes. Since neurons respond to NMDA but not to H₂S, NMDA was used to stimulate neurons (Fig. 2 A). After the neurons responded to NMDA, Ca²⁺ waves occurred in neighboring astrocytes (Fig. 2A, c and e of glial responses). To examine whether the Ca²⁺ waves induced by NMDA are mediated by the neuronal excitation, the effect of Na⁺ channel blocker tetrodotoxin (TTX) on Ca²⁺ waves was tested. TTX suppresses the Ca²⁺ waves induced by NMDA (Fig. 2B ), indicating that NMDA causes the neuron excitation that induces Ca²⁺ waves in astrocytes. The Ca²⁺ waves in astrocytes were not induced by NMDA in cultures of astrocytes in the absence of neurons. These observations suggest that neuronal excitation is required to induce the Ca²⁺ waves in astrocytes.

View larger version (37K): [in this window] [in a new window]   Figure 2. Ca2+ waves in astrocytes induced by neuronal excitation are blocked by La3+ and Gd3+. A) Calcium imaging of responses of neurons to NMDA and subsequent Ca2+ waves induced in astrocytes. The top left end panel shows the coculture of neurons and glia stained with Calcium Green-1, and the remaining top panels (a–f) shows the calcium imaging of the culture. The second and third panels show responses of neurons and glia, respectively, of the corresponding time of the top panels. 200 µM NMDA applied by superfusion activates only neurons (arrows in panel b). Ca2+ waves were subsequently induced in the surrounding astrocytes (c, e). This type of Ca2+ wave is not induced in pure astrocyte cultures by NMDA (data not shown). Only astrocytes respond to 200 µM NaHS (f). The plain fluorescent images of cultures stained with Calcium Green-1 (the left panel). B) Suppression of Ca2+ waves by TTX. Ca2+ waves induced by NMDA are suppressed by 2 µM TTX (P<0.04 by the Student t test, n=6). C) La3+ and Gd3+ completely suppress Ca2+ waves induced by NMDA. Ca2+ waves induced in 6 individual astrocytes were superimposed. 200 µM NMDA, 10 µM La3+ (upper panel) and Gd3+ (lower panel) were bath applied.

To examine the involvement of H₂S in the induction of Ca²⁺ waves, the effect of La³⁺ or Gd³⁺ on Ca²⁺ waves induced by NMDA was tested. The Ca²⁺ waves induced by NMDA were completely suppressed by 10 µM La³⁺ or 10 µM Gd³⁺ (Fig. 2C ). These observations suggest that H₂S released in response to neuronal excitation may increase intracellular Ca²⁺ and induce Ca²⁺ waves in neighboring astrocytes.

CONCLUSIONS AND SIGNIFICANCE

Although H₂S enhances the induction of hippocampal LTP, the mechanism by which H₂S modulates synaptic activity is not well understood. H₂S enhances the responses of neurons to glutamate in hippocampal slices, but H₂S alone does not induce the increase in intracellular Ca²⁺ in neurons in cocultures of neurons and glia (Fig. 2A ). In contrast, astrocytes respond to H₂S alone and H₂S elicits Ca²⁺ waves (Fig. 2A ). These observations suggest that H₂S enhances the responses to glutamate in neurons and induces Ca²⁺ waves in astrocytes.

Glial cells communicate with surrounding cells by increasing intracellular concentrations of Ca²⁺ and propagate the signal as Ca²⁺ waves that occur spontaneously or in response to a variety of stimuli. Cocultures of glia and neurons show Ca²⁺ oscillations as well as intercellular Ca²⁺ waves. The Ca²⁺ waves often appear to be initiated at sites of contact with neurons, suggesting that glial Ca²⁺ waves are initiated by neuronal excitation. Since astrocytes elicit intercellular Ca²⁺ waves by electrical stimulation as well as by the bath application of NMDA in mixed cultures of neurons and astrocytes, it has been suggested that astrocytes respond directly to a neurotransmitter released from neurons excited by NMDA or by electrical stimulation. The present observations show that NMDA induces Ca²⁺ waves in astrocytes in the presence of neurons (Fig. 2) but not in the absence of neurons (data not shown). In addition, TTX suppressed the Ca²⁺ waves induced by NMDA in the mixed cultures (Fig. 2B ), suggesting the neuronal excitation is required to release H₂S from astrocytes and to induce Ca²⁺ waves. Since La³⁺ and Gd³⁺ block Ca²⁺ waves and inhibit Ca²⁺ channels, La³⁺ and Gd³⁺ may inhibit the exocytosis of glutamate or some factor from neurons when neurons are stimulated by NMDA. However, La³⁺ and Gd³⁺ block H₂S initiated waves in pure astrocyte cultures, showing that Ca²⁺ is probably involved in the propagation step.

In conclusion, H₂S released in response to neuronal excitation may activate Ca²⁺ channels to induce Ca²⁺ waves in the neighboring astrocytes (Fig. 3 ). H₂S may therefore mediate signals between neurons and glia.

View larger version (34K): [in this window] [in a new window]   Figure 3. H2S induces Ca2+ waves in astrocytes. H2S is released from neurons or glia by neuronal excitation and increases the intracellular concentrations of Ca2+ by activating Ca2+ channels of astrocytes and to a lesser extent causing the release from intracellular Ca2+ stores. Elevated intracellular Ca2+ triggers the induction of Ca2+ waves that propagate to the neighboring astrocytes.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1052fje

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