Glutamate-induced calcium signals stimulate CO production in piglet astrocytes

Abstract

Glutamate-stimulated, astrocyte-derived carbon monoxide (CO) causes cerebral arteriole dilation by activating smooth muscle cell large-conductance Ca(2+)-activated K(+) channels. Here, we examined the hypothesis that glutamate activates heme oxygenase (HO)-2 and CO production via the intracellular Ca(2+) concentration ([Ca(2+)](i))/Ca(2+)-calmodulin signaling pathway in newborn pig astrocytes. The major findings are: 1) glutamate stimulated Ca(2+) transients and increased steady-state [Ca(2+)](i) in cerebral cortical astrocytes in primary culture, 2) in astrocytes permeabilized with ionomycin, elevation of [Ca(2+)](i) concentration-dependently increased CO production, 3) glutamate did not affect CO production at any [Ca(2+)](i) when the [Ca(2+)](i) was held constant, 4) thapsigargin, a sarco/endoplasmic reticulum Ca(2+)-ATPase blocker, decreased basal CO production and blocked glutamate-induced increases in CO, and 5) calmidazolium, a calmodulin inhibitor, blocked CO production induced by glutamate and by [Ca(2+)](i) elevation. Taken together, our data are consistent with the hypothesis that glutamate elevates [Ca(2+)](i) in astrocytes, leading to Ca(2+)- and calmodulin-dependent HO-2 activation, and CO production.

Fig. 1.

Effects of glutamate, hemin, and intracellular Ca²⁺ concentration ([Ca²⁺]_i) on carbon monoxide (CO) production by freshly isolated astrocytes from newborn pigs. Astrocytic CO production is stimulated by glutamate (A, the nos. of experiments were, from left to right, 29, 5, 11, 20, and 5, respectively), hemin (n = 4; B), and [Ca²⁺]_i elevation (in astrocytes permeabilized with 20 μM ionomycin; n = 12; C). Values are means ± SE. *P < 0.05 compared with control.

Fig. 2.

Effect of glutamate on [Ca²⁺]_i in piglet astrocytes in primary culture. A: a representative trace of glutamate-induced elevation of [Ca²⁺]_i. B: a representative trace of thapsigargin blockage of glutamate-stimulated [Ca²⁺]_i elevation. C: group data of A and B (n = 9). ΔF(340/380), the ratio changes of fura 2. Values are means ± SE. *P < 0.05 compared with control.

Fig. 3.

Effects of [Ca²⁺]_i on glutamate-stimulated CO production and heme oxygenase (HO)-2 activity. [Ca²⁺]_i was controlled by permeabilizing the cells with ionomycin (20 μM) and regulating medium Ca²⁺. A: effects of [Ca²⁺]_i clamped constant at different levels on glutamate-induced CO production in freshly isolated astrocytes. To cancel out the intracellular Ca²⁺-induced CO changes and isolate just the glutamate-induced changes, the y-axis is the ratio of CO production with and without glutamate (100 μM, n = 8). In the astrocytes with constant [Ca²⁺]_i, glutamate did not change CO production, and the ratio is ∼1. Without ionomycin, glutamate increases intracellular Ca²⁺ concentration (Fig. 2). CO production increased in response to glutamate [last bar, extracellular Ca²⁺ concentration ([Ca²⁺]_e)]. B: effects of [Ca²⁺]_i on HO-2 activity in freshly isolated astrocytes. [Ca²⁺]_i elevation increased CO production (n = 6). Elevation of [Ca²⁺]_i increased CO production from exogenous substrate (10 μM hemin, n = 6). Values are means ± SE. *P < 0.05 compared with control.

Fig. 4.

Effects of chromium mesoporphyrin (CrMP), an inhibitor of HO, on CO production by freshly isolated astrocytes from newborn piglets. CrMP (20 μM) reduced basal CO production and blocked elevation of CO production caused by glutamate (100 μM, n = 4). Values are means ± SE. *P < 0.05 compared with the basal CO production in the first bar. †P < 0.05 compared with CO production caused by glutamate alone.

Fig. 5.

Effects of thapsigargin on glutamate (100 μM)- and hemin (10 μM)-induced CO production by astrocytes in primary culture. Thapsigargin (2 μM) blocked CO production stimulated by glutamate (n = 6; A) but not by hemin (n = 6; B). Values are means ± SE. *P < 0.05 compared with control. †P < 0.05 compared with glutamate. ‡P < 0.05 compared with thapsigargin alone.

Fig. 6.

Effect of calmidazolium (CZCl) on astrocytic CO production by freshly isolated astrocytes from newborn pigs. CZCl (40 μM) blocked glutamate (10 μM) stimulation of CO production (n = 10; A). CZCl inhibited CO production at all [Ca²⁺]_i >0.1 μM (n = 6; B). Values are means ± SE. *P < 0.05 compared with control. †P < 0.05 compared with glutamate.