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 2ϩ -activated K ϩ channels. Here, we examined the hypothesis that glutamate activates heme oxygenase (HO)-2 and CO production via the intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i)/ Ca 2ϩ -calmodulin signaling pathway in newborn pig astrocytes. The major findings are: 1) glutamate stimulated Ca 2ϩ transients and increased steady-state [Ca 2ϩ ]i in cerebral cortical astrocytes in primary culture, 2) in astrocytes permeabilized with ionomycin, elevation of [Ca 2ϩ ]i concentration-dependently increased CO production, 3) glutamate did not affect CO production at any [Ca 2ϩ ]i when the [Ca 2ϩ ]i was held constant, 4) thapsigargin, a sarco/endoplasmic reticulum Ca 2ϩ -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 2ϩ ]i elevation. Taken together, our data are consistent with the hypothesis that glutamate elevates [Ca 2ϩ ]i in astrocytes, leading to Ca 2ϩ -and calmodulin-dependent HO-2 activation, and CO production. cerebral circulation; heme oxygenase; calmodulin; gasotransmitter; neurovascular coupling IN THE NEUROVASCULAR UNIT, astrocytes act as intermediaries between neurons and arterioles to regulate vascular tone in response to neuronal activity. Release of glutamate from presynaptic neurons increases blood flow to match metabolic demands; such coupling is called functional hyperemia. Functional hyperemia has been investigated extensively, and many astrocyte-derived vasoactive mediators, including ADP (40, 41), ATP (34, 36), epoxyeicosatrienoic acids (1-3), K ϩ (11, 13, 33), and prostacyclin (42), appear to be involved, depending on experimental models. We have shown that carbon monoxide (CO) is another critical regulator of cerebrovascular tone in newborn piglets (19,24,26,31,39).CO dilates newborn pig pial arterioles (22,25). CO causes vasodilation by stimulating Ca 2ϩ sparks and large-conductance Ca 2ϩ -activated K ϩ (BK Ca ) channels and by increasing effective coupling between Ca 2ϩ sparks and BK Ca channels in arterial smooth muscle cells (17,18,25,38). Physiologically, CO is produced by heme oxygenase (HO)-catabolized breakdown of heme to CO, iron, and biliverdin (27). Of three HO isoforms, only HO-2 is expressed under basal conditions in newborn piglet brain (31, 32). In piglets, endogenous HO-2 can produce sufficient CO to induce cerebral arteriole dilation (23).Our previous studies in piglets demonstrated that glutamate stimulates astrocytic CO production in primary-cultured astrocytes and in vivo (24,26,31). We also demonstrated that astrocytes release CO and astrocyte-derived CO causes glutamate-induced pial arteriolar dilation in vivo (24), supporting a regulatory role for astrocytic CO in neurovascular coupling in the newborn brain. Indeed, in brain slices from newborn piglets, we demonstrated that glut...

Use of Randomized Submaximal Glutamate Stimulus to Interpret Glial Effects on Neuronal Calcium Dynamics

“…In separate experiments, astrocytes in the cultures were identified on the basis of immunocytochemical staining with glial fibrillary acidic protein (GFAP) and [Ca 2+ ] i responses to direct glutamate stimulation (data not shown). We found that astrocytes demonstrated [Ca 2+ ] i responses to glutamate consistently only in the high micromolar and low millimolar range, which is consistent with a number of previous reports [11][12][13]. The ROIs were then used to measure fluorescence intensity over time in the specified area and created a data set based on ROIs.…”

Functional role of astrocyte glutamate receptors and carbon monoxide in cerebral vasodilation response to glutamate

“…In piglet cerebral vascular endothelial cells, iGluRs are functionally linked to HO-2 activation via protein tyrosine kinase-mediated posttranslational modifications of the enzyme by glutamate without involvement of PKC, CK-2, or Ca 2ϩ /calmodulin (15,16,25). In cortical astrocytes, Ca 2ϩ /calmodulin-dependent mechanisms are involved in rapid regulation of HO-2 activity by glutamate (37). However, the details of these cell-and signal-specific mechanisms and events leave much to be resolved.…”

“…Freshly isolated astrocytes or astrocytes cultured on Cytodex beads were incubated with glutamate and iGluR agonists or antagonists in Krebs solution inside sealed amber vials (2.0 ml) that contained Krebs buffer and the internal standard of the isotopically labeled CO ( 13 C 18 O) for 1 h at 37°C. The headspace gas CO concentration was determined by gas chromatography/mass spectrometry analysis as described previously (37). The components of the headspace gas were separated on a molecular sieve-coated capillary column (CP-Molsieve 5Å column) (Varian, Palo Alto, CA), and the CO peak was quantitatively detected based on the peak areas, with the mass-to-charge ratios corresponding to 12 C 16 O and 13 C 18 O, respectively.…”

Enteral Supplements of a Carbon Monoxide Donor CORM-A1 Protect against Cerebrovascular Dysfunction Caused by Neonatal Seizures

“…4,8,18,19 Postictal cerebral vascular responses to glutamate (10 − 4 mol/L) and the AMPA receptor agonist quisqualic acid (10 − 4 mol/L) were reduced by 60% to 70% compared with the intact control group (Figure 2). Heme oxygenase expressed in cerebral vascular endothelium and cortical astrocytes is an important contributor to cerebrovascular dilator function.…”