Astrocyte-derived CO is a diffusible messenger that mediates glutamate-induced cerebral arteriolar dilation by activating smooth muscle Cell KCa channels

Abstract

Astrocyte signals can modulate arteriolar tone, contributing to regulation of cerebral blood flow, but specific intercellular communication mechanisms are unclear. Here we used isolated cerebral arteriole myocytes, astrocytes, and brain slices to investigate whether carbon monoxide (CO) generated by the enzyme heme oxygenase (HO) acts as an astrocyte-to-myocyte gasotransmitter in the brain. Glutamate stimulated CO production by astrocytes with intact HO-2, but not those genetically deficient in HO-2. Glutamate activated transient K(Ca) currents and single K(Ca) channels in myocytes that were in contact with astrocytes, but did not affect K(Ca) channel activity in myocytes that were alone. Pretreatment of astrocytes with chromium mesoporphyrin (CrMP), a HO inhibitor, or genetic ablation of HO-2 prevented glutamate-induced activation of myocyte transient K(Ca) currents and K(Ca) channels. Glutamate decreased arteriole myocyte intracellular Ca2+ concentration and dilated brain slice arterioles and this decrease and dilation were blocked by CrMP. Brain slice arteriole dilation to glutamate was also blocked by L-2-alpha aminoadipic acid, a selective astrocyte toxin, and paxilline, a K(Ca) channel blocker. These data indicate that an astrocytic signal, notably HO-2-derived CO, is used by glutamate to stimulate arteriole myocyte K(Ca) channels and dilate cerebral arterioles. Our study explains the astrocyte and HO dependence of glutamatergic functional hyperemia observed in the newborn cerebrovascular circulation in vivo.

Fig. 1

Glutamate (20µmol/L) elevates transient K_Ca current frequency in cerebrovascular smooth muscle cells in contact with astrocytes (A) but not in cerebrovascular smooth muscle cells alone (B). Summarized data are shown in C. n=7 and n=12 for smooth muscle cells alone and with astrocytes, respectively. *P<0.05 compared to zero.

Fig. 2

Glutamate (20µmol/L) activates K_Ca channels in cerebrovascular smooth muscle cells in contact with astrocytes (A) but not in cerebrovascular smooth muscle cells alone (B). Summarized data are shown in C. n=6 and n=13 for smooth muscle cells alone and with astrocytes, respectively. *P<0.05 compared to zero.

Fig. 3

Chromium mesoporphyrin (20µmol/l)(CrMP) pretreatment of astrocytes blocks glutamate (20µmol/L) activation of transient K_Ca current frequency (A&B, n=8) and K_Ca channel activity (C&D, n=6) in smooth muscle cells in contact with astrocytes.

Fig. 4

A: HO-2 protein expression in HO-2^+/+ and HO-2^−/− mice. B: Glutamate (20µmol/L) stimulates CO production by HO-2^+/+ mouse astrocytes but not by HO-2^−/− mouse astrocytes (n=3 separate mouse brain preparations each for HO-2^+/+ and HO-2^−/− with 5 or 6 tubes for each preparation). *P<0.05 compared to basal production.

Fig. 5

Glutamate activates transient K_Ca current frequencies and single channel activity in piglet cerebral microvascular smooth muscle cells in contact with mouse HO-2^+/+ (A& B, n=8) but not HO-2^−/− (C& D, n=6) astrocytes. *P<0.05 compared to without glutamate.

Fig. 6

Glutamate (30 µmol/l) decreases intracellular Ca²⁺ in brain slice arteriole smooth muscle and this effect is blocked by chromium mesoporphyrin (CrMP, 20µmol/l). A. Representative traces of fura-2 fluorescence ratio in an arteriole of an untreated brain slice and a slice treated with CrMP. B. Collated data of brain slice arteriole responses to glutamate without and with CrMP (n=13 and 11, respectively). *P<0.05 compared to diameter prior to glutamate. # P<0.05 compared to without CrMP.

Fig. 7

Chromium mesoporphyrin (CrMP, 20µmol/l), paxilline (20µmol/l), and L-2-alpha aminoadipic acid (L-AAA, 2 mmol/l) block glutamate (30µmol/L)-induced dilation of piglet cerebral cortical brain slice arterioles. A. Representative trace illustrating the effect of glutamate on the diameter of an arteriole in an untreated slice (a) and a slice treated with CrMP (b). B. Representative trace of glutamate-induced arteriolar dilation and the effect of paxilline on that dilation. C. Collated data of brain slice arteriole responses to glutamate without and with CrMP or paxilline (n=26, 11, and 11, respectively). D. Brain slice arteriolar diameter responses to glutamate and sodium nitroprusside (SNP, 140µmol/L) following treatment with D-AAA (n=10) or L-AAA (n=9). *P<0.05 compared to control diameter. # P<0.05 compared to without CrMP, paxilline, or L-AAA, as appropriate.