Functional expression of constitutive nitric oxide synthases regulated by voltage‐gated Na⁺ and Ca²⁺ channels in cultured human astrocytes

Abstract

We report the functional characterization of constitutive nitric oxide synthase(s) (NOS) such as neuronal and endothelial NOS in cultured human astrocytes. Exposure of cultured human astrocytes to 1 microM veratridine or 50 mM KCl produced a pronounced increase in a calmodulin-dependent NOS activity estimated from cGMP formation. The functional expression of voltage-gated Na(+) channel, which is estimated by the response to veratridine, appeared to be earlier (at second day in culture) than that of voltage-gated Ca(2+) channels, which are estimated by the response to the KCl stimulation (at fourth day in culture). The KCl-evoked NO synthesis was totally reversed by L-type Ca(2+) channel blockers such as nifedipine and verapamil, but not by omega-conotoxin GVIA, an N-type Ca(2+) channel blocker, or omega-agatoxin IVA, a P/Q-type Ca(2+) channel blocker. In addition, verapamil abolished the KCl-induced increase in the intracellular free Ca(2+) concentration. RT-PCR analysis revealed that mRNA for neuronal and endothelial NOS was expressed in human astrocytes. In addition, Western blot analysis and double labeling of NOS and glial fibrillary acidic protein (GFAP) showed that cultured human astrocytes expressed neuronal NOS and endothelial NOS as well as the alpha(1) subunit of Ca(2+) channel. These results suggest that human astrocytes express constitutive NOS that are regulated by voltage-gated L-type Ca(2+) channel as well as Na(+) channel.

Activity-dependent NFATc3 nuclear accumulation in pericytes from cortical parenchymal microvessels

“…Different sources of NO have been demonstrated in the nervous system. Neurons, astrocytes, and endothelial cells have been shown to express constitutive NOS (neuronal and endothelial NOS isoforms) (6,34,36,40,49,50). Activation of the group I mGluRs in astrocytes increases intracellular Ca 2ϩ , which, in turn, results in the release of Ca 2ϩ -dependent vasoactive substances such as prostaglandins and NO (56).…”

“…In the brain, NO is a potent signal released by neurons, astrocytes, and endothelial cells (6,34,36,40). A recent study from our laboratory showed that NFATc3 nuclear accumulation in extracerebral VSMCs depends on both Ca 2ϩ influx and PKG activation through endothelium-derived NO (17).…”

“…The rise in astrocytic [Ca 2ϩ ] i enhances the release of several vasoactive substances, some of which have been implicated in vessel maturation (i.e., prostanoids and NO) (33,36,40). Importantly, the activation of gene expression is necessary for the proper establishment of the cerebral microcirculation, and intercellular signaling is an essential step in this process.…”

Nitric oxide and metastatic cell behaviour

“…(102) On the other hand, there is emerging evidence that VGSC activity may control NO production. (103) In strongly metastatic Mat-LyLu rat prostate cancer cell line, inhibition of VGSC activity with the highly specific blocker tetrodotoxin increased NO release. (104) For the way forward, in order to achieve better and more consistent understanding of the effects of NO in metastasis, the following would be essential.…”

Παράγοντες Που Επηρεάζουν Την Απελευθέρωση Του Μονοξειδίου Του Αζώτου (NO) Στους Αεραγωγούς

“…Τα αποτελέσματα των πειραμάτων αυτών δείχνουν ότι σε χαμηλή συγκέντρωση ασβεστίου (Σχήμα 10) ή σε παρουσία βεραπαμίλης (Σχήμα 11) στο διάλυμα, η παρουσία του L-NAME δεν μεταβάλλει την ανταποκρισιμότητα των ΛΜΙ στην ACh υποδηλώνοντας ότι σε τάση ηρεμίας 2.5 γραμμαρίων η παραγωγή του NO προάγεται από την είσοδο ασβεστίου από τον εξωκυττάριο χώρο κυρίως μέσω τασεοεξαρτώμενων διαύλων ασβεστίου. Κατά συνέπεια, η παραγωγή του NO, απαιτεί φυσιολογική εξωκυττάρια συγκέντρωση ασβεστίου και εξαρτάται από την είσοδο στο κύτταρο ιόντων ασβεστίου μέσω διαύλων ιόντων εξαρτώμενων από την τάση τύπου L [163] και άρα οι αποκλειστές διαύλων ιόντων ασβεστίου τύπου L [164,165] μπορούν να επηρεάσουν την έκφραση και/ή την δραστηριότητα της NOS. Τα αποτελέσματά μας δείχνουν ότι η επίδραση της τάσης ηρεμίας στην ανταποκρισιμότητα των ΛΜΙ στην ACh είναι ασβεστιοεξαρτώμενη.…”