Adrenomedullin Stimulates Nitric Oxide Production from Primary Rat Hypothalamic Neurons: Roles of Calcium and Phosphatases

Abstract

Adrenomedullin (ADM) in the brain plays important roles in the maintenance of homeostasis. Although in vivo evidence has suggested that nitric oxide (NO) mediates ADM's effects in the brain, mechanisms for ADM stimulation of NO production in neurons have not been identified. In the present study, primary hypothalamic neurons were used to characterize ADM-induced NO production and to study the underlying mechanisms. Using Calcium Orange/4-amino-5-methylamino-2Ј,7Ј-difluorofluorescein fluorescence live cell imaging, we found that ADM (1 or 10 nM, 5 min) significantly elevated [Ca 2ϩ ] i and NO production in a concentration-dependent manner. Ca 2ϩ and NO responses induced by 10 nM ADM were abolished by pretreatment with 50 M 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid-acetoxymethyl ester (BAPTA-AM), an intracellular Ca 2ϩ chelator, or protein kinase A (PKA) inhibitors 5 M N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and 50 M Rp-cAMP. Furthermore, the ADM-induced NO production was significantly attenuated by a protein phosphatase 1/2A inhibitor, okadaic acid (OA; 0.1 M), or calcineurin inhibitors, tacrolimus (FK506) (1 M) and cyclosporin A (CsA; 0.1 M). Using Western blotting, we found that ADM significantly decreased phosphorylation of neuronal nitric-oxide synthase (nNOS) at serine 847. This dephosphorylation was inhibited by 0.1 M OA, 1 M FK506, 0.1 M CsA, or 5 M H-89, and attenuated by 50 M BAPTA-AM. These results suggest that, in hypothalamic neurons, ADM elevates [Ca 2ϩ ] i via PKA-associated mechanisms. The PKA/Ca 2ϩ cascade leads to protein phosphatase (PP) 1/PP2A-and calcineurin-mediated dephosphorylation of nNOS. We hypothesize that the Ca 2ϩ increase and nNOS dephosphorylation contribute to activation of nNOS and production of NO in hypothalamic neurons.

Adrenomedullin acts in the lateral parabrachial nucleus to increase arterial blood pressure through mechanisms mediated by glutamate and nitric oxide

“…Our findings support the role of NO and glutamate in mediating at least some of ADM's central effects on MAP in the rostral ventrolateral medulla and paraventricular nucleus of anesthetized rats, as inhibition of NO synthesis or blockade of glutamate ionotropic receptors in these centers abolished ADM's effects (45,49). Furthermore, our in vitro studies confirm that ADM stimulates NO production in SK-N-SH human neuroblastoma cells (48) and in primary rat hypothalamic neuron cultures (47). ADM also regulates the activity of L-type calcium channels to facilitate calcium entry into the cell (48) to potentially contribute to a greater activation of glutamate NMDA receptors (4,35) and to stimulation of NO synthesis (21).…”

Involvement of PKA-dependent upregulation of nNOS–CGRP in adrenomedullin-initiated mechanistic pathway underlying CFA-induced response in rats

“…The potential role of the cAMP/PKA signaling pathway was investigated on the basis of data suggesting its role in mediating the effects of AM (Brain and Grant, 2004;Lopez and Martinez, 2002). AM has been shown to activate cAMP/PKA in peripheral tissues (Chiu et al, 2010;Kuwasako et al, 2010;Sakurai, 2009;Shimekake et al, 1995) as well as in the central nervous system (Ho et al, 2008;Takata et al, 2009;Xu and Krukoff, 2007). Moreover, cAMP and PKA have been found to be responsible for nNOS induction or activation in various cell types (Boissel et al, 2004;Ferrer et al, 2004;Ohnishi et al, 2008), including the central neurons (Xu and Krukoff, 2007).…”

“…AM has been shown to activate cAMP/PKA in peripheral tissues (Chiu et al, 2010;Kuwasako et al, 2010;Sakurai, 2009;Shimekake et al, 1995) as well as in the central nervous system (Ho et al, 2008;Takata et al, 2009;Xu and Krukoff, 2007). Moreover, cAMP and PKA have been found to be responsible for nNOS induction or activation in various cell types (Boissel et al, 2004;Ferrer et al, 2004;Ohnishi et al, 2008), including the central neurons (Xu and Krukoff, 2007). Consistent with these studies, our results show that AM-induced increase in nNOS level was inhibited by the PKA inhibitor H-89 at a concentration (2.5 μM) that did not alter constitutive nNOS level in control ganglion explants.…”

Activation of protein kinase A and C prevents recovery from persistent depolarization produced by oxygen and glucose deprivation in rat hippocampal neurons

“…In rat hypothalamic neurons, adrenomedullin-induced nitric oxide (NO) release is blocked by PKA inhibitors H-89 and Rp-cAMP (Xu and Krukoff 2007). The NO scavengers carboxy-2-phenyl-4, 4, 5, 5-tetramethyl-imidazoline-1-oxyl-3-oxide and hemoglobin and a NO synthase inhibitor, N G -nitro-L-arginine, restore the membrane toward the preexposure level after superfusion of oxygen-and glucose-deprived medium in rat hippocampal CA1 neurons (Onitsuka et al 1998).…”