Elsevier

Neuropharmacology

Volume 41, Issue 7, December 2001, Pages 895-906
Neuropharmacology

Prolonged exposure to YC-1 induces apoptosis in adrenomedullary endothelial and chromaffin cells through a cGMP-independent mechanism

https://doi.org/10.1016/S0028-3908(01)00131-9Get rights and content

Abstract

YC-1, a benzyl indazole derivative, is an NO-independent direct activator of soluble guanylyl cyclase (sGC), which presents a synergistic action with NO in stimulating cGMP synthesis. These properties have served to suggest YC-1 as an attractive therapeutic agent by permitting the reduction of nitrovasodilator dosage and regulating endogenous cGMP metabolism. Here we studied the effect of prolonged exposure of adrenomedullary endothelial and chromaffin cells to YC-1. We found that YC-1 increased cGMP in the two types of cells and this action was blocked by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Cells underwent apoptotic death in association with increased caspase-3-like activity, DNA fragmentation, cytoskeletal disorganization and changes in membrane permeability after prolonged incubation with YC-1. Caspase-3-like protease activity and DNA fragments in the cytoplasm were increased in a dose-dependent manner by 16 h YC-1 treatment. The specific and cell permeable caspase-3-like protease inhibitor DEVD-CHO effectively inhibited YC-1-mediated caspase-3-like activation and DNA fragmentation. Moreover, YC-1 also induced cell shape changes accompanied by actin filament disorganization and alterations in membrane permeability. Cells incubated for 24 h with YC-1 showed damaged membranes by binding to nucleic acid of a dye excluded by the intact plasma membrane of live cells. YC-1 also induced a decrease in the intracellular non-specific esterase activity, another indication of cell toxicity. Apoptotic phenomena were not prevented by the presence of ODQ although it effectively inhibited the YC-1-elicited cGMP increases. These findings indicate that YC-1 induces apoptosis by activating caspase-3-like protease through a mechanism independent of sGC activation.

Introduction

YC-1 (3-5′-hydroxymethyl-2′-furyl) 1-benzylindazole) has been described as a NO-independent and direct activator of soluble guanylyl cyclase (sGC). YC-1 inhibits several phosphodiesterase (PDE) activities (Ko et al., 1994, Friebe et al., 1998, Galle et al., 1999) thus increasing synthesis of cGMP and decreasing both cGMP and cAMP break down in different cell types (Galle et al., 1999). This compound has also been identified as an inhibitor of platelet aggregation (Ko et al., 1994, Wu et al., 1995) and adhesion (Wu et al., 1997). YC-1 also inhibits vascular smooth muscle contraction and proliferation (Yu et al., 1995, Mülsch et al., 1997), mimicking many of the NO actions ( Ignarro et al., 1986, Palmer et al., 1987, Yang et al., 1994). On the other hand, YC-1 not only activates sGC by increasing the KM for GTP and the Vmax but also potentiates in a synergistic way the action of NO and CO as sGC activators by decreasing the dissociation rate of these compounds (Mülsch et al., 1997, Friebe and Koesling, 1998). However, the effect of YC-1 seems to be dependent upon the cell type and perhaps also on the experimental conditions. Since whereas it does not change the time course of the magnitude of contraction in rat ventricular heart muscle and does not increase cGMP in isolated rat cardiomyocytes (Wegener et al., 1997) it induces apoptosis in adult rat cardiomyocytes via cGMP signalling (Taimor et al., 2000). YC-1 increases cGMP levels in platelets and smooth muscle by a direct activation of sGC without any effect on the production of NO (Wu et al., 1995). Nevertheless, in bovine aorta endothelial cells (BAEC) and in human umbilical vein endothelial cells (HUVEC) YC-1 stimulates NO production through stimulating calcium entry (Wohlfart et al., 1999), increasing cGMP production by direct activation of sGC and by stimulating NO production, which in turns also activates sGC to synthesize cGMP (Wohlfart et al., 1999). On the other hand, it has also been shown that YC-1 is a potent cGMP-independent inhibitor of PGE2 formation in macrophages (Abate et al., 2000). Moreover, Sheng-Nan et al., (2000) have shown that YC-1 has dual effects on IK(Ca) in GH3 cells, and they are independent of the activation of sGC, indicating that this compound may therefore act in a cGMP-independent way.

Different NO donor compounds have been clinically used for over 100 years for the treatment of different vascular and heart diseases, however, these compounds carry the risk of generating tolerance and cross tolerance after prolonged usage (Thadani, 1996), making it necessary to suppress the treatment for several periods (Luke et al., 1987). In general, high levels of NO exert cytotoxic action by diverse molecular mechanisms (Billiar, 1995, Moncada and Higgs, 1995). Since YC-1 is a highly effective vasodilator compound with a prolonged duration of action, the use of YC-1 with therapeutic purpose might avoid this problem. Furthermore, YC-1 boosts the effect of NO and its use might permit the reduction of nitrovasodilator dosage, avoiding the toxic effects due to high NO concentrations (Mülsch et al., 1997). However, much more work is necessary to clarify the effect of this compound in different cell types, and its long-term action.

The present study focuses on the effect of prolonged exposure to YC-1 at concentrations that activate sGC and cause vasorelaxation (Galle et al., 1999) in two different cell types: endothelial and chromaffin cells from bovine adrenal medulla. Chromaffin cells have been largely employed as a neural model, express all the elements of the NO/cGMP pathway, and cGMP increase leads to an inhibition of catecholamine release (Rodrı́guez-Pascual et al., 1996). On the other hand, adrenal vascular endothelial cells have been used to study the capacity of NO to induce apoptosis (López-Collazo et al., 1997). In this study, YC-1 was found to activate caspase-3-like protease activity and induce apoptosis in these two cellular types. Different biochemical and morphological changes associated with apoptosis such as DNA release to the cytoplasm and DNA-laddering, cytoskeletal organization and changes in cellular membrane permeability have been studied.

Section snippets

Isolation and culture of bovine adrenal endothelial and chromaffin cells

Cells were obtained after digestion of bovine adrenal glands with collagenase (EC 3.4.24.3) in retrograde perfusion as previously described (Rodrı́guez-Pascual et al., 1995a). Briefly, glands supplied by a local slaughterhouse were trimmed of fat, cannulated through the adrenal vein and washed with Ca2+/Mg2+-free saline buffer, containing (in mM) NaCl 154, KCl 5.6, NaHCO3 3.6, glucose 5, and HEPES 5, pH 7.4. Digestion was performed with a 0.2% collagenase plus 0.5% bovine serum albumin

YC-1 elicits increases on intracellular cGMP in chromaffin and endothelial cells and ODQ effectively inhibits them

Since YC-1 shows varying efficacy in increasing cGMP in different cell types, our first experiments were designed to determine the capacity of YC-1 to increase cGMP in both cell types and to establish whether ODQ, the specific sGC inhibitor (Garthwaite et al., 1995), was able to abolish any increase, thus determining if ODQ was equally effective in the two types of cells.

As is shown in Fig. 1A, YC-1 effectively increased cGMP in chromaffin cells in a concentration-dependent manner, and although

Discussion

The results of this study demonstrate that prolonged exposure to YC-1 caused caspase-3-like protease activation and elicited apoptotic events in two different cellular preparations, endothelial and chromaffin cells from bovine adrenal medulla, through a mechanism independent of its capacity to stimulate sGC and cGMP synthesis.

YC-1 was able to increase cGMP in both chromaffin and endothelial cells, and had a synergistic effect with NO, as has been previously reported (Mülsch et al., 1997, Friebe

Acknowledgements

This study was supported by grants from Comunidad de Madrid (08.5/0025.1/1999) and Ministerio de Educación y Cultura de España (PM96-0053 y PM99-0058). Rut Ferrero was supported by a fellowship from de Spanish Ministerio de Educación y Cultura. We thank Ann Burton for reading and correcting the English of this manuscript. We thank Dr Boscá and his group for their help in measuring caspase-3 activity.

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