Phasic cardiovascular responses to mevinphos are mediated through differential activation of cGMP/PKG cascade and peroxynitrite via nitric oxide generated in the rat rostral ventrolateral medulla by NOS I and II isoforms

Abstract

The organophosphate insecticide mevinphos (Mev) acts on the rostral ventrolateral medulla (RVLM), where sympathetic vasomotor tone originates, to elicit phasic cardiovascular responses via nitric oxide (NO) generated by NO synthase (NOS) I and II. We evaluated the contribution of soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) cascade and peroxynitrite in this process. PKG expression in ventrolateral medulla of Sprague–Dawley rats manifested an increase during the sympathoexcitatory phase (Phase I) of cardiovascular responses induced by microinjection of Mev bilaterally into the RVLM that was antagonized by co-administration of 7-nitroindazole or N^ω-propyl-l-arginine, two selective NOS I inhibitors or 1-H-[1,2,4]oxadiaolo[4,3-a]quinoxalin-1-one (ODQ), a selective sGC antagonist. Co-microinjection of ODQ or two PKG inhibitors, KT5823 or Rp-8-Br-cGMPS, also blunted the Mev-elicited sympathoexcitatory effects. However, the increase in nitrotyrosine, a marker for peroxynitrite, and the sympathoinhibitory circulatory actions during Phase II Mev intoxication were antagonized by co-administration of S-methylisothiourea, a selective NOS II inhibitor, Mn(III)-tetrakis-(4-benzoic acid) porphyrin, a superoxide dismutase mimetic, 5,10,15,20-tetrakis-N-methyl-4′-pyridyl)-porphyrinato iron (III), a peroxynitrite decomposition catalyst, or l-cysteine, a peroxynitrite scavenger. We conclude that sGC/cGMP/PKG cascade and peroxynitrite formation may participate in Mev-induced phasic cardiovascular responses as signals downstream to NO generated respectively by NOS I and II in the RVLM.

Introduction

Whereas organophosphate poisons represent the oldest agents used in chemical warfare (McCauley et al., 2001), they are currently attracting renewed attentions because of their potential deployment by militias or terrorists (Lallement et al., 1999). The clinical presentations of organophosphate poisons are generally believed to entail over-stimulation of peripheral and central synapses by the accumulated acetylcholine that results from acetylcholinesterase inhibition (Tafuri and Roberts, 1987). Detailed cellular and molecular mechanisms of the cardiovascular responses to organophosphate poisons, particularly in relationship to central circulatory regulation, however, are relatively lacking.

Mevinphos (3-[dimethoxyphosphinyl-oxyl]-2-butenoic acid methyl ester; Mev) is the most commonly used organophosphate poison for suicidal purposes in Taiwan (Chuang et al., 1996). The US Environmental Protection Agency has placed it in Toxicity Category I, indicating the greatest degree of acute toxicity for oral, dermal and inhalation effects. Like other organophosphate insecticides, manifestations of Mev poisoning vary from salivation, muscle fasciculation to stupor, respiratory failure or death (Bardin and van Eeden, 1990). A prolonged corrected Q-T interval has been related to a higher rate of respiratory failure and mortality (Chuang et al., 1996). As an organophosphate of the P = O type, Mev exerts direct inhibition on acetylcholinesterase in the brain (Takahashi et al., 1991). Our laboratory has identified that the rostral ventrolateral medulla (RVLM), where premotor sympathetic neurons that are responsible for the maintenance of vasomotor tone are located (Ross et al., 1984), is a site of action for Mev (Chang et al., 2001, Yen et al., 2001, Yen et al., 2004). Based on results from power spectral analysis of systemic arterial pressure (SAP) signals, we previously (Chang et al., 2001, Yen et al., 2001) designated the two distinct phases of cardiovascular responses elicited by intravenous administration or microinjection of Mev into the RVLM as Phase I (sympathoexcitatory) and Phase II (sympathoinhibitory) Mev intoxication. We further showed (Chang et al., 2001, Chan et al., 2004b) that Mev intoxication may result from nitric oxide (NO) produced on activation of the M2 subtype of muscarinic receptors by the accumulated acetylcholine in the RVLM. Very recently (Chan et al., 2004b), we demonstrated that NO produced by NO synthase (NOS) I (neuronal NOS) and II (inducible NOS) in the RVLM plays respectively a sympathoexcitatory and sympathoinhibitory role in the phasic cardiovascular responses during Mev intoxication.

The soluble guanylyl cyclase(sGC)/cGMP/protein kinase G (PKG) cascade is a well-known intracellular signaling pathway for the physiological actions of NO (Arnold et al., 1977, Knowles et al., 1989). Mediation of the cytotoxic cardiovascular actions of NO by peroxynitrite, a potent reactive oxidant formed by the reaction between superoxide anion and NO was also reported (Beckman et al., 1990, Garcia-Estan et al., 2002). Of particular note is that peroxynitrite is primarily responsible for the fatal sympathoinhibition induced by overproduction of NO after activation of NOS II in the RVLM during experimental endotoxemia (Chan et al., 2002). It follows that sGC/cGMP/PKG cascade and peroxynitrite formation may participate in the phasic cardiovascular responses during Mev intoxication as signals downstream to NO generated respectively by NOS I and II in the RVLM. This hypothesis is validated in the present study, based on results obtained from pharmacological intervention, in conjunction with Western blot analysis and cardiovascular evaluations.