KMUP-1 ameliorates monocrotaline-induced pulmonary arterial hypertension through the modulation of Ca²⁺ sensitization and K⁺-channel

Abstract

Aims

This study investigates the actions of KMUP-1 on RhoA/Rho-kinase (ROCK)-dependent Ca²⁺ sensitization and the K⁺-channel in chronic pulmonary arterial hypertension (PAH) rats.

Main methods

Sprague–Dawley rats were divided into control, monocrotaline (MCT), and MCT + KMUP-1 groups. PAH was induced by a single intraperitoneal injection (i.p.) of MCT (60 mg/kg). KMUP-1 (5 mg/kg, i.p.) was administered once daily for 21 days to prevent MCT-induced PAH. All rats were sacrificed on day 22.

Key findings

MCT-induced increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy were prevented by KMUP-1. In myograph experiments, KCl (80 mM), phenylephrine (10 µM) and K⁺ channel inhibitors (TEA, 10 mM; paxilline, 10 µM; 4-AP, 5 mM) induced weak PA contractions in MCT-treated rats compared to controls, but the PA reactivity was restored in MCT + KMUP-1-treated rats. By contrast, in β-escin- or α-toxin-permeabilized PAs, CaCl₂-induced (1.25 mM, pCa 5.1) contractions were stronger in MCT-treated rats, and this action was suppressed in MCT + KMUP-1-treated rats. PA relaxation in response to the ROCK inhibitor Y27632 (0.1 μM) was much higher in MCT-treated rats than in control rats. In Western blot analysis, the expression of Ca²⁺-activated K⁺ (BK_Ca) and voltage-gated K⁺ channels (Kv2.1 and Kv1.5), and ROCK II proteins was elevated in MCT-treated rats and suppressed in MCT + KMUP-1-treated rats. We suggest that MCT-treated rats upregulate K⁺-channel proteins to adapt to chronic PAH.

Significance

KMUP-1 protects against PAH and restores PA vessel tone in MCT-treated rats, attributed to alteration of Ca²⁺ sensitivity and K⁺-channel function.

Introduction

Pulmonary vasoconstriction is considered to be an early component of the pulmonary hypertensive process. Pulmonary arterial hypertension (PAH) is a fatal disease, often affecting young people. PAH is characterized by increasing vascular pressure and progressive structural remodeling in pulmonary arteries (PA) (Kimura et al., 1998, Cowan et al., 2000, McLaughlin & McGoon, 2006). Its pathogenesis is suggested by the findings of increased production of TXA2, reduction of lung eNOS and increased RhoA/Rho-kinase (ROCK) in the PA (Böhm and Pernow 2007). PAH is regulated by nitric oxide (NO), which results in vasodilatation and proliferation in PA (Nelin and Hoffman 1998). Hypoxia-induced decreases in eNOS expression are mediated by ROCK and suggest that ROCK inhibitors may have therapeutic benefits in patients with hypoxia-induced pulmonary hypertension (Takemoto et al., 2002, Gao et al., 2007).

In pulmonary and systemic vessels, tone is regulated by a variety of mechanisms acting through Ca²⁺-dependent, Ca²⁺-independent pathways, or both. K⁺ channels are currently considered especially important Ca²⁺-dependent pathways in the pulmonary circulation (Cogolludo et al., 2003, Bonnet & Archer, 2007). Several studies indicate that activation of the RhoA/ROCK pathway in Ca²⁺-independent pathways contributes to both vasoconstriction and vascular remodeling associated with PAH such as is induced by chronic hypoxia and monocrotaline (MCT) (Uehata et al., 1997, Ward et al., 2004).

RhoA-dependent Ca²⁺-sensitization plays an important role in sustained vasoconstriction in the PA and in vascular beds. ROCK-dependent myosin light chain phosphatase (MLCP) inhibition is responsible for both RhoA-dependent Ca²⁺-sensitization and agonist-induced stimulation in smooth muscle cells (SMCs). RhoA/ROCK serves as a point of convergence of various signaling cascades contributing to the development of PAH. Not surprisingly, the ROCK inhibitors Y27632 and fasudil can be used to treat PAH (Abe et al., 2004, Oka et al., 2007). In contrast, vascular smooth muscle (VSM) relaxation can result from a decrease in cytosolic Ca²⁺-concentration and/or reduced contractile apparatus sensitivity to Ca²⁺. Additionally, stimulation of cGMP also decreases cytosolic Ca²⁺ by Ca²⁺-lowering mechanisms and causes Ca²⁺-desensitization by activating the MLCP (Sauzeau et al. 2000). Thus, agents like sildenafil and fasudil can be used to treat PAH by enhancing cGMP and inhibiting ROCK, respectively, which contributes to the prevention of increased PA pressure and progressive structural remodeling in PAs (Itoh et al., 2004, Abe et al., 2004, Pauvert et al., 2004).

We demonstrated that KMUP-1 (7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) (Fig. 1) activates NO release resulting in an increase of cGMP level (Wu et al. 2006); the enhanced cGMP is similar to the phosphodiesterase-5 inhibitor sildenafil. Recently, KMUP-1 was shown to enhance cGMP and inhibit ROCK in prostate smooth muscles (Liu et al. 2007). In this study, we investigated whether KMUP-1, a theophylline-based derivative, inhibited MCT toxin-induced chronic PAH via cGMP-dependent inhibition of ROCK proteins and modulation of K⁺ channels. The combination of cGMP-enhancing and ROCK-suppressing actions provided by KMUP-1 could treat MCT-induced PAH. The main objective of this study was to further investigate the mechanisms by which KMUP-1 prevents MCT-induced chronic PAH via regulation of RhoA/ROCK-dependent Ca²⁺ sensitization and K⁺-channel function in rat PAs.