Possible role of SERCA in the monocrotaline rat model of pulmonary hypertension

Monocrotaline (MCT) injection provides a well-established model of pulmonary arterial hypertension (PAH) in rats. Pulmonary arteries (PA) from MCT-treated rats exhibit prolonged recovery after being stimulated to constrict with high K+ (1). This was proposed to result from reduced smooth muscle cell (SMC) sodium calcium exchanger (NCX) activity, but its mechanism has not been investigated. The aim of this study was to determine the mechanism of this slowed recovery by testing the possible roles of SMC depolarization, loss of NCX activity and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). A single injection of monocrotaline (60mg/kg) or saline was given to rats, the progression of pulmonary hypertension (PAH) was monitored and the rats killed when symptoms appeared. Intra-pulmonary arteries (PA) were dissected from the lungs, mounted in a small vessel myograph in physiological solution containing (in mM): NaCl 112, KCl 5, KH2PO4 0.5, Na2HPO4 0.5, CaCl2 1.8, MgCl2 1, HEPES 10, glucose 11; pH7.3; continually aerated at 37°C. Data are given as mean ± s.e.m and analysis was performed with two-way ANOVA followed by Bonferroni’s post hoc test or unpaired Student’s T-test as applicable. PA were constricted with 50 mM KCl or 1 µM phenylephrine (PE) then washed and the time for half maximal recovery (t1/2) measured. In KCl stimulated arteries from MCT rats t1/2 was increased 13-fold in comparison with controls (498±23 s vs 38±3 s, n=10-13; p<0.001). When the vessels were pre-constricted with PE, t1/2 was increased 3-fold in MCT-treated PA (458±70 s, n=5) compared with control PA (155±19 s, n=6, p=0.004). Inducing SMC depolarization in control arteries by raising the extracellular [K+] to 15mM did not affect the recovery rate, although 1 µM levcromakalim applied to MCT-treated PA to induce hyperpolarization reduced the recovery t1/2 from 546±64 s to 65±3 s (n=3, p<0.05). To investigate the role of NCX, extracellular Na+ was removed, monensin (20µM) was applied to load cells with Na+, or the NCX reverse mode inhibitor KB-R7943 (10µM) was applied. These approaches all failed to alter the recovery rate from PE constriction in both control and MCT-PA. In contrast, blocking SERCA activity with thapsigargin (5µM) caused the recovery t1/2 in KCl-constricted, control PA to be prolonged from 160±30 s to 525±45 s (n=3), thus mimicking the effect of MCT. In conclusion the recovery of PA following a constrictor stimulus is slowed after MCT treatment, regardless of the stimulus. It is not caused by SMC depolarization or loss of NCX activity, but may be due to loss of SERCA activity and slowed removal of Ca2+ from the SMC cytosol.

Ito KM et al. (2000). Am J Physiol 279: H1786-H1795