Investigation Of The Role Of Multidrug Resistance Proteins (MRPs) In Vascular Homeostasis

Background: Cellular levels of cyclic GMP (cGMP) are tightly controlled by synthetic and degradative mechanisms (e.g. soluble and particulate guanylate cyclases, phosphodiesterase). Cellular extrusion of cGMP is a potentially important mechanism in regulating cGMP-dependent signals, driven by a family of multidrug resistance proteins (MRPs). Herein, we investigated if pharmacologicalinhibition of MRPs modulates vascular reactivity, vascular smooth muscle proliferation, and systemic haemodynamics.

Methods: Vascular studies: Mouse (male, C57BL/6) thoracic aortic rings were set up in organ baths for the measurement of isometric tension. Concentration-response relationships were determined in pre-contracted (phenylephrine, EC80) tissues for the MRP inhibitors, MK571 (1nM-30µM) and probenecid (PB; 1µM-30mM), and for atrial natriuretic peptide (ANP; 10pM-1µM) and the NO-donor Spermine-NONOate (Sp-NO; 1nM-3µM) in the absence and presence of threshold concentrations of MK571 (3µM) and PB (300µM). Proliferation: Primary human coronary arterial smooth muscle cells (hCASMC) were treated with MK571 (30nM-30µM), and ANP (1µM) or the NO-donor DETA-NONOate (D-NO; 10µM)in the absence and presence of MK571 (30nM). Cell number was counted in a blinded manner every 24 h for 96 h using a haemocytometer. The cells and supernatant from the 24 h timepoint were used to measure intra/extracellular levels of cGMP by ELISA. In vivo haemodynamics: Mice (male, C57BL/6)were implanted with radio telemetric probes and mean arterial blood pressure (MABP) recorded under control conditions or following addition of MK571 (25mg/kg/day) to the drinking water 24 h before recording commenced.

Results: Vascular studies:MK571 and PB produced concentration-dependent relaxation of mouse aorta; sub threshold concentrations of MK571 and PB enhanced vessel relaxations to ANP and Sp-NO (see table below; *P<0.05, **P<0.01, ***P<0.001). Proliferation: MK571 produced concentration-dependent reductions in hCASMC proliferation (e.g. fold increase at 96 h; Ctrl: 1.9±0.3, +MK571 30µM: 1.0±0.1***; n=5). Moreover, the anti-proliferative effects of ANP and D-NO were enhanced significantly when combined with a sub threshold concentration of MK (30nM; Control: 2.4±0.5, ANP: 1.9±0.4, ANP+MK: 1.7±0.3**, D-NO: 2.0±0.4, D-NO+MK: 1.6±0.2***; n=5). The intraextra -cellular [cGMP]ratio was decreased by ANP (Control: 0.4±0.06, +ANP: 0.13±0.02*) and this was reversed in the presence of MK571 (0.25±0.078); a similar pattern was observed with D-NO. In vivo haemodynamics: MK571 caused a significant drop in MABP (Control: 117.5±0.83, +MK571: 115.7±0.76**, n=5)that was particularly prominent in the dark(active) phase.

Conclusion: These data suggest that extrusion by MRPs contributes to the dynamic equilibrium regulating intracellular levels of cGMP. MRPsmay represent a further target amenable to drug intervention for promoting cGMP signalling in the treatment of cardiovascular disease.

Supported by the Medical Research Council