Vascular effects of divalent manganese as a cofactor of soluble Guanylyl Cyclase

Abstract

OBJECTIVES: Divalent manganese (Mn2+) is a cofactor for soluble guanylyl cyclase (sGC), the enzyme producing cyclic GMP in vascular smooth muscle causing relaxation. Mn2+ competes with magnesium (Mg2+) to activate sGC. These divalent cations can bias the activity of the enzyme to produce cyclic nucleotides other than cyclic GMP, in particular cyclic AMP and cyclic IMP. Cyclic IMP, preferably produced in the presence of Mg2+(Beste KY et al, 2012), can cause contraction in precontracted pig arteries (Chen Z et al, 2014). The objective of the present study was to identify the mechanisms by which Mn2+, compared to Mg2+ [used in standard physiological solutions at millimolar concentrations (Liu JQ et al, 2006)], affects endothelium-dependent and –independent relaxations by influencing intracellular levels of cyclic nucleotides. METHODS: Ex vivo studies were conducted using isolated rat aortae of Sprague-Dawley rats of 12–14 weeks of age and porcine coronary arteries (collected at the local abattoir). Arteries were cut into rings and used for measurement of vascular reactivity in conventional organ chambers using pharmacological inhibitors of endothelial nitric oxide (NO) synthase (L-NAME), of cyclooxygenases (indomethacin), of endothelin-receptors (bosentan) and an antioxidant (apocynin). In some rings, the endothelium was removed through insertion of a wooden toothpick. After obtaining an optimal rest tension, the rings were treated with millimolar concentrations of Mn2+ or Mg2+ for forty-five minutes and precontracted with phenylephrine (rat aortae) or U46619 (porcine arteries). Subsequently, concentration-dependent responses were obtained with acetylcholine (rat arteries), bradykinin (porcine arteries) and the NO-donor sodium nitroprusside (SNP). In parallel, in vitro experiments were conducted using cultured porcine coronary artery smooth muscle cells (PCASMCs). The cells were exposed to millimolar concentrations of Mn2+ or Mg2+ in the presence or absence of a sGC-activator (YC-1). Intracellular cyclic GMP levels were measured using a commercially available ELISA kit. RESULTS: In vivo, millimolar ranges of Mn2+ significantly decreased endothelium-dependent relaxations to acetylcholine in rat aortae and to bradykinin in porcine coronary arteries compared to physiological levels of Mg2+. This effect was abolished by endothelium-removal. In addition, L-NAME partially reversed the effect of Mn2+, while indomethacin, bosentan and apocynin did not. Mn2+ also decreased, but to a smaller extent, endothelium-independent relaxations to SNP in both porcine coronary arteries and rat aortae. In parallel, the in vitro study showed that Mn2+ alone or in combination with YC-1 blocks the production of cyclic GMP. CONCLUSION: Taken into conjunction, the experiments show that Mn2+ at millimolar levels decreases endothelium-dependent and –independent relaxations compared to physiological levels of Mg2+. To the author’s surprise, this is likely explained through a decrease in cyclic GMP production. The effect of Mn2+ is endothelium-dependent and mediated (in the case of endothelium-dependent relaxations) by endothelial NO synthase, but not by cyclooxygenases, endothelin or oxidative stress. The findings indicate that in the presence of Mn2+ NO is a contractile signal and must lead to production of cyclic nucleotides other than cyclic GMP to cause contraction, similar to what is reported under hypoxic conditions.