Tacrolimus reduces nitric oxide synthase function by binding to FKBP rather than by its calcineurin effect

Abstract

Hypertension develops in many patients receiving the immunosuppressive drug tacrolimus (FK506). One possible mechanism for hypertension is a reduction in vasodilatory nitric oxide. We found that tacrolimus and a calcineurin autoinhibitory peptide significantly decreased vascular calcineurin activity; however, only tacrolimus altered intracellular calcium release in mouse aortic endothelial cells. In mouse aortas, incubation with tacrolimus increased protein kinase C activity and basal endothelial nitric oxide synthase phosphorylation at threonine 495 but reduced basal and agonist-induced endothelial nitric oxide synthase phosphorylation at serine 1177, a mechanism known to inhibit synthase activity. While this decreased nitric oxide production and endothelial function, the calcineurin autoinhibitory peptide had no such effects. Inhibition of ryanodine receptor opening or protein kinase C blocked the effects of tacrolimus. Since it is known that the FK506 binding protein (FKBP12/12.6) interacts with the ryanodine receptor to regulate calcium release, we propose this as the mechanism by which tacrolimus alters intracellular calcium and endothelial nitric oxide synthase rather than by its effect on calcineurin. Our study shows that prevention of the tacrolimus-induced intracellular calcium leak may attenuate endothelial dysfunction and the consequent hypertension.

Infiltrating T lymphocytes in the kidney increase oxidative stress and participate in the development of hypertension and renal disease

“…The observation in the SD rats is consistent with previous reports (43) and is likely a result of effects of this agent to decrease nitric oxide production by calcineurin-independent effects (9). In contrast to the prohypertensive effects in normal rats, however, administration of tacrolimus to the Dahl SS rat decreased infiltration of T cells in the kidney, decreased renal oxidative stress, and attenuated sodium-dependent hypertension and renal disease in these rats.…”

Protein Kinase CβII-Mediated Phosphorylation of Endothelial Nitric Oxide Synthase Threonine 495 Mediates the Endothelial Dysfunction Induced by FK506 (Tacrolimus)

“…However, we found that the acute effects of FK506 on eNOS were completely independent of calcineurin inhibition (Cook et al, 2009). Thus, it still remains to be elucidated which of the four cPKC isoforms (␣, ␤ I, ␤ II , and ␥) is responsible for the phosphorylation of eNOS at Thr495 in response to FK506.…”

“…Phosphorylation of Ser1177, Ser633, and Tyr83 stimulates eNOS activity, whereas phosphorylation at Thr495 and Ser116 inhibits eNOS activity (Mount et al, 2007;Fulton et al, 2008). We found that FK506, via inhibition of its intracellular target FK506 binding protein 12 (FKBP12), causes a concentrationdependent increase in eNOS Thr495 phosphorylation, decreases vasodilation, and increases blood pressure (Long et al, 2007;Cook et al, 2009). This is caused by the displacement of FKBP12 from intracellular calcium channels (ryanodine receptors), leading to an intracellular Ca 2ϩ leak that activates the conventional protein kinase C (cPKC) isoforms.…”

“…Post-transplant hypertension is positively correlated with an increased incidence of allograft failure and is a major limitation in the use of FK506 (Opelz et al, 1998;Mange et al, 2000). We and others have shown previously that a decrease in the production of the potent vasodilator nitric oxide (NO) by endothelial cells contributes to the hypertension caused by FK506 (De Lima et al, 1999;Takeda et al, 1999;Long et al, 2007;Cook et al, 2009). However, the exact molecular mechanisms remain unknown.…”

“…This is caused by the displacement of FKBP12 from intracellular calcium channels (ryanodine receptors), leading to an intracellular Ca 2ϩ leak that activates the conventional protein kinase C (cPKC) isoforms. Furthermore, administration of Gö6976 [5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo [3,4-c]carbazole-12-propanenitrile], a cPKC inhibitor, prevented the FK506-induced increase in Thr495 phosphorylation and restored NO production and vasodilatory responses (Cook et al, 2009).…”