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.

Abstract-Chronic treatment with the immunosuppressive drug rapamycin leads to hypertension; however, the mechanisms are unknown. Rapamycin binds FK506 binding protein 12 and its related isoform 12.6 (FKBP12/12.6) and displaces them from intracellular Ca 2ϩ release channels (ryanodine receptors) eliciting a Ca 2ϩ leak from the endoplasmic/sarcoplasmic reticulum. We tested whether this Ca 2ϩ leak promotes conventional protein kinase C-mediated endothelial NO synthase phosphorylation at Thr495, which reduces production of the vasodilator NO. Rapamycin treatment of control mice for 7 days, as well as genetic deletion of FKBP12.6, increased systolic arterial pressure significantly compared with controls. Untreated aortas from FKBP12.6 Ϫ/Ϫ mice and in vitro rapamycin-treated control aortas had similarly decreased endothelium-dependent relaxation responses and NO production and increased endothelial NO synthase Thr495 phosphorylation and protein kinase C activity. Inhibition of either conventional protein kinase C or ryanodine receptor restored endothelial NO synthase Thr495 phosphorylation and endothelial function to control levels. Rapamycin induced a small increase in basal intracellular Ca 2ϩ levels in isolated endothelial cells, and rapamycin or FKBP12.6 gene deletion decreased acetylcholine-induced intracellular Ca 2ϩ release, all of which were reversed by ryanodine. These data demonstrate that displacement of FKBP12/12.6 from ryanodine receptors induces an endothelial intracellular Ca 2ϩ leak and increases conventional protein kinase C-mediated endothelial NO synthase Thr495 phosphorylation leading to decreased NO production and endothelial dysfunction. This molecular mechanism may, in part, explain rapamycin-induced hypertension.

These data suggest that increased superoxide production by NADPH oxidase, peroxynitrite degradation of BH4, and uncoupled eNOS contribute to endothelial dysfunction in a rat model of pregnancy-induced hypertension.

Objectives-FK506Binding Protein 12 and its related isoform 12.6 (FKBP12/12.6) stabilize a closed state of intracellular Ca 2ϩ release channels (ryanodine receptors [RyRs]), and in myocytes removal of FKBP12/12.6 from RyRs alters intracellular Ca 2ϩ levels. The immunosuppressive drugs rapamycin and FK506 bind and displace FKBP12/12.6 from RyRs, and can also cause endothelial dysfunction and hypertension. We tested whether rapamycin and FK506 cause an intracellular Ca 2ϩ leak in endothelial cells and whether this affects endothelial function and blood pressure regulation. Methods and Results-Rapamycin or FK506 concentration-dependently caused a Ca 2ϩ leak in isolated endothelial cells, decreased aortic NO production and endothelium-dependent dilation, and increased systolic blood pressure in control mice. Rapamycin or FK506 at 10 mol/L abolished aortic NO production and endothelium-dependent dilation. Similar results were obtained in isolated endothelial cells and aortas from FKBP12.6 Ϫ/Ϫ mice after displacement of FKBP12 with 1 mol/L rapamycin or FK506. In hypertensive FKBP12.6 Ϫ/Ϫ mice, systolic blood pressures were further elevated after treatment with either rapamycin or FK506. Blockade of the Ca 2ϩ leak with ryanodine normalized NO production and endothelium-dependent dilation. Conclusions-Complete removal of FKBP12 and 12.6 from endothelial RyRs induces an intracellular Ca 2ϩ leak which may contribute to the pathogenesis of endothelial dysfunction and hypertension caused by rapamycin or FK506.

These results suggest that dietary isolated soy protein and isoflavones have no effect on bone and the vagina during premenopausal period, but may have an adverse effect on the uterus.