Calcineurin Subject

The regulation of eNOS activity by phosphorylation is a well documented subject as the eNOS protein possess several consensus sequences for phosphorylation by protein kinase A (PKA), protein kinase C (PKC), protein kinase B (Akt/PKB) and AMP-activated protein kinase (AMPK). The eNOS enzyme has been reported to be phosphorylated on threonine, serine and t50 osine residues in response to various agonists. Many studies have shown that phosphorylation of eNOS on serine 1177 leads to an activation of eNOS [26-29], whereas phosphorylation on threonine 495 inactivates eNOS as this site is in the Ca Vcalmodulin binding domain [26]. Fleming et al. (2001) demonstrated that bradykinin (100 nM) increased eNOS activity in both porcine coronary artery endothelial cells (PCAE) and HUVEC via dephosphorylation of threonine 495 and phosphorylation of serine 1177 [30]. The bradykinin-induced phosphorylation of serine 1177 was abolished in the presence of a calmodulin dependent kinase II inhibitor whilst the dephosphorylation of threonine 495 was abolished by a protein phosphatase I inhibitor [30]. Harris et al. (2001) documented in BAEC that bradykinin (1 pM)-induced eNOS activity was mediated by activation of Akt/PKB [31] (see section 1.2.2) resulting in NOS phosphorylation at serine 1179 (bovine sequence) and a de-phosphorylation at threonine 497 mediated by calcineurin phosphatase. Typically, phosphorylation of eNOS at either of these sites is coordinated with dephosphorylation at the alternate site. 

As most PPIs rely predominantly on fhe CYP2C19 pathway of metabohsm, it is understandable why these drugs are safe in studies examining normal subjects. However, since mutations in the CYP2C19 pathway exist that render the patients "poor metabolizers", a potential interaction between PPIs and calcineurin inhibitors may occur as more PPI metabohsm is shifted to the CYP3A4 enzyme. 

This would be analogous to the change that results from alkyl substitution that is, transition states become more associative in the continuum from monoesters to triesters. Although relatively few phosphatases have been subjected to serious scrutiny of their transition states, in the cases that have been reported, this prediction has not been borne out. The reactions catalyzed by AP proceeds through loose transition states that are not significantly altered from those in solution, both in its phosphatase and in its promiscuous sulfatase activities. " Results with A-phosphatase and with calcineurin, which both catalyze phosphoryl transfer to a metal-coordinated hydroxide nucleophile, also provide no evidence of a significantly different transition state. Protein tyrosine phosphatases (PTPs), which do not contain metal ion cofactors but have a conserved arginine residue and proceed via a phosphocysteine intermediate, similarly catalyze phosphoryl transfer via a transition state similar to the one in solution. ... 

The oxidation state of the Fe ion of calcineurin is still the subject of some debate. In a recent study, Klee and colleagues proposed that the functional redox state was the reduced Fe -Zn form [48]. This proposal was based on data which showed that a portion of calcineurin activity in crude brain extract was susceptible to inactivation following incubation with Ca + and calmodulin and that inactivation could be prevented by the addition of superoxide dismutase. Inactivation was more pronounced imder aerobic than in anaerobic conditions. Additionally, incubation with and calmodulin in the presence of Snm ascorbate protected calcineurin from inactivation while reactivation could be achieved by addition of 0.5 mM Fe(NH4)2(S04)2 in the presence of 5mM dithiothreitol and 5mM ascorbate. These data were taken to propose that the active redox state was Fe +-Zn " and that inactivation was due to oxidation of the cluster to the Fe +-Zn + state. Direct confirmation of the oxidation states was not provided. 

Further studies are necessary to resolve the question of which oxidation state is represented in vivo. Indeed, Yu et al. considered the possibility that the isoform of calcineurin in rat brain lysate that is subject to oxidative inactivation is not an Fe-Zn enzyme but rather a diiron isoform of calcineurin [35]. As noted above, the diiron form of calcineurin has access to three different redox states. Inactivation upon exposure to Ca +/calmodulin could be the result of oxidation of the metal cluster to the Fe +-Fe + state. The conditions used to reactivate calcineurin in crude brain lysates [48] are essentially the same as those which reactivate purple acid phosphatase, i.e. incubation in the presence of mild reducing agents ((e.g. 10-... 

Of 1471 adult renal transplant recipients, 205 were switched from calcineurin inhibitors to sirolimus (n = 88) or everolimus ( = 117) [29 ]. Six (2.9%) developed pneumonitis, one associated with sirolimus and five with everolimus. Median times from conversion to the onset of pneumonitis were 34 days in four patients (range 24—46 days) and 491 days in 2 subjects (range 454—528 days). The most common symptoms were dry cough ( = 6), fever ( = 5), and dyspnea ( = 4). Imaging tests showed lower lobe involvement in aU cases. Bronchoalveolar lavage in four patients showed ljmiphocj4 ic alveolitis. All recovered completely after drug withdrawal. 

---