Calcineurin (CaN), a member of ser/thr protein phosphatase, was cloned from bovine retina. The peptide sequence of CaN A subunit is consisted of 511 amino acid residues. A 10 amino acid (A-T-V-E-A-I-E-A-D-E-A) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni2+ was found to be the strongest stimulator while Zn2+ was found to inhibit CaN phosphatase activity. Mn2+ was a relatively less effective stimulator compared to Ni2+. Fe2+ was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe2+ slightly inhibited bovine brain CaN activity. The residues at 97-201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407-456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known auto inhibitory domain at 457-480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics compared to bovine brain CaN A.

Calcineurin (CaN), a member of ser/thr protein phosphatase, was cloned from bovine retina. The peptide sequence of CaN A subunit is consisted of 511 amino acid residues. A 10 amino acid (A-T-V-E-A-I-E-A-D-E-A) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni2+ was found to be the strongest stimulator while Zn2+ was found to inhibit CaN phosphatase activity. Mn2+ was a relatively less effective stimulator compared to Ni2+. Fe2+ was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe2+ slightly inhibited bovine brain CaN activity. The residues at 97-201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407-456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known auto inhibitory domain at 457-480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics compared to bovine brain CaN A.

Plasma membrane calcium/calmodulin-dependent calcium ATPases (PMCAs) are high affinity calcium pumps regulating many calcium-dependent processes and advances in its characterisation have discovered that it may play a novel role in signal transduction pathways. It was the aim of this work to further characterise and confirm the role PMCA plays in regulating calcium/calmodulin-dependent signal transduction pathways. PMCA4 has already been shown to inhibit the NFAT family of transcription factors by its interaction with calcineurin A in mammalian cells when ectopically expressed. This prompted the investigation into other isoforms of PMCA that may interact with the calcium/calmodulin-dependent calcineurin, to determine if this interaction was isoform-specific in a variety of cell lines. Endogenous proteins were isolated by immunoprecipitation with calcineurin A antibody and the presence of PMCA isoforms was determined by western blot using isoform-specific antibodies. This work has demonstrated that the PMCA and calcineurin interaction occurs in vitro at endogenous levels in MCF-7 human breast adenocarcinoma cells and endothelial cells and is isoform specific, predominantly for PMCA2. The characterisation of the PMCA2b-calcineurin A interactive domain was performed and it was demonstrated that PMCA2b significantly inhibits the NFAT/calcineurin pathway. These results indicate that PMCA2 is important in regulating the calcineurin/NFAT pathway in tissues where it is highly expressed. This work also demonstrates that the Flag-tagged, characterised interaction domain of PMCA2 with calcineurin, F-PMCA(462-684) when overexpressed, can disrupt the inhibitory PMCA2/calcineurin interaction in endothelial cells and significantly increase calcineurin activity. The expression of PMCA in endothelial cells prompted the investigation of calcium/calmodulin-dependent proteins in endothelial cells as evidence for the important role of PMCA in regulating signal transduction pathways. Nitric oxide synthases have been shown to be regulated by PMCA4 in cardiac cells. To further characterise the regulation of NOS by PMCA, this work shows that there is a novel molecular interaction between endogenous eNOS and the plasma membrane calcium ATPase (PMCA) in HUVEC primary endothelial cells. PMCA2 has been identified as the major isoform interacting with eNOS in endothelial cells. The interaction between the two proteins has been mapped to the region 735-934 of eNOS and 462-684 of human PMCA2b. NO production was found to be inhibited by ectopic expression of PMCA2b in HUVEC cells. Moreover, disruption of the interaction between endogenous PMCA and eNOS by overexpression of theFlag-tagged, PMCA2b interaction domain, F-PMCA2(462-684), significantly increased NO levels in activated HUVEC endothelial cells. In summary, these results offer strong evidence for a novel functional interaction between endogenous PMCA and eNOS in endothelial cells, suggesting a role for endothelial PMCA2 as a negative modulator of eNOS activity, and, therefore, NO-dependent signal transduction pathways. Overall this is a novel discovery which clearly demonstrates that PMCA is an important regulator of calcium/calmodulin-dependent signal transduction pathways in various cell types. Parts of this work have been published; ‘Holton, M., Yang, D., Wang, W., Mohamed, T.M., Neyses, L. and Armesilla, A. (2007) The interaction between endogenous calcineurin and the plasma membrane calcium-dependent ATPase is isoform specific in breast cancer cells. FEBS letter. 581(21), 4115-4119.’ and presented at ‘The 14th congress of calcium binding proteins, La Palma, Canary Islands, Spain. 2007’ and ‘The 25th Conference of the European Society on Microcirculation (August 26-29, 2008, Budapest, Hungary).’

Since the discovery of cyclosporine in 1971, calcineurin inhibitors have played a critical role in the therapeutic suppression of the immune response. Patients receiving solid organ transplants rely heavily on these medications to prevent the acute and chronic rejection of allografted tissue. Introduction of tacrolimus, the most frequently prescribed calcineurin inhibitor, has lead to improved clinical outcomes for organ transplant recipients; however, little improvement has been noted in lung transplantation. Difficulties with current oral dosing regimens for lung transplant patients stem primarily from drug systemic toxicity, heightened risk of invasive infection, and erratic oral bioavailability. We have proposed that pulmonary delivery of a tacrolimus formulation with improved solubility can provide high lung concentrations, while limiting corresponding systemic levels associated with toxicity. Chapter 2 investigates the pulmonary administration of tacrolimus dispersion for nebulization to lung transplanted rats. Resulting lung and blood levels were determined by appropriate bioanalytical methods. Limited systemic absorption was seen after pulmonary delivery, resulting in a 50 to 1 lung to blood concentration ratio. A 28 day safety and stability evaluation of tacrolimus dispersion for nebulization was conducted in Chapter 3. Results showed no signs of toxicity in Sprague Dawley rats and proved the stability of tacrolimus powder for dispersion for 3 months. For cases of severe asthma, immunosuppression is also necessary to restore normal lungs function and is typically treated with corticosteroids. Corticosteroids, however, are well known for their untoward side effects and can prove ineffective in severe asthmatics that have developed corticosteroid resistance. Chapter 4 investigates the use of tacrolimus dispersion for nebulization for prophylactic treatment of asthma. Efficacy was determined in an asthma-induced animal model by quantification of inflammatory cells and signaling chemicals. In Chapter 5, tacrolimus powder for inhalation is investigated in a novel dry powder inhalation platform. Respirable particles are produced when bulk particles (500 [micrometer]) comprising a matrix of drug/excipient are sheared apart by a marketed inhalation device to produce particles of the appropriate geometric diameter (50 [micrometer]). Biocompatible material with brittle properties were found to produce fine particle fractions (FPF) up to 70.3% and total emitted doses (TED) higher than 95%. / text

Immunosuppressants that target the serine/threonine phosphatase calcineurin are commonly administered following organ transplantation. Their chronic use is associated with reduced insulin secretion and new onset diabetes in a subset of patients, suggestive of pancreatic β cell dysfunction. Calcineurin plays a critical role in the activation of CREB, a key transcription factor required for β cell function and survival. CREB activity in the islet is activated by glucose and cAMP, in large part due to activation of Crtc2, a critical coactivator for CREB. Previous studies have demonstrated that Crtc2 activation is dependent on dephosphorylation regulated by calcineurin. In this study, we sought to evaluate the impact of calcineurin-inhibiting immunosuppressants on Crtc2-CREB activation in the primary β cell. In addition, we further characterized the role and regulation of Crtc2 in the β cell. We demonstrate that Crtc2 is required for glucose dependent up-regulation of CREB target genes. The phosphatase calcineurin and kinase regulation by LKB1 contribute to the phosphorylation status of Crtc2 in the β cell. CsA and FK506 block glucose-dependent dephosphorylation and nuclear translocation of Crtc2. Overexpression of a constitutively active mutant of Crtc2 that cannot be phosphorylated at Ser171 and Ser275 enables CREB activity under conditions of calcineurin inhibition. Furthermore, β cells lacking Crtc2 display impaired glucose-stimulated insulin secretion and cell survival. Together, these results demonstrate that phosphorylation of Crtc2 plays a critical role in regulating CREB activity and contributes to β cell dysfunction and death caused by chronic immunosuppression.

Thesis (Ph. D.)--University of Washington, 2006. / Includes bibliographical references (leaves 180-197).

Immunosuppressants that target the serine/threonine phosphatase calcineurin are commonly administered following organ transplantation. Their chronic use is associated with reduced insulin secretion and new onset diabetes in a subset of patients, suggestive of pancreatic β cell dysfunction. Calcineurin plays a critical role in the activation of CREB, a key transcription factor required for β cell function and survival. CREB activity in the islet is activated by glucose and cAMP, in large part due to activation of Crtc2, a critical coactivator for CREB. Previous studies have demonstrated that Crtc2 activation is dependent on dephosphorylation regulated by calcineurin. In this study, we sought to evaluate the impact of calcineurin-inhibiting immunosuppressants on Crtc2-CREB activation in the primary β cell. In addition, we further characterized the role and regulation of Crtc2 in the β cell. We demonstrate that Crtc2 is required for glucose dependent up-regulation of CREB target genes. The phosphatase calcineurin and kinase regulation by LKB1 contribute to the phosphorylation status of Crtc2 in the β cell. CsA and FK506 block glucose-dependent dephosphorylation and nuclear translocation of Crtc2. Overexpression of a constitutively active mutant of Crtc2 that cannot be phosphorylated at Ser171 and Ser275 enables CREB activity under conditions of calcineurin inhibition. Furthermore, β cells lacking Crtc2 display impaired glucose-stimulated insulin secretion and cell survival. Together, these results demonstrate that phosphorylation of Crtc2 plays a critical role in regulating CREB activity and contributes to β cell dysfunction and death caused by chronic immunosuppression.

Background: Cyclosporine is an immunosuppressive agent used to treat immune-mediated and inflammatory diseases in dogs. We have developed a pharmacodynamic (PD) assay that measures interleukin-2 (IL-2) produced by activated T cells to measure the immunosuppressive effects of cyclosporine. Hypothesis/objectives: Our retrospective study extracted data from samples submitted to our laboratory to obtain descriptive statistics, to determine whether assay results predicted treatment effectiveness, and to determine whether cyclosporine formulation or breed affected PD responses. Animals: 1,110 samples were analyzed over 4 years. Methods: Extracted data was analyzed to determine whether there was a relationship between assay results and clinical control, and whether either formulation or breed affected results. Results: We found no relationship between assay results and control of signs, and found that breed did not affect results. At comparable doses, proprietary modified cyclosporine was more immunosuppressive than proprietary non-modified cyclosporine, and both proprietary and generic modified formulations had similar efficacy.

Learning and memory in the brain are thought to be dependent on synaptic plasticity. In response to sensory input, synapses can be strengthened or weakened, known as long-term potentiation or long-term depression (LTD), respectively. Transient receptor potential vanilloid 1 (TRPV1) has been shown to mediate a novel form of presynaptic LTD in hippocampal interneurons. TRPV1 is currently being heavily studied in the PNS and being targeted by pharmaceuticals for its anti-nociceptive and anti-inflammatory properties. However, much less is known regarding TRPV1 function in the CNS, including the signal mechanism mediating hippocampal LTD despite its obvious importance. Here we performed whole-cell voltage clamp electrophysiology experiments from CA1 hippocampal interneurons to identify this signaling mechanism. Because calcineurin (CaN) is reported to be linked to multiple forms of synaptic plasticity, we hypothesized that TRPV1 activates presynaptic CaN, which is required for this presynaptic LTD. In order to distinguish between presynaptic and postsynaptic CaN activity we added the specific CaN inhibitors cyclosporin A (CsA) or FK-506 to the bath to block CaN activity ubiquitously in the slice, both presynaptically and postsynaptically, and to the internal solution to block CaN only in the postsynaptic neuron. CsA or FK-506 present in the internal solution, blocking only postsynaptic CaN, showed no effect on TRPV1-dependant LTD. Bath application of CsA or FK-506, inhibiting CaN in the presynaptic neuron as well, blocked LTD elicited by both a high frequency stimulation protocol (P < 0.05) and by direct TRPV1 activation with specific agonists resiniferotoxin and capsaicin (P < 0.05). This demonstrates that CsA and FK506 block both high frequency stimulation induced LTD and also TRPV1 specific depression. We are thus able to show that calcineurin is required for this form of presynaptic TRPV1 mediated LTD in the hippocampus. This finding is the first to demonstrate a TRPV1-induced signaling mechanism in CA1 hippocampus.

No / The Notch and Calcineurin/NFAT pathways have both been implicated in control of keratinocyte differentiation. Induction of the p21WAF1/Cip1 gene by Notch 1 activation in differentiating keratinocytes is associated with direct targeting of the RBP-J¿ protein to the p21 promoter. We show here that Notch 1 activation functions also through a second Calcineurin-dependent mechanism acting on the p21 TATA box-proximal region. Increased Calcineurin/NFAT activity by Notch signaling involves downregulation of Calcipressin, an endogenous Calcineurin inhibitor, through a HES-1-dependent mechanism. Besides control of the p21 gene, Calcineurin contributes significantly to the transcriptional response of keratinocytes to Notch 1 activation, both in vitro and in vivo. In fact, deletion of the Calcineurin B1 gene in the skin results in a cyclic alopecia phenotype, associated with altered expression of Notch-responsive genes involved in hair follicle structure and/or adhesion to the surrounding mesenchyme. Thus, an important interconnection exists between Notch 1 and Calcineurin-NFAT pathways in keratinocyte growth/differentiation control.