Beta-adrenergic regulation of the heart expressing the Ser1700A/Thr1704A mutated Cav1.2 channel.

Beta-adrenergic stimulation of the heart increases ICa. PKA dependent phosphorylation of several amino acids (among them Ser 1700 and Thr 1704 in the carboxy-terminus of the Cav1.2 α1c subunit) has been implicated as decisive for the ß-adrenergic up-regulation of cardiac ICa. Mutation of Ser 1700 and Thr 1704 to alanine results in the Cav1.2PKA_P2-/- mice. Cav1.2PKA_P2-/- mice display reduced cardiac L-type current. Fractional shortening and ejection fraction in the intact animal and ICa in isolated cardiomyocytes (CM) are stimulated by isoproterenol. Cardiac specific expression of the mutated Cav1.2PKA_P2-/- gene reduces Cav1.2 α1c protein concentration, ICa, and the ß-adrenergic stimulation of L-type ICa in CMs. Single channels were not detected on the CM surface of the cCav1.2PKA_P2-/- hearts. This outcome supports the notion that S1700/1704 is essential for expression of the Cav1.2 channel and that isoproterenol stimulates ICa in Cav1.2PKA_P2-/- CMs.

Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida.

Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in Escherichia coli each of the woodrat proteins gave the characteristic maximum at 450nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system.

Thrombocytosis as a response to high interleukin-6 levels in cGMP-dependent protein kinase I mutant mice.

OBJECTIVE: The purpose of this study was to investigate the influence of cGMP-dependent kinase I (cGKI) on platelet production. APPROACH AND RESULTS: We used hematology analyser to measure platelet counts in conventional cGKI-null mutants (cGKI(L1/L1)), gene-targeted cGKIα/ß rescue mice (referred to as cGKI-smooth muscle [SM]) in which cGKI expression is specifically restored only in SM, platelet factor 4-Cre(tg/+); cGKI(L2/L2) mice in which the cGKI protein was specifically deleted in the megakaryocyte/platelet lineage and cGKI-deficient bone marrow-chimeras. Thrombocytosis was detected in cGKI(L1/L1) and in cGKI-SM. In contrast, neither platelet factor 4-Cre(tg/+); cGKI(L2/L2) nor cGKI-deficient bone marrow-chimeras displayed a thrombocytosis phenotype, indicating that the high platelet count in cGKI(L1/L1) and cGKI-SM mutants is attributable to loss of an extrinsic signal rather than reflecting an intrinsic defect in megakaryopoiesis. Cytometric analyses further showed that stimulation of bone marrow-derived wild-type megakaryocytes in vitro using serum preparations obtained from cGKI-SM mutants strongly accelerated megakaryopoiesis, suggesting that the high platelet count develops in response to serum factors. Indeed, using ELISA assay, we found elevated levels of interleukin-6, a known stimulator of thrombopoiesis, in cGKI-SM mutant serum, whereas interleukin-6 levels were unaltered in platelet factor 4-Cre(tg/+); cGKI(L2/L2) mice and cGKI-deficient bone marrow-chimeras. Accordingly, antibody-mediated blockade of interleukin-6 normalized platelet counts in cGKI-SM mice. CONCLUSIONS: Abnormal cGMP/cGKI signaling in nonhematopoietic cells affects thrombopoiesis via elevated interleukin-6 production and results in thrombocytosis in vivo. Dysfunction of cGMP/cGKI signaling in nonhematopoietic cells contributes to a high platelet count, which is potentially associated with thrombosis.