Exogenous C-type natriuretic peptide restores normal growth and prevents early growth plate closure in its deficient rats.

Signaling by C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B, is a pivotal stimulator of endochondral bone growth. We recently developed CNP knockout (KO) rats that exhibit impaired skeletal growth with early growth plate closure. In the current study, we further characterized the phenotype and growth plate morphology in CNP-KO rats, and the effects of exogenous CNP in rats. We used CNP-53, an endogenous form of CNP consisting of 53 amino acids, and administered it for four weeks by continuous subcutaneous infusion at 0.15 or 0.5 mg/kg/day to four-week old CNP-KO and littermate wild type (WT) rats. We demonstrated that CNP-KO rats were useful as a reproducible animal model for skeletal dysplasia, due to their impairment in endochondral bone growth. There was no significant difference in plasma bone-turnover markers between the CNP-KO and WT rats. At eight weeks of age, growth plate closure was observed in the distal end of the tibia and the calcaneus of CNP-KO rats. Continuous subcutaneous infusion of CNP-53 significantly, and in a dose-dependent manner, stimulated skeletal growth in CNP-KO and WT rats, with CNP-KO rats being more sensitive to the treatment. CNP-53 also normalized the length of long bones and the growth plate thickness, and prevented growth plate closure in the CNP-KO rats. Using organ culture experiment of fetal rat tibia, gene set enrichment analysis indicated that CNP might have a negative influence on mitogen activated protein kinase signaling cascades in chondrocyte. Our results indicated that CNP-KO rats might be a valuable animal model for investigating growth plate physiology and the mechanism of growth plate closure, and that CNP-53, or its analog, may have the potential to promote growth and to prevent early growth plate closure in the short stature.

Involvement of MCP-1 in tubulointerstitial fibrosis through massive proteinuria in anti-GBM nephritis induced in WKY rats.

We investigated participation of monocyte chemoattractant protein-1 (MCP-1) in tubulointerstitial fibrosis and correlation between MCP-1 and proteinuria in Wistar-Kyoto (WKY) rats with glomerulonephritis induced by anti-glomerular basement membrane (anti-GBM) antibody. WKY rats showed marked proteinuria and severe glomerular crescent formation at 7 days post antibody injection. At 28 days, tubulointerstitial fibrotic lesions were observed, followed by sustained heavy proteinuria and severe tubulointerstitial fibrosis at 56 days. Histological examination revealed that the overlapped immunoreactivities of MCP-1, rat albumin, and p65NF-kappaB were detected in the same tubular segments of nephritic kidney, and a significant positive correlation was observed between proteinuria and MCP-1 expression in the tubulointerstitial fibrosis. ED-1- and CD8-positive cells were also abundant, and there was a good correlation between monocyte/macrophage recruitment and MCP-1 expression in the tubulointerstitial area. These results suggest that MCP-1 participates in the progression of tubulointerstitial fibrosis, through massive albuminuria, which is accompanied by marked monocyte/macrophage recruitment.

Voltage-sensitive oxonol dyes are novel large-conductance Ca2+-activated K+ channel activators selective for beta1 and beta4 but not for beta2 subunits.

The large-conductance Ca(2+)-activated K(+) (BK) channel is activated by both the increase of intracellular Ca(2+) concentration and membrane depolarization. The BK channel plays crucial roles as a key molecule in the negative feedback mechanism regulating membrane excitability and cellular Ca(2+) in various cell types. Here, we report that a widely used slow-response voltage-sensitive fluorescent dye, bis(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(4)(3)], is a potent BK channel activator. The application of DiBAC(4)(3) at concentrations of 10 nM and higher significantly increased whole-cell BK channel currents in human embryonic kidney 293 cells expressing rat BK channel alpha and beta1 subunits (rBKalphabeta1). In the presence of 300 nM DiBAC(4)(3), the activation voltage of the BK channel current shifted to the negative direction by approximately 30 mV, but the single-channel conductance was not affected. DiBAC(4)(3) activated whole-cell rBKalphabeta1 and rBKalphabeta4 currents in the same concentration range but partially blocked rBKalphabeta2 currents. The BK channel alpha subunit alone and some other types of K(+) channels examined were not markedly affected by 1 microM DiBAC(4)(3). Structure-activity relationship analyses revealed that a set of oxo- and oxoanion-moieties in two 1,3-dialkylbarbituric acids, which are conjugated by oligomethine, is the novel skeleton for the beta-subunit-selective BK channel-opening property of DiBAC(4)(3) and related oxonol compounds. This conjugated structure may be located stereochemically in one plane. These findings provide a molecular and structural basis for understanding the regulatory mechanism of BK channel activity by an auxiliary beta subunit and will be fundamental to the development of beta-selective BK channel openers.

Activation of large-conductance, Ca2+-activated K+ channels by cannabinoids.

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca2+-activated K+ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the alpha-subunit of the BK channel (BK-alpha), both alpha- and beta1-subunits (BK-alphabeta1), or both alpha- and beta4-subunits (BK-alphabeta4) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-alphabeta1 within a similar concentration range. Because methAEA could potentiate BK-alpha, BK-alphabeta1, and BK-alphabeta4 with similar efficacy, the beta-subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK-alpha and HEK-BK-alphabeta1 did not change intracellular Ca2+ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB1 antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB2 agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed.