cyclic-gmp and beta-glycerophosphoric-acid

The aim of the present study was to investigate the changes in heme-oxygenase (HO)-carbon monoxide (CO)-cyclic guanosine monophosphate (cGMP) pathway in clacified rat vascular smooth muscle cells (VSMCs).. Calcification of cultured rat VSMCs was induced by incubation of VSMCs with beta-glycerophosphate. Cellular calcium content, ALP activities and (45)Ca uptake were measured. HO activity, HbCO formation and content of cGMP in VSMCs were determined. Immunocytochemistry for HO-1 expression was observed.. In comparison of control VSMCs, the cellular calcium content, ALP activity and (45)Ca uptake in calcified VSMCs were obviously increased. Immunocytochemistry showed that HO-1 expression was weak and not well distributed in calcified cells as compared to non-calcified VSMCs, but interestingly, there was stronger staining in calcified nodules than in VSMCs. Compared with VSMCs, HO-1 activity in calcified cells decreased by 42.7% [36.4 +/- 2.8 pmol (mg Pr x h)(-1) vs 63.5 x 5.3 pmol (mg Pr x h)(-1), p < 0.01], and HbCO formation decreased by 39.2% (3.38 x 0.69 micromol/mg Pr vs 5.56 +/- 0.48 micromol/mg Pr, p < 0.05). The cGMP content in calcified VSMCs was 78.1% lower than that of non-calcified VSMCs (4.3 +/- 0.51 vs 19.6 +/- 1.2 pmol/mg, p < 0.01).. The results showed that HO-CO-cGMP pathway in calcified vascular cells obviously changed, which might contribute to disturbance of vascular function.

Topics: Animals; Calcinosis; Calcium; Carbon Monoxide; Carboxyhemoglobin; Cell Differentiation; Cells, Cultured; Cyclic GMP; Glycerophosphates; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Osteoblasts; Rats; Rats, Inbred Strains

To clarify the regulating mechanism of vascular calcification, the investigators observed the effects of three vasoactive peptides, adrenomedullin (ADM), C-type natriuretic peptide (CNP), and parathyroid hormone-related peptide (PTHrP) on calcification in rat vascular smooth muscle cells (VSMCs). Beta-glycerophosphate stimulated growth and calcification in VSMCs. Adrenomedullin and CNP lowered beta-glycerophosphate-induced increase in VSMC growth. All three vasoactive peptides attenuated the increases of 45Ca accumulation, calcium content, and alkaline phosphatase activity in calcified VSMCs. As for comparing the inhibitory effects, the strongest was PTHrP. Both ADM and PTHrP increased cyclic adenosine monophosphate (cAMP) content in calcified VSMCs, but CNP upregulated cyclic guanosine monophosphate (cGMP) content. The PKA inhibitor PKAI completely reversed the inhibition of ADM on cell growth and all inhibitory effects of PTHrP on the parameters of calcification. The PKG inhibitor H8, however, strongly antagonized all the inhibitory effects of CNP on calcification. These data suggested that beta-glycerophosphate-induced calcification in VSMCs was inhibited by ADM, CNP, and PTHrP. Adrenomedullin and PTHrP inhibited VSMC calcification partially through the cAMP/PKA pathway, whereas CNP inhibited VSMC calcification through the cGMP/PKG pathway. This study could be of help in understanding the pathogenesis of vascular calcification, and providing new target for clinical treatment of cardiovascular diseases associated with vascular calcification.

Topics: Adrenomedullin; Alkaline Phosphatase; Animals; Aorta; Calcinosis; Calcium; Cell Count; Cell Division; Cells, Cultured; Culture Media; Cyclic AMP; Cyclic GMP; Glycerophosphates; Male; Muscle, Smooth, Vascular; Natriuretic Peptide, C-Type; Parathyroid Hormone-Related Protein; Peptides; Rats; Rats, Sprague-Dawley

Involvement of protein kinases in the stimulation of cGMP-inhibited cAMP phosphodiesterase (PDE) activity by orthovanadate (vanadate) was studied. When the fat pads were incubated with 2 mM vanadate or 10 nM insulin, the stimulation of myelin basic protein kinase (MBPK) activity in the particulate by vanadate reached a maximum at 60 min. In contrast, insulin showed a transient increase at 20 min. A 60-min incubation of the fat pads with vanadate stimulated all activities of protein tyrosine kinase (PTK), MBPK, and PDE in the particulate, in a similar dose-dependent manner. Amiloride, a PTK inhibitor, inhibited the stimulations of three enzymes by vanadate in a similar concentration range. Enzyme fractions, which were separated from the solubilized particulate, were subjected to the immunoblot analysis. A fraction of MBPK was identified to contain a major protein of mol wt (44K) and a minor one (42K), both of which are immunoreactive with a mitogen-activated protein kinase (MAPK) antibody. The partially purified PDE activity was stimulated by the addition of the partially purified MBPK. The further stimulation was observed with the PTK-activated MBPK. These results suggest that vanadate stimulates in part the PDE activity through the activation of the particulate MBPK, probably MAPKs, by PTK sensitive to vanadate.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adipose Tissue; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cyclic GMP; Enzyme Activation; Fluorides; Glycerophosphates; Glycogen Synthase Kinase 3; Male; Models, Biological; Potassium Compounds; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Vanadates