Influence of glycosylation inhibitors on dihydropyridine binding to cardiac cells

In primary cultures of neonatal rat heart cells we found a linear correlation between the number of L-type calcium channel-specific dihydropyridine (DHP) binding sites and spontaneous beating frequency (v).Formation of glycoproteins in tissue culture was suppressed by different inhibitors of N-glycosylation. This inhibition alters to a different extent the binding of the DHP ligand (+)-[methyl-³H]PN 200-110 and v. The most severe but reversible effect occurs at 6 g/ml tunicamycin (B_max 45% and v 6%, resp., of control), a slight increase in B_max at 0.1–0.5 mM castanospermine and 0.05–2.5 mM deoxymannojirimycin. The other inhibitors gave no significant alterations of B_max.     

Primary cultures of cardiac muscle cells as models for investigation of protein glycosylation

Primary cardiac cell cultures of newborn rats containing approximately 50% (by cell number) spontaneously contracting cardiomyocytes were used to study the role of protein N-glycosylation for the binding of dihydropyridine (DHP) to the voltage-dependent L-type calcium channel. This binding is not influenced by the accompanying non-muscle cells.Exposure of the cells up to 6 g/ml of the N-glycosylation inhibitor tunicamycin for a 44 h period resulted in a decrease of the specific DHP binding sites (B_max) to 46.0 ± 17.2% of the untreated control. Similar effects were observed after enzymatic deglycosylation using N-glycosidase F (PNGase F). The results suggest that a posttranslational modification of parts of the cardiac L-type Ca⁺⁺ channel by N-glycosylation is an important determinant for the binding of Ca⁺⁺ antagonists of the DHP-type to the ₁ subunit which itself is not glycosylated. The results suggest a participation of N glycosylation in the assembling of the subunits to the functional channel and/or its turnover. However, a possible effect of tunicamycin on the expression of the Ca channel as an alternative mechanism cannot be excluded.     

Characterization of endogenous phosphorylation in isolated cardiac sarcolemma

The cardiac sarcolemma contains kinases which catalyze the incorporation of 32P-phosphate into acid stable and acid precipitable membrane components of low molecular weight. The phosphorylation is not influenced by cyclic AMP or calmodulin. Analysis of phosphorylation products using proteolytic digestion, organic solvent extraction, thin layer chromatography and gel filtration reveals both polypeptides and lipids as kinase substrates. Polypeptides are phosphorylated at their serine and threonine residues, while lipid phosphorylation gives rise to 32P-labelled phosphatidylinositol phosphates and some nonidentified compounds. Phosphorylated polypeptides and phosphorylated lipids do not separate in SDS polyacrylamide gel electrophoresis. On the basis of the fast time course of 32P-phosphate incorporation, it may be supposed that endogenous phosphorylation may play a role in the short term regulation of the cardiac sarcolemmal function.     

Microbiological study of lyophilized dairy kefir

Preservation of the intrinsic inhibitory power of dairy kefir after lyophilization by testing several desiccation substrates and several regeneration media was studied. The tested cryoprotectors were ribitol, sodium glutamate and glycerol. After lyophilization, the pellets of kefir were regenerated in water or milk. Glycerol was the best cryoprotector because of its high efficiency and its low cost, white sodium glutamate was unsatisfactory.