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H R de Jonge

Publications and source records attributed to H R de Jonge.

69 records · Page 4Linked to original sources
PubMed1980

Separation of basolateral plasma membranes from smooth endoplasmic reticulum of the rat enterocyte by zonal electrophoresis on density gradients.

Basolateral plasma membranes of rat small intestinal epithelium were purified by density gradient centrifugation followed by zonal electrophoresis on density gradients. Crude basolateral membranes were obtained by centrifugation in which the marker enzyme, (Na+ + K+)-ATPase, was enriched 10-fold with respect to the initial homogenate. The major contaminant was a membrane fraction derived from smooth endoplasmic reticulum, rich in NADPH-cytochrome c reductase activity. The crude basolateral membrane preparation could be resolved into the two major components by subjecting it to zonal electrophoresis on density gradients. The result was that (Na+ + K+)-ATPase was purified 22-fold with respect to the initial homogenate. Purification with respect to mitochondria and brush border membranes was 35- and 42-fold, respectively. Resolution of (Na+ + K+)-ATPase from NADPH-cytochrome c reductase by electrophoresis was best with membrane material from adult rats between 180 and 250 g. No resolution between the two marker enzymes occurred with material from young rats of 125 to 140 g. These results demonstrate that zonal electrophoresis on density gradients, a simple and inexpensive technique, has a similar potential to free-flow electrophoresis.

Animals↗
PubMed1977

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.

Adenosine Diphosphate↗
PubMedDate not supplied

Signalling by cGMP-dependent protein kinases.

The second messenger cGMP is a major intracellular mediator of the vaso-active agents nitric oxide and natriuretic peptides. The principal targets of cGMP are (i) phosphodiesterases, resulting in interference with the cAMP-signalling pathway, (ii) cGMP-gated cation channels, and (iii) cGMP-dependent protein kinases (cGKs). Only two mammalian isotypes of cGK have been described so far: type I cGK, consisting of an alpha and a beta isoform, presumably splice variants of a single gene, and identified as the most prominent cGK isotype in the cardio-vascular system; and type II cGK, expressed mainly in the intestine, the kidney and the brain. High levels of cGK I are found in vascular smooth muscle cells, endothelial cells and platelets. In these cells, cGK I is thought to counteract the increase in contraction provoked by Ca-mobilizing agonists, to reduce endothelial permeability and to inhibit platelet aggregation, respectively. Relatively low levels of cGK I are found in cardiomyocytes. In this cell type, cGK is implicated in the negative inotropic effect of cGMP, presumably through modulation of Ca channels and by diminishing the Ca-sensitivity of contractile proteins.

Animals↗
PubMedDate not supplied

Expression and regulation of chloride channels in neonatal rat cardiomyocytes.

Using an 125I- efflux assay, we have studied the expression of various types of chloride channels in isolated neonatal rat cardiomyocytes. Three different classes of anion conductances were distinguished: (1) a Ca(2+)-sensitive Cl- conductance, triggered upon stimulation of the cells with endothelin-1 or Ca(2+)-ionophore; (2) a cAMP/protein kinase A-operated Cl- conductance, activated by addition of forskolin. This anion channel could be identified as the Cystic Fibrosis Transmembrane conductance Regulator (CFTR-CI- channel) by Western blotting as well as by its enhanced activity in cultures pretreated with the tyrosine kinase inhibitor genistein; (3) a distinct class of cell volume-regulated Cl- channels, potentiated in the presence of endothelin-1 or the phosphotyrosine phosphatase inhibitor pervanadate. The potential role of each class of Cl- channels in the generation and/or modulation of action potentials as well as in maintaining cell volume is discussed.

1-Methyl-3-isobutylxanthine↗
PubMedDate not supplied

Regulation of the cell swelling-activated chloride conductance by cholesterol-rich membrane domains.

AIM: The role of high cholesterol-containing microdomains in the signal transduction cascade leading to the activation of volume-regulated anion channels (VRACs) was studied. METHODS: Osmotic cell swelling-induced efflux of 125I- was determined in human epithelial Intestine 407 cells and in skin fibroblasts obtained from healthy controls or Niemann-Pick type C (NPC) patients. Cellular cholesterol content was modulated by pre-incubation with 2-hydroxypropyl-beta-cyclodextrin in the presence of acceptor lipid vesicles. RESULTS: Osmotic cell swelling of human Intestine 407 cells leads to the rapid activation of a compensatory anion conductance. Treatment of the cells with cyclodextrin enhanced the response to submaximal hypotonic stimulation by approx. twofold, but did not further increase the efflux elicited by a saturating stimulus. In contrast, the volume-sensitive anion efflux was markedly inhibited when cholesterol-loaded cyclodextrin was used. Potentiation of the response by cholesterol depletion was maintained in caveolin-1 deficient Caco-2 colonocytes as well as in sphingomyelinase-treated Intestine 407 cells, indicating that cholesterol-rich microdomains are not crucially involved. However, treatment of the cells with progesterone, an inhibitor of NPC1-dependent endosomal cholesterol trafficking, not only markedly reduced the hypotonicity-provoked anion efflux, but also prevented its potentiation by cyclodextrin. In addition, the volume-sensitive anion efflux from human NPC skin fibroblasts was significantly smaller when compared with control fibroblasts. CONCLUSIONS: The results support a model of regulatory volume decrease involving recruitment of volume-sensitive anion channels from intracellular compartments to the plasma membrane.

2-Hydroxypropyl-beta-cyclodextrin↗