PubMed · 611849
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J Bommer, V Stossberg, H Schmidt-Gayk, J Hackeng. [Parathyroid gland function in hyperthyroidism].. https://pubmed.ncbi.nlm.nih.gov/611849/
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We have investigated Ca2+-induced exocytosis from human neutrophils using the whole cell patch-clamp capacitance technique. Microperfusion of Ca2+ buffer solutions (<30 nM to 5 mM free Ca2+) through the patch-clamp pipette revealed a biphasic activation of exocytosis by Ca2+. The first phase was characterized by high affinity (1.5-5 microM) and low apparent cooperativity (<=2) for Ca2+, and the second phase by low affinity (approximately 100 microM) and high cooperativity (>6). Only the second phase was accompanied by loss of myeloperoxidase, suggesting that the low-affinity exocytosis reflected release of peroxidase-positive (primary) granules, while the high-affinity exocytosis reflected release of peroxidase-negative (secondary and tertiary) granules. At submaximal Ca2+ concentrations, only a fraction of a given granule population was released. This submaximal release cannot simply be explained by Ca2+ modulation of the rate of exocytosis, and it suggests that the secretory response of individual cells is adjusted to the strength of the stimulus. The Ca2+ dependence of the high- and low-affinity phases of neutrophil exocytosis bears a resemblance to endocrine and neuronal exocytosis, respectively. The occurrence of such high- and low-affinity exocytosis in the same cell is novel, and suggests that the Ca2+ sensitivity of secretion is granule-, rather than cell-specific.
The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H2O2), compound I, contains a porphyrin pi-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the tryptophan residue (Trp-191) and oxidizes the redox partner, cytochrome c. To investigate the roles of the amino acid residue near the heme active center in discriminating the function of the peroxidases in these two enzymes, we prepared a CcP-like HRP mutant, F221W (Phe-221 --> Trp). Although the rapid spectral scanning and stopped-flow experiments confirmed that the F221W mutant reacts with H2O2 to form the porphyrin pi-cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin pi-cation radical disappeared rapidly, and were converted to the compound II-type spectrum. The EPR spectrum of the resultant species produced by reduction of the porphyrin pi-cation radical, however, was quite different from that of compound II in HRP, showing typical signals from a Trp radical as found for CcP. The sequential radical formation from the porphyrin ring to the Trp residue implies that the proximal Trp is a key residue in the process of the radical transfer from the porphyrin ring, which differentiates the function of peroxidases.
Activation of the mitogen-activated protein kinase (Erk) and c-Jun terminal kinase is a well-documented mechanism for the seven transmembrane spanning receptors. We have previously shown that thrombin stimulation of the T-leukemic cell line Jurkat induced a transient increase in [Ca2+]i and tyrosine phosphorylation of several cellular proteins. Here, we have analyzed p42-44 MAPK, JNK and p38 MAPK activation using Jurkat T-cell lines deficient in either the tyrosine kinase p56Lck (JCaM1) or the tyrosine phosphatase CD45 (J45.01). Our results demonstrate that p56Lck and CD45 exert a negative control on thrombin-induced p38 MAPK activation and [Ca2+]i release in Jurkat cells. Thrombin receptor expression was identical on the different cell lines as assessed by FACS analysis. Tyrosine phosphorylation of p38 MAPK was drastically increased after thrombin stimulation of JCaM1 or J45.01 cells, as compared with parental cells (JE6.1). P42-44 MAPK and JNK activity also enhanced after thrombin treatment of JE6.1 and JCaM1 cell lines, whereas basal kinase activity was higher in J45.01 cells and was not further stimulated by thrombin. Thrombin and thrombin receptor agonist peptide-induced [Ca2+]i mobilization paralleled p38 MAPK activation in JCaM1 and J45.01 cells. Moreover, reconstitution of J45.01 and JCaM1 cell lines with either CD45 or Lck is accompanied by restoration of a normal thrombin-induced [Ca2+]i response and p38MAPK phosphorylation. These data show that a component of the T-cell receptor signaling pathway exerts a negative control on thrombin-induced responses in Jurkat T cells. Accordingly, we found that thrombin enhanced tyrosine phosphorylation of p56Lck and decreased p56Lck kinase activity in J45.01 cells. Our results are consistent with a negative role for p56Lck on thrombin-induced [Ca2+]i release and p38 MAPK activation in Jurkat T-cell lines.