PubMed · 8965745
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1996-12-06. Calcium supplements.. https://pubmed.ncbi.nlm.nih.gov/8965745/
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Increasing evidence suggests that a continuous release of histamine from mast cells occurs in the airways of asthmatic patients and that histamine may modulate functions of other inflammatory cells such as macrophages. In the present study histamine (10(-9)-10(-6) M) increased in a concentration-dependent fashion the basal release of beta-glucuronidase (EC(50) = 8.2 +/- 3.5 x 10(-9) M) and IL-6 (EC(50) = 9.3 +/- 2.9 x 10(-8) M) from human lung macrophages. Enhancement of beta-glucuronidase release induced by histamine was evident after 30 min and peaked at 90 min, whereas that of IL-6 required 2-6 h of incubation. These effects were reproduced by the H(1) agonist (6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptane carboxamide but not by the H(2) agonist dimaprit. Furthermore, histamine induced a concentration-dependent increase of intracellular Ca(2+) concentrations ([Ca(2+)](i)) that followed three types of response, one characterized by a rapid increase, a second in which [Ca(2+)](i) displays a slow but progressive increase, and a third characterized by an oscillatory pattern. Histamine-induced beta-glucuronidase and IL-6 release and [Ca(2+)](i) elevation were inhibited by the selective H(1) antagonist fexofenadine (10(-7)-10(-4) M), but not by the H(2) antagonist ranitidine. Inhibition of histamine-induced beta-glucuronidase and IL-6 release by fexofenadine was concentration dependent and displayed the characteristics of a competitive antagonism (K(d) = 89 nM). These data demonstrate that histamine induces exocytosis and IL-6 production from human macrophages by activating H(1) receptor and by increasing [Ca(2+)](i) and they suggest that histamine may play a relevant role in the long-term sustainment of allergic inflammation in the airways.
The effect of fendiline, an anti-anginal drug, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteosarcoma cells was explored by using fura-2 as a Ca(2+) indicator. Fendiline at concentrations between 1 and 200 microM increased [Ca(2+)](i) in a concentration-dependent manner and the signal saturated at 100 microM. The Ca(2+) signal was inhibited by 65+/-5% by Ca(2+) removal and by 38+/-5% by 10 microM nifedipine, but was unchanged by 10 microM La(3+) or verapamil. In Ca(2+)-free medium, pre-treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) store inhibited fendiline-induced intracellular Ca(2+) release. The Ca(2+) release induced by 50 microM fendiline appeared to be independent of IP(3) because the [Ca(2+)](i) increase was unaltered by inhibiting phospholipase C with 2 microM U73122. Collectively, the results suggest that in MG63 cells fendiline caused an increase in [Ca(2+)](i) by inducing Ca(2+) influx and Ca(2+) release in an IP(3)-independent manner.
To visualize Ca(2+)-dependent protein-protein interactions in living cells by fluorescence readouts, we used a circularly permuted green fluorescent protein (cpGFP), in which the amino and carboxyl portions had been interchanged and reconnected by a short spacer between the original termini. The cpGFP was fused to calmodulin and its target peptide, M13. The chimeric protein, which we have named "pericam," was fluorescent and its spectral properties changed reversibly with the amount of Ca(2+), probably because of the interaction between calmodulin and M13 leading to an alteration of the environment surrounding the chromophore. Three types of pericam were obtained by mutating several amino acids adjacent to the chromophore. Of these, "flash-pericam" became brighter with Ca(2+), whereas "inverse-pericam" dimmed. On the other hand, "ratiometric-pericam" had an excitation wavelength changing in a Ca(2+)-dependent manner. All of the pericams expressed in HeLa cells were able to monitor free Ca(2+) dynamics, such as Ca(2+) oscillations in the cytosol and the nucleus. Ca(2+) imaging using high-speed confocal line-scanning microscopy and a flash-pericam allowed to detect the free propagation of Ca(2+) ions across the nuclear envelope. Then, free Ca(2+) concentrations in the nucleus and mitochondria were simultaneously measured by using ratiometric-pericams having appropriate localization signals, revealing that extra-mitochondrial Ca(2+) transients caused rapid changes in the concentration of mitochondrial Ca(2+). Finally, a "split-pericam" was made by deleting the linker in the flash-pericam. The Ca(2+)-dependent interaction between calmodulin and M13 in HeLa cells was monitored by the association of the two halves of GFP, neither of which was fluorescent by itself.