| Research Abstract |
1. Effect of Atrial Natriuretic Peptide on Cytosolic Free Calcium
We developed the method of measuring intracellular calcium using fluorescent calcium indicators (fura-2 & indol). Atrial natriuretic peptide (ANP) decreased either the resting level of intracellular calcium or sustained increases in cytosolic calcium induced by vasoconstrictive hormones, angiotensin II and vasopressin. ANP also decreased an increase in intracellular calcium evoked by high potassium depolarization. On the other hand, the initial increases in cytosolic free calcium induced by the hormones were not inhibited by ANP. Calcium antagonists, nicardipine and nifedipine, inhibited the increase in cytosolic free calcium induced by high potassium depolarization, whereas they did not affect increases in intracellular calcium brought about by angiotensin II and vasopressin. The results indicate that ANP can decrease intracellular calcium level and it is suggested that ANP stimulates a calcium-extrusion active transport
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in vascular smooth muscle cells.
2. Intracellular Compartmetalization of Fura-2 Demonstrated by Laser-Excitation Fluorescence Microscopy
When we examined cytosolic free calcium level in individual cell using laser-excitation fluorescence microscopy, we observed inhomogenous distribution of fura-2 dye. Subcellurar fluorescence of fura-2 dye appeared spotty or filamentous and resembled in shapes the intracellular organelles. Using either an analysis by high performance liquid chromatography or fluorescence spectra analysis, we examined fura-2/AM metabolism in smooth muscle cells. It has been shown that no other than fura-2 was found in the soluble fraction of the cell lysates, whereas membrane fraction contains fura2/AM or its unidentified lipophilic metabilites, In conclusion, we should take into account these problems ( intracellular compartmentalization of fura-2 or fura-2 metabolism ) in measuring intracellular calcium level in individual vascular smooth muscle cell.
3. Separation of Inositol phosphates by High Performance Liqu Chromatography : Effect of Bradykinin.
We observed bradykinin-induced increase in cytosolic free vascular smooth muscle cells. In order to examine the mechanis we examined the effect of bradykinin on phosphoinositide hydrol sis. We newly developed the method of separation of inosit phosphates using high pressure liquid chromatography. By t method, we separated isomers of inositol monophosphate, isome of inositol bisphosphate, inositol (1,3,4)trisphosphate a inositol ( 1, 4, 5 ) trisphosphate, inositol (1, 3, 4, 5 ) tetrakisphosphate, inositol pentakisphosphate and inosit hexakisphosphate, Bradykinin stimulated rapid formation inositol (1, 4, 5) trisphosphate, followed by formation of inosit tetrakisphosphate and inositol (1, 3, 4) trisphosphate. It idicat that bradykinin induces an increase in cytosolic free calci mediated by inositol (1, 4, 5) trisphosphate
4. Relationship between Bradykinin-Induced Increase in Calci and Prostaglandin Synthesis.
Bradykinin stimulates an increase in cytosolic free calcium vascular smooth muscle cells. Bradykinin also stimulate prostacyclin synthesis in the cells. To clarify the ti courserelationship between prostacyclin synthesis and calc increase induced by bradykinin, we developed a perfused monolay syst
em which enables to monitor simulataneously the level cytosolic calcium ( using fura-2 method ) and proxtacycl production in the perfusion solution. The result clearly show that bradykinin simultaneously induces an increase in cytosol free calcium and prostacyclin synthesis. It is suggested the calcium is supplied by bradykinin (mediated by phoshpholipase pathway) to phospholipase A which is ready to be activated by t bradykinin-receptor complex, resulting in prostacycin synthesi Less
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