Biomedical subjects
Publications and source records attributed to A Wada.
The "elution-band relaxation method" has been applied to the analysis of the cis-trans kinetics isomerization of the proline of L-Ala-L-Pro using reversed-phase liquid chromatography. A procedure suitable for cases where neither isomer can be injected separately is described.
To clarify the signal transduction mechanism of angiotensin II in renal glomeruli, we studied the effect of the hormone on phospholipid metabolism using isolated rat glomeruli. Stimulation of the glomeruli pulse-chase labeled with [3H]glycerol by angiotensin II caused a rapid (within 15 s) breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) with a concurrent production of 1,2-diacylglycerol. This effect of angiotensin II was in a dose-dependent manner within the range from 10(-12) M to 10(-6) M, and was inhibited by saralasin. Angiotensin II also decreased the 3H radioactivity of PIP slightly only at 15 s and increased that of phosphatidic acid after 15 s, with no significant effect upon the labelings of phosphatidylinositol (PI), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) within 1 min. The change in phospholipid metabolism by angiotensin II was similar when the glomeruli were labeled with [32P]orthophosphate: the decrease in the labeling of PIP2 and the increase in the labeling of phosphatidic acid after 15 s. In addition, 32P labeling of PI increased after 2 min. These results suggest that angiotensin II, after binding to glomerular receptors, induces initial PIP2 hydrolysis to diacylglycerol and subsequent resynthesis of PIP2 through phosphoinositide turnover.
Angiotensin II-induced change in inositol phosphates were studied in cultured rat mesangial cells prelabeled with [3H]myo-inositol. By using anion-exchange high performance liquid chromatography, we could analyzed the change in inositol mono-, bis-, and tris-phosphate more rapidly and easily with higher resolution than the previously reported methods. Angiotensin II rapidly increased inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate within 15 sec, followed by an increase in inositol 1-monophosphate at 30 sec. Angiotensin II-induced increases in inositol phosphates were dose-dependent and completely blocked by saralasin. These results indicate that angiotensin II induces the production of inositol phosphates including inositol 1,4,5-trisphosphate, an intracellular Ca2+-releasing factor, in cultured rat mesangial cells.
Incubation of 11-deoxycorticosterone with a cytochrome P-450(11)beta-reconstituted system yielded, in addition to corticosterone and 18-hydroxy-11-deoxycorticosterone, a new steroid product. The retention time of the new product was identical with that of authentic 19-hydroxy-11-deoxycorticosterone on high performance liquid chromatography (HPLC). The turnover number of 19-hydroxy-11-deoxycorticosterone formation was 7.0 mol/min/mol P-450. When a large amount of cytochrome P-450(11)beta was used for the reaction and the products were analyzed by HPLC, the 19-hydroxy-11-deoxycorticosterone peak disappeared from the chromatogram and concomitantly new unidentified peaks appeared. These results suggest that 19-hydroxy-11-deoxycorticosterone was further metabolized to other steroids by cytochrome P-450(11)beta. Therefore, we next incubated 19-hydroxy-11-deoxycorticosterone with cytochrome P-450(11)beta and analyzed the reaction products by HPLC. The above-mentioned unidentified peaks appeared again in the chromatogram. The retention time of one of the peaks coincided with that of authentic 19-oxo-11-deoxycorticosterone. This peak substance was purified by repeated HPLC and subjected to mass spectrometry and 1H NMR analyses. Its field desorption mass spectrum (FD-MS) showed a M+ peak at m/e 344. The 1H NMR spectrum showed the signal of an aldehyde proton instead of those of hydroxymethyl protons at the C-19 position. These results suggest that cytochrome P-450(11)beta can catalyze the 19-hydroxylation of 11-deoxycorticosterone, and the 19-hydroxy-11-deoxycorticosterone produced is further oxidized at the C-19 position to 19-oxo-11-deoxycorticosterone.
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The binding characteristics of [3H]saxitoxin and its binding site were examined in bovine adrenal medullary cells. These cells showed a specific binding of [3H]saxitoxin which was saturable and reversible. Scatchard analysis showed a single class of high-affinity binding sites with an equilibrium dissociation constant of 5.8 nM and a maximum binding capacity of 427.2 fmoles/10(7) cells (124.2 fmoles/mg of cell protein). A Hill plot revealed that there were no co-operative interactions among the binding sites. Unlabeled saxitoxin inhibited the specific binding of [3H]saxitoxin as well as veratridine-induced 22Na influx with a similar potency as did tetrodotoxin. However, veratridine, aconitine and scorpion venom, at concentrations that increased 22Na influx, did not inhibit [3H]saxitoxin binding. These results indicate that saxitoxin binds to a specific site on voltage-dependent Na channels and inhibits the influx of 22Na. [3H]Saxitoxin would be useful for the detailed analysis of voltage-dependent Na channels in adrenal medullary cells.
To investigate the K permeability mechanism(s) in cultured bovine adrenal medullary cells, we measured the effects of high K, veratridine and carbachol on 86Rb efflux from the 86Rb preloaded cells. In non-stimulated cells, the basal efflux of 86Rb into Krebs-Ringer phosphate buffer containing 5.6 mM K proceeded gradually at the rate of 0.7% of cell 86Rb per min. High K caused a rapid 86Rb efflux; it was considerably reduced in Ca free medium. Mn, Co and Mg strongly inhibited high K-induced 45Ca influx and 86Rb efflux. Veratridine induced a sustained 86Rb efflux; it was inhibited by tetrodotoxin and abolished in Na free sucrose medium, but little affected in Ca free medium. Carbachol evoked a rapid and transient efflux of 86Rb; it amounted to 16.9% of cell 86Rb during 1 min. Carbachol-induced 86Rb efflux was inhibited by hexamethonium and d-tubocurarine. Nicotine caused 86Rb efflux, but muscarine had no effect. Carbachol-induced 86Rb efflux was substantially reduced in Na free sucrose medium, but little affected in Ca free medium. Mn, Co and Mg strongly reduced carbachol-induced 45Ca influx, but they did not appreciably alter carbachol-induced 22Na influx and 86Rb efflux. These results suggest that adrenal medullary cells have, at least, three distinct types of K permeability mechanisms: (1) basal K efflux, (2) Ca dependent K efflux, and (3) Na dependent K efflux. It seems that nicotine receptors mediate K efflux by increasing Na influx via nicotinic receptor-associated ionic channels rather than Ca influx via voltage dependent Ca channels.
We determined the partial primary structures of and identified the genes for new basic proteins A and B in Escherichia coli ribosomal 50S subunits, found by means of an improved two-dimensional gel electrophoresis method. The sequence up to the 17th amino acid of protein B was in agreement with that of the X gene in the spc operon. The gene for protein A was searched for in the GenBank data base using the sequence up to the 35th amino acid, and was found at a locus between infC and rplT. The base sequence indicated that protein A contained 64 amino acids and had a molecular weight of 6,984. We conclude that proteins A and B are intrinsic ribosomal proteins, and propose calling their genes, rpmI and rpmJ, respectively.
Incubation of cultured bovine adrenal medullary cells in Na+-free sucrose medium or in Na+-free Cs+ medium enhanced the synthesis of 14C-catecholamines from [14C]tyrosine about two- to threefold or sixfold, respectively. The increment of 14C-catecholamine synthesis produced by Na+-free medium was partially dependent on the presence of Ca2+ in the medium. Dibutyryl cyclic AMP also stimulated the synthesis of 14C-catecholamines in adrenal medullary cells, and the effects of Na+ removal and dibutyryl cyclic AMP (5 mM) on the synthesis were almost additive. The intracellular pH measured by using a weak acid 5,5-dimethyloxazolidine-2,4-dione was 7.14 in control cells and when Na+ was replaced by sucrose or Cs+, it shifted down to 6.56 or 5.66, respectively. The fall in intracellular pH and the stimulation of 14C-catecholamine synthesis were similarly dependent on the concentration of Na+ in the medium. The optimal pH of soluble tyrosine hydroxylase was 5.5-6.0 both in control cells and in cells incubated in Na+-free medium. These results suggest that removal of extracellular Na+ increases the synthesis of catecholamines, at least in part, by shifting the intracellular pH toward the optimal pH of tyrosine hydroxylase.
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In bovine adrenal medullary cells we investigated the effects of antidepressants on ionic channels and secretion of catecholamines. Tricyclic (imipramine, amitriptyline and nortriptyline) and tetracyclic (maprotiline and mianserin) antidepressants inhibited carbachol-induced influx of 22Na, 45Ca and secretion of catecholamines (IC50, 14-96 microM). Influx of 22Na, 45Ca and secretion of catecholamines due to veratridine also were inhibited by these drugs (IC50, 10-17 microM). However, antidepressants did not suppress high concentration of K-induced 45Ca influx and catecholamine secretion, suggesting that antidepressants do not inhibit voltage-dependent Ca channels. [3H]Imipramine bound specifically to adrenal medullary cells. Binding was saturable, reversible and with two different equilibrium dissociation constants (13.3 and 165.0 microM). Tricyclic and tetracyclic antidepressants competed for the specific binding of [3H]imipramine at the same concentrations as they inhibited 22Na influx caused by carbachol or veratridine. Carbachol, d-tubocurarine, hexamethonium, tetrodotoxin, veratridine and scorpion venom did not inhibit the specific binding of [3H]imipramine. These results suggest that tricyclic and tetracyclic antidepressants bind to two populations of binding sites which are functionally associated with nicotinic receptor-associated ionic channels and with voltage-dependent Na channels, and inhibit Na influx. Inhibition of Na influx leads to the reduction of Ca influx and catecholamine secretion caused by carbachol or veratridine.
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In cultured bovine adrenal medulla cells, monensin caused the release of catecholamines simultaneous with the influx of 22Na to the cells. The release of catecholamines by monensin was dependent on Na but not on Ca in the medium. Release of catecholamines and the influx of 22Na caused by monensin were not inhibited by tetrodotoxin. Monensin did not cause the release of dopamine beta-hydroxylase from the cells showing that monensin caused the release of catecholamines by a nonexocytotic mechanism. Similarly, in isolated chromaffin granules, monensin caused Na-dependent release of catecholamines, simultaneously with the influx of 22Na to the granules. Basing on these findings, monensin seems to cause a nonexocytotic release of catecholamines by acting as an Na ionophore both at cell membranes and chromaffin granule.
We investigated the release of catecholamines by barium (Ba) in digitonin-permeabilized bovine adrenal medullary cells, to find out whether Ba acts directly or through the mobilization of endogenous calcium (Ca). We also tried to ascertain whether the release of catecholamines from the permeabilized cells occurs in the same manner as from the non-permeabilized control cells. In the permeabilized cells, a rise in either free Ca or Ba caused a saturable release of catecholamines. The maximal release of catecholamines caused by Ba was greater than that caused by Ca, suggesting that Ba directly triggered the release process. Release of catecholamines by Ba was accompanied by the release of dopamine beta-hydroxylase, both in the permeabilized and control cells, showing that Ba causes an exocytotic release of catecholamines. The concentration of Ba which was required for the release of catecholamines in the permeabilized cells agreed with that required in control cells. We conclude that Ba can substitute Ca in triggering the exocytotic release of catecholamines from the adrenal medullary cells. Permeabilized cells provide accurate information on the direct action of Ba in these cells.
The values of the cytologic features of individual cells and cellular patterns in aspirated materials in the diagnosis of 49 hepatocellular carcinomas (HCC) were investigated. Excellent cytologic specimens were obtained by percutaneous aspiration biopsy with a heparinized fine 22-gauge needle. In the well-differentiated type of HCC, a correct diagnosis of malignancy was difficult from the cytologic features of individual cells because of their resemblance to normal hepatocytes. In contrast, in moderately differentiated and poorly differentiated types of HCC, a correct diagnosis of malignancy was easily made from the features of individual cells, but there was little or no cytologic evidence of the hepatic origin of the cells. Comparison of histologic and cytologic findings in aspirated materials obtained from the same patients showed that the cellular patterns seen in cytologic specimens faithfully reflected the histologic structures of HCC. Various characteristic cellular patterns were recognized only in specimens obtained from patients with HCC, but not in those from patients with benign liver diseases. These cellular patterns were very useful not only for diagnosis of malignancy, but also for identification of the hepatic origin of cells. A combination of the features of individual cells and of characteristic cellular patterns raised the diagnostic rates for well-, moderately, and poorly differentiated types of HCC to 90.5%, 100%, and 100%, respectively.
The effects of ketamine, an intravenous anesthetic, on 22Na influx, 45Ca influx and catecholamine secretion were investigated in cultured bovine adrenal medullary cells. Ketamine inhibited carbachol-induced 45Ca influx and catecholamine secretion in a concentration-dependent manner with a similar potency (IC50 40 microM). Ketamine also reduced veratridine-induced 45Ca influx and catecholamine secretion (IC50 260 microM) but did not affect high K-induced 45Ca influx and catecholamine secretion. The influx of 22Na caused by carbachol or by veratridine was suppressed by ketamine with a concentration-inhibition curve similar to that of 45Ca influx and catecholamine secretion. Inhibition by ketamine of the carbachol-induced influx of 22Na, 45Ca and secretion of catecholamines was not reversed by the increased concentrations of carbachol. These observations indicate that ketamine, at clinical concentrations, can inhibit nicotinic receptor-associated ionic channels and that the inhibition of Na influx via the receptor-associated ionic channels is responsible for the inhibition of carbachol-induced Ca influx and catecholamine secretion. At higher concentrations, the anesthetic also inhibits voltage-dependent Na channels but has no effect on voltage-dependent Ca channels.
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The possible involvement of phospholipase A2 (PLA2) in the release of catecholamines was examined in a cell-free model system, using isolated chromaffin granules and plasma membranes of adrenal medulla cells. Plasma membranes treated with PLA2 in the presence of Ca2+ caused lysis of chromaffin granules which was also dependent on Ca2+. This finding suggests that Ca2+ acts in two steps of exocytosis, namely in the transformation of plasma membranes into a lytic form by PLA2 and in the interaction of chromaffin granules with plasma membranes. These findings show good agreement with recently reported findings in leaky adrenal medulla cells, and suggest the involvement of PLA2 in the release of catecholamines.