We have used stage-specific assays for ATP-dependent priming and for Ca(2+)-activated triggering in the absence of ATP to examine the effects of myosin light chain kinase (MLCK) inhibitors, ML-9 and ML-7, and calmodulin antagonists, W-7 and trifluoperazine (TFP), on regulated exocytosis in beta-escin-permeabilized bovine adrenal chromaffin cells. Ca2+ (0.1-30 microM) induced a significantly greater secretion of catecholamines in the presence of MgATP (2 mM) than in the absence of MgATP. ML-9 (30 and 100 microM), ML-7 (30 and 100 microM), W-7 (30 and 100 microM) and TFP (10 and 30 microM) inhibited the Ca(2+)-induced catecholamine secretion in the presence of MgATP, but did not affect the catecholamine response to Ca2+ in the absence of MgATP. In intact cells all these compounds inhibited catecholamine secretion in responses to acetylcholine (100 microM) and high K+ (40 mM). The results obtained in permeabilized cells suggest that the calmodulin-MLCK system plays an essential role in the ATP-requiring priming stage but not in the Ca2(+)-triggered fusion step in the exocytotic process in bovine adrenal chromaffin cells.
Abstract. The effects of SEA0400, a selective inhibitor of the Na + / Ca 2+ exchanger (NCX), on Na + -dependent Ca 2+ uptake and catecholamine (CA) release were examined in bovine adrenal chromaffin cells that were loaded with Na + by treatment with ouabain and veratridine. SEA0400 inhibited Na Ca2+ uptake and CA release, with the IC 50 values of 40 and 100 nM, respectively. The IC 50 values of another NCX inhibitor KB-R7943 were 1.8 and 3.7 µM, respectively. These results indicate that SEA0400 is about 40 times more potent than KB-R7943 in inhibiting NCX working in the reverse mode. In intact cells, SEA0400 and KB-R7943 inhibited CA release induced by acetylcholine and DMPP. The IC 50 values of SEA0400 were 5.1 and 4.5 µM and the values of KB-R7943 were 2.6 and 2.1 µM against the release induced by acetylcholine and DMPP, respectively, indicating that the potency of SEA0400 is about a half of that of KB-R7943 in inhibiting the nicotinic receptor-mediated CA release. The binding of [ 3 H]nicotine with nicotinic receptors was inhibited by SEA0400 (IC 50 = 90 µM) and KB-R7943 (IC 50 = 12 µM). From these results, it is concluded that unlike KB-R7943, SEA0400 has a potent and selective action on NCX in bovine adrenal chromaffin cells.
1. Differential secretion of adrenaline (Adr) and noradrenaline (NA) in response to various secretagogues was studied in bovine adrenal chromaffin cells. 2. Acetylcholine (ACh; 3-300 mumol/L), 1,1-dimethyl-4-phenyl-piperazinum (DMPP; 1-100 mumol/L), high K+ (20-60 mmol/L), calcimycin (1-100 mumol/L), histamine (0.3-30 mumol/L) and angiotensin (Ang)II (0.3-30 mumol/L) induced the secretion of a 1.3-2-fold greater percentage of NA stores than Adr stores in intact cells. 3. In beta-escin-permeabilized cells, Ca2+ (0.1-30 mumol/L) induced a greater secretion of Adr and NA in the presence of MgATP (2 mmol/L) than in the absence of MgATP. The percentage of NA secreted was 1.4- and 1.5-fold greater than that of Adr in the presence and absence of MgATP, respectively. 4. The ATP-independent phase of the Ca(2+)-dependent exocytosis is thought to be associated with the final step that ultimately leads to fusion, while the ATP-dependent phase is thought to be associated with the vesicle priming reaction. Therefore, the preferential secretion of NA in response to ACh, DMPP, high K+, calcimycin, histamine and AngII may be due, at least in part, to the greater effectiveness of Ca2+ in producing exocytosis in NA-containing cells.
1. The Na(+)/Ca(2+) exchanger (NCX) exchanges Na+ and Ca(2+) bidirectionally through the forward mode (Ca(2+) extrusion) or the reverse mode (Ca(2+) influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na(+)-loaded cells were prepared by treatment with 100 micromol/L ouabain and 50 micromol/L veratridine. Incubation of Na(+)-loaded cells with Na(+)-free solution in the presence of the Ca(2+) channel blockers nicardipine (3 micromol/L) and omega-conotoxin MVIIC (0.3 micromol/L) caused Ca(2+) uptake and catecholamine release. 2. The Na(+)-dependent Ca(2+) uptake and catecholamine release were inhibited by 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400; 1 micromol/L) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]isothiourea (KB-R7943; 10 micromol/L), both NCX inhibitors. These results indicate that the Na(+)-dependent responses are mostly due to activation of the NCX working in the reverse mode. 3. In addition, we examined the effects of PKC inhibitors and an activator on the NCX-mediated Ca(2+) uptake and catecholamine release. Bisindolylmaleimide I (0.3-10 micromol/L) and chelerythrine (3-100 micromol/L), both PKC inhibitors, inhibited NCX-mediated responses. In contrast, phorbol 12,13-dibutyrate (0.1-10 micromol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na(+)-dependent catecholamine release, had a little or no effect on high K(+)-induced catecholamine release in intact cells or on Ca(2+)-induced catecholamine release in beta-escin-permeabilized cells. 4. These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.
Aim: Static blood (SB) is an important pathological concept of Kampo medicine. Although tongue findings are very useful in the diagnosis of SB, the relation between the severity of SB and tongue color (TC) is unclear. The purpose of this study was to evaluate the relation between the severity of SB and TC obtained by the use of a Tongue image analyzing system (TIAS). Methods: We analyzed data from 227 new patients (age 20-85) who visited the Kyushu University Hospital Kampo Clinic between 2013 and 2018. The SB score was calculated based on the diagnostic criteria by Terasawa et al. and classified into three groups: non-static blood (NSB) ≤20 points, SB 20.5 to 39.5 points, severe static blood (SSB) ≥40 points. TC was measured by the device-independent international commission on Illumination 1976L*a*b* color space standards at four points: (i) edge; (ii) posterior; (iii) middle; and (iv) apex. Results: SB and SSB occurred significantly more frequently in women and patients were of younger age than in the NSB group (P < 0.05). Analysis of TC found 1L* and 4L* to be significantly lower (P < 0.05), and 1a*, 2a*, 3a*, and 4a* significantly higher in SSB than in NSB (P < 0.05). The cutoff value of 2a* and the combination of 1L* + 1a* for SSB prediction obtained from the ROC curve were 2a*
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