Asuka Baba

HMG-CoA reductase inhibitors pravastatin, lovastatin and simvastatin suppress delayed rectifier K+-channel currents in murine thymocytes

BACKGROUND Since lymphocytes predominantly express delayed rectifier K(+)-channels (Kv1.3) that trigger lymphocyte activation, statins, which exert immunosuppressive effects, would affect the channel currents. METHODS Employing the patch-clamp technique in murine thymocytes, we examined the effects of statins on Kv1.3-channel currents and the membrane capacitance (Cm). RESULTS Pravastatin significantly suppressed the pulse-end currents of the channels. Lovastatin and simvastatin also suppressed the peak currents, significantly decreasing the Cm. CONCLUSIONS This study demonstrated for the first time that statins inhibit thymocyte Kv1.3-channels. The slow inactivation patterns induced by lovastatin and simvastatin may be associated with their accumulation in the plasma membranes.

Suppressive effects of diltiazem and verapamil on delayed rectifier K+-channel currents in murine thymocytes

BACKGROUND Lymphocytes predominantly express delayed rectifier K(+)-channels (Kv1.3) in their plasma membranes, and these channels play crucial roles in the lymphocyte activation and proliferation. Since diltiazem and verapamil, which are highly lipophilic Ca(2+) channel blockers (CCBs), exert relatively stronger immunomodulatory effects than the other types of CCBs, they would affect the Kv1.3-channel currents in lymphocytes. METHODS Employing the standard patch-clamp whole-cell recording technique in murine thymocytes, we examined the effects of these drugs on the channel currents and the membrane capacitance. RESULTS Both diltiazem and verapamil significantly suppressed the peak and the pulse-end currents of the channels, although the effects of verapamil were more marked than those of diltiazem. Both drugs significantly lowered the membrane capacitance, indicating the interactions between the drugs and the plasma membranes. CONCLUSIONS This study demonstrated for the first time that CCBs, such as diltiazem and verapamil, exert inhibitory effects on Kv1.3-channels expressed in lymphocytes. The effects of these drugs may be associated with the mechanisms of immunomodulation by which they decrease the production of inflammatory cytokines.

Olopatadine inhibits exocytosis in rat peritoneal mast cells by counteracting membrane surface deformation.

Backgroud/Aims: Besides its anti-allergic properties as a histamine receptor antagonist, olopatadine stabilizes mast cells by inhibiting the release of chemokines. Since olopatadine bears amphiphilic features and is preferentially partitioned into the lipid bilayers of the plasma membrane, it would induce some morphological changes in mast cells and thus affect the process of exocytosis. Methods: Employing the standard patch-clamp whole-cell recording technique, we examined the effects of olopatadine and other anti-allergic drugs on the membrane capacitance (Cm) in rat peritoneal mast cells during exocytosis. Using confocal imaging of a water-soluble fluorescent dye, lucifer yellow, we also examined their effects on the deformation of the plasma membrane. Results: Low concentrations of olopatadine (1 or 10 µM) did not significantly affect the GTP-γ-S-induced increase in the Cm. However, 100 µM and 1 mM olopatadine almost totally suppressed the increase in the Cm. Additionally, these doses completely washed out the trapping of the dye on the cell surface, indicating that olopatadine counteracted the membrane surface deformation induced by exocytosis. As shown by electron microscopy, olopatadine generated inward membrane bending in mast cells. Conclusion: This study provides electrophysiological evidence for the first time that olopatadine dose-dependently inhibits the process of exocytosis in rat peritoneal mast cells. Such mast cell stabilizing properties of olopatadine may be attributed to its counteracting effects on the plasma membrane deformation in degranulating mast cells.

Benidipine suppresses in situ proliferation of leukocytes and slows the progression of renal fibrosis in rat kidneys with advanced chronic renal failure.

Background/Aims: Leukocytes, such as lymphocytes and macrophages, predominantly express delayed rectifier K+ channels (Kv1.3) in their plasma membranes. In our previous study, the overexpression of these channels in leukocytes was strongly associated with their proliferation in kidneys and the progression of renal fibrosis in advanced-stage chronic renal failure (CRF). Since benidipine, a long-acting 1,4-dihydropyridine Ca2+ channel blocker, is also highly potent as a Kv1.3 channel inhibitor, it could exert therapeutic efficacy in advanced CRF. Methods: Male Sprague-Dawley rats that underwent 5/6 nephrectomy followed by a 14-week recovery period were used as the model of advanced CRF. Benidipine hydrochloride (5 mg/kg) was started at 8 weeks after nephrectomy and orally administered daily for 6 weeks. The histopathological features of the kidneys were examined in vehicle-treated and benidipine-treated CRF rat kidneys. Cellular proliferation of leukocytes and the cortical expression of proinflammatory cytokines were also examined. Results: In CRF rat kidneys, Kv1.3 channels began to be overexpressed in leukocytes as early as 8 weeks after nephrectomy. In the cortical interstitium of benidipine-treated CRF rat kidneys, both immunohistochemistry and real-time PCR demonstrated decreased expression of fibrotic markers. Benidipine treatment significantly reduced the number of proliferating leukocytes within the cortical interstitium and decreased the expression of cell cycle markers and proinflammatory cytokines. Conclusion: This study demonstrated for the first time that benidipine slowed the progression of renal fibrosis in rat kidneys with advanced CRF. Kv1.3 channels overexpressed in leukocytes were thought to be the most likely therapeutic targets of benidipine in decreasing the number of proliferating leukocytes and repressing the production of inflammatory cytokines.

Anti-Allergic Drugs Tranilast and Ketotifen Dose-Dependently Exert Mast Cell- Stabilizing Properties

Background: Anti-allergic drugs, such as tranilast and ketotifen, inhibit the release of chemokines from mast cells. However, we know little about their direct effects on the exocytotic process of mast cells. Since exocytosis in mast cells can be monitored electrophysiologically by changes in the whole-cell membrane capacitance (Cm), the absence of such changes by these drugs indicates their mast cell-stabilizing properties. Methods: Employing the standard patch-clamp whole-cell recording technique in rat peritoneal mast cells, we examined the effects of tranilast and ketotifen on the Cm during exocytosis. Using confocal imaging of a water-soluble fluorescent dye, lucifer yellow, we also examined their effects on the deformation of the plasma membrane. Results: Relatively lower concentrations of tranilast (100, 250 µM) and ketotifen (1, 10 µM) did not significantly affect the GTP-γ-S-induced increase in the Cm. However, higher concentrations of tranilast (500 µM, 1 mM) and ketotifen (50, 100 µM) almost totally suppressed the increase in the Cm, and washed out the trapping of the dye on the surface of the mast cells. Compared to tranilast, ketotifen required much lower doses to similarly inhibit the degranulation of mast cells or the increase in the Cm. Conclusions: This study provides electrophysiological evidence for the first time that tranilast and ketotifen dose-dependently inhibit the process of exocytosis, and that ketotifen is more potent than tranilast in stabilizing mast cells. The mast cell-stabilizing properties of these drugs may be attributed to their ability to counteract the plasma membrane deformation in degranulating mast cells.

Salicylate inhibits thrombopoiesis in rat megakaryocytes by changing the membrane micro-architecture.

Background/Aims: Salicylate causes drug-induced immune thrombocytopenia. However, some clinical studies indicate the presence of additional mechanisms in the drug-induced thrombocytopenia, by which the platelet production from megakaryocytes may directly be affected. Since salicylate is amphiphilic and preferentially partitioned into the lipid bilayers of the plasma membrane, it can induce some structural changes in the megakaryocyte membrane surface and thus affect the process of thrombopoiesis. Methods: Employing the standard patch-clamp whole-cell recording technique, we examined the effects of salicylate on the membrane capacitance in rat megakaryocytes. Taking electron microscopic imaging of the cellular surface, we also examined the effects of salicylate on the membrane micro-architecture of megakaryocytes. Results: Salicylate significantly decreased the membrane capacitance of megakaryocytes, indicating the decreased number of invaginated plasma membranes, which was not detected by the fluorescent imaging technique. As shown by electron microscopy, salicylate actually halted the process of pro-platelet formation in megakaryocytes. Conclusion: This study demonstrated for the first time that salicylate inhibits the process of thrombopoiesis in megakaryocytes, as detected by the decrease in the membrane capacitance. Salicylate-induced changes in the membrane micro-architecture are thought to be responsible for its effects.

Mast cell involvement in the progression of peritoneal fibrosis in rats with chronic renal failure

Peritoneal fibrosis is a serious complication in patients with end stage renal disease (ESRD), especially those undergoing long‐term peritoneal dialysis therapy. Since the peritoneum is a major site of mast cell accumulation, and since mast cells are known to facilitate the progression of organ fibrosis, they would also contribute to the pathogenesis of peritoneal fibrosis. The aim of this study was to reveal the involvement of mast cells in the progression of peritoneal fibrosis in chronic renal failure.

Amphipath-induced plasma membrane curvature controls microparticle formation from adipocytes: novel therapeutic implications for metabolic disorders.

Microparticles produced from the membrane surface of adipocytes promote lipid biosynthesis and angiogenesis in adipose tissues. Thus, they are deeply associated with the onset of metabolic disorders. Despite our understanding of their roles in physiological or pathological responses, we know little about the mechanism by which microparticles are produced from adipocytes. Based on our previous studies using rat megakaryocytes or mast cells during exocytosis, we proposed that membrane curvature induced by amphiphilic reagents, such as chlorpromazine or salicylate, facilitate or inhibit the formation of microparticles. Since the plasma membranes in adipocytes share many common biophysiological features with those in megakaryocytes or mast cells during exocytosis, the same stimulatory or inhibitory mechanism of microparticle formation would exist in adipocytes. Therefore, we hypothesize here that amphiphilic reagents would also change the membrane curvature in adipocytes, and that such changes would facilitate or inhibit the microparticle formation from adipocytes. Our hypothesis is unique because it sheds light for the first time on the physiological mechanism by which microparticles are produced in adipocytes. It is also important because the idea could have novel therapeutic implications for metabolic disorders that are triggered by increases in the microparticle formation.

Chlorpromazine-induced changes in membrane micro-architecture inhibit thrombopoiesis in rat megakaryocytes.

Chlorpromazine often causes severe and persistent thrombocytopenia. Several clinical studies have suggested the presence of an as-yet-unknown mechanism in this drug-induced thrombocytopenia, by which the platelet production from megakaryocytes may directly be affected. As we previously demonstrated in rat peritoneal mast cells or adipocytes, chlorpromazine is amphiphilic and preferentially partitioned into the lipid bilayers of the plasma membrane. Therefore, it can induce some structural changes in the megakaryocyte membrane surface and thus affect the process of thrombopoiesis. In the present study, employing the standard patch-clamp whole-cell recording technique, we examined the effects of chlorpromazine on the membrane capacitance and Kv1.3-channel currents in rat megakaryocytes. By electron microscopic imaging of the cellular surface, we also examined the effects of chlorpromazine on the membrane micro-architecture of megakaryocytes. Chlorpromazine markedly decreased the membrane capacitance of megakaryocytes, indicating the decreased number of invaginated plasma membranes, which was not detected by the fluorescent imaging techniques. As shown by electron microscopy, chlorpromazine actually changed the membrane micro-architecture of megakaryocytes, and was likely to halt the process of pro-platelet formation in the cells. This drug persistently decreased the membrane capacitance and almost totally and irreversibly inhibited the Kv1.3-channel currents in megakaryocytes. This study demonstrated for the first time that chlorpromazine is likely to inhibit the process of thrombopoiesis persistently in megakaryocytes, as detected by the long-lasting decrease in the membrane capacitance and the irreversible suppression of the Kv1.3-channel currents. Chlorpromazine-induced changes in the membrane micro-architecture are thought to be responsible for its persistent effects.

Less contribution of mast cells to the progression of renal fibrosis in Rat kidneys with chronic renal failure.

Chronic renal failure (CRF) is histopathologically characterized by tubulointerstitial fibrosis in addition to glomerulosclerosis. Although mast cells are known to infiltrate into the kidneys with chronic inflammation, we know little about their contribution to the pathogenesis of renal fibrosis associated with CRF. The aim of this study was to reveal the involvement of mast cells in the progression of renal fibrosis in CRF.