Prazosin Potentiates Mast Cell-Stabilizing Property of Adrenaline.

BACKGROUND/AIMS: Adrenaline quickly inhibits the release of histamine from mast cells. Besides ß2-adrenergic receptors, several in vitro studies also indicate the involvement of α-adrenergic receptors in the process of exocytosis. Since exocytosis in mast cells can be detected electrophysiologically by the changes in the membrane capacitance (Cm), its continuous monitoring in the presence of drugs would determine their mast cell-stabilizing properties. METHODS: Employing the whole-cell patch-clamp technique in rat peritoneal mast cells, we examined the effects of adrenaline on the degranulation of mast cells and the increase in the Cm during exocytosis. We also examined the degranulation of mast cells in the presence or absence of α-adrenergic receptor agonists or antagonists. RESULTS: Adrenaline dose-dependently suppressed the GTP-γ-S-induced increase in the Cm and inhibited the degranulation from mast cells, which was almost completely erased in the presence of butoxamine, a ß2-adrenergic receptor antagonist. Among α-adrenergic receptor agonists or antagonists, high dose prazosin, a selective α1-adrenergic receptor antagonist, significantly reduced the ratio of degranulating mast cells and suppressed the increase in the Cm. Additionally, prazosin augmented the inhibitory effects of adrenaline on the degranulation of mast cells. CONCLUSION: This study provided electrophysiological evidence for the first time that adrenaline dose-dependently inhibited the process of exocytosis, confirming its usefulness as a potent mast cell-stabilizer. The pharmacological blockade of α1-adrenergic receptor by prazosin synergistically potentiated such mast cell-stabilizing property of adrenaline, which is primarily mediated by ß2-adrenergic receptors.

Brain Leukocytes as the Potential Therapeutic Target for Post-COVID-19 Brain Fog.

After recovering from the acute phase of coronavirus disease 2019 (COVID-19), many patients struggle with additional symptoms of long COVID during the chronic phase. Among them, the neuropsychiatric manifestations characterized by a short-term memory loss and inability to concentrate are called "brain fog". Recent studies have revealed the involvement of "chronic neuro-inflammation" in the pathogenesis of brain fog following COVID-19 infection. In the COVID-related brain fog, similarly to neurodegenerative disorders caused by neuro-inflammation, brain leukocytes, such as microglia and lymphocytes, are hyperactivated, suggesting the overexpression of delayed rectifier K+-channels (Kv1.3) within the cells. In our previous patch-clamp studies, drugs, such as antihistamines, statins, nonsteroidal anti-inflammatory drugs, antibiotics and anti-hypertensive drugs, suppressed the Kv1.3-channel activity and reduced the production of pro-inflammatory cytokines. Additionally, newer generation antihistamines, antibiotics and corticosteroids strongly stabilize mast cells that directly activate microglia in the brain. Taking such pharmacological properties of these commonly used drugs into account, they may be useful in the treatment of COVID-related brain fog, in which the enhanced innate and adaptive immune responses are responsible for the pathogenesis.

Pyridoxine Synergistically Potentiates Mast Cell-Stabilizing Property of Ascorbic Acid.

BACKGROUND/AIMS: Besides their physiological properties, vitamins, such as vitamin C (ascorbic acid) and B6 (pyridoxine), ameliorate the symptoms of allergic disorders. Because exocytosis in mast cells can be detected electrophysiologically by the changes in the membrane capacitance (Cm), its continuous monitoring in the presence of these vitamins would determine their mast cell-stabilizing, anti-allergic properties. METHODS: Employing the whole-cell patch-clamp technique in rat peritoneal mast cells, we examined the effects of ascorbic acid and pyridoxine on the degranulation of mast cells and the increase in the Cm during exocytosis. RESULTS: Both ascorbic acid and pyridoxine dose-dependently suppressed the GTP-γ-S-induced increase in the Cm and inhibited the degranulation from mast cells. Surprisingly enough, relatively low concentrations of pyridoxine (1, 2 mM) synergistically enhanced the suppressive effect of 2 mM ascorbic acid on mast cell degranulation. CONCLUSION: These results provided electrophysiological evidence for the first time that ascorbic acid and pyridoxine inhibited the process of exocytosis in a dose-dependent manner. At relatively lower concentrations, these vitamins were not enough to stabilize mast cells. However, such concentrations of pyridoxine synergistically potentiated the mast cell-stabilizing property of ascorbic acid.

Targeting ACE2 as a potential prophylactic strategy against COVID-19-induced exacerbation of chronic kidney disease.

Patients with chronic kidney disease (CKD) are at higher risk for severe coronavirus disease 2019 (COVID-19). Such patients are more likely to develop "COVID-19-induced acute kidney injury (AKI)", which exacerbates the pre-existing CKD and increases the mortality rate of the patients. COVID-19-induced AKI is pathologically characterized by acute tubular necrosis and the interstitial infiltration of proinflammatory leukocytes. In our rat model with advanced CKD, immunohistochemistry for angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) demonstrated their strong expression in the cytoplasm of damaged proximal tubular cells and the infiltrating leukocytes within the cortical interstitium, which overlapped with the lesions of COVID-19-induced AKI. Since ACE2 and TMPRSS2 are enzymes that facilitate the viral entry into the cells and trigger the onset of cytokine storm, the renal distribution of these proteins in advanced CKD was thought to be responsible for the development of COVID-19-induced AKI. Concerning such mechanisms, the pharmacological blockade of ACE2 or the use of soluble forms of the ACE2 protein may halt the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells. This would protect against the COVID-19-induced exacerbation of pre-existing CKD by preventing the development of AKI.

Effects of indacaterol on the LPS-evoked changes in fluid secretion rate and pH in swine tracheal membrane.

An acquired dysregulation of airway secretion is likely involved in the pathophysiology of chronic bronchitis and chronic obstructive pulmonary disease (COPD). Nowadays, it is widely known that several kinds of long-acting bronchodilators reduce the frequency of COPD exacerbations. However, limited data are available concerning the complementary additive effects on airflow obstruction. Using an optical method and a selective pH indicator, we succeeded in evaluating the gland secretion rate and the pH in swine tracheal membrane. A physiologically relevant concentration of acetylcholine (ACh) 100 nM induced a gradual increase in the amount of gland secretion. Lipopolysaccharides (LPS) accelerated the ACh-induced secretory responses up to around threefold and lowered the pH level significantly. Long-acting ß2-agonists (LABAs) including indacaterol (IND), formoterol, and salmeterol restored the LPS-induced changes in both the hypersecretion and acidification. The subsequent addition of the long-acting muscarine antagonist, glycopyrronium, further increased the pH values. Two different inhibitors for cystic fibrosis transmembrane conductance regulator (CFTR), NPPB and CFTRinh172, abolished the IND-mediated pH normalization in the presence of both ACh and ACh + LPS. Both immunofluorescence staining and western blotting analysis revealed that LPS downregulated the abundant expression of CFTR protein. However, IND did not restore the LPS-induced decrease in CFTR expression on Calu-3 cells. These findings suggest that the activation of cAMP-dependent HCO3- secretion through CFTR would be partly involved in the IND-mediated pH normalization in gland secretion and may be suitable for the maintenance of airway defense against exacerbating factors including LPS.

Potential prophylactic efficacy of mast cell stabilizers against COVID-19 vaccine-induced anaphylaxis.

To fight against coronavirus disease 2019 (COVID-19), the vaccination is currently the most effective approach. However, in addition to common systemic side effects, the vaccines can cause serious allergic reactions or anaphylaxis. In anaphylaxis, the exposure to the allergen causes a sudden release of chemical mediators from mast cells, for which adrenaline is the drug of first choice. In our previous basic studies, in addition to adrenaline, anti-allergic drugs (olopatadine, loratadine, tranilast and ketotifen), antibiotics (clarithromycin), corticosteroids (hydrocortisone and dexamethasone) and certain food constituents (caffeine and catechin) inhibited the process of exocytosis and showed their effectiveness as highly potent mast cell stabilizers. In these studies, since mast cells were pre-incubated with these drugs or the food constituents before exocytosis was induced, the findings strongly indicated their prophylactic efficacy in stabilizing mast cells. Considering such pharmacological properties of these commonly prescribed medications or the food constituents, their prophylactic use may potentially be beneficial in preventing anaphylaxis caused by COVID-19 vaccination.

TLR7 agonist attenuates acetylcholine-induced, Ca2+ -dependent ionic currents in swine tracheal submucosal gland cells.

NEW FINDINGS: What is the central question of this study? Does Toll-like receptor 7 (TLR7) have any direct effects on Ca2+ -dependent physiological function of tracheal submucosal gland cells? What is the main finding and its importance? TLR7 is co-localized with SERCA2 in tracheal submucosal gland cells and causes a rapid attenuation of acetylcholine (ACh)-induced, Ca2+ -dependent ionic currents through the activation of SERCA2-dependent Ca2+ clearance. TLR7 is abundantly expressed in the airways of both swine and healthy human subjects, but is significantly downregulated in chronic obstructive pulmonary disease (COPD) airways. These findings suggest that a dysfunction of TLR7 in COPD removes the brake on ACh-induced serous secretion during viral infections, resulting in prolonged airway hypersecretion, and that it is one of the triggers of COPD exacerbations. ABSTRACT: Airway surface fluids are mainly secreted from submucosal glands (SMGs) and play important roles in the defence of airways via the activation of mucociliary transport. Toll-like receptor 7 (TLR7) recognizes and eliminates single stranded RNA (ssRNA) viruses through the induction of innate immunity. However, there is no obvious connection between TLR7 and mucus secretion, aside from TLR7 recognizing ssRNA viruses, which are often associated with airway hypersecretion in chronic obstructive pulmonary disease (COPD). Here, we investigated whether TLR7 has any direct effects on the Ca2+ -dependent physiological function of tracheal SMG cells. Patch-clamp analyses revealed that TLR7 ligand inhibited the acetylcholine (ACh)-induced ionic currents in isolated tracheal SMG cells. Intracellular calcium assays and pharmacological analyses revealed that TLR7 attenuated the transient rises in the intracellular calcium concentration evoked by ACh by activating sarco/endoplasmic reticulum Ca2+ -ATPase 2 (SERCA2). Immunofluorescence staining and immunohistochemical staining revealed that TLR7 was co-localized with SERCA2. These findings suggest that the activation of TLR7 during viral infections contributes to the rapid attenuation of ACh-induced ionic currents through an increase in SERCA2-dependent Ca2+ clearance in healthy airway SMG cells. Our study also revealed that TLR7 expression was significantly downregulated in COPD airways. Based on these findings, we speculate that a dysfunction of TLR7 may not only have an adverse effect on the elimination of these viruses but also remove the brake on ACh-induced serous secretion, resulting in prolonged hypersecretion and acting as one of the triggers of COPD exacerbations.

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

AIM: 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. METHODS: Using a rat model with CRF resulting from 5/6 nephrectomy, we examined the histopathological features of the kidneys and the infiltration of mast cells into the renal interstitium. By treating the rats with a potent mast cell stabilizer, tranilast, we also examined the involvement of mast cells in the progression of renal fibrosis associated with CRF. RESULTS: The CRF rat kidneys were characterized by the wide staining of collagen III and increased number of myofibroblasts, indicating the progression of renal fibrosis. Compared to T-lymphocytes or macrophages, the number of tryptase-positive mast cells was much smaller within the fibrotic kidneys and they did not proliferate in situ. The mRNA expression of mast cell-derived fibroblast-activating factors was not increased in the renal cortex isolated from CRF rat kidneys. Treatment with tranilast did not suppress the progression of renal fibrosis, nor did it ameliorate the progression of glomerulosclerosis and the interstitial proliferation of inflammatory leukocytes. CONCLUSIONS: This study demonstrated for the first time that mast cells are neither increased nor activated in the fibrotic kidneys of CRF rats. Compared to T-lymphocytes or macrophages that proliferate in situ within the fibrotic kidneys, mast cells were less likely to contribute to the progression of renal fibrosis associated with CRF.

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.

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-x03B3;-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.

Clarithromycin Dose-Dependently Stabilizes Rat Peritoneal Mast Cells.

BACKGROUND: Macrolides, such as clarithromycin, have antiallergic properties. Since exocytosis in mast cells is detected electrophysiologically via changes in membrane capacitance (Cm), the absence of such changes due to the drug indicates its mast cell-stabilizing effect. METHODS: Employing the whole-cell patch clamp technique in rat peritoneal mast cells, we examined the effects of clarithromycin on Cm during exocytosis. Using a water-soluble fluorescent dye, we also examined its effect on deformation of the plasma membrane. RESULTS: Clarithromycin (10 and 100 µM) significantly inhibited degranulation from mast cells and almost totally suppressed the GTP-x03B3;-S-induced increase in Cm. It washed out the trapping of the dye on the surface of mast cells. CONCLUSIONS: This study provides for the first time electrophysiological evidence that clarithromycin dose-dependently inhibits the process of exocytosis. The mast cell-stabilizing action of clarithromycin may be attributable to its counteractive effect on plasma membrane deformation induced by exocytosis.

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

BACKGROUND/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.

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.

Usefulness of targeting lymphocyte Kv1.3-channels in the treatment of respiratory diseases.

T lymphocytes predominantly express delayed rectifier K(+)-channels (Kv1.3) in their plasma membranes. Patch-clamp studies revealed that the channels play crucial roles in facilitating the calcium influx necessary to trigger lymphocyte activation and proliferation. Using selective channel inhibitors in experimental animal models, in vivo studies further revealed the clinically relevant relationship between the channel expression and the development of chronic respiratory diseases, in which chronic inflammation or the overstimulation of cellular immunity in the airways is responsible for the pathogenesis. In chronic respiratory diseases, such as chronic obstructive pulmonary disease, asthma, diffuse panbronchiolitis and cystic fibrosis, in addition to the supportive management for the symptoms, the anti-inflammatory effects of macrolide antibiotics were shown to be effective against the over-activation or proliferation of T lymphocytes. Recently, we provided physiological and pharmacological evidence that macrolide antibiotics, together with calcium channel blockers, HMG-CoA reductase inhibitors, and nonsteroidal anti-inflammatory drugs, effectively suppress the Kv1.3-channel currents in lymphocytes, and thus exert anti-inflammatory or immunomodulatory effects. In this review article, based on the findings obtained from recent in vivo and in vitro studies, we address the novel therapeutic implications of targeting the lymphocyte Kv1.3-channels for the treatment of chronic or acute respiratory diseases.

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

AIM: 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. METHODS: Using a rat model with chronic renal failure (CRF) resulting from 5/6 nephrectomy, we examined the histopathological features of the rat peritoneum and compared them to those of age-matched sham-operated rat peritoneum. By treating the CRF rats with a potent mast cell stabilizer, tranilast, we also examined the involvement of mast cells in the progression of peritoneal fibrosis. RESULTS: The CRF rat peritoneum was characterized by the wide staining of collagen III and an increased number of myofibroblasts, indicating the progression of fibrosis. Compared to sham-operated rat peritoneum, the number of toluidine blue-stained mast cells was significantly higher in the fibrotic peritoneum of CRF rats. The mRNA expression of fibroblast-activating factors and stem cell factor was significantly higher in peritoneal mast cells obtained from CRF rats than in those obtained from sham-operated rats. Treatment with tranilast significantly suppressed the progression of peritoneal fibrosis in CRF rats. CONCLUSIONS: This study demonstrated for the first time that the number of mast cells was significantly increased in the fibrotic peritoneum of CRF rats. The proliferation of mast cells and their increased activity in the peritoneum were thought to be responsible for the progression of peritoneal fibrosis.

Roles of lymphocyte kv1.3-channels in the pathogenesis of renal diseases and novel therapeutic implications of targeting the channels.

Delayed rectifier K(+)-channels (Kv1.3) are predominantly expressed in T lymphocytes. Based on patch-clamp studies, the channels play crucial roles in facilitating the calcium influx necessary to trigger lymphocyte activation and proliferation. Using selective channel inhibitors in experimental animal models, in vivo studies then revealed the clinically relevant relationship between the channel expression and the pathogenesis of autoimmune diseases. In renal diseases, in which "chronic inflammation" or "the overstimulation of cellular immunity" is responsible for the pathogenesis, the overexpression of Kv1.3-channels in lymphocytes promotes their cellular proliferation and thus contributes to the progression of tubulointerstitial fibrosis. We recently demonstrated that benidipine, a potent dihydropyridine calcium channel blocker, which also strongly and persistently inhibits the lymphocyte Kv1.3-channel currents, suppressed the proliferation of kidney lymphocytes and actually ameliorated the progression of renal fibrosis. Based on the recent in vitro evidence that revealed the pharmacological properties of the channels, the most recent studies have revealed novel therapeutic implications of targeting the lymphocyte Kv1.3-channels for the treatment of renal diseases.

Glucocorticoid mediates the transcription of OAT-PG, a kidney-specific prostaglandin transporter.

OAT-PG is a kidney-specific prostaglandin transporter and exclusively expressed at the basolateral membrane of proximal tubules in rodent kidneys. We previously reported that OAT-PG was dominantly expressed in the male kidney similar to the other SLC22 family proteins as organic anion transporter (OAT) 1 and OAT3. Recently, Wegner et al. revealed that a transcription factor, B-cell CLL/lymphoma 6 (BCL6), is associated with the male-dominant expressions of OAT1 and OAT3 in the rat kidney. Here, we performed the luciferase assay to investigate whether OAT-PG is also transcriptionally regulated by BCL6. However, the promoter activity of OAT-PG was not directly affected by BCL6 overexpression nor the testosterone treatment, suggesting that different regulatory mechanisms underlie the male-dominant transcriptional regulation of OAT-PG compared to those of OAT1 and OAT3. We newly found that adrenalectomy (Adx) of male rat caused a significant reduction of OAT-PG expression without any significant changes in the OAT1 and OAT3 expressions, and it was recovered by the dexamethasone administration. Furthermore, the renocortical PGE2 concentration was markedly increased in Adx male rat, concomitant with the downregulation of OAT-PG, and it was reduced to the basal level by dexamethasone treatment. In the luciferase assay, dexamethasone stimulated OAT-PG promoter activity but not OAT1. The luciferase activity responsiveness to dexamethasone was significantly reduced by the deletion of glucocorticoid response elements in the OAT-PG promoter region. These results suggest that glucocorticoid plays an important role in the regulation of the renocortical PGE2 concentration by the transcriptional regulation of OAT-PG in the rat kidney.

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 Ca(2+) 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.

Amphipaths differentially modulate membrane surface deformation in rat peritoneal mast cells during exocytosis.

BACKGROUND/AIMS: Salicylate and chlorpromazine exert differential effects on the chemokine release from mast cells. Since these drugs are amphiphilic and preferentially partitioned into the lipid bilayers of the plasma membranes, they 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 salicylate and chlorpromazine on the membrane capacitance (Cm) during exocytosis in rat peritoneal mast cells. Using confocal imaging of a water-soluble fluorescent dye, lucifer yellow, we also examined their effects on plasma membrane deformation of the cells. RESULTS: Salicylate dramatically accelerated the GTP-γ-S-induced increase in the Cm immediately after its application, whereas chlorpromazine significantly suppressed the increase. Treatment with salicylate increased the trapping of the dye on the cell surface, while treatment with chlorpromazine completely washed it out, indicating that both drugs induced membrane surface deformation in mast cells. CONCLUSION: This study demonstrated for the first time that membrane amphipaths, such as salicylate and chlorpromazine, may oppositely modulate the process of exocytosis in mast cells, as detected by the changes in the Cm. The plasma membrane deformation induced by the drugs was thought to be responsible for their differential effects.

Decreased expression of a novel prostaglandin transporter, OAT-PG, facilitates renocortical PGE2 accumulation during rat pregnancy.

BACKGROUND: Prostaglandin (PG)-specific organic anion transporter (OAT-PG) is a recently identified renal transporter involved in the local clearance of prostaglandin E2 (PGE2). Since the renal biosynthesis of PGE2 is not increased during pregnancy, this transporter expression would affect the gestational changes in the renal PGE2 content. METHODS: Kidneys from rats at different gestational stages were used to examine gestational changes in the renocortical PGE2 concentration. The renal expression of OAT-PG and the enzymes for PGE2 synthesis was also examined sequentially, together with the gestational changes in renal renin production. RESULTS: The renocortical PGE2 concentration was significantly increased during midterm to late pregnancy, with a maximum increase of 47.6 ± 11.5% from the virgin value. Although the expression of the enzymes, such as cyclooxygenases and PG synthases, was not increased, that of OAT-PG was significantly decreased throughout pregnancy, inversely correlating with changes in the renocortical PGE2 concentration. Renal renin production was significantly increased during pregnancy. CONCLUSION: This study demonstrated for the first time that the tissue PGE2 concentration was increased in pregnant rat kidneys, which may be associated with the gestational rise in glomerular filtration rate. The decreased expression of OAT-PG was thought to be responsible for the increased tissue PGE2 content.