MIXED PATHOPHYSIOLOGIES OF LAMELLAR MACULAR HOLES AND RELATED DISEASES: A Multimodal Optical Coherence Tomography-Based Study.

PURPOSE: To investigate the characteristics of mixed pathophysiologies in lamellar macular holes (LMHs) and related diseases using multimodal optical coherence tomography. METHODS: Overall, 126 eyes diagnosed with LMH, epiretinal membrane foveoschisis, or macular pseudohole using the horizontal B-scan image according to the definition proposed by Hubschman et al in 2020 were analyzed using multimodal optical coherence tomography imaging including horizontal and vertical 5-line B-scan, radial scan, and macular three-dimensional volume scan images. If at least two diagnostic criteria for LMH, epiretinal membrane foveoschisis, or macular pseudohole were satisfied in these scans, the patient was diagnosed as having a "mixed type." Retinal traction force was quantitatively evaluated by measuring the maximum depth of the retinal folds using en-face images. RESULTS: Mixed types constituted 34.1% of the cases. The LMH-related mixed group demonstrated intermediate characteristics between the epiretinal membrane foveoschisis/macular pseudohole and true LMH groups in terms of retinal traction and LMH-specific features and had a significant positive correlation between the maximum depth of the retinal folds and mean M-CHARTS scores (P = 0.034). CONCLUSION: A thorough optical coherence tomography analysis is necessary to accurately diagnose LMH and related diseases. A significant positive correlation was observed between the maximum depth of the retinal folds and the degree of metamorphopsia in the LMH-related mixed group.

INVESTIGATION OF THE PATHOPHYSIOLOGY OF EPIRETINAL MEMBRANE FOVEOSCHISIS: Analysis of Longitudinal Changes in Visual Functions, Retinal Structures, and Retinal Traction Force.

PURPOSE: To analyze the pathophysiology of epiretinal membrane foveoschisis (FS) by evaluating the longitudinal changes in visual function and several optical coherence tomography parameters. METHODS: The medical records of 33 consecutive patients (35 eyes) with untreated epiretinal membrane foveoschisis were retrospectively reviewed. Best-corrected visual acuity, M-CHARTS score, and optical coherence tomography parameters including epiretinal membrane area, maximum depth of retinal folds, FS area, and FS circularity were evaluated. RESULTS: A wide range of FS area changes was observed at the final follow-up visit (59.68%-240.45% of the baseline FS area). In the FS enlargement group, best-corrected visual acuity and mean M-CHARTS scores significantly worsened and maximum depth of retinal folds significantly increased over time, whereas in the FS non-enlargement group, no significant change was observed in the best-corrected visual acuity, mean M-CHARTS scores, or maximum depth of retinal folds during the follow-up period. Multivariate logistic regression analyses revealed that maximum depth of retinal folds (odds ratio: 1.05, 95% confidence interval: 1.00-1.10, P = 0.048) and FS circularity (odds ratio: 0.91, 95% confidence interval: 0.83-1.00, P = 0.043) were significantly associated with FS enlargement. CONCLUSION: Epiretinal membrane foveoschisis encompasses diverse pathophysiologies. Since visual functions do not worsen in some cases, monitoring the changes in visual functions and retinal morphology over time is recommended to determine surgical indications.

OBJECTIVE AND QUANTITATIVE ESTIMATION OF THE OPTIMAL TIMING FOR EPIRETINAL MEMBRANE SURGERY ON THE BASIS OF METAMORPHOPSIA.

PURPOSE: To establish an objective and quantitative biomarker of metamorphopsia in epiretinal membranes (ERMs) and determine the optimal timing for ERM surgery. METHODS: Retrospectively, 172 eyes with ERM were reviewed. Retinal folds because of tangential traction by ERM were visualized by en-face optical coherence tomography. The maximum depth of retinal folds (MDRF) within the parafovea was quantified. Metamorphopsia was quantified by M-CHARTS. The change in the distance between the retinal vessels after ERM surgery and the preoperative total depth of retinal folds between the vessels were quantified using en-face optical coherence tomography and optical coherence tomography angiography. RESULTS: Significant correlations were observed between preoperative MDRF and M-CHARTS scores before and at 6 months after surgery (r = 0.617 and 0.460, respectively; P < 0.001) and change in the distance between the retinal vessels after ERM surgery and preoperative total depth of retinal folds between the vessels (r = 0.471; P = 0.013). The preoperative MDRF values at which M-CHARTS scores were 0.5 before and 6 months after the surgery were 69 µm and 118 µm, respectively. CONCLUSION: The MDRF is an objective and quantitative biomarker of metamorphopsia in ERM. To maintain patients' quality of vision, ERM surgery may be performed when the preoperative MDRF ranges between 69 µm and 118 µm.

ADVERSE EFFECT OF MACULAR INTRARETINAL HEMORRHAGE ON THE PROGNOSIS OF SUBMACULAR HEMORRHAGE DUE TO RETINAL ARTERIAL MACROANEURYSM RUPTURE.

PURPOSE: To investigate the clinical course of submacular hemorrhage associated with ruptured retinal arterial macroaneurysm using swept-source optical coherence tomography. METHODS: This study included 23 eyes of 23 consecutive patients diagnosed with submacular hemorrhage associated with ruptured retinal arterial macroaneurysm. Cases underwent displacement of submacular hemorrhage (vitrectomy + subretinal injection of tissue plasminogen activator + air tamponade) and were followed up for 6 months after surgery. Localization of the preoperative hemorrhage and its effect on preoperative and postoperative best-corrected visual acuity, central retinal thickness, and continuity of the ellipsoid zone were measured. RESULTS: Macular intraretinal hemorrhage (IRH) was observed in 17 eyes (73.9%, IRH [+] group) and was not observed in 6 eyes (26.1%, IRH [-] group). The IRH (+) group showed worse postoperative best-corrected visual acuity values compared with the IRH (-) group (0.89 ± 0.47 in logarithm of the minimal angle of resolution units, Snellen equivalent 20/155 and 0.16 ± 0.23, 20/29, respectively; P < 0.01), smaller central retinal thickness values (97.7 ± 53.5 µm, 173.0 ± 32.3 µm, respectively; P < 0.01), and a higher rate of ellipsoid zone disruption (100%, 33.3%, respectively; P < 0.01). CONCLUSION: Patients with preoperative macular IRH showed lower postoperative visual acuity and worse macular contour after submacular hemorrhage displacement compared with patients without macular IRH.

Microperimetry-guided inverted internal limiting membrane flap site selection to preserve retinal sensitivity in macular hole with glaucoma.

Purpose: In cases of macular hole (MH) that is difficult to close, including large, chronic, or highly myopic cases, the inverted internal limiting membrane (ILM) flap technique is often preferred and yields favorable surgical outcomes as compared to those yielded by conventional ILM peeling. However, no consensus exists on the optimal location and area for peeling and inverting the ILM, since multiple alternative methods have been reported alongside the original method. Several adverse effects associated with ILM peeling have been documented, including mechanical impairment of the retinal nerve fiber layer and decreased retinal sensitivity. Particularly, when glaucoma is concomitant, the retinal nerve fiber layer is fragile, raising concerns about a decrease in retinal sensitivity. Consequently, in patients with large MH alongside glaucoma, the goal is to select a procedure that maximizes the closure rate of the MH while minimizing any negative impact on glaucomatous visual field impairment. However, a technique for this purpose has not yet been validated. Observations: A woman in her 60s presented with visual impairment (20/50), metamorphopsia, and central scotoma of unknown onset in the right eye. A full-thickness MH accompanied by epiretinal proliferation (EP) was identified, with a minimum diameter of 506 µm. Although a retinal nerve fiber layer defect was not evident on ophthalmoscopy, thinning of the ganglion cell complex (GCC), extending from the superotemporal aspect of the optic disc, was observed on optical coherence tomography. Both microperimetry and static visual field testing revealed reduced retinal sensitivity in the thinned GCC areas. A pars plana vitrectomy combined with cataract surgery was performed to address her condition. The EP was embedded into the foveal cavity. On the basis of the microperimetry results, the ILM within the absolute scotoma region was peeled on the superotemporal side of the fovea to create a flap, which was then placed over the MH. A gas tamponade was applied, and the patient was maintained in a prone position postoperatively. The MH was successfully closed after the surgery, resulting in visual improvement (20/25). No decline in retinal sensitivity after the surgery was observed. Conclusions and importance: Determining the location and area of the inverted ILM flap on the basis of microperimetry results is a promising patient-tailored strategy for treating MH concomitant with glaucoma while preventing further ILM peeling-associated reduction in the retinal sensitivity.

A factor for predicting simultaneous internal limiting membrane peeling during epiretinal membrane removal: swept-source optical coherence tomography-based evaluation of epiretinal membrane adhesion to the retina.

PURPOSE: To investigate preoperative factors associated with simultaneous internal limiting membrane (ILM) peeling during epiretinal membrane (ERM) removal. STUDY DESIGN: Observational cross-sectional study. METHODS: We retrospectively reviewed 60 eyes with idiopathic ERM that underwent vitrectomy. The gap between the ERM and ILM was visualized using en face optical coherence tomography. The depth and width of the ERM-ILM gap at the initiation site of ERM removal were measured, and the relationship between preoperative factors including these parameters and simultaneous ILM peeling during ERM removal was investigated. RESULTS: The ILM was simultaneously peeled during ERM removal in 30 eyes, but not in the other 30 eyes. Age was significantly higher (P = 0.017) and the width of the ERM-ILM gap was significantly smaller (P < 0.001) in the simultaneous ILM peeling (+) group than in the simultaneous ILM peeling (-) group. Multivariate logistic regression analysis confirmed the width of the ERM-ILM gap as a significant negative predictor for simultaneous ILM peeling (odds ratio, 0.992; 95% confidence interval, 0.986-0.997; P = 0.003). Receiver operating characteristic curve analysis of the width of the ERM-ILM gap revealed that the optimal cutoff for predicting simultaneous ILM peeling was 187.1 µm. CONCLUSION: The small width of the ERM-ILM gap at the initiation site of ERM removal was significantly associated with simultaneous ILM peeling, indicating that the adhesion strength between the ERM and ILM at the initial ERM grasping site determines whether simultaneous ILM peeling will occur during ERM removal.

Quantitative Analyses of Retinal Traction Force and Metamorphopsia in Lamellar Macular Hole and Related Diseases.

Purpose: To investigate the involvement of retinal traction in the pathogenesis of lamellar macular hole (LMH) and related diseases based on OCT-based consensus definition. Design: Retrospective, observational study. Participants: Seventy-two eyes with LMH, epiretinal membrane foveoschisis (ERM-FS), or macular pseudohole (MPH). Methods: To quantitatively evaluate the involvement and strength of retinal traction in their pathogenesis, retinal folds were visualized with en face OCT imaging, and the maximum depth of the parafoveal retinal folds (MDRF) was measured. Metamorphopsia was quantified by measuring the minimum visual angle of dotted lines needed to cause it to disappear using M-CHARTS (Inami). Main Outcome Measures: Maximum depth of retinal folds and M-CHARTS scores. Results: Of the 72 eyes, 26 were classified as having LMH, 25 as having ERM-FS, and 21 as having MPH. Parafoveal retinal folds were observed in 7 (26.9%) eyes with LMH, 25 (100%) with ERM-FS, and 21 (100%) with MPH. The MDRF (7.5 ± 17.6 µm) was significantly smaller in LMH than in ERM-FS (86.3 ± 31.4 µm) and MPH (74.5 ± 24.6 µm) (both P < 0.001), whereas no significant difference in MDRF between MPH and ERM-FS was observed (P = 0.43). A significant positive correlation between MDRF and M-CHARTS scores was observed in ERM-FS and MPH (P = 0.008 and 0.040, respectively) but not in LMH (P = 0.073). Conclusions: Retinal traction was significantly weaker in the LMH group than in the ERM-FS and MPH groups. The MDRF was significantly associated with the degree of metamorphopsia in the ERM-FS and MPH groups. These results provide insights into the diseases' pathophysiology and treatment strategy.

Epiretinal Membrane Impairs the Inner Retinal Layer in a Traction Force-Dependent Manner.

Purpose: To investigate the relationship between retinal traction force and impairment of the inner retinal layer in patients with epiretinal membrane (ERM). Design: Nonrandomized, retrospective consecutive case series. Participants: Two hundred nine eyes of 201 patients with idiopathic ERM who underwent vitrectomy for idiopathic ERM were enrolled. Methods: Retinal folds caused by ERM were visualized using en face OCT, and the maximum depth of retinal folds within the parafovea (MDRF) was measured. Focal macular electroretinogram (ERG) was used to measure the amplitude and implicit time of each component for the ERM eyes and the normal fellow eyes. B-scan OCT images were used to measure the thicknesses of the inner nuclear layer (INL) and outer nuclear layer (ONL) + outer plexiform layer (OPL). Expression of α-smooth muscle actin (α-SMA) in surgically removed ERM specimens was quantified by reverse-transcription polymerase chain reaction. Main Outcome Measures: We analyzed the relationship between MDRF and the relative amplitudes of focal macular ERG (affected eye/fellow eye), the relationships between MDRF and the mean INL thickness and ONL+OPL thickness, comparison of INL thickness and ONL+OPL thickness for each area when cases were classified according to MDRF localization in the ETDRS chart, and the relationship between MDRF and the relative expression of α-SMA in the ERM specimens. Results: The MDRF significantly correlated with the relative amplitudes (affected eye/fellow eye) of b-waves and oscillatory potentials (r = -0.657, P = 0.015; r = -0.569, P = 0.042, respectively) and the mean INL thickness and ONL+OPL thickness (r = 0.604, P < 0.001; r = 0.210, P = 0.007, respectively). However, only the INL thickness progression rate was significantly correlated with the MDRF progression rate (r = 0.770, P < 0.001). On case stratification by localization of MDRF based on the ETDRS chart, in regions other than temporal regions, the INL thickness was significantly greater in regions with MDRF than in other regions. The MDRF significantly correlated with α-SMA expression in the ERM specimens (r = 0.555, P = 0.009). Conclusions: The findings suggest that ERM impairs the inner retinal layer in a traction force-dependent manner. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Pioglitazone opposes neurogenic vascular dysfunction associated with chronic hyperinsulinaemia.

BACKGROUND AND PURPOSE: We previously demonstrated that chronic hyperinsulinaemia induced by drinking high levels of fructose augments adrenergic nerve-mediated vasoconstriction and suppresses vasodilatation mediated by calcitonin gene-related peptide (CGRP)-containing (CGRPergic) vasodilator nerves. In this study, the effects of pioglitazone on vascular responses induced by stimulation of adrenergic nerves, CGRPergic nerves and vasoactive agents were investigated in pithed rats given 15% fructose solution to drink (FDR). EXPERIMENTAL APPROACH: To assess the effect of pioglitazone on the altered vascular responsiveness in the hyperinsulinaemic state in vivo, changes in vascular responses to spinal cord stimulation (SCS) and intravenous bolus injections of noradrenaline, angiotensin II and CGRP were evaluated in pithed control rats and FDR either untreated or treated with pioglitazone. KEY RESULTS: In the pithed FDR, vasoconstrictor responses to SCS and to injections of noradrenaline and angiotensin II were significantly greater than those of pithed control rats. In pithed FDR with artificially increased blood pressure and blockade of the autonomic ganglia, the vasodilator responses to SCS and CGRP injection were significantly smaller than those of pithed control rats. Oral administration of pioglitazone to FDR for two weeks markedly decreased plasma levels of insulin, triglycerides and blood glucose. In FDR pioglitazone diminished the augmented vasoconstrictor responses to SCS, noradrenaline and angiotensin II, and ameliorated the decrease in vasodilator responses to SCS. CONCLUSIONS AND IMPLICATIONS: The present results suggest that pioglitazone improves not only insulin resistance, but also the dysfunctions in vascular control regulated by adrenergic and CGRPergic nerves in the hyperinsulinaemic state.

Angiotensin II type 2 receptors facilitate reinnervation of phenol-lesioned vascular calcitonin gene-related peptide-containing nerves in rat mesenteric arteries.

The present study was designed to investigate involvement of angiotensin II (Ang II) type 2 receptors (AT2 receptors) in restoration of perivascular nerve innervation injured by topical phenol treatment. Male Wistar rats underwent in vivo topical application of 10% phenol around the superior mesenteric artery. After phenol treatment, animals were subjected to immunohistochemistry of the third branch of small arteries, Western blot analysis of AT2 receptor protein expression in dorsal root ganglia (DRG) and studies of mesenteric neurogenic vasoresponsiveness. Ang II (750 ng/kg/day), nerve growth factor (NGF; 20 microg/kg/day) and PD123,319 (AT2 receptor antagonist; 10 mg/kg/day) were intraperitoneally administered for 7 days using osmotic mini-pumps immediately after topical phenol treatment. Losartan (AT1 receptor antagonist) was administered in drinking water (0.025%). Phenol treatment markedly reduced densities of both calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) and neuropeptide Y (NPY)-LI-containing fibers. NGF restored densities of both nerve fibers to the sham control level. Coadministration of Ang II and losartan significantly increased the density of CGRP-LI-fibers but not NPY-LI-fibers compared with saline control. The increase of the density of CGRP-LI-fibers by coadministration of Ang II and losartan was suppressed by adding PD123,319. Coadministration of Ang II and losartan ameliorated reduction of CGRP nerve-mediated vasodilation of perfused mesenteric arteries caused by phenol treatment. The AT2 receptor protein expression detected in DRG was markedly increased by NGF. These results suggest that selective stimulation of AT2 receptors by Ang II facilitates reinnervation of mesenteric perivascular CGRP-containing nerves injured by topical phenol application in the rat.

Permeability increase in black lipid membrane induced by compound 48/80.

When compound 48/80, a potent histamine liberator, was added in the aqueous phase facing the black lipid membrane, the conductivity of the membrane was remarkably increased. Although valinomycin displayed a distinct selectivity for K+ movement, such selection for ionic permeability was not observed in the case of compound 48/80.

Analysis of the mechanism of histamine release induced by substance P.

Substance P causes release of histamine from rat peritoneal mast cells; the structure-activity relationship shows that N-terminal residue is essential and the hydrophobic region of C-terminal plays an important role. Electrical conductivity of black lipid membrane containing phosphatidic acid was augmented by substance P. In this case, N-terminal residues and C-terminal hydrophobicity were also unavoidable. The partitioning of substance P into the organic phase increased in the presence of phosphatidic acid. The CD spectrum of substance P was changed from the unordered form to beta-form by coexistence of phosphatidic acid/PC liposomes in the medium. The addition of calcium or magnesium in the test solution is effective to prevent either of these phenomena. These findings indicate that substance P probably binds to negatively charged sites of membrane lipids, and subsequent penetration of C-terminal into the hydrophobic core of lipid bilayer may induce an increase of membrane permeability and the following histamine release.

Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein.

Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 micrograms/ml both in the presence and in the absence of extracellular Ca2+. After reverse-phase high-performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pI values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPs was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 x 10(5) clones, a positive clone, which encoded a full length of pre-proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre-proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.

Comparison of the amino acid and nucleotide sequences between human and two guinea pig major basic proteins.

By means of reverse-phase HPLC, 2 different proteins were obtained from apparently purified pig eosinophil major basic protein (MBP) and these proteins were named GMPB1 and GMBP2. It was revealed that these 2 components of MBP have similar molecular weights and pI values, although the amino acid compositions were slightly different. In the previous study, we cloned and sequenced GMPB1 cDNA. Here we obtained another clone by plaque hybridization using a screening probe synthesized by means of polymerase chain reaction. After sequencing, it became apparent that this clone corresponded to GMBP2. As in the case of GMBP1, the cDNA of GMBP2 encoded pre-proGMBP2 with 3 domains; signal peptide, acidic pro-portion, and mature GMBP2. By comparing the sequences of GMBP1 and GMBP2, it was revealed that the proteins were quite similar to each other. In addition, their sequences also resembled those of human MBP, especially in the basic domain of mature protein; but no such similarity existed in the pro-portion. Although the molecular weights determined by SDS-PAGE of guinea pig and human MBPs were 11,000 and 9,300, respectively, the calculated molecular weights of these 3 MBPs were all 13.8 kDa. The calculated pI values of GMBP1, GMBP2 and human MBP were 11.7, 11.3 and 11.6, respectively. By means of Harr plot analysis, it was revealed that the amino acid sequences, not only in signal peptides but also in the basic domains of mature proteins, were well conserved between guinea pig and human MBPs.

Cortisol secretion induced by substance P from bovine adrenocortical cells and its inhibition by calmodulin inhibitors.

When primary cultured bovine adrenocortical cells were treated with substance P (SP) at concentrations higher than 10 pM, cortisol output increased in a dose-dependent fashion. Although other neurokinins, such as neurokinin A (NKA) and neurokinin B (NKB), were also effective in secreting cortisol, SP was the most potent among the tested neurokinins, the potency order being SP greater than NKA much greater than NKB. This suggests that the NK-1 type receptor on adrenocortical cells may be the site of action of SP on cortisol secretion. The maximal response in SP-induced cortisol secretion was comparable to that elicited by adrenocorticotropic hormone (ACTH). SP-induced cortisol secretion was dependent upon extracellular Ca2+ concentrations, and 45Ca2+ uptake into adrenocortical cells treated with SP was long-lasting. While, in the case of ACTH, 45Ca2+ uptake proceeded transiently, the increase in intracellular cAMP content was much greater compared with that of SP. Although KT-5720, an inhibitor of protein kinase A, inhibited potently ACTH-induced cortisol secretion, SP-induced secretin was not affected by this inhibitor at all. On the other hand, calmodulin inhibitors, such as calmidazolium, trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, were not more effective in inhibiting SP-induced cortisol secretion than secretion induced by ACTH. The present study indicates that SP may be one of the physiological stimulants of cortisol secretion and that an increase in intracellular Ca2+ concentration and the subsequent activation of calmodulin may precede SP-induced cortisol secretion.

de novo synthesis of calmodulin binding protein in substance P-induced steroidogenesis in bovine adrenocortical cells.

In order to clarify the mechanism of substance P (SP)-induced cortisol secretion from bovine adrenocortical (BAC) cells, protein synthesis at the early stage of SP-stimulation in BAC cells was investigated. Both SP and adrenocorticotropic hormone (ACTH) increased [3H]leucine uptake into BAC cells in a dose-dependent fashion. Although the SP-induced [3H]leucine uptake precedes the cortisol secretion, ACTH was slower in inducing [3H]leucine uptake and cortisol secretion. Protein synthesis inhibitors, actinomycin D and cycloheximide, were potent in inhibiting the SP-induced cortisol secretion. SDS-PAGE analysis, revealed that a 240 kDa protein is newly synthesized in BAC cells in response to SP but not ACTH. It was also indicated that the production of this 240 kDa protein was elicited about 30 min after stimulation by SP. Moreover, A23187 and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also caused a rapid [3H]leucine uptake and production of 240 kDa protein. In contrast, dibutyryl cAMP did not induce the synthesis of this 240 kDa protein. Calmidazolium, a calmodulin inhibitor, effectively inhibited not only [3H]leucine uptake but also 240 kDa protein production due to SP. On the other hand, KT-5720, an inhibitor of protein kinase A, had no effect on [3H]leucine uptake or 240 kDa production. Using the [125I]calmodulin-membrane overlay method, it was found that the 240 kDa protein was a newly synthesized calmodulin binding protein. From the present study, it was concluded that the de novo synthesis of this 240 kDa protein may be intimately related to the cortisol secretion in SP-stimulated BAC cells associated with an activation of the Ca-calmodulin pathway.

Involvement of the microtubular system in the endothelin-1 secretion from porcine aortic endothelial cells.

The effects of certain microtubule-disrupting agents on endothelin-1 (ET-1) secretion from porcine aortic endothelial cells were studied. When endothelial cells were treated with thrombin (1 unit/mL), a significant increase in ET-1 secretion was detected in the incubation medium, while ET-1 secretion in the medium was diminished when the cells were treated simultaneously with either colchicine or vinblastine (10(-8)-10(-6) M). In such cases, however, the ET-1 content detected in the cells increased dose-dependently in accordance with the concentrations of the microtubule-disrupting agents. The intracellular accumulation of ET-1 was observed both in mitochondrial and microsomal fractions. On the other hand, thrombin produced a significant increase in polymerized tubulin content without affecting the total tubulin content. A thrombin-induced increase in the intracellular Ca2+ concentration of endothelial cells was inhibited by treatment with either colchicine or vinblastine. These results seem to indicate that the microtubular system may play an important role in ET-1 secretion from endothelial cells.

Histamine-induced differentiation of HL-60 cells. The role of cAMP and protein kinase A.

When HL-60 cells were stimulated with histamine, a significant differentiation of the cells toward neutrophils was elicited. Histamine increased phagocytic activity, but it reduced myeloperoxidase activity of HL-60 cells. Histamine-induced differentiation in HL-60 cells was inhibited not only by H2 antagonists, such as cimetidine, ranitidine and famotidine, but also by an inhibitor of protein kinase A (A kinase), KT-5720. Histamine increased the cAMP level and A kinase activity in HL-60 cells; both increases preceded the cell differentiation. Histamine also enhanced phosphorylation of a 160 kD protein in HL-60 cells, while H2 antagonists and KT-5720 inhibited this phosphorylation. The results of the present study indicate that an activation of A kinase via H2 receptor stimulation may cause the phosphorylation of a 160 kD protein and that this phosphorylation is probably involved in the process leading to differentiation of HL-60 cells.

Role of microtubules on Ca2+ release from the endoplasmic reticulum and associated histamine release from rat peritoneal mast cells.

In order to study the role of cytoskeletons on histamine release from mast cells, the effects of cytoskeleton-inhibiting agents were investigated. Since neither colchicine, vinblastine nor cytochalasin D was effective in inhibiting the IP3 formation, it is possible that neither microtubules nor microfilaments of rat peritoneal mast cells participate in the initial membrane events of the histamine release. However, both colchicine and vinblastine, but not cytochalasin D, were effective in inhibiting Ca2+ release from the intracellular Ca store. It was accordingly suggested that the microtubules, rather than microfilaments, are intimately related to the Ca2+ releasing process from the endoplasmic reticulum. The fluorescence intensity of the mast cells stained with FITC-labeled anti-tubulin antibody reflects the amount of tubulin polymers inside the cell, and colchicine treatment decreased the fluorescence intensity, indicating that colchicine is effective in depolymerizing the microtubules of rat mast cells. By contrast, the amount of tubulin polymer in the mast cells increased by compound 48/80, indicating that the rearrangement of microtubules took place in the mast cells, leading to histamine release. When permeabilized mast cells were exposed to potassium antimonate solution, microtubules attached themselves to the endoplasmic reticulum and many Ca antimonate dots were observed. From the present results, it was concluded that microtubules play an important role in the processes leading to Ca2+ release from the intracellular Ca store and subsequent histamine release.

The pathway responsible for EEG synchronization and effect of histamine on this system.

Electrical stimulation (3 Hz, 0.5 volts) to the midbrain reticular formation of conscious rats induced significant increase of EEG power densities (synchronization) recorded at the frontal cortex (FCOR), nucleus ventralis thalami (VE), or nucleus medialis centralis thalami (CM). Significant synchronization was also observed in the FCOR when electrical stimulation was applied to the VE and CM. When ipsilateral and bilateral VEs were electrocoagulated, no EEG synchronization was observed in the FCOR and CM. Intracerebroventricular administration of histamine (Hi) caused a marked suppression of FCOR EEG synchronization in both CM-lesioned and normal rats through H1 receptors. EEG synchronization in FCOR was not induced in ipsilateral or bilateral VE-lesioned rats after RF stimulation. When Hi (1 microgram) was injected into the VE of normal rats, EEG synchronization of FCOR was markedly reduced after RF or VE stimulation. No such changes were induced when Hi was injected into the CM.