Transient outward K+ current reduction prolongs action potentials and promotes afterdepolarisations: a dynamic-clamp study in human and rabbit cardiac atrial myocytes.

Human atrial transient outward K(+) current (I(TO)) is decreased in a variety of cardiac pathologies, but how I(TO) reduction alters action potentials (APs) and arrhythmia mechanisms is poorly understood, owing to non-selectivity of I(TO) blockers. The aim of this study was to investigate effects of selective I(TO) changes on AP shape and duration (APD), and on afterdepolarisations or abnormal automaticity with ß-adrenergic-stimulation, using the dynamic-clamp technique in atrial cells. Human and rabbit atrial cells were isolated by enzymatic dissociation, and electrical activity recorded by whole-cell-patch clamp (35-37°C). Dynamic-clamp-simulated I(TO) reduction or block slowed AP phase 1 and elevated the plateau, significantly prolonging APD, in both species. In human atrial cells, I(TO) block (100% I(TO) subtraction) increased APD(50) by 31%, APD(90) by 17%, and APD(-61 mV) (reflecting cellular effective refractory period) by 22% (P < 0.05 for each). Interrupting I(TO) block at various time points during repolarisation revealed that the APD(90) increase resulted mainly from plateau-elevation, rather than from phase 1-slowing or any residual I(TO). In rabbit atrial cells, partial I(TO) block (∼40% I(TO) subtraction) reversibly increased the incidence of cellular arrhythmic depolarisations (CADs; afterdepolarisations and/or abnormal automaticity) in the presence of the ß-agonist isoproterenol (0.1 µm; ISO), from 0% to 64% (P < 0.05). ISO-induced CADs were significantly suppressed by dynamic-clamp increase in I(TO) (∼40% I(TO) addition). ISO+I(TO) decrease-induced CADs were abolished by ß(1)-antagonism with atenolol at therapeutic concentration (1 µm). Atrial cell action potential changes from selective I(TO) modulation, shown for the first time using dynamic-clamp, have the potential to influence reentrant and non-reentrant arrhythmia mechanisms, with implications for both the development and treatment of atrial fibrillation.

Distribution of antioxidant enzymes in the normal aged human conjunctiva: an immunocytochemical study.

BACKGROUND: The purpose of the study was to map the precise location of four antioxidant enzymes in the normal human conjunctiva. METHODS: Conjunctival tissue (seventeen specimens) from seven patients was processed for light microscopy and immunogold electron microscopy. Antibodies were used to label glutathione peroxidase, glutathione S-transferase (acidic and neutral forms) and copper/zinc superoxide dismutase. RESULTS: All layers of the epithelium exhibited labelling for all four enzymes, although there were differences in labelling intensities and distribution between the enzymes. Wing cells showed more intense labelling for glutathione peroxidase, glutathione S-transferase (neutral form) and copper/zinc superoxide dismutase than did basal and superficial cells. The strongest immunoreactivity was detected for glutathione S-transferase (acidic) and was equally strong in the superficial and wing cells. Wing cells demonstrated more immunoreactivity in the cell nuclei with antibodies against all four enzymes. A high degree of labelling was also observed in the microplicae and actin cortex of the superficial cells. Leukocytes, fibroblasts, endothelial cells and erythrocytes within the stroma exhibited minimal labelling. CONCLUSION: The subtle changes in distribution of antioxidant enzymes in the normal human conjunctival epithelium most probably reflect exposure to free radicals in the tear film and are based on the different proliferative and functional activities of each of the anatomical layers.

Microvilli elongate in response to hydrogen peroxide and to perturbations of intracellular calcium.

Using scanning electron microscopy and fluorescence microscopy, we have found that apical microvilli of diverse cell types, including nonepithelial cells, elongate in culture in response to the oxidative stress of hydrogen peroxide. The microvilli induced in culture on retinal pigment epithelial cells display a 30-nm axial periodicity similar to that described for stable microvilli of intestinal brush border. Microvilli can also be induced to elongate by chelating intracellular Ca2+ and by the Ca(2+)-uptake inhibitor thapsigargin. Thus a response of microvillar protrusion occurs widely and may be related to depletion of intracellular calcium stores.

Antioxidant enzymes in the human iris: an immunogold study.

AIMS: To determine the nature and extent of the antioxidant enzyme system in the human iris. METHODS: The fine structural distribution of five antioxidant enzymes (acidic, neutral, and basic glutathione S-transferase (GST), Cu/Zn superoxide dismutase (Cu/Zn SOD), and glutathione peroxidase) was determined by immunogold labelling of ultrathin sections of tissue from six eyes appropriately fixed for immunocytochemistry and embedded in LR white resin. RESULTS: Both Cu/Zn SOD and acidic GST were localised to all constituent cells of the iris. Glutathione peroxidase and basic GST were localised to erythrocytes alone, and labelling for neutral GST was absent. CONCLUSIONS: Dense labelling for acidic GST could be linked with the previously documented presence of large quantities of polyunsaturated fatty acids in the iris and/or the presence of xenobiotic substances in the aqueous humour.

Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat.

In order to determine whether spinothalamic neurons in the lumbar spinal cord of the rat process neurokinin-1 (substance P) receptors, we injected cholera toxin B subunit into the thalamus and carried out dual-labelling immunocytochemistry to search for neurons that were immunoreactive with antibodies to cholera toxin and neurokinin-1 receptor. We examined 356 spinothalamic neurons in transverse sections and found that 35% of these were neurokinin-1 receptor-immunoreactive. Double-labelled cells made up the majority of the spinothalamic population in lamina I and the lateral spinal nucleus, and were also present in laminae III-V and the area around the central canal. On the side contralateral to the injection site, 77% of spinothalamic neurons in lamina I also showed neurokinin-1 receptor immunoreactivity, while 33% of those in laminae III-V and 14% of the ventromedial group possessed the receptor. Several of the double-labelled neurons with cell bodies in laminae III and IV had dendrites which could be followed dorsally into the superficial dorsal horn. These results indicate that substance P released from nociceptive primary afferents into the superficial dorsal horn is likely to act on spinothalamic tract neurons in lamina I, and also on those with cells bodies in laminae III-IV and long dorsal dendrites.

Hydrogen peroxide induces microvilli on human retinal pigment epithelial cells in culture.

We have found that hydrogen peroxide (10(-4)-10(-2) M) rapidly induces microvilli on separate cells and confluent sheets of human retinal pigment epithelium in culture. t-butyl hydroperoxide and sodium arsenite do not induce microvilli. A role for hydrogen peroxide as an intercellular messenger has previously been proposed in the inflammatory response, in which hydrogen peroxide from phagocytes may signal to vascular endothelial cells. Our observations thus provide a second example of the induction of what may be a physiological response by this potentially toxic agent. In the retina, hydrogen peroxide released from illuminated photoreceptors may elongate the microvilli which extend into the spaces between them. Increased numbers of microvilli and their protrusion further into the photoreceptor layer may enhance various interactions between the two cell types, including the antioxidant functions of the epithelium.

Human scleral elastic system: an immunoelectron microscopic study.

An immunocytochemical study was conducted on elastic components in the sclera of seven aged human eyes. By conventional electron microscopy, elastic tissue consists of three distinct fibre types--elastic fibres, elaunin fibres, and oxytalan fibres. The distribution of six components associated with the elastic system (elastin, amyloid P component, laminin, fibronectin, gp 115, and vitronectin) were studied by immunogold transmission electron microscopy. The codistribution of amyloid P component and laminin was further studied by double immunolabelling. Both elastic and elaunin fibres contained elastin. The microfibrillar sheaths of elastic fibres labelled for amyloid P component, those of elaunin fibres for amyloid P and laminin, and those of oxytalan fibres for laminin only. No labelling was observed for fibronectin, gp 115, and vitronectin. In terms of the proteins investigated, the biochemical profile of the three fibre types was not completely identical and was manifest as different affinities in the binding of serum amyloid P component and an association with laminin.

Immunogold study of non-collagenous matrix components in normal and exfoliative iris.

The present investigation was undertaken to determine if some of the components of exfoliation material in iris tissue were unique to exfoliation or were part of normal iris architecture. Eleven normal iris specimens and 10 exfoliative iris specimens were processed for cryoultramicrotomy and London resin white embedding. Immunogold electron microscopy was used to investigate the fine structural distribution of amyloid P component, elastin, entactin, fibronectin, gp115, and vitronectin in normal iris and their association with exfoliation material. Exfoliation material was positive for amyloid P component and possibly gp115, neither of which were present in normal iris tissue. Elastin and fibronectin were present in the normal iris stroma but were not associated with exfoliation material. The distribution of amyloid P component in the vessel lumen and wall led to the conclusion that amyloid P is a serum contaminant. The presence of gp115 in exfoliation material represents the synthesis of a component novel to the iris vascular cell synthetic repertoire.

Corneal endothelial cell abnormalities in an early stage of the iridocorneal endothelial syndrome.

A corneal disc, obtained from a 52-year-old woman suffering from an early stage of the iridocorneal endothelial syndrome (ICE), was investigated by various morphological techniques to analyse the structural variations in the endothelial cells and to identify the collagen types within the abnormal layer of Descemet's membrane. Scanning electron microscopy of the posterior corneal surface revealed a mosaic of (a) flat hexagonal cells resembling irregular but normal endothelial cells, and (b) rounded hexagonal (ICE) cells with numerous surface microvilli. Degenerative changes were present in each cell type, but were more common in the flat hexagonal cells which contained intracytoplasmic spaces. By transmission electron microscopy the flat hexagonal cells exhibited many of the features of normal endothelial cells in terms of organelles and intercellular attachments, but lateral invaginations were absent. The ICE cells differed in that the apical surface was covered by microvilli and the cytoplasm contained tonofilaments, which were also observed by light microscopic immunocytochemical staining. Most commonly, intercellular attachments were rudimentary in both types of cell and intercellular spaces were dilated, but desmosomes were sometimes prominent in the ICE cells where interdigitations were pronounced. In some sectors, the basal surface of the ICE cells was indented by deposition of clumps of fibrillar collagenous material. An immunocytochemical study of the abnormal posterior deposits localised type IV collagen to the amorphous matrix and collagen types III and V, but not type I, to the collagen fibril bundles. Mononuclear inflammatory cells were identified between the ICE cells in the monolayer. The evidence suggests that some of the flat hexagonal cells were undergoing a degenerative change while others were transforming into ICE cells.

Immuno-electron labelling of matrix components in congenital hereditary endothelial dystrophy.

Two corneal buttons were obtained from a patient with congenital hereditary endothelial dystrophy (CHED) at the ages of 2.5 years (right eye) and 14 years (left eye) and were studied by light and electron microscopy including immunogold labelling for collagen types I-V and laminin. The posterior collagenous layer (PCL) of Descemet's membrane contained collagen types I, III-V, and laminin: the latter was also localised to fine-banded and granular material in the posterior non-banded zone (PNBZ). Comparison of the endothelium 2.5 years and 14 years revealed occasional dystrophic changes in the former and extensive dystrophic changes in the latter. The distribution of collagen types I, III and V within the PCL supports previous morphological observations of fibroblast-like change of the endothelium in CHED. Persisting endothelial properties were manifest as positive labelling of type IV collagen and laminin. An excessive amount of laminin found in PNBZ and PCL is another stress-related endothelial reaction.

Collagens in the aged human macula.

Immunogold cytochemistry was used to investigate the fine structural distribution of collagen types I-VI in Bruch's membrane and choroid of the aged human macula. Macular tissue was obtained from ten eyes, and processed for cryoultramicrotomy and London Resin white embedding. Striated collagen fibrils within the inner and outer collagenous layers were found to contain collagen types I, III and V. In addition, type V collagen was also present in the basement membrane of the choriocapillaris. Gross thickening of the choriocapillaris basement membrane was attributed to the deposition of type IV collagen. However, type IV collagen appeared to be absent from the basement membrane of the retinal pigment epithelium. The interesting location of type VI collagen on the choroidal side of the choriocapillaris suggested that its function is to anchor the choriocapillaris onto the choroid. The collagens studied were absent from fibrous banded material, long-spacing collagen, the elastic layer and amorphous granular material. It was concluded that, of the collagen types studied, only the deposition of type IV collagen contributes to the age-related thickening of Bruch's membrane.

Morphology of iris vasculopathy in exfoliation glaucoma.

Iris tissue obtained from 26 consecutive patients operated upon for exfoliation glaucoma and control iris tissue from 26 age-matched subjects operated upon for primary open angle glaucoma was used to investigate the iris vasculopathy associated with exfoliation glaucoma. By light microscopy exfoliation material was discerned by increased density of the perivascular matrix in affected vessels. By transmission electron microscopy exfoliation vasculopathy was divided into 4 grades. Grade I was characterized by focal accumulation of exfoliation material without evidence of cellular degeneration. In grade II, exfoliation material accumulation was accompanied by degeneration of vascular supporting cells; endothelial cells were unaffected. In grade III, endothelial cells exhibited degenerative changes and in grade IV, exfoliation material occupied an acellular vascular wall (ghost vessel). It is suggested that in iris vessels the synthesis of exfoliation material can be attributed primarily to the vascular supporting cells.

Prevalence, diagnostic features, and response to trabeculectomy in exfoliation glaucoma.

BACKGROUND: The "true" prevalence and clinical attributes of exfoliation glaucoma remain controversial. The authors studied these characteristics in glaucoma patients requiring trabeculectomy. METHODS: One hundred consecutive patients undergoing trabeculectomy for open-angle glaucoma were investigated by clinical examination (biomicroscopy and gonioscopy) and classified into three categories: exfoliation glaucoma, possible exfoliation glaucoma, and primary open-angle glaucoma (POAG). A definitive diagnosis of exfoliation glaucoma was provided by pathologic examination of iris tissue. RESULTS: All 22 patients with clinical evidence of exfoliation glaucoma and 4 of 18 patients with possible exfoliation glaucoma on clinical examination had ultrastructural evidence of exfoliation material. The prevalence of exfoliation glaucoma, therefore, was 26%. The clinical examination for the diagnosis of exfoliation glaucoma had an 85% sensitivity rate and a 100% specificity rate. In comparison with POAG, patients with exfoliation glaucoma had higher untreated intraocular pressure (IOP), higher IOP with medical therapy, and shorter duration of medical therapy. They were more often operated on for unacceptably high IOP. Exfoliation glaucoma patients exhibited significantly lower IOP after surgery. CONCLUSION: Exfoliation glaucoma is common in patients requiring trabeculectomy for open-angle glaucoma. This condition differs from POAG by a poorer response to medical therapy and a better response to trabeculectomy.

Collagens in the aged human macular sclera.

Scleral tissue from the region of the human macula was studied by the immunogold labeling technique (cryoultramicrotomy and LR white resin embedding) in an attempt to identify the fine structural distribution of collagen types I-VI. Labeling of the striated collagen fibrils suggested colocalisation of collagen types I, III and V with type V occurring at the fibril surface. Both types V and VI collagen were localised to filamentous strands in the interfibrillar matrix. Collagen types II and IV were absent from the scleral stroma.

Type IV collagen and laminin in Bruch's membrane and basal linear deposit in the human macula.

Tissue obtained from the macula in 10 human eyes (53-77 years) was used for an investigation into the extracellular matrices of the retinal pigment epithelium (RPE), Bruch's membrane, and the choriocapillaris. The ultrastructural distribution of type IV collagen and laminin was documented using immunogold labelling. Labelling for type IV collagen was strongly positive in all the specimens in the basement membranes of the choriocapillaris but not that of the RPE where labelling was either weak or absent. Laminin was localised to deposits of granular material in Bruch's membrane but was absent from the basement membrane of the RPE and the choriocapillaris. Basal linear deposit, observed in three cases, demonstrated labelling for laminin but not for type IV collagen. The series was too small for correlation of these morphological changes with age.

Extracellular matrix in aged human ciliary body: an immunoelectron microscope study.

Tissue from nine human eyes (ages 52-78 yr) was used to investigate the fine structural distribution of collagens I-VI and laminin in the ciliary body using the immunogold antibody labeling technique. The anterior segments of the specimens were normal, and the eyes were removed in treatment of choroidal melanoma. The basement membranes of the ciliary epithelium contained collagens I, III, and IV. Laminin was in greater concentration in the outer part of the nonpigmented epithelial basement membrane, and the distribution suggested a washout effect. The zonular apparatus labeled intensely with laminin. In contrast, laminin was not present in the basement membrane of the myocytes in the ciliary body. These cells were sheathed in a basement membrane that contained types I, III, and IV collagen. Plaque-like structures of slightly different morphology (a, filamentous; b, granular; c, amorphous) were found in the tendinous insertions, and subtypes a and b were strongly labeled with laminin. The basement membranes of the vessels contained types I and IV collagen, but laminin labeling was inconclusive. The major finding was that the lamina densa in the basement membranes of various sites labeled for collagens I, III, and IV. Striated collagen fibrils in the stroma were labeled for types I and III. Collagen subtypes V and VI were not identified in significant quantity in any of the regions examined.

An immunoelectron microscope study of the aged human lens capsule.

The distribution of types I-IV collagen and laminin was studied in seven aged human lens capsules using the immunogold EM technique on LR White embedded tissue. Samples were taken from the anterior, equatorial and posterior regions. Labelling for type II collagen was not observed. Type IV collagen was evenly distributed throughout the thickness of the capsule but was absent from the zonules. However, an unexpected finding was strong labelling for types I and III collagen, again evenly distributed throughout the capsule. The presence of type III collagen makes the lens capsule unique among ocular basement membranes. Laminin was present in linear densities, zonular lamellae and zonular fibres, suggesting that linear densities are an integral part of the zonular apparatus.

Iris vasculopathy in exfoliation syndrome. An immunocytochemical study.

Samples of control iris tissue obtained from seven enucleated eyes and nine exfoliative iridectomy specimens were prepared for an immunocytochemical study of the matrix in the walls of iris vessels. The distribution of collagen types I, IV and laminin was studied in normal and exfoliative vessels. Laminin was an integral component of exfoliation material and was present mainly in the matrix of the outer surface of normal vascular cohort cells. The laminin content in the latter location was reduced in vessels in which exfoliation aggregates were not visible. Collagen type IV was absent from exfoliation material. While type I was present in the deposits, it was considered to represent a residue of native normal tissue. Exfoliation deposits appeared to stimulate the synthesis of collagen types I and IV at an early stage of the disease.

Immunogold ultrastructural localization of collagens in the aged human outflow system.

Immunogold labeling was applied at the ultrastructural level to the identification of collagen types I, II, III, V, and VI in the human trabecular meshwork obtained from 11 surgically enucleated globes. Both London resin white embedding and cryoultramicrotomy were used, and for types V and VI, the latter provided more sensitive localization. Type II collagen was not identified. Types I and III were localized to the striated collagen fibrils of the trabecular core, the basement membrane of the trabecular beams, and loose aggregates in the juxtacanalicular tissue. Types V and VI formed a fine network around the striated fibrils in the trabecular cores and linkage strands to the basement membranes. The outer wall of Schlemm's canal contained collagens I, III, V, and VI. Long-spacing collagen and elastin-like material did not label with any of the antibodies used in this study.