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.

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.

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.

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.

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 localisation of laminin in normal and exfoliative iris.

Immunoelectron microscopic studies of exfoliative iris tissue (seven specimens) revealed the presence of laminin in the fibrillar component of exfoliation material. The immunogold label was uniformly distributed on the exfoliation fibres. Deposition of laminin labelled exfoliation material in the dilator muscle was a noteworthy feature, as was an apparent depletion of laminin in the basement membranes of ostensibly unaffected vessels. In control iris tissue (five enucleated eyes) laminin was identified in the basement membrane round vascular contractile cells, but not beneath the endothelium.

Ultrastructural distribution of collagen types I-VI in aging human retinal vessels.

Retinal vessels from freshly enucleated human eyes were classified into three stages of the hyalinisation process. The distribution of collagen types I-VI within the vessel walls was studied ultrastructurally by immunogold labelling combined with the tissue preparation techniques of cryoultramicrotomy and London resin white embedding. Collagen types I, III, IV, V, and VI were found in large vessels, types I, IV, and V plus small amounts of III and VI in small vessels, and types I, IV, and V in capillaries. Hyalinised vessel walls consisted mainly of types I, IV, and VI collagen.

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.

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.

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.

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.

The stereochemical structure of disodium DL-glycerol 3-phosphate hexahydrate, the D isomer of which is an inhibitor of triose phosphate isomerase.

The stereochemistry of dl-glycerol 3-phosphate was studied by X-ray-crystallographic techniques. All the bond lengths and angles are within normally accepted limits except the ester bond, which is one of the largest yet noted, being 0.1637nm. The conformation of the molecule is such that an intramolecular hydrogen bond is formed between the hydroxyl group on the beta-carbon atom and the phosphate group. The crystal, which was grown by alcohol diffusion into an aqueous solution, is held together by sodium co-ordination and a complex system of hydrogen bonds. A table of the observed and calculated structure factors, F(obs.) and F(calc.), has been deposited as Supplementary Publication 50010 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972) 126, 5.

Immunocytochemical localisation of collagens (I-V) in the human iris.

In this study we investigated the distribution of collagen types I-V in the human iris at the fine-structural level using cryoultramicrotomy and London Resin White plastic embedding. Collagen type I was shown to be present in the basement membrane of iris vessels, in contrast to type III, which was absent; both types I and III were present in the iris stroma. Collagen type IV was a major component of basement membranes of vascular cells, myoepithelial cells, fibroblasts and epithelial cells. Types II and V were absent. Both cryo and plastic embedding techniques produced closely comparable results.

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.

Crystalloid material in cells of the murine mononuclear phagocyte system.

In an attempt to throw further light on the nature and distribution of crystalloid material in macrophages, tissues from a variety of locations in 9 normal adult mice of 36-46 g body weight were studied by light and electron microscopy. Crystalloid inclusions were widely distributed in mononuclear phagocytic cells, being present in macrophages of the bone marrow, spleen, lung and ileum and in the Kupffer cells of the liver. They were not observed in the macrophages of the thymus, parotid lymph node or skin nor were they seen within the lymphatic nodules of Peyer's patches. The observed distribution of crystalloid material seems consistent with its suggested origin from granulocyte breakdown.

Immunogold localization of type IV collagen and laminin in the aging human outflow system.

Tissue from the outflow system of six surgically enucleated aged eyes was used for an ultrastructural immunocytochemical study of the distribution of laminin and type IV collagen. The immunogold technique provided precise localization of laminin beneath lining endothelial cells of the inner wall of Schlemm's canal. Laminin labelling was absent in the trabecular beams. Type IV collagen was found in the basement membranes of the trabecular beams and in fine filamentous basement membrane material in the cribriform layer. Electron dense plaques in the cribriform layer labelled positively for laminin in the outer coarse fibrillar zone but not in the electron dense core. Long-spacing collagen was negative for type IV collagen.

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 fine structural localization of extracellular matrix components in aged human cornea. I. Types I-IV collagen and laminin.

Using the immunogold technique combined with cryoultramicrotomy and London Resin white (LR white) embedding, we studied the fine structural distribution of types I-IV collagen and laminin in corneal tissue from seven enucleated human eyes (age range, 63-78 years). Type II collagen was not identified in any corneal layer. Type I and type III collagen were distributed in a similar fashion in striated collagen fibrils in Bowman's layer and in the stroma. Type IV collagen was located only in the posterior non-banded region of Descemet's membrane. Laminin was identified in subepithelial anchoring plaques and the sub-endothelial region of Descemet's membrane in accordance with its recognized adhesive function.

Immunogold fine structural localization of extracellular matrix components in aged human cornea. II. Collagen types V and VI.

Using immunogold immunocytochemical techniques we studied the distribution of collagen types V and VI in corneal tissue from seven enucleated human eyes (age range, 63-78 years). Results obtained by cryoultramicrotomy were marginally more intense than those obtained using London Resin white (LR white) embedding. Type V collagen was present in the striated collagen fibrils in Bowman's layer, in the stroma and in a thin, non-banded anterior zone of Descemet's membrane. Our results suggest that types I, III and V collagen co-distribute in striated collagen fibrils. By contrast, type VI collagen was located in fine filaments in the interfibrillar matrix of the stroma, in Bowman's layer and in the anchoring plaques of the sub-epithelial basement-membrane complex. This implies an importance in epithelial adhesion which was previously unsuspected. Keratocyte bodies were electron-dense, amorphous extracellular deposits of matrix-like material, and these were labelled with types III, V and VI collagen antibodies. Long-spacing collagen was observed in the corneal stroma, and this deposit did not contain any of the collagen types studied.