Upregulation of the cardiac monocarboxylate transporter MCT1 in a rat model of congestive heart failure.

BACKGROUND: Cardiac metabolism becomes more dependent on carbohydrates in congestive heart failure (CHF), and lactate may be used as an important respiratory substrate. Monocarboxylate transporter 1 (MCT1) promotes cotransport of lactate and protons into and out of heart cells and conceivably flux of lactate between cells, because it is abundantly present in the intercalated disk. METHODS AND RESULTS: Six weeks after induction of myocardial infarction (MI) in Wistar rats, left ventricular end-diastolic pressures were >15 mm Hg, signifying CHF. MCT1 and connexin43 protein levels in CHF were 260% and 20%, respectively, of those in sham-operated animals (Sham), and the corresponding mRNA signals were 181% and not significantly changed, respectively. Confocal laserscan immunohistochemistry and quantitative immunogold cytochemistry showed that MCT1 density was much higher in CHF than in Sham both at the surface membrane and in the intercalated disk. In CHF, a novel intracellular pool of MCT1 appeared to be associated with cisternae, some close to the T tubules. In contrast, connexin43 particles, seen exclusively at gap junctions, were substantially fewer. Maximum lactate uptake was 107+/-15 mmol. L(-1). min(-1) in CHF and 42+/-6 mmol. L(-1). min(-1) in Sham cells (P<0.05). The K(m) values were between 7 and 9 mmol/L (P=NS). CONCLUSIONS: In cardiomyocytes from CHF rats, (1) the amount of functional MCT1 in the sarcolemma, including in the intercalated disk, is increased several-fold; (2) a new intracellular pool of MCT1 appears; (3) another disk protein, connexin43, is much reduced; and (4) increased reliance on lactate and other monocarboxylates (eg, pyruvate) could provide tight metabolic control of high-energy phosphates.

PALIREL, a computer program for analyzing particle-to-membrane relations, with emphasis on electron micrographs of immunocytochemical preparations and gold labeled molecules.

Many vital substances, such as receptors, transporters, and ion channels, in cells occur associated with membranes. To an increasing extent their precise localization is demonstrated by immunocytochemical methods including labeling with gold particles followed by electron microscopy. PALIREL has primarily been developed to facilitate such research, enabling rapid analysis of topographic relations of particles (gold or others) to neighboring linear interfaces (membranes). After digitization of membranes and particles, the program particularly allows computation of (1) the particle number and number per unit length of membrane, in individual bins (membrane lengths) interactively defined along the membrane; (2) the distance of each particle from the membrane; (3) the particle number, and the density (number per micron2), in zones defined along (over and under) the membrane; and (4) the particle number and density in "zonebins" resulting from zones and bins being defined simultaneously. If there occurs, somewhere in the membrane, a segment of different nature, such as a synapse, the quantitative data may be had separately for that and the adjoining parts of the membrane. PALIREL allows interactive redefinition of bins, zones, or objects (particle-line files) while other definitions are retained. The results can be presented on the screen as tables and histograms and be printed on request. A dedicated graphic routine permits inspection on screen of lines, particles, zones, and bins. PALIREL is equally applicable to biological investigations of other kinds, in which the topographic relations of points (structures represented as points) to lines (boundaries) are to be examined. PALIREL is available from the authors on a noncommercial basis.

High resolution immunogold analysis reveals distinct subcellular compartmentation of protein kinase C gamma and delta in rat Purkinje cells.

High resolution immunogold cytochemistry was used to investigate the subcellular distribution of protein kinase C gamma and delta in Purkinje cells of the rat cerebellum. Postembedding incubation with an antibody raised to a peptide sequence near the C-terminus of protein kinase C gamma resulted in strong labelling along the dendrosomatic plasma membrane. A quantitative analysis indicated that this labelling reflected the existence of two pools of protein kinase C gamma; one membrane associated pool and one cytoplasmic pool located within 50 nm of the plasma membrane. The labelling along the plasma membrane showed a pronounced and abrupt increase when moving from the cell body into the axon initial segment. Gold particles signalling protein kinase C gamma were also enriched in putative Purkinje axon terminals in the dentate nucleus. The only organelle showing a consistent immunolabelling for protein kinase C gamma was the Golgi apparatus where the gold particles were restricted to the trans face. Protein kinase C gamma immunoreactivity also occurred in the Purkinje cell spines, with an enrichment in or near the postsynaptic density. Antibodies to protein kinase C delta produced a very different labelling pattern in the Purkinje cells. Most of the gold particles were associated with rough endoplasmic reticulum, particularly with those cisternae that were located close to the nucleus or in the nuclear indentations. No significant protein kinase C delta immunolabelling was detected at the plasma membrane or in Purkinje cell spines. The present data point to a highly specific compartmentation of the two major protein kinase C isozymes in Purkinje cells and suggest that these isozymes act on different substrates and hence have different regulatory functions within these neurons.

Cellular and subcellular expression of the monocarboxylate transporter MCT1 in rat heart. A high-resolution immunogold analysis.

An antibody to the C-terminus of the monocarboxylate transporter MCT1 was used to study the precise cellular and subcellular distribution of this transporter in rat heart. Postembedding immunogold procedures revealed that the labeling in the heart was restricted to cardiomyocytes and concentrated along the plasma membrane, including the transverse tubules. Gold particles occurred with highest densities in intercalated disks, where they avoided desmosomes and gap junctions. Labeling was also associated with plasmalemmal invaginations having ultrastructural features typical of caveolae. Internal membrane compartments were unlabeled. Quantitative analyses following postembedding labeling showed that the distribution of gold particles across the plasma membrane was nearly symmetrical, indicating that the C-terminus of the transporter is situated very close to the cell membrane. In preembedding immunogold experiments, the gold particles were localized at the external aspect of the plasma membrane, suggesting that the C-terminus is extracellular. From the present data, it can be concluded that even under basal conditions the majority of the MCT1 molecules in heart is present in the myocyte plasma membrane, implying that there is a constitutive functional expression of this transporter. It follows that the increased transmembrane flux of lactate during exercise or in pathological conditions such as ischemia must be a result of altered substrate gradients rather than of translocation of MCT1 molecules to the plasma membrane.

The central nucleus of the inferior colliculus in rat: a Golgi and computer reconstruction study of neuronal and laminar structure.

The cellular basis for the laminar structure of the central nucleus of the inferior colliculus has been investigated by computer-assisted 3-D reconstruction of Golgi impregnated dendritic arbors, sampled from serial sections of resin embedded material of adult rat. Two types of flattened neurone, defined here as flat (F) and less flat (LF), are described as contributing to the pattern. The dendritic arbors of F neurones had a smaller absolute thickness (mean about 50 microns) and a denser arbor. They were strikingly parallel regionally and formed laminae mostly one cell thick. The laminae appeared to be separated by interlaminar compartments populated by the LF neurones. The arbors of the latter were thicker (mean about 100 microns) and less dense than those of the F neurones. The different density of the two types may, at least in part, be responsible for the corresponding difference in density of oriented dendrites within the laminae and interlaminar compartments. The orientation planes of F and LF arbors were roughly similar, but a consistent, slight difference in orientation between F and LF arbors is not excluded. Most of the F and LF arbors were elongated in parallel with the ventrolaterally to dorsomedially oriented long axis of the laminae. A few were instead oriented rostrocaudally or in intermediate directions. The interlaminar compartments appeared less distinct in the low than in the high frequency region. The latter region also differed from the former by having F neurones with a higher number of intermediate and terminal segments and a denser arbor. It is discussed whether the observed F and LF cells constitute two distinct cell types or are varieties of one type of neurone, with the morphological differences reflecting differences in location. Further characterization of the neurones on histochemical, hodological, and other criteria is required to settle this question.

Computer methods in neuroanatomy: determining mutual orientation of whole neuronal arbors.

In most neurons orientation can be recognized because their arbors are more or less polarized and/or flattened. These are morphological characteristics of great functional importance. This paper deals with three-dimensional display and mathematical definition of orientation planes and vectors in whole arbors. An orientation plane can be derived from the flattest rectangular prism with which it is possible to enclose the arbor, or may be found by best-fit least square determination (based on all digitized points of the arbor). Both approaches allow description and comparison, in quantitative terms, of the orientation of neurons under various normal, pathological or experimental conditions.

Cellular and subcellular redistribution of glutamate-, glutamine- and taurine-like immunoreactivities during forebrain ischemia: a semiquantitative electron microscopic study in rat hippocampus.

The effect of 20 min of ischemia on the cellular and subcellular distribution of glutamate, glutamine and taurine in the rat hippocampus was studied by means of an immunocytochemical procedure based on antisera raised against protein glutaraldehyde conjugates of the respective amino acids. Forebrain ischemia was induced by temporary occlusion of the common carotid arteries in rats with permanently occluded vertebral arteries. Within 90 s after removal of the carotid ligatures, the rats were perfused through the heart with a mixture of glutaraldehyde and paraformaldehyde. For semiquantitative electron microscopic analysis, ultrathin sections were incubated in a primary antiserum followed by a secondary antibody coupled to colloidal gold particles. The gold particle densities over different tissue compartments within the CA1 field and the mossy fiber zone of the hippocampus were determined by means of a specially designed computer program, and values from normal and ischemic animals were compared. It was found that in the astrocytes, the level of immunoreactivity for glutamine and taurine is unchanged or slightly decreased after ischemia, while that for glutamate is increased, particularly within the mitochondria (by about 100%). In contrast, pyramidal cell bodies display a reduced immunolabeling for all three amino acids following the ischemic episode. The results show that ischemia causes a redistribution of glutamate from neurons to glia. The observed increase in the glial immunolabeling for glutamate indicates that the capacity of the glial cells to metabolize glutamate is exceeded during ischemia. This glial response to ischemia has not previously been recognized and may play a role in the chain of events leading to "excitotoxic" cell death during or following an ischemic episode. The reduction of glutamate and taurine immunolabeling in neurons points to a possible amino acid efflux and is compatible with previous biochemical studies demonstrating an elevated extracellular level of these amino acids during ischemia.

MORFOREL, a computer program for two-dimensional analysis of micrographs of biological specimens, with emphasis on immunogold preparations.

The program is a tool for accelerating analysis of tissue components (profiles) as seen in micrographs, including electron micrographs with immunoreactive substances labelled with gold particles. Required equipment is a computer with digitizer and printer. From coordinates sampled around the profiles, area, perimeter and form factor are calculated; particles in profiles, when wanted, are counted to obtain particle densities. MORFOREL permits basic statistical calculations on primary data or on composite expressions based on them. Expressions can be saved on disk and retrieved. Primary and calculated data are readily output in a format readable by common commercial packages.

Computer programs for neuroanatomy: three-dimensional reconstruction and analysis of populations of cortical neurons and other bodies with a laminar distribution.

The program is designed for analysis of serial microtome sections containing biological structures (bodies, particles), each of which may be represented as one point. Any subset of sections may quickly be selected for reconstruction. In addition to rotation, translation, scaling etc. two particular features are offered. (1) Section-independent parts of reconstructions may be isolated ("windows") and then reconstructed, analyzed and subdivided, like the parent data. (2) Curved, bandshaped parts may be unfolded, for inspection of distribution pattern, determination of density etc. The program is written in Pascal (source 121 Kb, compiled program 65 Kb). User-friendliness, robustness and speed were key aims.

Pyramidal neurones of the dorsal cochlear nucleus: a Golgi and computer reconstruction study in cat.

The main projection neurones of the dorsal cochlear nucleus, termed pyramidal, bipolar or fusiform cells, have an apical dendritic arbor approaching the ependymal surface of the nucleus and a basal arbor oppositely directed. In Golgi-Del Rio-Hortega material these neurones were studied, with the light microscope, in nonconventional planes of sectioning oriented across or parallel to the main axis of the elongated nucleus. The pyramidal neurones were seen to be flattened across this axis. The size, shape and orientation of 21 cells from six blocks were studied in detail with computer-aided graphic reconstructions including stereo views. Camera lucida drawings of each cell (usually from several sections) were digitized to obtain x and y coordinates while z coordinates (depths in the tissue) were read from the fine focus knob during microscopy and typed interactively during digitization. The z values were corrected for the effects of refractive index differences in the optical system. Since it was the aim of this study to focus on some fundamental principles of structure and arrangement of pyramidal cells in the dorsal cochlear nucleus rather than on topographic variations, only the middle, regularly built part of the nucleus was examined. Towards the ends of the nucleus the architecture is less regular and will require separate analysis. Measurements of arbor and total cell height and of dendritic length are given. The height of the apical and basal arbor in individual cells showed considerable reciprocity. The total dendritic length was up to 8300 micron (average 6536 micron). The basal arbors always proved to be conspicuously flattened; roughly, the width varied between about 300 and 700 micron (average 489 micron) and the thickness between 65 and 105 micron (average 80 micron). The apical arbors were also often flattened but much less and with a greater variability than the basal arbors (average width 319 micron, thickness 115 micron). The two arbors of individual cells were practically coplanar, the arbor planes showing only moderate angularity (bend) and/or torsion relative to each other (angularity maximum 10 degrees, average 5 degrees; torsion maximum 18 degrees, average 6 degrees). The mutual orientation of cells from the same block was examined. The planes through the basal arbors proved to be very parallel, the differences in orientation angles being between 10 and 0 degrees with rare exceptions. Clearly flattened, apical arbors showed a somewhat greater spread.(ABSTRACT TRUNCATED AT 400 WORDS)

Electron microscopy of experimental axonal degeneration in photochemically modified Golgi preparations: a procedure for precise mapping of nervous connections.

A new version is presented of a procedure for bleaching (deimpregnating) silver chromate Golgi preparations through ultraviolet (UV) light irradiation followed by thiosulphate treatment, leaving only scattered particles of metallic silver. This version gives a better ultrastructure of the tissue than obtained before. Moreover, the results of studying a specific connection in such material are reported. The perforant path, connecting the entorhinal area with the hippocampus and fascia dentata, was transected in the rat and after a 2-day survival the brain was subjected to Golgi impregnation followed by the deimpregnation procedure. Blocks of tissue containing impregnated fascia dentata granule cell dendrites were selected for electron microscopy. Postsynaptic thickenings associated with degenerated boutons could be discerned within the deimpregnated dendrites, proving that the entorhino-dentate fibres end on the dendrites of the granule cells (smooth surfaces as well as spines) and that they end at asymmetric junctions (of Gray's type 1). While this conclusion was as expected from previous experimental studies carried out with different methods it is thought that the same strategy for cell-to-cell mapping of nervous connections may be used also in other parts of the central nervous system.