Voltage-clamp studies on the canine Purkinje strand

Purkinje strands were excised from the left and right ventricles of adult mongrel dogs and cut to lengths of less than 2.0 mm in order to apply the two-microelectrodes voltage-clamp technique. A sizeable fraction of these preparations fully recover following dissection, with resting potentials more negative than--80 mV and upstroke velocities faster than 290 V s-1. Analysis of the voltage response to small current pulses shows that the short Purkinje strands can be treated as simple finite one-dimensional cables with ends of infinite resistance. The average length constant is 2.5 mm. In keeping with the relatively long length constant, insertion of a third microelectrode along the strand demonstrates a high degree of longitudinal homogeneity of the voltage clamp. Analysis of the capacity transient gives an estimate of the total capacity, normalized to cylindrical surface area, of 11.5 muF cm-2. The final decay of the capacity transient is a single exponential with an average time constant of 1 ms. A second slower component to the final decay of the capacity transient is absent in solutions of normal conductivity as well as in solutions of reduced (13%) conductivity. This suggests that the extracellular series resistance may be relatively small. The magnitude of the K+ depletion current was estimated by measuring the ratio of depletion current to instantaneous current. This ratio averaged 10%. These two results are consistent with the morphometric data described in the accompanying paper, which show that the canine preparation has wider extracellular clefts than the ungulate preparation. The existence of the full complement of inward and outward currents, including the pacemaker current, is demonstrated. The presence of wide clefts does not affect the potential at which the pacemaker current reverses (about--107 mV in 4 mM [K+] Tyrode solution), since the pacemaker current reverses at approximately the same potential in the canine Purkinje preparation as it does in the ungulate.     

On the effect of unstirred layers on K+-activated electrogenic Na+ pumping in cardiac Purkinje strands.

Many studies of electrogenic Na+ pumping in Purkinje strands have involved intracellular Na+ loading by exposure to 0 mM K+, followed by reexposure to K+. For sheep Purkinje strands the K+ concentration for half-maximal stimulation (K0.5) in such studies is higher than K0.5 of canine Purkinje strands. A model was developed to determine if gradients in the K+ concentration of extracellular fluid layers during enhanced pump activity can account for the discrepancy. Pump activity was assumed linearly dependent on [Na+]i and dependent on [K+]o, according to Michaelis-Menten kinetics. The model simulated diffusion of K+ across unstirred layers and both depletion and accumulation of K+ in extracellular clefts of Purkinje strands during changes in the K+ concentration of the tissue bath. Errors in estimates of K0.5 occurred when delay in achieving a steady state extracellular K+ concentration was simulated. The simulations suggested that a linear relationship between pump current and intracellular Na+, a monoexponential decay of pump current, independence of the rate constants for the current decay on the initial Na+ load and holding potential, and apparent Michaelis-Menten K+ kinetics is not sufficient evidence against pump-induced interstitial K+ depletion having introduced errors in determination of K0.5. It is concluded that interstitial K+ depletion may account for the difference between determinations of K0.5 in sheep and canine Purkinje strands.     

Nexuses between the smooth muscle cells of the guinea-pig ileum

The circular musculature of the guinea-pig ileum has been studied by freeze-fracture to analyze quantitatively the gap junctions (nexuses) between its smooth muscle cells. The average cell surface area and cell volume are 5,074 micron 2 and 3,260 micron 3. The packing density of nexuses is 48/1,000 micron 2 of cell surface or approximately 244/muscle cell. Nexuses range in area from less than 0.1 to approximately 1.5 micron 2 and they occupy 0.212% of the cell surface. The average packing density of intramembrane particles or pits in nexuses is approximately 7,200/micron 2 of nexal surface, indicating that there may be approximately 77,000 intercellular channels in the full complement of nexuses of one muscle cell.     

Electrical properties of sheep Purkinje strands. Electrical and chemical potentials in the clefts

The impedence of sheep Purkinje strands, measured to 3-5 kHz, is interpreted with circuit models based on morphology. The strand is described as a one-dimensional electrical cable. Clefts between myocytes of the strand allow radial current to flow in parallel with current across the outer membrane. A lumped model of the clefts, in which all the cleft membrane is in series with 100 omega-cm2, fits only below 20 Hz. Two distributed models, pie and disk, fit at all frequencies with somewhat different (31%) luminal resistivities, but with similar membrane parameters. Series resistance representing the endothelial sheath is small. Simulations of voltage clamp experiments include measured linear parameters and nonlinear membrane channels, as well as radial variation of cleft concentration, membrane flux, voltage, and current. Cleft potential is drastically nonuniform when sodium current flows. Cleft potential is reasonably uniform when calcium and potassium currents flow, but the calcium and potassium concentrations change markedly, enough to turn off the calcium current, even if the calcium channel did not inactivate. We conclude that physiological current flows produce significant nonuniformities in electrochemical potentials in the clefts of this cardiac preparation.     

Lysosomal interconnections and horseradish peroxidase compartmentalization in three-dimensionally reconstructed bovine alveolar macrophages

Following a 1-h incubation of bovine alveolar macrophages in 1 to 2 mg/ml exogenous horseradish peroxidase (HRP), ultrathin sections revealed vacuolar interconnections among both labeled and unlabeled vacuoles constituting the lysosomal compartment. Four entire cells and their vacuolar components were subsequently computer resconstructed from serial transmission electron micrographs and measured using a morphometric technique. HRP-labeled and unlabeled vacuoles ranged in size from 0.5 micron to greater than or equal to 4.0 microns in diameter and occupied up to 25% of the cytoplasmic volume. HRP-containing vacuoles were distributed throughout each cell in a clumped distribution (P less than 0.05) and occupied up to 75% of the total vacuole compartment. Up to 60% of all vacuoles were interconnected through a series of openings formed by membrane fusions (average pore diameter 0.42 micron), which resulted in a labyrinth of vacuoles comprising up to 55% of the total volume of the lysosomal compartment. The area of open interconnections resulting from vacuolar fusions represented less than 1% of the total surface area of the lysosomal membrane. Rotation of a three-dimensionally reconstructed macrophage about the Y-axis revealed an interconnected vacuolar network of 75 fused vacuoles in a chain up to 21 microns in length. We have demonstrated that HRP-labeled vacuoles interconnect with each other as well as with preexisting unlabeled vacuoles. As a result of such interconnections, individual vacuoles become contributing members of a large, continuous, lysosomal compartment in bovine alveolar macrophages.     

Sheep cardiac Purkinje fibers: Configurational changes during the cardiac cycle

Previous attempts to study the cytoarchitecture of cardiac Purkinje fibers with the scanning electron microscope (SEM) have been limited by the surrounding dense connective tissue. In this study the connective tissue was removed by treatment with 8N HCl, after adult sheep hearts were fixed in diastole or systole and tissue taken for SEM and transmission electron microscopy (TEM). In SEM, Purkinje fibers freely anastomosed in false tendons and formed a subendocardial plexus. In systole, medium and small-sized Purkinje fibers formed deep clefts not observed in diastole. The clefts are thought to be due to sarcolemmal folding and fiber buckling and may therefore affect conduction. The myofibrils beneath the laterally apposed sarcolemmas of adjacent Purkinje cells when fixed in systole were often observed as tightly curved arches in series. Similar configurations with expanded arches were observed in diastole. The formation of arches by myofibrils is unique to Purkinje fibers and is interpreted as the mechanism responsible for their compliance to stretch. The significance of contraction in producing the observed geometric changes in Purkinje fibers and the implications of their cytoarchitecture with respect to conduction are discussed.     

Gap junctions and zonulae occludentes of hepatocytes during biliary atresia in the lamprey

Gap junctions and zonulae occludentes of hepatocytes were examined in thin sections and freeze-fracture replicas from livers of larval and juvenile adult lampreys and during the phase of metamorphosis when bile ducts and bile canaliculi disappear (biliary atresia). Larvae possess zonulae occludentes at the canaliculi which are composed of one to five (mean = 2.81) junctional strands that provide a bile-blood barrier. Morphometry demonstrates that during biliary atresia the decreases in number of junctional strands and apico-basal depth of the zonulae occludentes are accompanied by an increase in the frequency of gaps or interruptions in the strands and in a breakdown of the bile-blood barrier. The zonulae occludentes completely disappear during metamorphosis and are not found in the adult liver. Gap junctions of the larval liver occupy 1% of the surface of the plasma membrane and have a mean area of 0.167 micron 2 but, following an initial decline in these parameters during early biliary atresia, they rise sharply in later stages of metamorphosis and in adults are 3.2% and 0.502 micron 2, respectively. The events of alteration in junctional morphology during lamprey biliary atresia is in many ways comparable to the changes in gap junctions and zonulae occludentes during experimental and pathological intra- and extrahepatic cholestasis in mammals.     

Different response of cellular DNA content to cardiac hypertrophy in human and rat heart myocytes

1. Rat and human heart myocytes adapt to overload-induced hypertrophy differently. 2. Human myocyte nuclei respond with polyploidization and multinucleation, thus increasing the DNA content per myocyte from 20 to 40 pg. As a result, nuclear DNA content per 10,000 microns3 of cell volume decreases from 12 to 10 pg. 3. In rat hearts with aortic constriction nuclear DNA content remains constant (13 pg), and the DNA content per 10,000 microns3 of myocyte volume falls from 9 to 6 pg. 4. We hypothesize that "dilution" of nuclear DNA in the hypertrophied rat heart myocyte limits the capacity to hypertrophy (much less than 100%). 5. The human heart myocyte, which is able to compensate for dilution of nuclear DNA, may increase in size more than three-fold. 6. The lower limit of DNA content per unit of myocyte volume is 6 pg/10,000 microns3 in both species.     

The demonstration of close nerve-Purkinje fibre contacts in false tendons of sheep heart

Summary An observation of intimate nerve-Purkinje fibre associations in false tendons of sheep heart is reported. Nerve bundles were observed in deep clefts of Purkinje fibres, in channels running between coupled Purkinje cells and embedded within Purkinje cells, as well as in the outer connective tissue sheath. Most nerve terminals in these areas were filled with small clear vesicles and a few large dense-cored vesicles. Only a few axons with many small dense-cored vesicles were observed.Intimate associations (separation, 60 to 90 nm) between the Purkinje cell and nerve varicosity were observed in the deep clefts. Similar close appositions were also present where nerves were embedded in Purkinje cells. In these cases the Purkinje cell enclosing the nerve bundle formed intercellular junctions with its own sarcolemma.Elaborate sarcolemmal folds with multi-vesicular bodies were also frequently observed near nerve bundles and varicosities. The identity of the transmitter is unknown although the nerves forming intimate associations with Purkinje cells have a morphology typical of cholinergic nerves.     

Molecular architecture of the inner membrane of mitochondria from rat liver: a combined biochemical and stereological study

The molecular structure of mitochondria and their inner membrane has been studied using a combined approach of stereology and biochemistry. The amount of mitochondrial structures (volume, number, surface area of inner membrane) in a purified preparation of mitochondria from rat liver was estimated by stereological procedures. In the same preparation, the oxidative activity of the respiratory chain with different substrates and the concentration of the redox complexes were measured by biochemical means. By relating the stereological and biochemical data, it was estimated that the individual mitochondrion isolated from rat liver has a volume of 0.27 micron 3, an inner membrane area of 6.5 micron 2, and contains between 2,600 (complex I) and 15,600 (aa3) redox complexes which produce an electron flow of over 100,000 electrons per second with pyruvate as substrate. The individual redox complexes and the H+-ATPase together occur at a density of approximately 7,500/micron 2 and occupy approximately 40% of the inner membrane area. From the respective densities it was concluded that the mean nearest distance between reaction partners is small enough (70-200 A) to cause the formation of micro-aggregates. The meaning of these results for the mechanism of mitochondrial energy transduction is discussed.     

Decidual cells in the human ovary at term: II. Morphometric analysis of cytoplasmic processes and organelles

Morphometric analysis of human ovarian decidual cells was performed with a Videoplan computer, and mean values were established for the area and perimeter of cellular processes and organelles. Two-hundred forty electron micrographs representing 160 cells were analyzed. The mean decidual cell area was 218.7 microns2, of which 34.5 microns2 was occupied by the nucleus (15.8% of the cytoplasmic area); the nucleus contained 1.74 micron2 of nucleolar material (0.8%). The endoplasmic reticulum occupied 13.63 microns2 (6.2%). Mitochondria occupied 7.3 microns2 (3.3%) and the Golgi network 5.49 microns2 (2.5%). Decidual secretory bodies occupied 0.91 micron2 (0.42%) and cytoplasmic processes 1.89 micron2 (0.94%). The remainder of the cytoplasm, containing inclusions and cytoskeleton, represented 71% of the cell area. Perimeter measurements indicated an average decidual cell was surrounded by 87.8 microns of plasma membrane. The mean nuclear membrane measured 28.3 microns (representing 32.3% of the plasma membrane, pm, or 4.1% of total cellular membranes, cm). Outer mitochondrial membranes measured 156.6 microns (178% pm, 23.5% cm); endoplasmic reticulum membranes measured 350.3 microns (400% pm, 52.6% cm); Golgi membrane measured 30.77 microns (35% pm; 4.5% cm) and membrane surrounding secretory bodies measured 9.8 microns (11.2% pm; 1.4% cm). A mean of 280 secretory bodies per ovarian decidual cell was calculated. The plasma membranes of evaginated cytoplasmic processes represented 22.3% of the total pm (19.6 microns or 2.9% cm). A mean of seven such processes was observed per 87.8 microns of plasma membrane (160/cell). These morphometric data provide a baseline for comparisons of human ovarian decidual cells with uterine decidua, in vivo and in vitro, as well as with decidual cells of other species.     

Dicrete zones of electrical connection between Purkinje terminals and muscle fibers in dog ventricles]

Activity of Purkinje terminals (P) and neighbouring muscle (M) fibres (P -- M-pairs) in various regions of dog's right ventricle was recorded. It has been shown that transmission of stimulation from P-to M-fibres is abserved not over all endocardial surface, rather in descrete sites -- zones of P--M-interaction. The zones ellips like with the axes 300X100 micron, they are located relatively far from one another -- at the distance of 800-2000 microns. The total area of the zones equals 5% of endocardial surface. P-M delay is less within the zone of connection of P--M-fibres (4 msec) and greater outside the zone (up to 10 msec).     

Role of the membrane cortex in neutrophil deformation in small pipets.

The simplest model for a neutrophil in its "passive" state views the cell as consisting of a liquid-like cytoplasmic region surrounded by a membrane. The cell surface is in a state of isotropic contraction, which causes the cell to assume a spherical shape. This contraction is characterized by the cortical tension. The cortical tension shows a weak area dilation dependence, and it determines the elastic properties of the cell for small curvature deformations. At high curvature deformations in small pipets (with internal radii less than 1 micron), the measured critical suction pressure for cell flow into the pipet is larger than its estimate from the law of Laplace. A model is proposed where the region consisting of the cytoplasm membrane and the underlying cortex (having a finite thickness) is introduced at the cell surface. The mechanical properties of this region are characterized by the apparent cortical tension (defined as a free contraction energy per unit area) and the apparent bending modulus (introduced as a bending free energy per unit area) of its middle plane. The model predicts that for small curvature deformations (in pipets having radii larger than 1.2 microns) the role of the cortical thickness and the resistance for bending of the membrane-cortex complex is negligible. For high curvature deformations, they lead to elevated suction pressures above the values predicted from the law of Laplace. The existence of elevated suction pressures for pipets with radii from 1 micron down to 0.24 micron is found experimentally. The measured excess suction pressures cannot be explained only by the modified law of Laplace (for a cortex with finite thickness and negligible bending resistance), because it predicts unacceptable high cortical thicknesses (from 0.3 to 0.7 micron). It is concluded that the membrane-cortex complex has an apparent bending modulus from 1 x 10(-18) to 2 x 10(-18) J for a cortex with a thickness from 0.1 micron down to values much smaller than the radius of the smallest pipet (0.24 micron) used in this study.     

Effects of total parenteral nutrition on rat bile canaliculi: a stereologic analysis

OBJECTIVE: To quantitate, in a stereologic manner, changes in bile canalicular morphology before and after choleretic infusion of total parenteral nutrition (TPN) and to determine whether TPN produces changes in localized regions within the hepatic lobule. STUDY DESIGN: Livers were obtained from sham-operated on normal adult male rats (control) and from rats that received intravenous TPN solution containing 20% glucose and 3.5% Molipron F. The tissues, obtained by a rigorous sampling procedure, were systematically subjected to stereologic analysis. Measurements were made on electron micrographs at two levels of magnification by point, intersection and profile counts, and then volume, surface area and length were estimated per unit parenchymal volume. RESULTS: The surface area of the canalicular wall per parenchymal volume increased significantly (from 5.33 x 10(-2) to 6.73 x 10(-2) microns 2/micron 3) after TPN treatment, as did the length of microvilli (from 0.241 to 0.267 microns/micron 3). However, the volume of bile canalicular lumina per parenchymal volume (0.306% and 0.320%), total length of bile canaliculi (1.05 x 10(-2) and 1.06 x 10(-2) microns/micron 3) and diameter of microvilli (8.73 x 10(-2) and 8.94 x 10(-2) microns) remained constant. CONCLUSION: These results indicate that changes in canalicular shape and microvillus hypertrophy may cause lowering efficiency of the bile flow rate.     

To the problem of Campylobacter jejuni detectability in water

In a series of model experiments two isolation procedures for the detection of water-borne Campylobacter jejuni were compared: a standard culture in thioglycolate broth enriched with 7% defibrinated sheep blood and supplement C and a modified membrane filtration method in which the filter (porosity 0.45 microns) plated on campylobacter agar surface was removed after the first 24 hours of incubation and the plate further incubated for 48 hours. The recovery rates by the thioglycolate broth method were markedly less pronounced than those obtained by the modification of membrane filtration technique, especially in the case of water rich in organics. The best isolation parameters were achieved with water samples of at least 10 ml in volume.     

Isolation and morphology of single Purkinje cells from the porcine heart

Purkinje cells were isolated from both ventricles of young adult domestic pigs and examined by transmitted light or laser scanning confocal microscopy. Purkinje cells in free running Purkinje fibres were organised in multicellular strands where individual cells were tightly connected end-to-end and closely side-to-side. After isolation, single cells gradually lost the elongated appearance and became more rounded, but the cell membrane remained smooth and undamaged. The contractile material was not very dense and was seen most clearly in the submembraneous area. Staining of the cell membrane with the lipophilic fluorescent (lye di-8-ANNEPS, and visualization with confocal microscopy, confirmed that the cell surface membrane was smooth without blebs. This staining also showed that Purkinje cells had no transversal tubules. We reconstructed the three-dimensional geometry of the Purkinje cells and determined the cell size. The average values were 62 +/- 9 microm for length, 32 +/- 3 microm for width, and 41 +/- 4 microm for depth (n = 7). Calculated cross-section area and volume were 1047 +/- 167 microm2 and 47 +/- 14 pl. Compared to ventricular cells, the morphology of the Purkinje cells reflects their specific role in impulse conduction.     

Migration of polarized epithelial cells through permeable membrane substrates of defined pore size.

We have observed that cells of various epithelial lines exhibit the ability to migrate through permeable membrane substrates containing 3.0 microns pores. Scanning and transmission electron microscopic observations of Vero C1008 and Caco-2 cell lines grown on polycarbonate membranes containing 3.0 microns pores revealed extensive penetration of the filter and the establishment of virtually complete monolayers on the opposing surface. The migration of MDCK cells was also observed to occur under the same conditions; however, the extent of MDCK cell growth on the opposing surface was significantly less than observed for Vero C1008 and Caco-2 cells. Morphological differences were apparent between cells growing on the upper and lower faces of the filter membrane, although cells growing on both surfaces exhibited a polarized phenotype. The cells which invaded the filter were collected and maintained by serial passage. The passaged cells exhibited morphological differences and an altered rate of differentiation in comparison to the parental cell type, suggesting that the invasive cells represent a variant of the parental cell population. Studies using filters of different pore sizes indicated that cellular migration also occurs through pores of 2.0 microns diameter, but not through 1.0 micron (or smaller) pores. These observations have significant implications for studies involving the growth of epithelial cells on permeable membrane substrates containing large pores.     

The interrelationship of cesium, intracellular sodium activity, and pacemaker potential in cardiac Purkinje fibers

The actions of cesium (Cs) on intracellular sodium activity (aiNa), membrane potentials, and force were studied in sheep cardiac Purkinje and myocardial fibers superfused in vitro. In Purkinje fibers, Cs (2 mM) decreased diastolic depolarization, aiNa (-6.7%, p less than 0.005), and force (-28.0%, p less than 0.01). The effects of 4 and 8 mM Cs were more pronounced. In quiescent fibers, Cs (2-4 mM) also decreased aiNa (-17.3%, p less than 0.005) and induced an initial hyperpolarization (+5.6 +/- 1.3%, p less than 0.005) followed by a return toward control. Diastolic depolarization was almost abolished by driving the fibers at 180/min (diastole was very short) but still Cs decreased aiNa (-15.4%). Tetrodotoxin decreased aiNa (-16.2%, p less than 0.025) and reduced the Cs-induced fall in aiNa (-2.2%, p less than 0.05). In zero [K]o, Cs decreased aiNa and caused repolarization. In 0.1 mM strophanthidin, Cs did not decrease aiNa any longer and affected the membrane potential little. In quiescent myocardial fibers, Cs (4 mM) decreased aiNa (-12.6%, p less than 0.05) and transiently hyperpolarized (+2.1%). Rubidium (2 mM) decreased aiNa and resting potential in Purkinje fibers and in myocardial fibers and also decreased diastolic depolarization in Purkinje fibers. Thus, cesium and rubidium decrease aiNa and modify the membrane potential but not through a block of the inward pacemaker current If.     

Freeze-fracture studies on the subcommissural organ tight junction in gerbils and mice

Freeze-fracture studies on the tight junction of ependymal cells in the gerbil and mouse subcommissural organ (SCO) show an obvious species-specific variation. The tight junctional structure of the mouse SCO is composed of several strands (7.03 +/- 2.09 strands/cell) and occupies a total depth of 0.88 +/- 0.16 micron with a linear density of 7.12 +/- 2.11 strands/micron. The tight junction of the gerbil SCO is composed of three regions: (1) an apical region: made of 4 to 6 strands, oriented parallel to the free surface, with a high linear density (21.78 +/- 3.98 strands/micron) and small depth (0.049 +/- 0.009 micron); (2) a rather smooth and/or empty intermediate region, and (3) a basal region similar in morphology and morphometry to the junctional area of mouse SCO. These data indicate that the main difference in the SCO tight junction between the gerbil and the mouse is the presence of an apical region of high strand density in the former. We speculate that this apical region may play a role in maintaining the homeostasis of this CNS region in gerbils and possibly other desert animals, and may be part of a mechanism for survival in an extremely dry environment.     

Strain-induced reorientation of an intramuscular connective tissue network: implications for passive muscle elasticity

The most abundant intramuscular connective tissue component, the perimysium, of bovine M. sternomandibularis muscle was shown to be a crossed-ply arrangement of crimped collagen fibres which reorientate and decrimp on changing muscle fibre sarcomere length. Reorientation of perimysial strands was observed by light microscopy and identification of these strands as collagen fibres was confirmed by high-angle X-ray diffraction. Mean collagen fibre direction with respect to the muscle fibres ranged from approximately 80 degrees at sarcomere length = 1.1 micron to approximately 20 degrees at 3.9 microns. This behaviour was well described by a model of a crimped planar network surrounding a muscle fibre bundle of constant volume but varying length. Modelling of the mechanical properties of the perimysium at different sarcomere lengths produced a load-sarcomere length curve which was in good agreement with the passive elastic properties of the muscle, especially at long sarcomere lengths. It is concluded that the role of the perimysial collagen network is to prevent over-stretching of the muscle fibre bundles.