Intramyocardial Influences on Blood Flow Distributions in the Myocardial Wall

Flow velocity wave forms of coronary arterial inflow and venous outflow of myocardium are influenced by cardiac contraction and relaxation: arterial flow is exclusively diastolic; venous outflow is systolic. We first discuss the intramyocardial microvascular flow dynamics, then present some results of visualization of transmural microvessels by our needle-probe charge coupled device (CCD) microscope, along with an interpretation of the arteriolar and venular hemodynamics through a cardiac cycle. After describing a hierarchical system of coronary microvessels (small artery, arteriole, and capillary), we emphasize the importance of spatial heterogeneity of blood supply to myocardium with reference to a minimal vascular control unit (400 m). An understanding of mechanoenergetic interaction is fundamentally important to an understanding of intramyocardial coronary circulation, and the Physiome Project will provide powerful tools for understanding the integrated role of the intramyocardial microcirculation system. © 2000 Biomedical Engineering Society. PAC00: 8719Hh, 8719Ff, 8719Tt     

Parallel changes in red blood cell and renal Na−K-ATPase activity in adrenal and electrolyte disorders in the rat

To compare the activity of Na–K-ATPase in the red blood cells (RBCs) and in renal tissue in disorders of Na⁺ metabolism, the following groups of rats were studied: 1) control, intact rats, 2) adrenalectomized (ADX) rats, 3) intact rats treated with DOCA, 4) ADX DOCA-treated rats, 5) intact salt-loaded rats, 6) ADX salt-loaded rats, 7) intact dexamethasone-treated rats (DEXA), and 8) ADX DEXA-treated rats. After adrenalectomy (group 2) serum Na¹ decreased and serum K⁺ increased.Renal Na–K-ATPase in cortex, medulla and papilla of the control group was 44±2.7 mol Pi/mg prot/h, 128.2±5.9 and 44±3.2 respectively and in group 2 the enzyme activity was 32.5±2.0 (P<0.005), 81.7±4.5 (P<0.001) and 23.6±1.9 (P<0.001) respectively. RBCs Na–K-ATPase of control animals was 2.82±0.19 mol Pi/mg prot/h, while in group 2 the activity was 1.43±0.24 (P<0.001). DOCA treatment of ADX rats (group 4) normalized serum electrolytes and Na–K-ATPase activity in the renal cortex and papilla and in the RBCs. In the renal medulla the correction by DOCA was only partial. Salt loading of ADX rats (group 6) normalized serum electrolytes and Na–K-ATPase activity in the renal medulla and RBCs. Salt loading of normal rats increased RBC Na–K-ATPase to 3.72±0.36 (P<0.02) and medullary Na–K-ATPase to 185.6±9.8 (P<0.01). DEXA treatment of ADX rats (group 8) corrected only partially the abnormalities in serum electrolytes and Na–K-ATPase activity in the kidney and in the RBCs. These findings show, 1) parallel changes in the activity of Na–K-ATPase in the RBCs and in the kidney after adrenalectomy, 2) parallel changes in the enzyme activity in RBCs and in the kidney medulla after salt loading, and 3) correction towards normal of RBC Na–K-ATPase after ADX by NaCl and DOCA treatment.This study was supported by the Morton S. Kaufman Hemodialysis Foundation     

Scanning electron microscopy of red blood cells from eleven species of marsupial

Samples of red of blood cells (RBC), washed free of plasma, from eleven marsupial species were examined in a Jeol JSM-6300 F scanning electron microscope. The diameters of the RBC, lying completely flat or exactly on edge, were measured on photographs using a binocular enlarging optical system with a calibrated eye piece. RBC from the following species were studied: bandicoot (Isoodon macrourus), bilby (Macrotis lagotis sagitta), Bennett's wallaby (Macropus rufogriseus), Goodfellow's tree kangaroo (Dendrolagus goodfellowi), koala (Phascolarctos cinereus), parma wallaby (Macropus parma), red kangaroo (Macropus rufus), swamp wallaby (Wallabia bicolor), Tammar wallaby (Macropus eugenii), Tasmanian devil (Sarcophilus harrish) and whiptail wallaby (Macropus parryi). In all species the RBC were biconcave discs. The major morphological difference was in the size of the cells. Taking the human RBC as a reference (with a diameter of 8.0 m), the RBC of the Tasmanian devil, bilby, bandicoot and Goodfellow's tree kangaroo were 7 m in diameter, whereas those of the other marsupials ranged from 7.8 to 8.6 m.     

Perturbation of red blood cell flow in small tubes by white blood cells

The flow of blood in the microcirculation is facilitated by the dynamic reduction in viscosity (Fahraeus-Lindquist effect) resulting from the axial flow of deforming crythrocytes (RBCs) and from the decrease in the ratio of cell to vessel diameter. RBC velocity exceeds that of average fluid velocity; however the slower moving white blood cells (WBC) perturb flow velocity and the ratio of cell to vessel diameter by obstructing red cell flow through formation of trains of red cells collecting behind the white cell. This effect of white cells was studied quantitatively in a model in vitro tubes less than 10 m in diameter with the demonstration that flow resistance increases linearly with white cell numbers up to 1,000 WBC/mm³ at tube hematocrit of 17.7%. The increase in resistance exceeds the flow resistance of WBC and appears to relate directly to train formation. A mechanical model of train formation developed to predict WBC influence in flow resistance over the range of WBC studied reasonably fits observed WBC effects.     

Inhibitory effects of E-5110 on interleukin-1 generation from human monocytes

Effects of E-5110, a novel non-steroidal antiinflammatory drug, on interleukin-1 (IL-1) generation from human monocytes were studiedin vitro. E-5110 reduced the amounts of extra- and intracellular IL-1 activity induced by lipopolysaccharide (LPS, 1 g/ml) in a dose-dependent manner (1–10M). E-5110 also inhibited the IL-1 generation induced by antigen-antibody complexes, opsonized zymosan and silica particles. It was suggested that the inhibition of IL-1 generation by E-5110 was independent of the inhibitory effects on arachidonate cyclooxygenase and/or lipoxygenase because indomethacin, piroxicam, BW755C and AA861 had no effects on IL-1 generation. Hydrocortisone (IC₅₀:0.084 M), aurothioglucose (11.5 M) and lobenzarit (75.0 M), which are clinically effective antirheumatic drugs, also inhibited IL-1 generation, like E-5110 (1.21 M). It is expected that E-5110 will be superior to classical non-steroidal antiinflammatory drugs in medical treatment of rheumatoid arthritis.     

Netilmicin disk susceptibility tests: Effect of cations on the MIC correlates

When testingPseudomonas aeruginosa against netilmicin, MICs were markedly affected by the concentration of cations added to the test medium. A susceptible disk test result (zone15 mm) corresponded to MIC4.0g/ml in unsupplemented broth, 12g/ml in broth with half the usual amount of cations and 32 g/ml in broth with the recommended concentration of cations. Tests with 30g netilmicin disks best predicted susceptibility as determined by MICs in broth without added cations. When the MICs were determined in cation supplemented broth, the number of interpretive discrepancies increased to an unacceptably high level.     

The continuous measurement of tubular volume changes in response to step changes in contraluminal osmolality

A new method to measure time dependent (t) volume (V) changes in proximal straight tubules (PST) is described.V is calculated from diameter (d) measurements for which a video camera and an integrating circuit are used. A tubular image of high optical contrast is recorded with the TV camera such that the scan lines run crosswise to the tubule. The video signal is analyzed by a special processor which adds 225 tubular diameters of each TV frame and feeds this analog signal to a pen recorder. The fractional error ind measurements is 10^–3. Diameter changes of less than 0.05 m can be detected, as compared to the usual error of a single measurement of about 0.4 m.P _os ^cb , the osmotic water permeability of the contraluminal cell membrane was measured by setting up osmotic steps across it in less than 0.1 s and following the ensuing d/t. the time delay between solution change and the linear part of the osmotic response was 0.51±0.05 s.P _os ^cb was found to be 50.4 (±8.7)×10^–4 cm³·cm^–2 of basement membrane area ·s^–1·osmolar^–1.     

Enhanced stability of YEp plasmids in lager brewing yeasts is related to lager brewing yeast 2-μm DNA

Summary YEp plasmid stability in the presence of either Saccharomyces cerevisiae laboratory strain 2-m DNA, or lager brewing yeast 2-m DNA in the same genetic background, was compared under non-selective culture conditions. It was found that YEp plasmids were more stably maintained in the presence of lager 2-m DNA under these conditions. By construction of laboratory-lager 2-m DNA hybrid plasmids, an 867 bp StuI fragment of lager 2-m DNA was shown to be responsible for the enhanced stability of the YEp plasmid. Nucleotide substitutions at two sites were found by sequencing this region. It was also confirmed that increasing cell ploidy enhanced YEp stability under non-selective conditions.     

High frequency FLP-independent homologous DNA recombination of 2 μ plasmid in the yeast Saccharomyces cerevisiae

Summary The purpose of this work is to identify and quantitate in vivo 2 plasmid FLP-independent recombination in yeast, using a nonselective assay system for rapid detection of phenotypic expression of the recombination events. A tester plasmid was constructed such that in vivo recombination between 2 direct repeat sequences produces the resolution of the plasmid into two circular DNA molecules. This recombinational event is detected as a phenotypic shift from red to white colonies, due to the mitotic loss of the plasmid portion containing the yeast ADE8 gene in a recipient ade1 ade2 ade8 genetic background. In the absence of the 2 FLP recombinase and/or its target DNA sequence, recombination is not abolished but rather continues at a high frequency of about 17%. This suggests that the FLP-independent events are mediated by the chromosomally-encoded general homologous recombination system. We therefore conclude that the totality of 2 DNA recombination events occurring in FLP⁺ cells is the contribution of both FLP-mediated and FLP-independent events.     

Curing of Saccharomyces cerevisiae 2-μm DNA by transformation

Summary A general procedure for the curing of 2-m in Saccharomyces cerevisiae is described. The method is based on the displacement of endogenous 2-m DNA by the recombinant plasmid pMP78-1, which carries the yeast leu2 gene and the 2 -m DNA replicon, but cannot be maintained stably in a yeast cell without endogenous 2-m DNA. After transformation with pMP78-1 cells are grown selectively to displace 2-m DNA. During the non-selective growth which follows, plasmid pMP78-1 is lost and up to 100% of the cells completely lack plasmids. In conjunction with a kanamycin resistance marker, as present in plasmid pMP81, this method should be applicable to cure any wild-type yeast strain. The stability of recombinant plasmids in cir ⁺ and cir ⁰ strains has been compared.     

Papillary cystic tumours of the pancreas: An analysis by nuclear morphometry

Papillary cystic tumour (PCT) is a rare, low-grade malignant pancreatic neoplasm, in which the histological criteria for malignancy are still uncertain. We performed a histological examination of 3 metastasizing PCTs, while comparing them with 18 non-metastasizing PCTs, using a computed image analyser. The mean maximum nuclear diameter, the mean standard deviation (SD) of the nuclear diameter, the mean nuclear area and the nuclear-nonnuclear (N/NN) ratio obtained by the image analyser of the metastasizing PCTs (7.23 m, 2.21 m, 30.45 m², 36.41%) were all significantly larger than those of the non-metastasizing PCT (6.34 m, 1.59 m, 23.66 m², 23.74%;P<0.005,P< 0.005,P<0.005,P<0.001 respectively). However, there were no statistical differences in either the nuclear ellipsoidity or nuclear regularity. These results suggested that nuclear morphometry might be a useful parameter to define metastatic potential, in addition to histological variables such as venous invasion, nuclear grade and mitotic rate.     

Quantitative estimation of the area of luminal and basolateral membranes of rat parotid acinar cells: some physiological applications

Knowledge of luminal and basolateral acinar cell membrane areas of the secretory endpieces is a prerequisite for a detailed quantitative analysis of the ion transport involved in secretion of the primary saliva. In the present study, these areas were estimated in rat parotid acinar cells using standard stereological methods. A total of 480 micrographs — obtained by random sampling from eight glands from four rats — were analysed at a final magnification of 40000x. Expressed per unit cell volume, the area of the luminal acinar cell membrane was: 0.125 m² · m^–3 (SEM=0.027 m² · m^–3, n=4 animals) and the area of the basolateral membrane was: 1.54 m² · m^–3 (SEM=0.085 m² · m^–3, n=4 animals). These figures make it possible to perform a synthesis based upon different categories of experimental data, e.g. on ion fluxes, membrane potentials and single-channel conductances. Thus, we have estimated the density of open, low-conductance Cl^– channels in the luminal membrane — which are not readily accessible for direct, patch-clamp analysis — to be approximately 18 channels per m² in the stimulated state.     

Protein kinase C as mediator of arachidonic acid-induced decrease of neuronal M current

The M current, I _M, of NG108-15 neuroblastoma×glioma hybrid cells, a non-inactivating K⁺ current, is decreased by arachidonic acid (5–25 M), often after an initial transitory increase. To test the possibility that the decrease is caused by activation of protein kinase C (PKC) we used the PKC 19–31 peptide, which is an effective inhibitor of PKC. With 1 M peptide in the pipette solution the normally observed strong reduction of I _M by 1 M phorbol 12,13-dibutyrate (PDB) was almost totally prevented, indicating that PKC is completely inhibited; also the voltage dependence of the M conductance, g _M(V), was shifted to more negative membrane potentials. In the presence of 1 M peptide the effect of 25 M arachidonic acid on I _M was significantly reduced, suggesting that the effect, or at least a large part of it, is mediated by PKC.     

Effects of arachidonic acid on the gap junctions of neonatal rat heart cells

Myocytes were isolated from neonatal rat hearts and grown in tissue-culture dishes for 1–2 days. Spontaneously formed cell pairs were used to study the conductance of gap junctions. The experiments involved a double voltage-clamp approach and whole-cell, tight-seal recording. Exposure to arachidonic acid (AA) produced a quasi dose-dependent decrease in junctional conductance, g _j (binding constant, K _d=4 M; Hill coefficient, n = 0.75). AA-dependent uncoupling was reversible. Addition of 1 mg/ml albumin to the bath solution accelerated the recovery. During control, cell pairs exhibited a gradual decrease in g _j (16.4 % in 6 min). Exposure to 20 M 4-bromophenacyl bromide, a phospholipase inhibitor, suppressed the decay in g _j (1.8% in 6 min), suggesting that endogenous AA may be involved in spontaneous uncoupling. The effect of AA on g _j was specific. Arachidic acid (100 M) and arachidonamide (10 M), structural analogues of AA, had no effect on g _j. Currents recorded shortly before complete uncoupling caused by AA, or early during recovery from uncoupling, revealed random opening and closing of single channels. The single channel conductance, _j, was not affected by the concentration of AA (1 M–100 M). The mean _j turned out to be 33.5 pS. The results suggest that AA-dependent uncoupling was caused via decrease in open channel probability, presumably mediated by a direct action on channel proteins.     

Inhibitory effects ofγ-interferon on bradykinin-induced bone resorption and prostaglandin formation in cultured mouse calvarial bones

The effects of mouse recombinant-interferon (-IFN) and indomethacin on bone resorption stimulated by bradykinin, Lys-bradykinin, Met-Lys-bradykinin, des-Arg⁹-bradykinin and prostaglandin E₂ (PGE₂) have been studied using cultures of neonatal calvarial bones and analyzing the release of⁴⁵Ca from prelabelled bones as a paramenter of bone resorption. In addition, the effects of-IFN and indomethacin on formation of PGE₂ in bone cultures stimulated by bradykinin was analyzed. Indomethacin (1 mol/l) totally abolished bradykinin (1 mol/l) induced⁴⁵Ca release. The inhibitory effect of indomethacin could be fully reversed by addition of PGE₂ (1 mol/l).-IFN (1000 U/ml) almost totally inhibited⁴⁵Ca release stimulated by bradykinin (1 mol/l), but the inhibitory effect could only be partially overcome by PGE₂.-IFN and indomethacin also inhibited the stimulatory effects of Lys-bradykinin, Met-Lys-bradykinin and des-Arg⁹-bradykinin (1 mol/l) on⁴⁵Ca release. The stimulatory effects of PGE₂ (1 mol/l) on radioactive calcium mobilization was partially inhibited by-IFN (1000 U/ml), whereas indomethacin (1 mol/l) was without effect. The inhibitory effect of-IFN on⁴⁵Ca release stimulated by bradykinin and PGE₂ was dose-dependent with threshold for action at 3–30 U/ml. Comparative dose-response curves showed that-IFN was most potent as inhibitor of bradykinin induced⁴⁵Ca release. Bradykinin (1 mol/l) significantly stimulated PGE₂ formation by a mechanism that was completely inhibited by indomethacin (1 mol/l).-IFN (1000 U/ml) partially inhibited the stimulatory effect of bradykinin on PGE₂ formation. These data show that i)-IFN is a potent inhibitor of bone resorption induced by bradykinin and bradykinin analogues and ii) that the mechanism of action can be mainly explained by an inhibition of kinin induced prostaglandin biosynthesis. The results, however, also show that-IFN can inhibit bone resorption by mechanisms unrelated to prostaglandin formation.     

A camera-type diameter gauge applicable to small blood and lymph vessels

A camera-type diameter gauge was constructed and used in physiological experiments. The distinctive features of the design and use of the gauge may be summarized as follows. Installation of an image sensor in the focal plane of a single-lens reflex camera equipped with a set of extension tubes which makes it possible to measure outer diameters of blood and lymph vessels as small as 100 m. Using the gauge, we can distinguish a diameter change of about 12 m. Simultaneous measurements of the diameters of two different vessels can be made using two window signals generated independently at desired moments for appropriate periods within the video scans.     

Voltage-Sensitive Dye Mapping of Activation and Conduction in Adult Mouse Hearts

A custom-made apparatus based on a charge-coupled-device camera has been used to monitor changes in fluorescence from Langendorff-perfused adult mouse hearts stained with a voltage-sensitive dye, di-4-ANEPPS. With this approach it is possible to monitor activation of the ventricles at high temporal (375 s/frame) and spatial resolution 72 × 78pixels,100 ×100 m/pixel. In sinus rhythm, activation occurred with a complicated breakthrough pattern on both ventricles, and a total activation time of 3.51 ± 0.16ms (32 °C). A stimulus applied near the apex of the left ventricle resulted in a single activation wave front with a total activation time of 8.18 ± 0.25 ms. Pacing from a site near the middle of the left ventricular epicardial surface revealed anisotropic conduction, indicating that conduction occurs preferentially in the direction of the predominant fiber orientation. The total activation time in this configuration was 5.44 ± 0.24 ms. The difference in total activation time between sinus rhythm and epicardial stimulation suggests an important role for transmural conduction (the Purkinje system) in the mouse heart. These findings provide much of the necessary background needed for studying conduction abnormalities in genetically altered mice and suggest that the comparison of sinus rhythm and epicardial pacing can be used to reveal transmural conduction abnormalities. © 2000 Biomedical Engineering Society. PAC00: 8719Nn, 8719Hh, 8716Uv, 8764Ni     

Characterization of two components of the N-like,high-threshold-activated calcium channel current in differentiated SH-SY5Y cells

Retinoic acid differentiated SH-SY5Y cells exhibit only a high-threshold-activated (–30 to –20 mV) whole cell calcium channel current. When barium was used as the charge carrier, the high-threshold-activated current showed bi-exponential inactivation kinetics during a 500 ms voltage step from –90 to +10mV. The time constants of inactivation were approximately 75 and 750 ms. The fast inactivating component was more sensitive than the slow inactivating component to steady-state inactivation at depolarized holding potentials. The calcium channel current was inhibited by externally applied cadmium (10–300 M) and gadolinium (10–30 M) as well as by high concentrations of nickel and cobalt, Conus toxin (1 M) irreversibly blocked the calcium channel current. However, the dihydropyridine agonist, BAY K 8644 (3–10 M) and antagonists, nifedipine (3–10 M) and nimodipine (10 M) did not affect either component of the calcium channel current. Agents which blocked the calcium channel current did not exhibit any selectivity for the fast inactivating over the slow inactivating component of the current. These results indicate that whilst the calcium channel current recorded in differentiated SH-SY5Y cells can be classified on the basis of the blocking agents as being of the N type, the current shows more than one form of inactivation.     

Vergleichende Sauerstoffverbrauchsmessungen bei Kokzidienoocysten der Gattung Eimeria (Protozoa,Sporozoa)

Zusammenfassung Der Sauerstoffverbrauch sporulierender Oocysten von E. falciformis, E. tenella und E. contorta n.sp. Haberkorn, 1970 lag mit 14,9 bis 23,8 l/10⁵ Oocysten/72 Std deutlich unter dem von E. stiedai mit 31,1 l. Der unmittelbar im Anschluß an die Sporulation verminderte Sauerstoffverbrauch sank nach 3- bzw. 10monatiger Lagerung weiter ab. Bis zu einem Temperaturoptimum bei etwa 35°C verbrauchten sporulierte Oocysten von E. stiedai mit zunehmender Temperatur mehr Sauerstoff.

---

Comparative measurements of oxygen consumption in Eimeria oocysts (Protozoa, Sporozoa)

Summary The oxygen consumption of E. falciformis, E. tenella, E. contorta n.sp., and E. stiedai oocysts during sporulation ranged from 14.9–31.1 l/10⁵ oocysts/72 hrs. It decreased to 0.4–1.3 l/24 hrs after a storage period of three months. There was an increase in the oxygen consumption of sporulated E. stiedai oocysts measured from +4°–35°C.

     

The effect of EMD 57033, a novel cardiotonic agent,on the relaxation of skinned cardiac and skeletal muscle produced by photolysis of diazo-2, a caged calcium chelator

Summary EMD 57033 is thought to produce its potentiating effect by increasing the apparent calcium sensitivity of myofibrils. We have investigated the effect of 10M EMD 57033 on relaxation speed, induced by flash photolysis of 2mM diazo-2 (a caged Ca²⁺ chelator), in skinned semitendinosus frog muscle fibres and guineapig trabeculae. 10M EMD 57033 has no effect on the relaxation speed of semitendinosus fibres. In trabeculae, EMD 57033 slightly increases the relaxation speed slightly, in contrast to ADP which produces a slowing. 1mM ADP combined with 10M EMD 57033 slows relaxation but not to the degree seen with ADP alone. Like ADP, EMD 57033 increases the number of cross-bridges in the force producing state, but unlike ADP does not affect the transition rates involved in relaxation.