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B A Wittenberg

Publications and source records attributed to B A Wittenberg.

At least 37 records · Page 2Linked to original sources
PubMed1990

Intracellular calcium and high-energy phosphates in isolated cardiac myocytes.

The relationship between intracellular calcium (Cai) and high-energy phosphates was studied in adult cardiac myocytes. Cai and high-energy phosphates were measured in the same population of cells. Cai, reported by the fluorescence of fura-2, was maintained at normal levels in the presence of increased transsarcolemmal calcium gradients, up to 5 mM extracellular calcium concentration. Cai was experimentally elevated by increasing calcium influx from the extracellular medium and/or by diminishing calcium efflux by Na-Ca exchange. Under these conditions, cells contracted and relengthened repetitively. The regulation of high-energy phosphates was challenged by increasing ATP utilization and by inhibiting ATP synthesis. Cai regulation was not affected by inhibition of glycolysis or NADH oxidation, so long as ATP concentration remained unchanged. High-energy phosphates were not depleted in beating cells with intact NADH oxidation, but inhibition of NADH oxidation caused a significant drop in phosphocreatine, demonstrating the increased rate of ATP consumption during beating. In beating cells, as in the working heart, ATP supply is increased to meet ATP demand, and steady-state ATP and phosphocreatine concentrations remain unchanged.

Adenosine Triphosphate↗
PubMed1989

The kinetics of ligand binding to plant hemoglobins. Structural implications.

The rates of reaction of oxygen, carbon monoxide, and nitric oxide with 14 plant hemoglobins have been determined by relaxation and stopped-flow methods. The combination rates for oxygen lie between 0.12 and 0.26 x 10(9)/M.s, for carbon monoxide between 0.01 and 0.07 x 10(9)/M.s, and for nitric oxide between 0.12 and 0.25 x 10(9)/M.s. The dissociation velocities for oxygen range from 5 to 25/s, and for CO from 0.005 to 0.011 s. The oxygen dissociation constants range only from 36 to 78 nM. Nanosecond relaxation experiments show large differences between the proteins. Five have known primary structures which correlate closely with the nanosecond relaxations and less immediately with the millisecond reactions. The relevant amino acid substitutions are concentrated in the C-E interhelical region.

Amino Acid Sequence↗
PubMed1987

Morphology, composition, and function of struts between cardiac myocytes of rat and hamster.

The morphology, composition, and function of struts that interconnect the lateral surfaces of cardiomyocytes were examined in the hearts of rats and hamsters. Methods included brightfield and fluorescent light microscopy, secondary and backscatter scanning electron microscopy, and transmission electron microscopy in conjunction with silver stain, cationic dye, and antibody to type-I collagen. These studies reveal a twisted, beaded appearance and a complex substructure of collagen fibrils embedded in a ground substance that has a positive reaction with cationic dye. A hierarchy of patterns of branching and attachment was seen among intercellular struts ranging in diameter from 0.1 micron to several micron. The hypothesis that struts tether not only the surfaces but the contractile lattices of laterally adjacent myocytes is supported by the following: (a) the attachments of struts to the collagen weave of the sarcolemma, often lateral to the level of Z bands, (b) the presence of collagen type I in a composite material arrangement, (c) the relative dispositions and configurational changes of struts and myocyte surfaces in various physiological states and induced, non-physiological perturbations of cardiac muscle, (d) the corrugated sarcolemmas with infoldings near Z bands, and (e) the continuity of intracellular filaments from Z bands to the inner aspect of the sarcolemma in relaxed and contracted myocytes. Implications of struts acting as tethers and sites for storage of energy in the motions of myocytes during the cardiac cycle are discussed.

Animals↗
PubMed1987

Myoglobin-mediated oxygen delivery to mitochondria of isolated cardiac myocytes.

Myoglobin-mediated oxygen delivery to intracellular mitochondria is demonstrated in cardiac myocytes isolated from the hearts of mature rats. Myocytes are held at high ambient oxygen pressure, 40-340 torr (5-45 kPa); sarcoplasmic myoglobin is fully oxygenated. In this condition oxygen availability does not limit respiratory rate; myoglobin-facilitated diffusion contributes no additional oxygen flux and, since oxygen consumption is measured in steady states, the storage function of myoglobin vanishes. Carbon monoxide, introduced stepwise, displaces oxygen from intracellular oxymyoglobin without altering the optical spectrum of the largely oxidized intracellular mitochondria. A large part, about one-third, of the steady-state oxygen uptake is abolished by carbon monoxide blockade of myoglobin oxygenation. The myoglobin-dependent component of the oxygen uptake decreases linearly with decreasing fraction of intracellular oxymyoglobin, with a slope near unity. Studies using inhibitors of mitochondrial electron transport indicate that myoglobin-delivered oxygen uptake depends on electron flow through the mitochondrial electron transport chain. We conclude that cardiac mitochondria accept two additive simultaneous flows of oxygen: a flow of dissolved oxygen to cytochrome oxidase and a flow of myoglobin-bound oxygen to a mitochondrial terminus. Myoglobin-mediated oxygen delivery supports ATP generation by heart cells at physiological ambient oxygen pressure.

Animals↗
PubMed1986

The kinetics of the reactions of Parasponia andersonii hemoglobin with oxygen, carbon monoxide, and nitric oxide.

Hemoglobin I was isolated from nodules formed on the roots of Parasponia andersonii inoculated with Rhizobium strain CP 283. The rate of oxygen dissociation from Parasponia hemoglobin increases about 12-fold between pH 4 and 7, with apparent pK 6.4, to reach a limiting value of 14.8s-1. The optical spectrum of oxyhemoglobin in the visible region is also dependent on pH with pK near 6.4. The rate constant for oxygen combination with Parasponia hemoglobin increases about 7-8-fold between pH 4 and 7, with apparent pK 5.37, to reach a value of 1.67 X 10(8) M-1 s-1 at pH 7. The optical spectrum of deoxyhemoglobin in the visible region and the rate constant for carbon monoxide combination are also dependent on pH with apparent pK 5.65 and 5.75, respectively. The rate constant for carbon monoxide dissociation is independent of pH. The oxygen affinity of Parasponia hemoglobin, P50 = 0.049 torr at 20 degrees C, calculated from the kinetic constants at pH 7, is very great. At alkaline pH there is a prominent geminate reaction with oxygen and nitric oxide, with both subnanosecond and tens of nanosecond components. These reactions disappear at acid pH, with pK 6.4, and the effective quantum yield is reduced. In general, the reactions of Parasponia hemoglobin with oxygen and carbon monoxide resemble those of soybean leghemoglobin. In each, great oxygen affinity is achieved by unusually rapid oxygen combination together with a moderate rate of oxygen dissociation. We suggest that protonation of a heme-linked group with pK near 6.4 controls many properties of Parasponia oxyhemoglobin, and protonation of a group with pK near 5.5 controls many properties of Parasponia deoxyhemoglobin.

Carbon Monoxide↗
PubMed1986

Effect of calcium on the dissociation of the mature rat heart into individual and paired myocytes: electrical properties of cell pairs.

The dissociation of adult rat heart into individual, functionally intact, calcium-tolerant myocytes requires precise manipulation of extracellular calcium levels. Dissociation of intercellular connections is achieved by lowering extracellular calcium to micromolar levels for a short period. By imposing a very small increment in free calcium activity (from 14 to 17 microM) during this period, we achieve a significant yield of functionally intact pairs of myocytes still joined at the intercalated disc. We obtain fewer intact cells, but many of these are paired end to end. These findings permit us to describe some structural characteristics of intercellular connections between cardiac cells and to report unambiguous measurements of electrotonic coupling and dye transfer between rat cardiac cell pairs. We find that the strength of electrical coupling between cells isolated as pairs with intact junctional contacts is much greater than that measured between cell pairs that have formed new junctional contacts.

Animals↗
PubMed1985

Oxygen pressure gradients in isolated cardiac myocytes.

Intracellular oxygen pressure within intact isolated cardiac myocytes is studied as a function of steady state extracellular oxygen pressure. The fractional saturation of myoglobin with oxygen is used to report sarcoplasmic oxygen pressure. The fractional oxidation of cytochrome oxidase, the fractional oxidation of cytochrome c, the rate of respiratory oxygen uptake, and lactate accumulation are used to reflect the availability of oxygen at the inner mitochondrial membrane. These probes of mitochondrial function show no large change with decreasing extracellular oxygen pressure until that pressure is less than 2 torr and intracellular myoglobin is largely deoxygenated. Sarcoplasmic oxygen pressure in resting cells is nearly the same as extracellular oxygen pressure and is about 2 torr less in cells whose respiration has been increased 3.5-fold by mitochondrial uncoupling. Oxygen pressure at the mitochondrial inner membrane differs from sarcoplasmic oxygen pressure by no more than 0.2 torr and from extracellular oxygen pressure by no more than 2 torr. We conclude that differences of oxygen pressure within the cardiac myocyte are very small. This implies that most of the large, about 20 torr, difference in oxygen pressure between capillary lumen and mitochondria of the working heart must be extracellular. We conclude also that mitochondria of the cardiac myocyte become oxygen limited only when sarcoplasmic myoglobin is almost entirely deoxygenated.

Adenosine Triphosphate↗
PubMed1985

NMR studies of intracellular sodium ions in mammalian cardiac myocytes.

The unambiguous measurement of intracellular sodium ion [Na+]i by the noninvasive NMR technique offers a new opportunity to monitor precisely the maintenance and fluctuations of [Na+]i levels in intact cells and tissues. The anionic frequency shift reagent, dysprosium (III) tripolyphosphate, which does not permeate intact cells, when added to suspensions of intact adult rat cardiac myocytes, alters the NMR frequency of extracellular sodium ions, [Na+]o, leaving that of intracellular ions, [Na+]i, unaffected. Using 23Na NMR in conjunction with this shift reagent, we have determined NMR-visible intracellular Na+ ion concentration in a suspension of isolated cardiac myocytes under standard conditions with insulin and Ca2+ in the extracellular medium to be 8.8 +/- 1.2 mmol/liter of cells (n = 4). This value is comparable to that measured by intracellular ion-selective microelectrodes in heart tissue. Cardiac myocytes incubated for several hours in insulin-deficient, Ca2+-containing medium prior to NMR measurement exhibited a somewhat lower [Na+]i value of 6.9 +/- 0.5 mmol/liter of cells (n = 3). Reversible Na+ loading of the cells by manipulation of extracellular calcium levels is readily measured by the NMR technique. Incubation of myocytes in a Ca2+-free, insulin-containing medium causes a 3-fold increase in [Na+]i to a level of 22.8 +/- 2.6 mmol/liter of cells (n = 10). In contrast to cells with insulin, insulin-deficient myocytes exhibit a markedly lower level of [Na+]i of only 14.6 +/- 2.0 mmol/liter of cells (n = 4) in Ca2+-free medium. These observations suggest that insulin may stimulate a pathway for Na+ influx in heart cells.

Animals↗
PubMed1985

Some electrical and pharmacological properties of gap junctions between adult ventricular myocytes.

Ventricular myocytes were isolated from adult rat hearts using the technique of Wittenberg and Robinson (Cell Tissue Res. 216: 231-251, 1981). These cells exhibited morphology, input resistance, time constant, and excitability expected for cells in intact cardiac tissue. Pairs of these cells were electronically coupled, and junctional conductance was unaffected by transjunctional potential or hyperpolarization of both cells. Brief exposure of cell pairs to medium equilibrated with 100% CO2 or containing 0.1 mM octanol quickly and reversibly decreased junctional conductance. We conclude that gap junctions between pairs of ventricular myocytes possess physiological properties like those of junctions in many other tissues. This preparation will be useful in evaluating drug action on junctional communication in heart.

Animals↗
PubMed1985

Fate of intercellular junctions in isolated adult rat cardiac cells.

Freeze fracture and thin section techniques have revealed morphological changes in gap junctions and intercalated discs of adult rat myocytes following enzymatic dissociation. Cell separation leaves behind small vesicular remnants of formerly adjacent cells connected to the intact cell by gap junctions; in contrast, desmosomes cleave at the region of intercellular contact. Apparently, the next step in gap junction breakdown is internalization of the remnants. In thin section, lanthanum penetration reveals that the cleft of some apparently internalized gap junctions is in contact with the sarcolemma, while that of others is not. In freeze fracture replicas, cytoplasmic gap junctions frequently possess hexagonally packed domains of E-face pits separated by smooth regions that may correspond to separations of membranes of internalized junctions found in thin section. Study of material maintained overnight at 37 degrees C showed no surface junctional remnants; topologies of cytoplasmic gap junctions were generally complex, and concentric membrane vesicles were common. These observations suggest that enzymatic dissociation initiates a progressive, defined sequence of junctional internalization that begins with attached cell remnants and may have as the last determinable step the separation into single membranes inside the intact cell.

Animals↗
PubMed1985

Oxygen transport in isolated cardiac myocytes.

A preparation of isolated cardiac myocytes, retaining functional and structural properties of myocytes in the intact heart, has been used to measure intracellular oxygen pressure gradients. These are shallow and do not exceed 2 torr. Oxygen does not limit mitochondrial function in the cardiac myocyte until intracellular myoglobin is almost entirely deoxygenated. Most of the large oxygen pressure gradient in the working heart must be extracellular.

Animals↗
PubMed1984

Monoamine oxidase, an intracellular probe of oxygen pressure in isolated cardiac myocytes.

The activity of monamine oxidase, an enzyme located almost exclusively at the outer mitochondrial membrane, toward the substrate phenylethylamine is used to report the oxygen pressure at the outer mitochondrial membrane of intact cardiac myocytes isolated from hearts of adult rats. The rate of substrate oxidation, under the conditions used, follows the Michaelis-Menten relation, and accordingly can be used as a measure of the local chemical activity of dissolved oxygen. The oxygen pressure at the outer mitochondrial membrane of myocytes, at rest and after 2- to 3-fold stimulation of respiratory oxygen consumption, differs from the extracellular oxygen pressure by at most 2 torr. This implies that most of the large, about 20 torr, difference in oxygen pressure between capillary lumen and mitochondria of the working heart must be extracellular. At physiologically relevant concentrations of the substrates phenylethylamine and norepinephrine, monoamine oxidase activity is relatively insensitive to extracellular oxygen pressure in the range 155 to 8 torr, suggesting a limited role for regulation of biogenic amine oxidation by oxygen availability.

Animals↗
PubMed1983

Leghemoglobin. Kinetic, nuclear magnetic resonance, and optical studies of pH dependence of oxygen and carbon monoxide binding.

The rate of dissociation of oxygen from soybean oxyleghemoglobin a increases about 5-fold between pH 4 and pH 7, with apparent pK = 5.46 and n = 1. The rate of dissociation of carbon monoxide from carbon monoxyleghemoglobin a and the rates of combination of oxygen and carbon monoxide with ferrous leghemoglobin a are all invariant in this range of pH. The optical spectrum of oxyleghemoglobin in the visible region and the resonances of the four heme meso protons (protons of the bridge carbon atoms) in the NMR spectrum of oxyleghemoglobin are also dependent on pH with pK near 5.5. We suggest that protonation of the imidazole of the distal histidine residue (His 61) leads to formation of a hydrogen bond to the bound oxygen molecule which affects the electronic configuration or conformation of the heme and decreases the rate of oxygen dissociation.

Carbon Monoxide↗
PubMed1982

Mitochondrial function in the presence of myoglobin.

The effect of myoglobin on oxygen consumption and ATP production by isolated rat skeletal muscle mitochondria was studied under steady-state conditions of oxygen supply. A method is presented for the determination of steady-state oxygen consumption in the presence of oxygen-binding proteins. Oxygen consumed in suspensions of mitochondria was replenished continuously by transfer from a flowing gas phase. Liquid-phase oxygen pressure was measured with an oxygen electrode; the gas-phase oxygen concentration was held constant at a series of fixed values. Oxygen consumption was determined from the characteristic response time of the system and the difference in the steady-state gas- and liquid-phase oxygen concentrations. ATP production was determined from the generation of glucose 6-phosphate in the presence of hexokinase. During steady-state mitochondrial oxygen consumption, the oxygen pressure in the liquid phase is enhanced when myoglobin is present. Functional myoglobin present in the solution had no effect on the relation of mitochondrial respiration and ATP production to liquid-phase oxygen pressure. Myoglobin functions in this system to enhance the flux of oxygen into the myoglobin-containing phase. Myoglobin may function in a similar fashion in muscle by increasing oxygen flux into myocytes.

Adenosine Triphosphate↗
PubMed1981

Oxygen requirements, morphology, cell coat and membrane permeability of calcium-tolerant myocytes from hearts of adult rats.

The morphological, functional, and biochemical properties of freshly isolated heart muscle cells were examined. A reproducible method for the separation and purification of such cells isolated from adult rat heart was developed. It yields an average of 5 X 10(6) striated rectangular cells which retain normal morphology (range 2.5 to 11 X 10(6) and 4 X 10(6) calcium-tolerant cells (range 2.5 to 5.5 X 10(6) per heart. After purification, 85 to 95% of the cells retain normal morphology in solutions of calcium ion activity equal to 10 microM, and 65 to 79% of the cells are rectangular in solutions of calcium ion activity equal to 1 mM. Under the light microscope we were able to identify functionally intact individual cells that are calcium-tolerant and contract only in response to electrical stimulations, as well as dying myocytes that beat spontaneously. The examination of such cells under the electron microscope permitted us to address the question: What is the sequence of structural changes in a dying cell? The sarcomere lengths measured both in the living state and after preparation for electron microscopy are in the physiological range. In steady states of oxygen tension, respiration of the intact cells is undiminished from 50 torr to 2 torr. The oxygen tension for half maximal respiration is 0.15 torr. Therefore, the limitation of oxygen diffusion to the mitochondria of isolated heart muscle cells must be remarkably small.

Animals↗
PubMed1981

Isolated heart myocytes: ultrastructural case study technique.

We have developed a method for performing case studies of heart muscle cells enzymatically isolated from the ventricular walls of rats that is a simple and inexpensive adaptation of procedures developed for the examination of monolayers of attached, cultured cells. The technique represents a marked departure from published accounts of electron microscopic studies of pellets or monolayers from a population of potentially heterogeneous isolated myocytes. Here we report the method, which we have used under controlled conditions with 0 mmol and 1 mmol added CaCl2, to correlate sarcomere length and electrical stimulatibility in the living state with ultrastructural features that include the relative disposition of myofilaments and the integrity of the cell coat. The degree of shrinkage during the preparative steps in less than 5%, as directly determined from photographs of striations in the living, fixed, and embedded states.

Animals↗