Biomedical subjects
Publications and source records attributed to R N Pittman.
During the past year,several model systems have been developed to identify cellular and biochemical events involved in neuronal cell death, to investigate the role of bcl-2 in cell survival, and to characterize the relationship between cell death and the cell cycle.
Electrophoretic mobility-shift assays were used to characterize binding of nuclear proteins to consensus sequences for Sp1, E2F, octamer, and cAMP responsive enhancer element (CRE) during neuronal death in vitro after removal of nerve growth factor (NGF). Molecular events occurring prior to cell death in terminally differentiated PC12 cells could be divided into three phases: (i) within 2 hr of removing NGF, binding to the octamer sequence decreased, (ii) after 5-7 hr an increase in binding to CRE occurred; and (iii) after 14 hr (the point at which 50% of the cells are committed to die) a decrease in binding to the Sp1 sequence occurred. Assays performed with extracts from sympathetic ganglia indicated that changes in binding to CRE and octamer motifs also occurred during the period of developmental cell death in vivo. Double-stranded oligonucleotides were delivered to neurons to act as dominant negative "promoters" unable to couple to transcriptional events but capable of binding and sequestering transcription factors. Double-stranded but not single-stranded octamer oligonucleotides increased cell death of primary cultures of sympathetic neurons. Most of the induced neuronal cell death could be blocked with NGF, which is consistent with oligonucleotides activating an endogenous death program rather than having a nonspecific toxic effect. Other double-stranded oligonucleotides as well as a mutant octamer oligonucleotide had little or no effect on cell death. These data are consistent with the hypothesis that cell death results from a cascade of cellular and molecular events and that an early event in programmed neuronal cell death is a decrease in binding of transcription factor(s) to octamer motif sequences.
This study investigated the effect of temperature on the oxygen diffusion coefficient (DO2) of hamster retractor muscle from 11 to 37 degrees C. DO2 was measured using a non-steady-state technique, whereas muscle O2 consumption (VO2) was estimated after steady state was reached. DO2 was 0.84 +/- 0.04 x 10(-5) cm2/s at 11 degrees C and rose exponentially to 2.41 +/- 0.19 x 10(-5) cm2/s at 37 degrees C, producing a temperature coefficient for DO2 of 4.60%/degrees C for this temperature range. To measure DO2 directly at 37 degrees C, it was necessary to inhibit tissue VO2 with Amytal. The DO2 measurements made at 37 degrees C were significantly higher than previously reported values, which had been based on extrapolations from lower temperatures (6). Further analysis suggests a possible transition in the diffusion pathway between 23 and 30 degrees C, resulting in a DO2 higher than that previously expected. This larger DO2, together with a recently published value of oxygen solubility (alpha) (21), results in an in vitro Krogh's diffusion coefficient (KO2) that is 2.4 times larger than that previously reported (24) and therefore significantly reduces an order of magnitude discrepancy between in vitro and estimated in vivo KO2 values (24). Muscle VO2 was 0.35 ml O2.min-1.100 g-1 at 11 degrees C and increased with temperature, resulting in an activation energy of the rate-limiting reaction from the Arrhenius equation of -10.5 kcal/mol between 11 and 30 degrees C.
The in vivo effects on hemoglobin (Hb)-O2 affinity and tissue PO2 were investigated after intraperitoneal administration of 2-[4-(((dichloroanilino)-carbonyl)methyl)phenoxyl]-2-methyl propionic acid (RSR4; 150 mg/kg) or its 3,5-dimethyl derivative (RSR13; 300 mg/kg) in C3Hf/Sed mice. The Hb-O2 dissociation curve was plotted from tail vein blood samples using an O2 dissociation analyzer before and up to 160 min after compound administration. Twenty to 40 min after injection, the PO2 at 50% saturation of hemoglobin (Hb P50) increased by a mean of 25% (range 18-31%) after RSR4 and 53% (range 36-76%) after RSR13. Tissue PO2 was continuously measured using an O2 microelectrode in thigh muscle before and up to 40 min after RSR4 or RSR13 injection. Twenty to 40 min after administration, tissue PO2 increased by a mean of 78% (range 30-127%) after RSR4 and 66% (range 39-97%) after RSR13 administration in anesthetized mice. No change in tissue PO2 was seen in anesthetized controls.
Myoglobin (Mb) may facilitate O2 diffusion in muscle tissue, yet models of O2 transport are often simplified by ignoring the role of Mb. A recent analysis of O2 transport in hamster retractor muscle revealed a large discrepancy between the observed O2 diffusion from arterioles and that predicted by a mathematical model that did not include Mb. To establish whether this simplification was justified, we measured Mb content ([Mb]) in three hamster muscles that vary markedly in histochemical fiber type composition. [Mb] was determined spectrophotometrically using freshly excised tissues from hamsters of different ages (5-34 wk). [Mb] increased rapidly up to 15 wk of age and then rose more slowly. [Mb] in hamster muscles paralleled oxidative capacity. Our measurements of rat and dog muscles also show that [Mb] varies greatly among species and among muscles of a given species. The results indicate that in the hamster the variability in [Mb] with age and muscle should be taken into account when the potential role of Mb in studies on O2 transport is interpreted.
A model system has been established in which PC12 cells are converted to neuronal-like cells that undergo transcription-dependent cell death following removal of NGF. Nineteen sublines of PC12 cells were tested to establish parameters for making cells dependent on NGF for survival. In most sublines, a relatively small percentage of cells become dependent on NGF for survival, and following removal of NGF, most of the cells begin proliferating in serum-containing medium. In several sublines, however, a significant percentage of cells die following removal of NGF. One of these sublines, PC6-3, can be grown under conditions in which 90% of the cells undergo transcription-dependent cell death following removal of NGF in either serum-free or serum-containing medium. Fourteen hours after removing NGF, 50% of the cells are committed to die, while initial morphological signs of cell death as determined by time-lapse videomicroscopy occur 2-6 hr later and include loss of neurites followed by a 1-3 hr period of active membrane "blebbing" and protrusions. Cell death can be blocked by the RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximide, KCl, basic fibroblast growth factor, or dibutryl-cAMP, but not by epidermal growth factor, leupeptin, or the endonuclease inhibitor aurintricarboxylic acid (ATA). Removal of NGF activates an endonuclease that causes nucleosomal laddering of the DNA; however, endonuclease activity does not appear to be required for cell death. In agreement with previous studies (Batistatou and Greene, 1991; Rukenstein et al., 1991) demonstrating that naive PC12 cells undergo transcription-independent cell death when shifted into serum-free medium in the absence of growth factors, all cell lines tested except for one die when cultured in RPMI medium lacking growth factors. DNA fragmentation is a prominent feature of transcription-independent cell death, and death can be blocked with NGF, ATA, and dibutryl-cAMP but not with actinomycin D or KCl. The PC12 model system described here should be useful for identifying cell death genes and for characterizing cellular and molecular events in programmed neuronal cell death.
We have devised a method to display and directly evaluate red blood cell (rbc) dynamics in capillaries using the same dual camera intravital video microscopy system employed to determine rbc oxygen saturation (Ellis et al., 1990). Capillary images are recorded on videotape and an interactive graphics system is used for analysis. Data are sampled once a frame for 60 sec using a window (one pixel wide (0.93 micron) and 100 pixels high) positioned along the axis of a capillary. The resulting data are displayed as sequential space-time images 100 pixels high by 300 pixels wide (10 sec). The space-time images thus created represent the dynamics of the rbc's in a single comprehensive static image in which the rbc's appear as dark, diagonal bands separated by light bands representing plasma gaps. From these images one can obtain information on velocity of individual rbc's (micron/sec), lineal density of rbc's (rbc/mm), and rbc supply rate (rbc/sec). This information can be used to delineate the temporal and spatial heterogeneity of hemodynamics in capillary networks. These data can then be combined with coincident data on red blood cell oxygenation to provide a complete picture of oxygen transport in capillaries or it can be used alone as a tool for the evaluation of basic in vivo and in vitro rheological questions.
To investigate the suggestion of A. S. Popel, R. N. Pittman, and M. L. Ellsworth [Am. J. Physiol. 256 (Heart Circ. Physiol. 25): H921-H924, 1989] that Krogh's diffusion coefficient for O2 in vivo might be an order of magnitude higher than in vitro, O2 diffusion coefficient (DO2) and resting O2 consumption were measured on hamster retractor muscle in vitro and under near in vivo conditions where the muscle remained attached to the animal but the arterial inflow was occluded just before measurement. Experiments were performed on two groups of animals, differing in weight and age. We found that DO2 determined in vitro (extrapolated to 37 degrees C) was 1.81 +/- 0.12 x 10(-5) cm2/s for group I (smaller and younger), which was not significantly different from the value (2.00 +/- 0.08 x 10(-5) cm2/s) determined in group II. In both groups, DO2 under near in vivo conditions tended to be 10-15% larger than the value in vitro, although this trend did not reach statistical significance. It is unlikely that this trend is large enough to reconcile the inconsistency between theoretical and experimental determinations of O2 diffusion from the arteriolar network of this tissue.
DDT1-MF2 smooth muscle cells demonstrated a robust phospholipase C response to norepinephrine, as detected by inositol phosphate accumulation. A selective A1-adenosine receptor agonist, cyclopentyladenosine, caused only a minor stimulation of phospholipase C, which was eliminated in the absence of added extracellular calcium. The simultaneous addition of norepinephrine and cyclopentyladenosine resulted in a synergistic increase in phosphoinositide hydrolysis either in the absence or in the presence of external calcium. In the presence of external calcium and a calcium ionophore, and adenosine agonist caused a significant stimulation of phosphoinositide hydrolysis without the addition of norepinephrine. Influx of extracellular calcium through voltage-sensitive calcium channels did not appear to be required to observe an effect of cyclopentyladenosine, because neither calcium channel antagonists (nifedipine, verapamil, and LaCl3) nor a chelator of extracellular calcium (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid) were able to alter the degree of potentiation of norepinephrine-stimulated phosphoinositide hydrolysis due to the adenosine agonist. On the other hand, buffering of intracellular calcium concentration with the membrane-permeant calcium chelator quin2 blocked the potentiation. This blockade of potentiation by quin2 was reversed by the addition of extracellular calcium. Agents that stimulated cAMP production or membrane-permeable analogues of cAMP also blocked the action of the adenosine agonist to potentiate norepinephrine-stimulated phosphoinositide hydrolysis. This effect of cAMP was less pronounced in the presence of elevated extracellular calcium and was abolished in the presence of a calcium ionophore. When norepinephrine-stimulated calcium transients were quantitated using fura-2 fluorescence, a reduction in the amplitude of the calcium response was observed in the presence of forskolin. Conversely, both the amplitude and the duration of the calcium response were enhanced by the addition of the adenosine agonist. The results of these studies suggest that the mechanism by which adenosine receptors enhance the stimulation of phosphoinositide hydrolysis is dependent upon a rise in intracellular Ca2+ concentration resulting from the simultaneous activation of alpha 1-adrenergic receptors. The results further suggest that cAMP inhibits this mechanism by decreasing the norepinephrine-stimulated rise in intracellular Ca2+ concentration.
Recent studies indicate that mediation of neurite outgrowth by the glycoprotein laminin may be a significant factor in the outgrowth of neurites to their targets during embryogenesis. To further characterize the possible role of this extracellular matrix molecule during development, we have systematically measured several features of outgrowth by neonatal rat sympathetic neurons on different concentrations of laminin. Individual neurons, obtained by mechanical dissociation of superior cervical ganglia (SCG), were cultured at low density on laminin substrates ranging from 0.01 to 1.0 microgram/cm2. Outgrowth characteristics were subsequently analyzed for noninteracting cells in both fixed and live cultures. Data obtained from neurons fixed after 11 hr of culture showed approximately twofold increases in neurite initiation and outgrowth, and a twofold decrease in branching for a corresponding 100-fold increase in adsorbed laminin concentration. In time-lapse videomicroscopy observations, the root-mean square speed of growth cone movement increased from 60 to 90 microns/hr over the same range in concentration, while the persistence time remained constant at 0.10 hr. In general, neurite outgrowth parameters were relatively insensitive to changes in laminin concentration, supporting the idea that laminin is a permissive rather than an "instructive" substrate during development. Data obtained from fixed cultures were examined in terms of probability models to suggest possible mechanisms contributing to the dose-dependent effects observed.
A method has been developed for obtaining mixed primary cultures of dissociated epidermis enriched in Merkel cells. Merkel cells obtained from embryonic rat buccal pads were grown in serum-free medium and identified in vitro using a variety of histological and immunohistochemical markers. Quinacrine, a fluorescent amine, which has been used to identify Merkel cells in situ, labeled a morphologically distinct population of cells in vitro. Cells labeled with quinacrine had a large, phase bright nucleus with prominent nucleoli, surrounded by a phase dark perinuclear ring. Antibodies directed against neuron-specific enolase, another marker for Merkel cells in situ, and antibodies against a well-characterized neuroendocrine vesicle antigen also labeled this population of quinacrine fluorescent cells. Electron microscopic examination of our cultures indicated that cells containing characteristic features of Merkel cells including cytoplasmic dense-cored granules were present. A small but significant increase in the number of Merkel cells was observed over time in culture. Merkel cells supported the survival and outgrowth of both trigeminal ganglion sensory neurons and sympathetic neurons from the superior cervical ganglion in serum-free medium in the absence of exogenous nerve growth factor (NGF). Immunoblots probed with antibodies directed against NGF demonstrated that NGF was present in the medium taken from these cultures. NGF-like immunoreactivity colocalized to cells containing quinacrine fluorescence in situ and in vitro. Addition of antibodies directed against NGF to cocultures of Merkel cells and neurons decreased survival of sympathetic neurons by 90% and decreased survival of sensory neurons by 60%. These results suggest that Merkel cells are capable of providing trophic support for their normal complement of sensory neurons by producing NGF. Selective recognition of these targets was studied in vitro by characterizing the interactions between Merkel cells and growth cones from sensory or sympathetic neurons using both time-lapse videomicroscopy and standard morphometry of fixed cocultures. The majority of trigeminal ganglion sensory neurons (approximately 60%) extended growth cones onto clusters of Merkel cells. Neurites which contacted clusters of Merkel cells were significantly more highly branched than those growing on collagen. In contrast, the majority of sympathetic neurons (greater than 90%) failed to grow onto Merkel cells. Growth cones of sympathetic neurons often "collapsed" and retracted when contact was made with a cluster of Merkel cells. Fixation of Merkel cells with paraformaldehyde prior to coculture did not affect this difference between sensory and sympathetic neurite extension onto the Merkel cells. However, prior fixation of Merkel cells eradicated the apparent Merkel ce-induced branching of sensory neurites.(ABSTRACT TRUNCATED AT 400 WORDS)
Since most oxygen exchange in muscle is thought to occur by diffusion across the walls of capillaries, it is important to determine the spatial relationship between capillaries and muscle fibers. We have extended the work of Kayar et al. (Microvasc. Res 24: 326-341, 1982) to include other statistical tests that allow one to make stronger statements regarding the spatial pattern. Data for hamsters were obtained from two sartorius, three retractor, and five soleus muscle sections. Distances between all pairs of capillaries, distances between a capillary and its first nearest neighbor for all capillaries, and distances between random tissue sample points and the closest capillary were used to test the spatial arrangement of capillaries. The null hypothesis tested of complete spatial randomness of capillary locations was rejected in favor of a regular alternative in one each of the sartorius and retractor fields and in all five soleus fields. We formulated a geometric model, composed of a space-filling array of identical hexagonal muscle fibers with capillaries placed randomly at the juncture of three fibers or between two fibers, according to the observed relative probability of those occurrences. The model simulations of muscle fibers and capillaries were then analyzed by the same statistical tests used on the histological sections. The findings were similar in both cases, providing confidence that the assumptions of the model were sufficient approximations. The results of this study provide a basis for the placement of capillaries around muscle fibers in mathematical models of oxygen transport in capillary networks.
When the growth cone of a chick dorsal root ganglion (DRG) neurite contacts the neurite of a chick retinal ganglion cell in vitro, the growth cone typically responds by withdrawing its lamellipodia and filopodia and collapsing. We have used the fluorescent calcium indicator dye fura-2 and digital imaging microscopy to measure calcium levels within DRG growth cones and to determine whether changes in calcium levels are responsible for the collapse of growth cone morphology when a DRG growth cone contacts a retinal ganglion cell neurite. Calcium levels within DRG growth cones were stable during neurite outgrowth. Calcium was typically distributed homogeneously throughout the growth cone, though occasionally gradients of free calcium were present. When calcium gradients were observed, calcium levels appeared higher in the active veil regions than in the central core region. Calcium levels in DRG growth cones appeared to remain stable during the period of contact-mediated growth cone collapse. Low concentrations of the calcium ionophore ionomycin increased calcium levels two- to threefold without having any observable morphological effects on DRG growth cones. Likewise, depolarization with 15 mM KCl caused a transient two- to threefold increase in calcium levels without having any observable morphological effect. These results suggest that changes in calcium levels are not responsible for contact-mediated collapse of growth cone structure. A growth cone collapsing activity has been solubilized from embryonic chick brain (Raper and Kapfhammer, 1990). Application of this material to cultures of DRG neurons caused growth cones to collapse but had no effect on calcium levels within the growth cones. The crude growth cone collapsing activity was not blocked by the presence of cobalt, nickel, lanthanum, nifedipine, or reduced-calcium medium, suggesting that transmembrane calcium fluxes were not required for growth cone collapse. These results suggest that the morphological changes associated with the collapse of growth cone structure can be independent of changes in growth cone calcium levels, and that second messengers other than calcium are likely to be involved in the regulation of many growth cone behaviors.
Based on experimental data that show the presence of significant oxygen saturation gradients in precapillary arterioles, it has been suggested that the in vivo permeability to oxygen of resting striated muscle may be significantly higher than the corresponding in vitro value obtained in unperfused tissue samples (Popel et al., 1989b, Adv. expl. Med. Biol. 247, 215). The present study performs two analyses to further compare theoretical predictions with experimental data obtained under control conditions and during hemodilution and hemoconcentration. First, it is shown that, in principle, a capillary-perfused tissue layer with a thickness of a few hundred microns is necessary to convectively carry the experimentally determined amount of oxygen released by precapillary arterioles under control and hemodiluted conditions. This capacity to convect oxygen depends strongly on the resting tissue oxygen tension. Second, a more general version of a previous model (Weerappuli & Popel, 1989, J. Biomech. Eng. 111, 24) is used to examine whether changes made in the model parameters within the physiological range of values can explain the experimentally measured flux. The results show that the theoretical predictions can be made compatible with experimental observations if the in vivo permeability of perfused tissue to oxygen is assumed to be one to two orders of magnitude higher than the in vitro value. Furthermore, the predicted in vivo permeability for perfused tissue surrounding an arteriole varies with the arteriolar luminal oxygen tension and flow. This may be due to simplifying approximations made in the model or possible experimental artifacts. Alternatively, it could also be speculated that this variability indicates the flow dependency of the permeability of perfused tissue to oxygen.
We have developed a new video microspectrophotometric system for the in vivo determination of oxygen saturation in red blood cells in striated muscle capillaries. This method allows one to quantify changes in the oxygenation of small groups of red blood cells as they traverse the capillary. Simultaneous images of a single microscopic field are recorded using two silicon-intensified target cameras and high-resolution video recorders. One image is recorded at an oxygen-dependent wave-length (431 nm) and the other at an isosbestic wavelength (420 nm). Light intensities from 10 adjacent pixels aligned along the axis of the capillary from identical 10-s segments of the video-tapes are digitized once per frame. Both sets of data are redisplayed simultaneously as two-dimensional images (10 pixels high x 300 frames wide) using a graphics system. These images show alternating bright and dark bands corresponding to plasma gaps and red blood cells. Light intensities in the presence and absence of red blood cells are determined by positioning a window over the appropriate region of the graphics image. Optical densities of single red blood cells at the two wavelengths, OD431 and OD420, are computed as is their ratio (OD431/OD420), which is linearly related to oxygen saturation. In vivo calibration studies in capillaries of the hamster retractor muscle indicate that the error in measuring oxygen saturation with this technique is approximately 2.7% saturation for a group of 10 cells.
In the early part of this century, August Krogh proposed a model of oxygen transport in capillaries that assumes that all oxygen is delivered to the capillaries by convection from small terminal arterioles and lost from these capillaries by diffusion. This model and its consequences have been used extensively to interpret whole organ oxygen transport data in terms of diffusion between capillaries and tissues and to relate changes in microvascular hemodynamics to alterations in oxygen transport. We evaluated the appropriateness of such extrapolation by measuring oxygen saturation at discrete locations along the lengths of individual capillaries in the hamster cheek pouch retractor muscle. Our results indicate that the amount of oxygen lost from individual capillaries can be markedly affected by the presence of larger microvessels that frequently cross the capillary path. These larger vessels act either as a diffusive supply of oxygen for the red blood cells within the capillary or as an additional sink for the oxygen depending on the direction of the oxygen tension gradient. This transfer of oxygen between larger microvessels and capillaries attenuates the importance of capillary hemodynamics in oxygen exchange. Therefore, conclusions about local oxygen exchange that utilize only hemodynamic data from whole organ or microvascular experiments and the Krogh model will generally be invalid and should be viewed with caution.
We investigated the influence of isovolemic hemoconcentration on microcirculatory hemodynamics and oxygen transport in the hamster cheek pouch retractor muscle. In 17 hamsters, measurements of red blood cell velocity, hematocrit, vessel diameter, segment length (L), hemoglobin oxygen saturation (SO2), and longitudinal SO2 gradient (delta SO2/L) were made in four branching orders of arterioles before and after isovolemic exchange with packed red blood cells. Hemoconcentration increased systemic hematocrit from 50 to 65%; systemic blood gases were unchanged, but mean arterial blood pressure increased approximately 10 mmHg. Accompanying this change in systemic hematocrit the microcirculatory hematocrit increased from 40 to 50%, and red blood cell velocity and computed blood flow decreased approximately 40 and 30%, respectively. In addition, delta SO2/L was significantly increased in the four arteriolar branching orders compared with control values. We estimated that approximately 10% of the oxygen that diffused across the arteriolar network was consumed by the surrounding tissue with the remainder assumed to have diffused to venules and capillaries. After hemoconcentration, the proportion of this diffusional transfer increased by approximately 80%. Convective oxygen flow remained at its control level in the first-order arterioles and progressively decreased below control in the more distal branching orders. Our analysis of arteriolar oxygen diffusion indicated that tissue oxygenation was unchanged after hemoconcentration, a result that can be attributed to a combined effect of decreased red blood cell velocity, arteriolar vasodilation, increased precapillary oxygen loss, and a relatively modest reduction in convective oxygen flow to the capillaries.
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