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R N Pittman

Publications and source records attributed to R N Pittman.

At least 73 records · Page 4Linked to original sources
PubMed1989

Capillary spatial pattern and muscle fiber geometry in three hamster striated muscles.

The primary goal of this project was to elucidate the spatial pattern of capillaries in three hamster striated muscles according to statistical techniques of pattern analysis. The spatial pattern of capillaries and traditional measures of capillarity are important to understanding the supply and distribution of oxygen and nutrients in a tissue. Statistical tests based on the distance between nearest neighbor capillaries are the most sensitive for detecting regularity in a pattern. A mathematical model was created to simulate the observed muscle fields. The same statistical tests that were performed on the empirical data were performed on the modelled data. The results of the analysis of the modelled data agree sufficiently with those of the empirical data to justify overall confidence in the assumptions. Conclusions that may be drawn from this investigation are (1) the spatial pattern of capillaries tends to be more regular than random and never was there evidence for aggregation using the test statistics; (2) as many as 60-75% of capillaries are located at the corners of muscle fibers indicating that there is some preferential placement for capillaries, and (3) the model developed is a good first approximation to the real situation.

Animals↗
PubMed1989

Growth cone-growth cone interactions in cultures of rat sympathetic neurons.

Growth cones of sympathetic neurons from the superior cervical ganglia of neonatal rats were studied using video-microscopy to determine events following contact between growth cones and other cell surfaces, including other growth cones and neurites. A variety of behaviors were observed to occur upon contact between growth cones. Most commonly, one growth cone would collapse and subsequently retract upon establishing filopodial contact with the growth cone of another sympathetic neuron. Contacts resulting in collapse and retraction were often accompanied by a rapid and transient burst of lamellipodial activity along the neurite 30-50 microns proximal to the retracting growth cone. In no instances did interactions between growth cones and either fibroblasts or red blood cells result in the growth cone collapsing, suggesting that a specific recognition event was involved. On several occasions, growth cones were seen to track other growth cones, although fasciculation was rare. In some cases, there was no obvious response between contacting growth cones. Growth cone-growth cone contact was almost four times more likely to result in collapse and retraction than was growth cone-neurite contact (45% vs 12%, respectively). These observations suggest that the superior cervical ganglion may be composed of neurons with different cell surface determinants and that these determinants are more concentrated on the surface of growth cones than on neurites. These results further suggest that contact-mediated inhibition of growth cone locomotion may play an important role in growth cone guidance.

Animals↗
PubMed1989

Oxygen exchange in the microcirculation of hamster retractor muscle.

We determined percent hemoglobin oxygen saturation (SO2) in arterioles and venules of the hamster retractor muscle at rest. We found that SO2 decreased from 69.9 +/- 1.4% (SE) in large input arterioles (first order, 1A, ID = 60 +/- 3 micron) to flow-weighted values of 56.7% in small arterioles (4A, ID = 20 +/- 1 micron), 51.3% in small venules (4V, ID = 28 +/- 1 micron), and to 50.6 +/- 1.0% in large venules (1V, ID = 147 +/- 13 micron). Thus approximately two-thirds of the net decline in SO2 for this tissue occurred by diffusion of oxygen from arterioles, whereas only about one-third occurred by diffusion from capillaries. Furthermore, no net shunting of oxygen from the arterioles to the venules was detected as evidenced by the absence of any significant change in venular SO2. By determining the SO2 at upstream and downstream ends of arterioles in four consecutive branching orders (1A-4A), we found that the decrease in SO2 per unit length (delta SO2/L) increased approximately 20-fold from 1A to 4A. This increase in delta SO2/L was directly proportional to estimated luminal minus tissue oxygen tension and inversely proportional to red blood cell flow.

Algorithms↗
PubMed1989

Rate of oxygen loss from arterioles is an order of magnitude higher than expected.

The experimental data on oxygen flux from arterioles in the hamster cheek pouch retractor muscle [L. Kuo and R. N. Pittman, Am. J. Physiol. 254 (Heart Circ. Physiol. 23): H331-H339, 1988] were analyzed under the assumption that the permeability to oxygen is the same in both perfused and unperfused tissue; permeability is defined as the product of the diffusion and solubility coefficients. However, our analysis indicated that the observed oxygen flux was inconsistent with this assumption and that permeability to oxygen of a blood-perfused tissue may be an order of magnitude higher than previously assumed.

Animals↗
PubMed1989

Neurite penetration into collagen gels requires Ca2+-dependent metalloproteinase activity.

Cultured sympathetic neurons from neonatal rats release a Ca2+-dependent metalloproteinase capable of degrading native and denatured type I collagen. A large fraction of the metalloproteinase activity is released from growth cones of growing neurites, which suggests that this proteinase may be involved in neurite penetration into collagen-rich extracellular matrices during development. In the present study, we investigated the possibility that neurite penetration into 3-dimensional collagen gels requires the Ca2+-dependent metalloproteinase. Sympathetic neurons were plated on top of collagen gels and the neuronal metalloproteinase activity was inhibited with the collagenase inhibitor HSCH2CH[CH2CH(CH3)2]-CO-Phe-Ala-NH2 (HS-LFA). HS-LFA inhibited the Ca2+-dependent metalloproteinase activity, penetration of neurites into collagen gels and release of 3H from 3H-collagen gels in a dose-dependent manner over very similar concentration ranges (EC50 = 150-200 nM). Highly significant correlations existed between neurite penetration into collagen gels and metalloproteinase activity (r = 0.99), and metalloproteinase activity and release of 3H from 3H-collagen gels (r = 0.99). Although HS-LFA inhibited the growth of neurites within collagen gels, it had no effect on neurite outgrowth on collagen films. Thus, the neuronal Ca2+-dependent metalloproteinase appears to be required for neurite penetration into and growth within 3-dimensional collagen matrices, but not for growth along a 2-dimensional collagen substrate.

Animals↗
PubMed1989

Neuronal plasminogen activators: cell surface binding sites and involvement in neurite outgrowth.

Sympathetic neurons release both urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA). A number of inhibitors of serine proteases have been tested to determine their effects on neurite outgrowth from rat sympathetic neurons. Some inhibitors increase neurite outgrowth while others have little or no effect on outgrowth. Inhibition of plasminogen activator (PA) activity but not other serine protease activity correlates with the increase in neurite outgrowth (uPA, r = 0.89; tPA, r = 0.86; plasmin, r = 0.015; thrombin, r = 0.025). Antibodies that inhibit uPA activity increase neurite outgrowth, while antibodies that bind to uPA but do not inhibit activity do not alter outgrowth. Time-lapse videomicroscopy of neurite outgrowth indicates that about 85% of the neurites increase their rate of outgrowth following exposure to inhibitors of PA. Routinely, 1-2 min after exposure of a growth cone to an inhibitor, there is an increase in lamellipodial activity at the leading edge of the growth cone and a decrease in lamellipodial activity on the sides and base of the growth cone. The increase in the rate of outgrowth combined with the decrease in lamellipodial activity on the sides of the growth cones results in neurites being very long and straight in the presence of inhibitors (persistence time P = 3.7 and 15.3 hr for controls and in the presence of inhibitors of PA, respectively). PAs released from sympathetic neurons and PC12 cells interact with 3 different binding sites on the cell surface: (1) an inhibitor of serine proteases (including uPA and tPA) is bound to the surface via a heparinase-sensitive site; (2) a uPA-selective binding site is present in patches on the bottom surface of PC12 cells; and (3) a tPA-selective binding site with high affinity (KD = 23 +/- 10 nM) and high capacity (340,000 +/- 130,000 sites/neuron) for 125I-tPA is homogeneously distributed over the entire surface. Data in the present study are consistent with PA being involved in neurite outgrowth and open the possibility of other PA-dependent functions occurring when tPA and/or uPA interacts with cell surface binding sites.

Amino Acid Chloromethyl Ketones↗
PubMed1988

Assessment and impact of heterogeneities of convective oxygen transport parameters in capillaries of striated muscle: experimental and theoretical.

Convective oxygen transport parameters were determined in arteriolar (n = 5) and venular (n = 5) capillary networks in the hamster cheek pouch retractor muscle. Simultaneously determined values of red blood cell velocity, lineal density, red blood cell frequency, hemoglobin oxygen saturation (SO2), oxygen flow (QO2), longitudinal SO2 gradient, and diameter were obtained in a total of 73 capillaries, 39 at the arteriolar ends of the network (arteriolar capillaries) and 34 at the venular ends (venular capillaries). We found that the hemodynamic variables were not different at the two ends. However, not unexpectedly, SO2 and QO2 were significantly higher at the upstream end of arteriolar capillaries (60.8 +/- 9.8 (SD)% and 0.150 +/- 0.081 pl/sec, respectively) compared with the downstream end of venular capillaries (39.9 +/- 13.6% and 0.108 +/- 0.095 pl/sec, respectively). Heterogeneities in red blood cell velocity, lineal density, SO2, and QO2, assessed by their coefficients of variation, were significantly greater in venular capillaries. To evaluate the impact of these heterogeneities on oxygen exchange, we incorporated these unique experimental data into a mathematical model of oxygen transport which accounts for variability in red blood cell frequency, lineal density, inlet SO2, capillary diameter, and, to some degree, capillary flow path lengths. An unexpected result of the simulation is that only the incorporation of variability in capillary flow path lengths had any marked effect on the heterogeneity in end-capillary SO2 in resting muscle due to extensive diffusional shunting of oxygen among adjacent capillaries. We subsequently evaluated, through model simulations, the effect of these heterogeneities under conditions of increased flow and high oxygen consumption. Under these conditions, the model predicts that heterogeneities in the hemodynamic parameters will have a marked effect on oxygen transport in this muscle.

Animals↗
PubMed1988

Effect of hemodilution on oxygen transport in arteriolar networks of hamster striated muscle.

Experiments were performed on the hamster cheek pouch retractor muscle to investigate the influence of isovolemic hemodilution on microcirculatory hemodynamics and the rate of oxygen transport to striated muscle. In 23 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 plasma. Hemodilution decreased systemic hematocrit from 52 to 33%. In first- through fourth-order arterioles, this degree of hemodilution resulted in an average decrease in microcirculatory hematocrit from 42 to 28% and average increases in red blood cell velocity, computed blood flow, and systemic arterial PO2 of 50, 30, and 10%, respectively. In addition, delta SO2/L was significantly smaller in second-, third-, and fourth order arterioles compared with control values. It was estimated that approximately 84% of the oxygen that diffused across the arteriolar network was transferred by diffusion to nearby venules and capillaries; the remaining oxygen was consumed by the surrounding tissue. Following hemodilution, the magnitude of diffusional transfer declined to 73%. Oxygen flow remained at its control level in the first-order arterioles and progressively increased above control with increasing branching order. The increased oxygen delivery to the capillary network after limited hemodilution can be attributed to a compensatory increase in blood flow, an increase in systemic arterial blood oxygenation, and a decrease in precapillary oxygen loss.

Algorithms↗
PubMed1987

Analysis of vascular pattern and dimensions in arteriolar networks of the retractor muscle in young hamsters.

A quantitative analysis of the distribution of microvascular blood flow and oxygen delivery requires a detailed description of the vascular network geometry. The distributions of lengths and diameters were determined in terminal arteriolar networks of the cheek pouch retractor muscle of young (34 +/- 2 days) hamsters. We compared the Strahler centripetal vessel ordering scheme, which assigns lowest order to the capillaries and proceeds upstream toward the larger vessels, with the centrifugal ordering scheme, which begins with the input arteriole and proceeds downstream toward the capillaries. The terminal networks of the retractor muscle typically contain 2 to 4 Strahler orders and 2 to 6 centrifugal orders. The coefficients of variation of diameter and length are smaller for Strahler ordering than for centrifugal ordering. In addition, for Strahler ordering, we found that the sequence of number of vessels obeyed Horton's law. We have compared three different methods of calculating the bifurcation, diameter, and length ratios. As an alternative method for analyzing network topology, we also studied the distribution of the number of segments on each pathway from the inlet of a network to a capillary. The information obtained from this analysis is useful for the mathematical modeling of flow in the microvascular network.

Animals↗
PubMed1987

Relationship between mitochondrial volume density and capillarity in hamster muscles.

Mitochondrial volume density and lipid droplet-volume density were stereologically determined from electron micrographs of muscle fibers from three hamster muscles: retractor, sartorius, and soleus. The number of capillaries around a fiber, length of capillary-fiber contact, and muscle fiber area were also measured. Glycolytic fibers of the retractor and sartorius had larger cross-sectional areas, lower mitochondrial-volume densities, fewer subsarcolemmal aggregates of mitochondria, and lower capillary-fiber contact length in comparison to oxidative fibers of the retractor and soleus. Values for mitochondrial volume density in the different muscles correlated well (r = 0.97) with resting O2 consumption. The mitochondrial volume densities for each muscle correlated well (r = 0.99) with O2 diffusion coefficients of these muscles. Our results indicate that an analysis of the adequacy of O2 supply to an individual muscle fiber must take into account an interplay among fiber size, percent of the muscle fiber perimeter in contact with a capillary (capillary-fiber contact), and O2 demand of the fiber estimated by mitochondrial volume density.

Animals↗
PubMed1987

Measurement of hemoglobin oxygen saturation in capillaries.

We present a computer-aided videodensitometric method for the determination of oxygen saturation in red blood cells flowing through capillaries of the hamster cheek pouch retractor muscle. The optical density (OD) of red blood cells is determined at two wavelengths. At the first, 431 nm, there is a maximum difference between absorption by oxygen deoxyhemoglobin. At the second, 420 nm, absorption is equal for the two absorbing species (isosbestic wavelength). In capillaries of the retractor muscle a relationship between oxygen saturation (S) and the following OD ratio was obtained as S = -1.71 (OD431/OD420) + 2.20. The error (95% confidence interval) in oxygen saturation associated with a determination of the OD ratio is estimated to be +/- 4.8%. The computerization of the method employs a frame-by-frame analysis of the light intensity over a selected capillary segment. The light intensity waveform along the segment is digitized and the minimum (I) and maximum (I0) light intensities are used to compute an optical density (OD = log10 [I0/I]). These minimum and maximum intensities correspond to the presence and absence of a red blood cell, respectively. The method permits the off-line analysis of videotaped scenes and provides a means of assessing the extent of temporal and spatial heterogeneity of oxygen saturation in selected capillary networks. The method has been developed for use in capillaries in transilluminated striated muscle but should be generally applicable to the measurement of capillary oxygen saturation in other tissues.

Animals↗
PubMed1987

Characterization of an inhibitor of neuronal plasminogen activator released by heart cells.

A basic understanding of growth cone dynamics and developmental events involving growth cones requires an understanding of the function and regulation of molecules associated with and released by growth cones. Rat sympathetic neurons in culture release a urokinase-like plasminogen activator from their distal processes and/or growth cones (Pittman, 1985a). When sympathetic neurons are grown in cocultures with heart cells, however, plasminogen activator activity is not detected. The absence of plasminogen activator activity in cocultures of sympathetic neurons and heart cells appears to be due to the release of an inhibitor of plasminogen activator by heart cells. This inhibitor has a molecular weight of approximately 50 kDa in the presence of SDS and apparent molecular weights of approximately 50 and greater than 2000 kDa under native conditions. A significant fraction of the large-molecular-weight form of the inhibitor is converted to the smaller form following treatment with heparinase. Extremely stable complexes of 68 and 80 kDa are formed between the heart inhibitor and the plasminogen activator, urokinase, such that the complexes withstand boiling in SDS/mercaptoethanol. The data are consistent with the formation of an 80 kDa urokinase-inhibitor complex in the presence of heparan sulfate proteoglycan and a 68 kDa complex in the absence of heparan sulfate proteoglycan. A highly purified preparation of the heart inhibitor produces a 2- to 3-fold increase in neurite outgrowth from sympathetic neurons. These data indicate that the activity of the plasminogen activator released by sympathetic neurons can be regulated by a normal target tissue and that this regulation may result in increased neurite outgrowth from the neurons.

Animals↗
PubMed1986

In vivo photometric analysis of hemoglobin.

Since virtually all the oxygen carried by blood at normal hematocrit is reversibly bound to red blood cell hemoglobin, the distribution of oxygen within the microcirculation can be determined from measurements of hemoglobin concentration and hemoglobin oxygen saturation in vessels of the network. Photometric methods that rely on light absorption and scattering properties of blood are described. Criteria for selecting the wavelengths needed to analyze hemoglobin in the microcirculation are specified. Two theoretical descriptions of light absorption and scattering, multiple scattering theory and photon diffusion theory, are applied to the problem. Practical approaches to the determination of hemoglobin concentration and oxygen saturation in the microcirculation follow from these theoretical formulations. Technical aspects of microscope photometry including light sources, microscopy, and detection systems are described with special emphasis on the problem of glare. The importance of in vitro as well as in vivo calibrations is stressed, and several recent applications of a working system are discussed. Current problems as well as future developments of this methodology are delineated as a guide to future work in this area.

Animals↗
PubMed1986

Estimation of red cell flow microvessels: consequences of the Baker-Wayland spatial averaging model.

The dual sensor cross-correlation method of H. Wayland and P.C. Johnson [1967), J. Appl. Physiol. 22, 333-337) has become a standard technique for determining the velocity of red blood cells (RBCs) in glass tubes and blood vessels. M. Baker and H. Wayland [1974), Microvasc. Res. 7, 131-143) found that under a variety of conditions the ratio of dual sensor velocity at the centerline of a glass tube to the blood velocity averaged over the lumen was close to 1.6. They provided an explanation of this factor based on spatial averaging of RBC velocity vertically through the tube as well as laterally across the face of the sensor. Their spatial averaging model could also account for the apparent blunting of RBC velocity profiles determined with the dual sensor technique. We used Baker and Wayland's spatial averaging model to calculate how the above velocity ratio depends on sensor size. A nonlinear relation between the velocity ratio and sensor size was found such that the velocity ratio varied from 1.6 to 1.33 as the ratio of sensor width to vessel or tube diameter was varied from 0 to 1. These results also hold for vessels or tubes of elliptic cross section. Some investigators have found that the velocity of red cells near the walls of blood vessels can be a substantial fraction of centerline velocity which suggests that RBC velocity distributions can be blunter than a Poiseuille distribution. We repeated the above calculation for blunted parabolic profiles and we found that the velocity ratio ranged from 1 for plug flow to 1.6 for Poiseuille flow. These calculations show that reliable estimates of RBC flow from dual sensor centerline velocity measurements require one to take into account the relative size of the sensor and blood vessel diameter as well as the bluntness of the RBC velocity distribution.

Animals↗
PubMed1986

Evaluation of photometric methods for quantifying convective mass transport in microvessels.

We evaluated the accuracy of blood (Qb), hemoglobin (QHb) and O2 (QO2) flow determinations in arterioles (30-89 micron ID) and venules (30-140 micron ID) of the hamster retractor muscle by testing flow conservation in straight segments (for Qb and QHb) and at bifurcations (for Qb, QHb, and QO2). We found absolute values of relative discrepancies in flow conservation of greater than 30% in straight sections and of greater than 20% at bifurcations. To understand these results, we evaluated the assumptions implicit in the flow calculations by determining the intraluminal profiles of red blood cell velocity, hemoglobin concentration, and O2 saturation. We found that these assumptions were not generally satisfied since only 59% of velocity profiles, 70% of hemoglobin concentration profiles, and 70% of O2 saturation profiles were symmetric about the vessel axis with the velocity profiles more blunt than would be expected for Poiseuille flow. We reevaluated flow conservation by taking into account the bluntness of the velocity profiles. By selecting vessels that had symmetric velocity and hemoglobin concentration profiles and by including the bluntness of the velocity profile, we obtained a significant improvement in the demonstration of flow conservation.

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