Oxygen distribution and consumption in the macaque retina

The oxygen distribution in the retina of six anesthetized macaques was investigated as a model for retinal oxygenation in the human retina in and adjacent to the fovea. P(O2) was measured as a function of retinal depth under normal physiological conditions in light and dark adaptation with O(2) microelectrodes. Oxygen consumption (Q(O2)) of the photoreceptors was extracted by fitting a steady-state diffusion model to P(O2) measurements. In the perifovea, the P(O2) was 48 +/- 13 mmHg (mean and SD) at the choroid and fell to a minimum of 3.8 +/- 1.9 mmHg around the photoreceptor inner segments in dark adaptation, rising again toward the inner retina. The P(O2) in the inner half of the retina in darkness was 17.9 +/- 7.8 mmHg. When averaged over the outer retina, photoreceptor Q(O2) (called Q(av)) was 4.6 +/- 2.3 ml O(2).100 g(-1).min(-1) under dark-adapted conditions. Illumination sufficient to saturate the rods reduced Q(av) to 72 +/- 11% of the dark-adapted value. Both perifoveal and foveal photoreceptors received most of their O(2) from the choroidal circulation. While foveal photoreceptors have more mitochondria, the Q(O2) of photoreceptors in the fovea was 68% of that in the perifovea. Oxygenation in macaque retina was similar to that previously found in cats and other mammals, reinforcing the relevance of nonprimate animal models for the study of retinal oxygenation, but there was a smaller reduction in Q(O2) with light than observed in cats, which may have implications for understanding the influence of light under some clinical conditions.     

Reappraisal of diffusion, solubility, and consumption of oxygen in frog skeletal muscle, with applications to muscle energy balance

Previously we tested the validity of the one-dimensional diffusion equation for O2 in the excised frog sartorius muscle and used it to measure the diffusion coefficient (D) for O2 in this muscle and the time course of its rate of O2 consumption (Qo2) after a tetanus (Mahler, 1978, 1979, J. Gen. Physiol., 71:533-557, 559-580, 73:159-174). A transverse section of the frog sartorius is in fact well fit by a hemi-ellipse with width divided by maximum thickness averaging 5.1 +/- 0.2. Using the previous techniques with the two-dimensional diffusion equation and this hemi-elliptical boundary yields a value for D that is 30% smaller than reported previously; the revised values at 0, 10, and 22.8 degrees C are 6.2, 7.9, and 10.8 X 10(-6) cm2/s, respectively. After a tetanus at 20 degrees C, Qo2 rose quickly to a peak and then declined exponentially, with a time constant (tau) approximately 15% faster than that reported previously; tau averaged 2.1 min in Rana temporaria and 2.6 min in Rana pipiens. A technique was devised to measure the solubility (alpha) of O2 in intact, respiring muscles, and yielded alpha (muscle)/alpha (H2O) = 1.26 +/- 0.04. With these modifications, the values for O2 consumption obtained with the diffusion method were in agreement with those measured by the direct method of Kushmerick and Paul (1976, J. Physiol. [Lond.]., 254:693-709). Using results from both methods, at 20 degrees C the ratio of phosphorylcreatine split during a tetanus to O2 consumption during recovery ranged from 5.2 to 6.2 mumol/mumol, and postcontractile ATP hydrolysis was estimated to be 13.6 +/- 4.1 (n = 3) nmol/mumol total creatine.     

Direct fluorescence measurement of diffusional water permeability in the vasopressin-sensitive kidney collecting tubule.

A fluorescence method has been developed for accurate and instantaneous measurement of transepithelial diffusional water permeability (Pd) in perfused kidney tubules based on the sensitivity of the fluorophore aminonapthelane trisulfonic acid (ANTS) to solution H2O/D2O content. The fluorescence of ANTS was 3.2-fold lower in an H2O buffer than in a D2O buffer. The response of ANTS fluorescence to a change in solution H2O/D2O content occurred in less than 1 ms and was due to a collisional quenching mechanism. Isolated cortical (CCT) and outer medullary (OMCT) collecting tubules from rabbit were perfused with an isosmotic D2O buffer at specified lumen flow rates (2-100 nl/min); tubules were bathed in isosmotic H2O or D2O buffers in which vasopressin (VP) could be added rapidly. Lumen fluorescence was monitored by quantitative epifluorescence microscopy at 380 +/- 5 nm excitation and greater than 530 emission wavelengths. Pd was determined from tubule geometry, lumen flow, ANTS fluorescence, and ANTS fluorescence vs. H2O/D2O calibration relation. The instrument response time for a change in bath H2O/D2O content was less than 4 s. At 37 degrees C, Pd values (mean +/- SE in cm/s x 10(4] were 6.4 +/- 1.0 (-VP, n = 9) and 14.3 +/- 1.1 (+250 microU/ml bath VP, n = 9) in the CCT, and 5.8 +/- 1.0 (-VP, n = 6) and 15.3 +/- 2.0 (+VP, n = 6) in the OMCT; at 23 degrees C, Pd was 5.1 +/- 0.6 (-VP, n = 4) and 7.8 +/- 0.6 (+VP, n = 4) in the CCT.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diffusion of water in cat ventricular myocardium

The rates of diffusion of tritiated water (THO) and [14C]sucrose across cat right ventricular myocardium were studied at 23 degrees C in an Ussing-type diffusion cell, recording the time-course of increase in concentration of tracer in one chamber over 4--6 h after adding tracers to the other. Sucrose data were fitted with a model for a homogeneous sheet of uneven thickness in which the tissue is considered to be an array of parallel independent pathways (parallel pathway model) of varying length. The volume of the sucrose diffusion space, presumably a wholly extracellular pathway, was 23% of the tissue or 27.4 +/-1.7% (mean +/- SEM; n=11) of the tissue water. The effective intramyocardial sucrose diffusion coefficient, D8, was 1.51 +/- 0.19 X 10(-6)cm2.s-1 (n=11). Combining these data with earlier data, D8 was 22.6 +/- 1.1% (n=95) of the free diffusion coefficient in aqueous solution D degrees 8. The parallel pathway model and a dead-end pore model, which might have accounted for intracellular sequestration of water, gave estimates of DW/D degrees W (observed/free) of 15%. Because hindrance to water diffusion must be less than for sucrose (where D8/D degrees 8=22.6%), this showed the inadequacy of these models to account simultaneously for the diffusional resistance and the tissue water content. The third or cell-matrix model, a heterogeneous system of permeable cells arrayed in the extracellular matrix, allowed logical and geometrically reasonable interpretations of the steady-state data and implied estimates of DW in the cellular and extracellular fluid of approximately 25% of the aqueous diffusion coefficient.     

Pulsed high-field gradient in vivo NMR spectroscopy to measure diffusional water permeability in Corynebacterium glutamicum

Pulsed high-field gradient in vivo NMR spectroscopy was used to measure diffusional water permeability in cell suspensions of the Gram-positive bacterium Corynebacterium glutamicum. Two different regions of H2O mobility were detected. One was characterized by the apparent coefficient of self-diffusion, D(1 app) = (4.6-12.7)x10(-8) cm(2) s(-1), depending on the observation time t. The other region was characterized by D(2) = 1.4x10(-5) cm(2) s(-1). The value of D(2) was similar to the diffusion coefficient of H2O in free water and in extracellular biological fluids. Restricted diffusion could be demonstrated for the slower process (D(1)). It was attributed to the cytoplasm of the cells. The membrane permeability, P(d H2O), for C. glutamicum was (4.8+/-0.4)x10(-3) cm s(-1). It compared favorably with values reported for human erythrocytes and was higher by a factor of about 100 compared to the diffusional permeability for ethanol, P(d ethanol), in Zymomonas mobilis. Addition of HgCl2, a water channel inhibitor in eukaryotes, decreased P(d H2O) in C. glutamicum by a factor of approximately 8. To our knowledge, these are the first functional studies of water transport in prokaryotes that yielded quantitative data, viz., transmembrane water permeability expressed through D(H2O) and P(d H2O).     

Calcium diffusion in transient and steady states in muscle.

Rates of diffusion through the extracellular space of thin sheets of myocardium from the right ventricular outflow tract of kittens were estimated at 23 degrees C for 45Ca2+ and an inert reference tracer, [14C]sucrose. The myocardial sheets were mounted in an Ussing chamber and equilibrated with Tyrode solution with varied calcium concentrations, Cao. The tracers were added to one side and their concentrations on the other side measured at 5-15-min intervals for 6 h. The apparent tracer diffusion coefficient for sucrose was 1.11 +/- 0.06 X 10(-6) cm2s-1 (mean +/- SEM, n = 74), 22% of the free diffusion coefficient; the lag time before reaching a steady state provided estimates of the intratissue volume of distribution or diffusion space of 0.41 +/- 0.15 ml/ml tissue (n = 74), a value compatible with expectations for extracellular fluid space. Over the range of Cao from 0.02 to 9.0 mM, the intratissue apparent diffusion coefficient for Ca, DCa, averaged 1.65 +/- 0.10 X 10(-6) cm2s-1, n = 74, which is 21% of the free DoCa, and was not influenced by Cao. Because transsarcolemmal Ca permeation is slow, DCa is the diffusion coefficient in the extracellular region. The paired ratios DCa/Ds averaged 1.32 +/- 0.05 (n = 67) for all levels of Cao but at physiologic or higher Cao averaged 1.45 +/- 0.07 (n = 39), close to the ratio of free diffusion coefficients, 1.53. Equations distinguishing transient from steady state diffusion were fitted to the data, showing that the apparent distribution volume of "binding sites" external to the diffusion pathway diminished at higher Cao in a fashion suggesting that a least two different Ca2+ binding sites were present.     

Diffusion and partition of solutes in cartilage under static load

We describe experimental apparatus, methodology and mathematical algorithms to measure diffusion and partition for typical small ionic solutes and inulin (a medium size solute) in statically loaded cartilage. The partition coefficient based on tissue water (K(H(2)O)) of Na(+) increased from 1.8 to 4.5 and for SO(4)(-2) decreased from 0.5 to 0.1, when the applied pressure was raised from zero to 22 atm K(H(2)O) of inulin decreased from 0.3 to 0.05, for an increase in pressure from zero to 11 atm. Our theoretical interpretation of the results is that the partition coefficient can be expressed as a function of fixed charge density (FCD) for both loaded and unloaded cartilage. The partition coefficient shows good agreement with the ideal Gibbs-Donnan equilibrium, particularly when FCD is based on extrafibrillar water (EFW). The diffusion coefficients, D also decreased with an increase in applied pressure; raising the pressure from 0 to 22 atm resulted in the following changes in the values of D: for Na(+) from 2.86 x 10(-6) to 1.51 x 10(-6) cm(2)/s, for SO(4)(-2) from 1.58 x 10(-6) to 7.5 x 10(-7) cm(2)/s, for leucine from 1.69 x 10(-6) to 8.30 x 10(-7) cm(2)/s and for inulin from 1.80 x 10(-7) to 3.30 x 10(-8) cm(2)/s. For the three small solutes (two charged and one neutral) the diffusion coefficient D is highly correlated with the fraction of fluid volume in the tissue. These experimental results show good agreement with the simple model of Mackie and Meares: hence solute charge does not affect the diffusion of small solutes under load. For inulin D & K show some agreement with a modified Ogston model based on two major components, viz., glycosaminoglycans (GAG) and core protein. We conclude that the changes in the partition and diffusion coefficients of small and medium size solutes in statically loaded cartilage can be interpreted as being due to the reduction in hydration and increase in FCD. The change in the latter affects the partition of small ionic solutes and the partition and diffusion of larger molecules. Our results throw light on the ionic environment of chondrocytes in loaded cartilage as well as on the transport of solutes through the matrix.     

The increase of oxygen consumption after a flash of light is tightly coupled to sodium pumping in the lateral ocellus of barnacle

In the lateral ocellus of the barnacle, we have tested the hypothesis that the transient increase of oxygen consumption (delta QO2) induced by light results from an increase in the rate of Na+ pumping. With a Na(+)-sensitive microelectrode, we measured the intracellular concentration of Na+ (Nai) in the photoreceptor cells. Nai was 17.6 +/- 1.2 mM (SE; n = 18) in darkness and it increased transiently by 10-20 mM after an 80-ms flash of intense light. The increase of Nai recovered in about the same time as the delta QO2, and the Na+/O2 ratio was 19.2 +/- 3.8 (SE; n = 6). Removing Na+ from the bath caused the delta QO2 to decrease by 79 +/- 3% (SE; n = 5). Exposure to 25 microM ouabain inhibited Na+ pumping and abolished the delta QO2. Removal of K+ from the bathing solution inhibited Na+ pumping in darkness, but mostly shortened the duration of the delta QO2; with a K(+)-sensitive microelectrode, we measured pericellular [K+] and found that it increased after the flash for about the same time as the delta QO2. Increasing Na+ pumping in darkness by reintroducing K+ in the bath or by injecting Na+ into one of the photoreceptor cells induced a delta QO2. Finally, intracellular injection of adenosine diphosphate and inorganic phosphate (ADP + Pi), the metabolic products of ATP splitting by the Na+ pump, also induced a delta QO2 in darkness. We conclude that all the results obtained are consistent with the formulated hypothesis.     

Erythrocyte-associated transients in capillary PO2: an isovolemic hemodilution study in the rat spinotrapezius muscle

Mathematical simulations of oxygen delivery to tissue from capillaries that take into account the particulate nature of blood flow predict the existence of oxygen tension (Po(2)) gradients between erythrocytes (RBCs). As RBCs and plasma alternately pass an observation point, these gradients are manifested as rapid fluctuations in Po(2), also known as erythrocyte-associated transients (EATs). The impact of hemodilution on EATs and oxygen delivery at the capillary level of the microcirculation has yet to be elucidated. Therefore, in the present study, phosphorescence quenching microscopy was used to measure EATs and Po(2) in capillaries of the rat spinotrapezius muscle at the following systemic hematocrits (Hct(sys)): normal (39%) and after moderate (HES1; 27%) or severe (HES2; 15%) isovolemic hemodilution using a 6% hetastarch solution. A 532-nm laser, generating 10-micros pulses concentrated onto a 0.9-microm spot, was used to obtain plasma Po(2) values 100 times/s at points along surface capillaries of the muscle. Mean capillary Po(2) (Pc(O(2)); means +/- SE) significantly decreased between conditions (normal: 56 +/- 2 mmHg, n = 45; HES1: 47 +/- 2 mmHg, n = 62; HES2: 27 +/- 2 mmHg, n = 52, where n = capillary number). In addition, the magnitude of Po(2) transients (DeltaPo(2)) significantly decreased with hemodilution (normal: 19 +/- 1 mmHg, n = 45; HES1: 11 +/- 1 mmHg, n = 62; HES2: 6 +/- 1 mmHg, n = 52). Results suggest that the decrease in Pc(O(2)) and DeltaPo(2) with hemodilution is primarily dependent on Hct(sys) and subsequent microvascular compensations.     

Effects of exercise training on acclimatization to hypoxia: systemic O2 transport during maximal exercise.

Acclimatization to hypoxia has minimal effect on maximal O2 uptake (Vo2 max). Prolonged hypoxia shows reductions in cardiac output (Q), maximal heart rate (HR-max), myocardial beta-adrenoceptor (beta-AR) density, and chronotropic response to isoproterenol. This study tested the hypothesis that exercise training (ET), which attenuates beta-AR downregulation, would increase HRmax and Q of acclimatization and result in higher Vo2 max. After 3 wk of ET, rats lived at an inspired Po2 of 70 Torr for 10 days (acclimatized trained rats) or remained in normoxia, while both groups continued to train in normoxia. Controls were sedentary acclimatized and nonacclimatized rats. All rats exercised maximally in normoxia and hypoxia (inspired Po2 of 70 Torr). Myocardial beta-AR density and the chronotropic response to isoproterenol were reduced, and myocardial cholinergic receptor density was increased after acclimatization; all of these receptor changes were reversed by ET. Normoxic Vo2 max (in ml.min-1.kg-1) was 95.8 +/- 1.0 in acclimatized trained (n = 6), 87.7 +/- 1.7 in nonacclimatized trained (P < 0.05, n = 6), 74.2 +/- 1.4 in acclimatized sedentary (n = 6, P < 0.05), and 72.5 +/- 1.2 in nonacclimatized sedentary (n = 8; P > 0.05 acclimatized sedentary vs. nonacclimatized sedentary). A similar distribution of Vo2 max values occurred in hypoxic exercise. Q was highest in trained acclimatized and nonacclimatized, intermediate in nonacclimatized sedentary, and lowest in acclimatized sedentary groups. ET preserved Q in acclimatized rats thanks to maintenance of HRmax as well as of maximal stroke volume. Q preservation, coupled with a higher arterial O2 content, resulted in the acclimatized trained rats having the highest convective O2 transport and Vo2 max. These results show that ET attenuates beta-AR downregulation and preserves Q and Vo2 max after acclimatization, and support the idea that beta-AR downregulation partially contributes to the limitation of Vo2 max after acclimatization in rats.     

Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo

Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1'-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine iodide (C14diI) as a fluorescent lipid probe. During this period of development the lateral diffusion coefficient of membrane lipids is consistently greater in the vegetal polar lobe area as compared to the animal plasma membrane area (on average 30%), demonstrating the existence of an animal-vegetal polarity in plasma membrane properties. At third cleavage, the differences between animal and vegetal plasma membrane region become even more pronounced; in the four animal micromeres the diffusion coefficient (D) and mobile fraction (MF) are 2.9 +/- 0.2 X 10(-9) cm2/sec and 51 +/- 2%, respectively, while in the four vegetal macromeres D = 5.0 +/- 0.3 X 10(-9) cm2/sec and MF = 78 +/- 2%. Superimposed upon the observed animal-vegetal polarity, the lateral diffusion in the polar lobe membrane area shows a cell-cycle-dependent modulation. The highest mean values for D are reached during the S phase (ranging from 7.0 to 7.8 X 10(-9) cm2/sec in the three cycles measured), while at the end of G2 phase and during early mitosis mean values for D have decreased significantly (ranging from 5.0 to 5.9 X 10(-9) cm2/sec). Diffusion rates in the animal membranes of the embryo are constant during the three successive cell cycles (D = 4.3-5.0 X 10(-9) cm2/sec), except for a peak at the S phase of the first cell cycle (D = 6.0 X 10(-9) cm2/sec). These results are discussed in relation with previously observed ultrastructural heterogeneities in the Nassarius egg plasma membrane. It is speculated that the observed animal-vegetal polarity in the organization of the egg membrane might play an important role in the process of cell diversification during early development.     

Effects of histamine on vascular resistance and protein permeability in the isolated dually perfused guinea-pig placenta

The possibility that histamine can affect both the vascular resistance and permeability of the isolated dually perfused guinea-pig placenta has been investigated. Change from control to histamine (2.7 x 10(-4)M) perfusion of the fetal circulation elicited a significant (P less than 0.01, paired 't' test) maximum increase of 1.17 +/- 0.14 (SEM) kPa in fetal perfusion pressure 3 min later, representing a 33% rise. This vasoconstriction was completely blocked by the H1 antagonist diphenhydramine (10(-4)M) but not by the H2 receptor antagonist cimetidine (10(-4)M). In the same experiments the clearance (calculated as the ratio of fetal to maternal perfusate concentration times fetal flow-rate) of a macromolecular tracer, anionic horseradish peroxidase from the maternal to fetal circulation was significantly increased (P less than 0.05, paired 't' test) when steady state (15-20 min of perfusion) values were compared, from 5.9 +/- 1.7 (SEM) microliter min-1 placenta-1 to 12.9 +/- 3.5 (SEM) microliter min-1 placenta-1 (n = 20) for control and histamine respectively. By contrast the steady state clearance (calculated as before) of a smaller hydrophilic tracer, 51Cr-EDTA, was not significantly affected, being 587 +/- 59 (SEM) microliter min-1 placenta-1 in control and 587 +/- 55 (SEM) microliter min-1 placenta-1 (n = 20) with histamine perfusion. When histamine was perfused simultaneously with an H1 or H2 antagonist there was no change in anionic horseradish peroxidase clearance. Electron microscopy of placentas perfused with histamine failed to reveal any obvious alteration in morphology or anionic horseradish peroxidase localisation as compared to placenta perfused without histamine. This study thus demonstrates that histamine may cause changes in the macromolecular permeability of the placenta as well as vasoconstriction of the placental vasculature.     

Molecular determinants of inactivation within the I-II linker of alpha1E (CaV2.3) calcium channels

Voltage-dependent inactivation of CaV2.3 channels was investigated using point mutations in the beta-subunit-binding site (AID) of the I-II linker. The quintuple mutant alpha1E N381K + R384L + A385D + D388T + K389Q (NRADK-KLDTQ) inactivated like the wild-type alpha1E. In contrast, mutations of alpha1E at position R378 (position 5 of AID) into negatively charged residues Glu (E) or Asp (D) significantly slowed inactivation kinetics and shifted the voltage dependence of inactivation to more positive voltages. When co-injected with beta3, R378E inactivated with tau(inact) = 538 +/- 54 ms (n = 14) as compared with 74 +/- 4 ms (n = 21) for alpha1E (p < 0.001) with a mid-potential of inactivation E(0.5) = -44 +/- 2 mV (n = 10) for R378E as compared with E(0.5) = -64 +/- 3 mV (n = 9) for alpha1E. A series of mutations at position R378 suggest that positively charged residues could promote voltage-dependent inactivation. R378K behaved like the wild-type alpha1E whereas R378Q displayed intermediate inactivation kinetics. The reverse mutation E462R in the L-type alpha1C (CaV1.2) produced channels with inactivation properties comparable to alpha1E R378E. Hence, position 5 of the AID motif in the I-II linker could play a significant role in the inactivation of Ca(V)1.2 and CaV2.3 channels.     

Diffusion and consumption of oxygen in the resting frog sartorius muscle

Adaptations of the method of Takahashi et al. (1966. J. Gen. Physiol. 50:317-333) were used to test the validity of the one-dimensional diffusion equation for O2 in the resting excised frog sartorius muscle. This equation is: (formula: see text) where x is the distance perpendicular to the muscle surface. t is time, P(x, t) is the partial pressure of O2,D and alpha are the diffusion coefficient and solubility for O2 in the tissue, and Q is the rate of O2 consumption. P(O, t), the time-course of PO2 at one muscle surface, was measured by a micro-oxygen electrode. Transients in the PO2 profile of the muscle were induced by two methods: (a) after an equilibration period, one surface was sealed off by a disc in which the O2 electrode was embedded; (b) when PO2 at this surface reached a steady state, a step change was made in the PO2 at the other surface. With either method, the agreement between the measured P(O, t) and that predicted by the diffusion equation was excellent, making possible the calculation of D and Q. These two methods yielded statistically indistinguishable results, with the following pooled means (+/- SEM): (formula: see text) At each temperature, D was independent of muscle thickness (range, 0.67-1.34 mm). The activation energy (EA) for diffusion of oxygen in muscle was -3.85 kcal/mol, which closely matches the corresponding value in water. Together with absolute values of D in water taken from the literature, the present data imply that (Dmuscle/DH2O) is in the range 0.59-0.69. This value, and that of EA, are in agreement with the theory of Wang (1954, J. Am. Chem. Soc. 76:4755-4763), suggesting that with respects to the diffusion of O2, to a useful approximation, frog skeletal muscle may be considered simply as a homogeneous protein solution.     

Low oxygen consumption in the inner retina of the visual streak of the rabbit

The oxygen requirements of different retinal layers are of interest in understanding the vulnerability of the retina to hypoxic damage in retinal diseases with an ischemic component. Here, we report the first measurements of retinal oxygen consumption in the visual streak of the rabbit retina, the region with the highest density of retinal neurons, and compare it with that in the less-specialized region of the retina underlying the vascularized portion of the rabbit retina. Oxygen-sensitive microelectrodes were used to measure oxygen tension as a function of retinal depth in anesthetized animals. Measurements were performed in the region of the retina containing overlying retinal vessels and in the center of the visual streak. Established mathematical analyses of the intraretinal oxygen distribution were used to quantify the rate of oxygen consumption in the inner and outer retina and the relative oxygen contributions from the choroidal and vitreal sides. Outer retinal oxygen consumption was higher in the visual streak than in the vascularized area (means +/- SE, 284 +/- 20 vs. 210 +/- 23 nl O2.min(-1) x cm(-2), P = 0.026, n = 10). However, inner retinal oxygen consumption in the visual streak was significantly lower than in the vascular area (57 +/- 4.3 vs. 146 +/- 12 nl O2 x min(-1) x cm(-2), P < 0.001). We conclude that despite the higher processing requirements of the inner retina in the visual streak, it has a significantly lower oxygen consumption rate than the inner retina underlying the retinal vasculature. This suggests that the oxygen uptake of the inner retina is regulated to a large degree by the available oxygen supply rather than the processing requirements of the inner retina alone.     

Basic properties and potential regulators of the apical K+ channel in macula densa cells

These studies examine the properties of an apical potassium (K+) channel in macula densa cells, a specialized group of cells involved in tubuloglomerular feedback signal transmission. To this end, individual glomeruli with thick ascending limbs (TAL) and macula densa cells were dissected from rabbit kidney and the TAL covering macula densa cells was removed. Using patch clamp techniques, we found a high density (up to 54 channels per patch) of K+ channels in the apical membrane of macula densa cells. An inward conductance of 41.1 +/- 4.8 pS was obtained in cell-attached patches (patch pipette, 140 mM K+). In inside- out patches (patch pipette, 140 mM; bath, 5 mM K+), inward currents of 1.1 +/- 0.1 pA (n = 11) were observed at 0 mV and single channel current reversed at a pipette potential of -84 mV giving a permeability ratio (PK/PNa) of over 100. In cell-attached patches, mean channel open probability (N,Po, where N is number of channels in the patch and Po is single channel open probability) was unaffected by bumetanide, but was reduced from 11.3 +/- 2.7 to 1.6 +/- 1.3 (n = 5, p < 0.02) by removal of bath sodium (Na+). Simultaneous removal of bath Na+ and calcium (Ca2+) prevented the Na(+)-induced decrease in N.Po indicating that the effect of Na+ removal on N.Po was probably mediated by stimulation of Ca2+ entry. This interpretation was supported by studies where ionomycin, which directly increases intracellular Ca2+, produced a fall in N.Po from 17.8 +/- 4.0 to 5.9 +/- 4.1 (n = 7, p < 0.02). In inside- out patches, the apical K+ channel was not sensitive to ATP but was directly blocked by 2 mM Ca2+ and by lowering bath pH from 7.4 to 6.8. These studies constitute the first single channel observations on macula densa cells and establish some of the characteristics and regulators of this apical K+ channel. This channel is likely to be involved in macula densa transepithelial Cl- transport and perhaps in the tubuloglomerular feedback signaling process.     

Oxygen distribution and consumption in the cat retina during normoxia and hypoxemia

Oxygen tension (PO2) was measured with microelectrodes within the retina of anesthetized cats during normoxia and hypoxemia (i.e., systemic hypoxia), and photoreceptor oxygen consumption was determined by fitting PO2 measurements to a model of steady-state oxygen diffusion and consumption. Choroidal PO2 fell linearly during hypoxemia, about 0.64 mmHg/mmHg decrease in arterial PO2 (PaO2). The choroidal circulation provided approximately 91% of the photoreceptors' oxygen supply under dark-adapted conditions during both normoxia and hypoxemia. In light adaptation the choroid supplied all of the oxygen during normoxia, but at PaO2's less than 60 mmHg the retinal circulation supplied approximately 10% of the oxygen. In the dark-adapted retina the decrease in choroidal PO2 caused a large decrease in photoreceptor oxygen consumption, from approximately 5.1 ml O2/100 g.min during normoxia to 2.6 ml O2/100 g.min at a PaO2 of 50 mmHg. When the retina was adapted to a rod saturating background, normoxic oxygen consumption was approximately 33% of the dark-adapted value, and hypoxemia caused almost no change in oxygen consumption. This difference in metabolic effects of hypoxemia in light and dark explains why the standing potential of the eye and retinal extracellular potassium concentration were previously found to be more affected by hypoxemia in darkness. Frequency histograms of intraretinal PO2 were used to characterize the oxygenation of the vascularized inner half of the retina, where the oxygen distribution is heterogeneous and simple diffusion models cannot be used. Inner retinal PO2 during normoxia was relatively low: 18 +/- 12 mmHg (mean and SD; n = 8,328 values from 36 profiles) in dark adaptation, and significantly lower, 13 +/- 6 mmHg (n = 4,349 values from 19 profiles) in light adaptation. Even in the dark-adapted retina, 30% of the values were less than 10 mmHg. The mean PO2 in the inner (i.e., proximal) half of the retina was well regulated during hypoxemia. In dark adaptation it was significantly reduced only at PaO2's less than 45 mmHg, and it was reduced less at these PaO2's in light adaptation.     

Oxygen diffusion in biological and artificial membranes determined by the fluorochrome pyrene

Quenching of pyrene fluorescence by oxygen was used to determine oxygen diffusion coefficients in phospholipid dispersions and erythrocyte plasma membranes. The fluorescence intensity and lifetime of pyrene in both artificial and natural membranes decreases about 80% in the presence of 1 atm O2, while the fluorescence excitation and emission spectra and the absorption spectrum are unaltered. Assuming the oxygen partition coefficient between membrane and aqueous phase to be 4.4, the diffusion coefficients for oxygen at 37 degrees C are 1.51 X 10(-5) cm2/s in dimyristoyl lecithin vesicles, 9.32 X 10(-6) cm2/s in dipalmitoyl lecithin vesicles, and 7.27 X 10(-6) cm2/s in erythrocyte plasma membranes. The heats of activation for oxygen diffusion are low (less than 3 kcal/degree-mol). A dramatic increase in the diffusion constant occurs at the phase transition of dimyristoyl and dipalmitoyl lecithin, which may result from an increase in either the oxygen diffusion coefficient, partition coefficient, or both. The significance of the change in oxygen diffusion below and above the phase transition for biological membranes is discussed.     

Oxymyohemerythrin: discriminating between O2 release and autoxidation

Myohemerythrin (Mhr) is a non-heme iron O2 carrier (with two irons in the active site) that is typically found in the retractor muscle of marine 'peanut' worms. OxyMhr may either release O2, or undergo an autoxidation reaction in which hydrogen peroxide is released and diferric metMhr is produced. The autoxidation reaction can also be promoted by the addition of certain anions to Mhr solutions. This work, using recombinant Themiste zostericola Mhrs, contrasts the results of environmental effects on these reactions. For the O2 release reaction, deltaVdouble dagger(21.5 degrees C) = +28+/-3 cm3 mol(-1), deltaHdouble dagger(1 atm) = +22+/-1 kcal mol(-1), and deltaSdouble dagger(1 atm) = +28+/-4 eu. The autoxidation reaction (pH 8.0, 21.5 degrees C, 1 atm) displays different kinetic parameters: deltaVdouble dagger = -8+/-2 cm3 mol(-1), deltaHdouble dagger = +24.1+/-0.7 kcal mol(-1), and deltaSdouble dagger = +1+/-1 eu. Autoxidation in the presence of sodium azide is orders of magnitude faster than solvolytic autoxidation. The deltaVdouble dagger parameters for azide anation and azide-assisted autoxidation reaction are +15+/-2 and +59+/-2 cm3 mol(-1), respectively, indicating that the rate-limiting steps for the Mhr autoxidation and anation reactions (including O2 uptake) are not associated with ligand binding to the Fe2 center. The L103V and L103N oxyMhr mutants autoxidize approximately 10(3)-10(5) times faster than the wild-type protein, emphasizing the importance of leucine-103, which may function as a protein 'gate' in stabilizing bound dioxygen.     

Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching

The diffusion coefficient of tubulin has been measured in the cytoplasm of eggs and embryos of the sea urchin Lytechinus variegatus. We have used brain tubulin, conjugated to dichlorotriazinyl-aminofluorescein, to inject eggs and embryos. The resulting distributions of fluorescence were perturbed by bleaching with a microbeam of light from the 488-nm line of an argon ion laser. Fluorescence redistribution after photobleaching was monitored with a sensitive video camera and photography of the television-generated image. With standard photometric methods, we have calibrated this recording system and measured the rates of fluorescence redistribution for tubulin, conjugated to dichlorotriazinyl-aminofluorescein, not incorporated into the mitotic spindle. The diffusion coefficient (D) was calculated from these data using Fick's second law of diffusion and a digital method for analysis of the photometric curves. We have tested our method by determining D for bovine serum albumin (BSA) under conditions where the value is already known and by measuring D for fluorescein-labeled BSA in sea urchin eggs with a standard apparatus for monitoring fluorescence redistribution after photobleaching. The values agree to within experimental error. Dcytoplasmtubulin = 5.9 +/- 2.2 X 10(-8) cm2/s; DcytoplasmBSA = 8.6 +/- 2.0 X 10(-8) cm2/s. Because DH2OBSA = 68 X 10(-8) cm2/s, these data suggest that the viscosity of sea urchin cytoplasm for protein is about eight times that of water and that most of the tubulin of the sea urchin cytoplasm exists as a dimer or small oligomer, which is unbound to structures that would impede its diffusion. Values and limitations of our method are discussed, and we draw attention to both the variations in D for single proteins in different cells and the importance of D for the upper limit to the rates of polymerization reactions.