Space Marker Research Articles

Effects of dextran on hippocampal brain slice water, extracellular space, calcium kinetics and histology

Hippocampal brain slices are valuable models for studying brain function but are compromised by several artifacts, including significant water gain and histologic injury, which occur under certain incubation conditions. Addition of colloid to Krebs-Ringer buffer (K-R) has been shown to eliminate water gain but has not achieved widespread acceptance. We confirm prior observations that dextran and PEG lessen the increase in slice mass during incubation in a dose-dependent manner with no water gain occurring at 4% concentrations. However, we also observe that addition of colloid to standard K-R induces severe neuronal pyknosis. Fortunately, the pyknosis can be eliminated by reduction in buffer osmolarity through adjustment of NaCl, producing markedly improved slice histology in dextran buffer, especially in the CA3 and CA4 regions of the hippocampus which are severely injured when incubated submerged in K-R at 37°C. Extracellular space markers are not affected by either colloid. The volume of distribution for 45Ca is much larger in dextran buffers than in K-R and variability of 45Ca kinetics is also reduced. In the presence of dextran, hypoxia induces significant slice water gain, a relatively selective histologic injury and an alteration of tissue Ca 2+ kinetics. Use of dextran buffers may eliminate many troubling brain slice artifacts.

Simultaneous measurement of 45Ca outflow and human placental lactogen release from placental explants

Kinetics of 45Ca outflow and human placental lactogen (hPL) release were characterized in human placental explants. Measurements of the rate of 45Ca and [ 3H]-sucrose (extracellular space marker) outflow from preloaded explants showed that, after a 40 min washout period, the 45Ca effluent radioactivity presumably originated from an intracellular compartment. This view was further supported by the La 3+ and temperature sensitivity of the 45Ca outflow. Moreover, the addition of ionomycin as well as an increase in the extracellular Ca 2+ or Ba 2+ concentration provoked a dose-dependent rise in both the 45Ca outflow and hPL release. There were no systematic temporal analogies between the pattern of 45Ca outflow and hPL release. Taken together, these observations suggest that the stimulation of 45Ca outflow reflects an increased rate of 40Ca entry. The present data also extend previous observations indicating that hPL release can be stimulated by Ca 2+ entry. Lastly, the ‘in vitro’ method described herein allows one to compare rapid changes in hPL release with associated ionic events and can be used to further document the relationships between placental secretory responses and cell calcium metabolism.

Intracellular volume measurement and detection of edema: multinuclear NMR studies of intact rat hearts during normothermic ischemia.

The present study describes the cell volume dynamics in intact rat hearts, during ischemia and after reperfusion. Cell volumes were measured in isolated hearts by either 13C or 59Co NMR of mannitol or cobalticyanide, respectively, as extracellular markers and 1H NMR of water as the aqueous space marker. A constant volume chamber was built inside a 15-mm NMR tube; the contents of the chamber were measured with and without a heart. The intracellular volume of isolated rat hearts was estimated to be 2.45 +/- 0.13 ml/g dry weight. In the perfused heart, adenosine triphosphate (ATP) and phosphocreatine (PCr) concentrations were calculated to be 12.2 +/- 0.7 and 16.1 +/- 1.0 mM, respectively. Consecutive volume measurements showed cell swelling of 16% during 30 min of ischemia, which was reduced at reperfusion to 7%. After 30 min of reperfusion, ATP and PCr concentrations were 4.5 +/- 0.8 and 8.1 +/- 0.9 mM. It is concluded that: (1) cell swelling is an ischemic event, which is partially reversed by reperfusion; and (2) continuous measurement of cell volumes provides intracellular molar concentrations of metabolites, which are the physiologically significant parameters.

Basolateral transport of taurine in epithelial cells of isolated, perfused Mytilus californianus gills.

We found that the basolateral surface of the gill epithelium of the marine mussel Mytilus californianus possesses a carrier-mediated process capable of concentrating taurine within epithelial cells. We used retrograde perfusion of gill sections to demonstrate the kinetics, specificity and ion-dependence of taurine transport. [3H]taurine was concentrated relative to a space marker ([14C]mannitol); this accumulation was blocked by the inclusion of 10 mmol l-1 unlabeled taurine in the perfusate. The drop in [3H]taurine uptake at increasing concentrations of unlabeled taurine was fitted to Michaelis-Menten kinetics and indicated a basolateral process with a taurine concentration at which transport is half-maximal (Kt) of 35.3 mumol l-1 and a maximal flux (Jmax) of 0.35 mumol g-1 wet mass h-1. Taurine accumulation on the apical surface had a higher affinity (Kt = 9.5 mumol l-1) and a higher maximum rate of transport (Jmax = 1.23 mumol g-1 h-1). Basolateral transport was inhibited by inclusion in the perfusate of 1 mmol l-1 of another beta-amino acid (beta-alanine), but not by inclusion of alpha-alanine, glutamic acid or betaine. The dependence of basolateral taurine transport on Na+ (when replaced with N-methyl-D-glucamine) was sigmoidal with an apparent Hill coefficient of 2.3, indicating that more than one Na+ is necessary for the transport of each taurine molecule. Complete substitution of Cl- in bathing media reduced taurine accumulation by 90% and 70% on the apical and basolateral surfaces, respectively. Taurine accumulation on both surfaces was reduced by only 20% when Cl- was reduced from 496 to 73 mmol l-1, suggesting that taurine uptake is not significantly influenced by the changes in Cl- concentration accompanying the salinity fluctuations normally encountered by mussels. We estimate that the various Na+ and Cl- gradients naturally encountered by epithelial cells are capable of providing ample energy to maintain a high intracellular concentration of taurine. We suggest that the ability of epithelial cells to accumulate taurine across the basolateral surface from the hemolymph plays a significant role in the intracellular regulation of this important osmolyte and may effect osmolality-dependent changes in the intracellular concentration of taurine.

Blood-to-lens Transport of Reduced Glutathione in an In Situ Perfused Guinea-pig Eye

Transport mechanisms of reduced glutathione (GSH) in intact eye are poorly understood. In this study, an in situ vascular eye perfusion (VEP) model was used to characterize the transport kinetics of circulating GSH into the aqueous humor and lens in guinea pigs. Radiolabeled [ 35S]GSH or [ 3H]GSH and [ 14C]sucrose (an extracellular space marker) were exposed to the blood-aqueous barrier up to 10 min, and uptake of tracers by the aqueous humor and lens was determined in the presence of different concentrations of unlabeled peptide as GSH, a gamma-glutamyl compound as a derivative of GSH (GSH monoethyl ester), and an inhibitor of gamma-glutamyl transpeptidase (GGT) activity. Plasma-aqueous and aqueous-lens compartmental unidirectional transport constant, K in, and the initial rapid volume of distribution. V i, were estimated by multiple-time-point graphic analysis. Our results indicated that both labeled GSH and sucrose entered the aqueous humor slowly at comparable rates with respective blood-aqueous K in values of 1·34 ± 0·12 and 1·25 ± 0·08 min -1 × 10 3. In contrast to blood-aqueous transport, GSH uptake by the lens was rapid, and the respective aqueous-lens K in values for labeled GSH and sucrose were 79·3 ±4·1 and 3·5 ± 0·7 min -1 × 10 3. Over 94% of plasma-derived GSH remained in its original molecular form of GSH in the lens, during the 10 min perfusion both with and without the GGT inhibitor, serine borate. The amount of [ 35S]GSH in lens anterior epithelium (dpm mg -1) was more than three times that of aqueous humor (dpm μl -1) within only 10 min of VEP. There was also significant accumulation of [ 35S]GSH in the interior cortex, as indicated by 10-min cortex/aqueous ratio of 0·65. A specific GGT independent GSH transport system was demonstrated in the lens in situ, with a K m of 26 ± 3 μM, and V max of 34 ± 3 pmol min -1 g -1 of whole lens tissue water. The lenticular influx of GSH was inhibited by GSH monoethyl ester with an affinity that was half that for GSH. It is concluded that: (a) uptake of plasma-derived GSH into the aqueous humor is by simple diffusion, and (b) cellular uptake of GSH by the lens is carrier-mediated via mechanism that is separate from the transpeptidation metabolic pathway.

Intracellular and extracellular spaces and the direct quantification of molar intracellular concentrations of phosphorus metabolites in the isolated rat heart using 31P NMR spectroscopy and phosphonate markers

To quantify metabolite and cation concentrations using NMR spectroscopy, the volumes of intracellular and extracellular spaces must be known. We describe a simple 31P NMR spectroscopic method that employs dimethyl methylphosphonate (DMMP) as a marker of total water space and phenylphosphonate (PPA) as a marker of extracellular space to determine intracellular and extracellular space volumes in the isolated, perfused rat heart. In vivo and in vitro radiolabel studies were used to verify this method. The difference between the total and extracellular water spaces, determined as milliliters/heart, gave the intracellular volume and allowed direct calculation of myocardial creatine phosphate, ATP, and inorganic phosphate concentrations, which were 13.4 mM, 10.1 mM, and 3.4 mM, respectively, for the glucose-perfused rat heart. The extracellular volume decreased by 84% in hearts subjected to 28 min total, global ischemia and increased by 15% during reperfusion. The method described allows the determination of intracellular energy metabolite concentrations in perfused rat heart directly from a single, fully relaxed 31P NMR spectrum.

Light-dependent hydration of the space surrounding photoreceptors in the cat retina.

We have studied the effect of retinal illumination on the concentration of the extracellular space marker tetramethylammonium (TMA+) in the dark-adapted cat retina using double-barreled ion-selective microelectrodes. The retina was loaded with TMA+ by a single intravitreal injection. Retinal illumination produced a slow decrease in [TMA+]o, which was maximal in amplitude in the most distal portion of the space surrounding photoreceptors, the subretinal space. The light-evoked decrease in [TMA+]o was considerably slower and of a different overall time course than the light-evoked decrease in [K+]o, also recorded in the subretinal space. [TMA+]o decreased to a peak at 38 s after the onset of illumination, then slowly recovered towards the baseline, and transiently increased following the offset of illumination. It resembled the light-evoked [TMA+]o decreases previously recorded in the in vitro preparations of frog (Huang & Karwoski, 1990, 1992) and chick (Li et al., 1992, 1994) but was considerably larger in amplitude, 22% compared with 7%. As in frog, where it was first recorded, the light-evoked [TMA+]o decrease is considered to originate from a light-evoked increase in the volume of the subretinal space (or subretinal hydration). A mathematical model accounting for [TMA+]o diffusion predicted that the volume increase underlying the response was 63% on average and could be as large as 95% and last for minutes. The estimated volume increase was then used to examine its effect on K+ concentration in the subretinal space. We conclude that a light-dependent hydration of the subretinal space represents a significant physiological event in the intact cat eye, which should affect the organization of the interphotoreceptor matrix, and the concentrations of all ions and metabolites located in the subretinal space.

31P Magnetic Resonance Spectroscopy Demonstrates Expansion of the Extracellular Space in the Skeletal Muscle of Starved Rats

Starvation significantly alters the distribution of body water. To study the effects of starvation on cellular energetics and water distribution in skeletal muscle, a novel 31P magnetic resonance technique (31P MRS) was developed to measure water compartments. After 31P MRS-visible water space markers which distribute in total body water (dimethyl methylphosphonate, DMMP) and extracellular water (phenylphosphonate, PPA) were infused intravenously, 31P MRS spectra were obtained from the gastrocnemius muscle of male virus-free Wistar rats at baseline and after starvation or ad libitum feeding for 4 days. Muscle water spaces were also measured using the chloride method and Nernst's equation. Muscle water contents as determined by drying were equivalent in the two groups. In vivo measurements of changes in DMMP relative to all of the MRS visible phosphates also demonstrated that the total water space was similar in control and starved rats. However, starvation significantly increased the ratio of PPA/DMMP (0.67 ± 0.05 vs 0.87 ± 0.04, Control vs Starvation; P < 0.001), and therefore the ratio of extracellular water to total water in the gastrocnemius. Furthermore, because muscle water contents were comparable between the groups, this expansion of the extracellular space was accompanied by contraction of the intracellular compartment in starved animals. Equivalent changes were detected in vitro using the chloride method. Lastly, phosphocreatine/ATP ratios, which measured changes in high-energy phosphate stores, decreased after starvation (4.09 ± 0.06 vs 3.61 ± 0.06; P < 0.001) and were inversely related to changes in PPA/DMMP (r = -0.61; P < 0.001).We conclude that starvation alters the distribution of water within skeletal muscle and these changes are related to the depletion of energy stores. These phenomena can be studied simultaneously in a noninvasive fashion using in vivo31P MRS and MRS-visible water space markers.

Membrane Properties of Antiviral Phospholipids Containing Heteroatoms in the Acyl Chains

Phospholipids containing heteroatoms in the lipid acyl chains, e.g., 1,2-bis(12-methoxydodecanoyl)-sn-3-phosphocholine (L-AC2), exhibit potent anti-HIV activity [Pidgeon, C., Markovich, R. J., Liu, M. D., Holzer, T., Novak, R., & Keyer, K. (1993) J. Biol. Chem. 268, 7773-7778]. AC2 is a synthetic chemical analog of the long-chain phospholipid, dimyristoylphosphatidylcholine (DMPC). Sonicated AC2 lipid dispersions would not entrap either Dextran-4000 or Mn2+ used as aqueous space markers. The lack of entrapment of aqueous space markers indicates that the AC2 structures do not contain an aqueous core that is the characteristic morphology of conventional lipid vesicles formed by sonication. Transmission electron microscopy (TEM) showed that sonicated AC2 lipid dispersions are small homogeneous particles approximately 70-100 A in diameter. 1H NMR experiments using Mn2+ as a broadening reagent indicated that Mn2+ was accessible to all of the AC2 phospholipid headgroups in the AC2 lipid particles formed by sonication. The temperature dependence of 1H spin-lattice (T1) relaxation time measurements revealed that the motional activation energies increased from the choline headgroup to the end of the acyl chains of AC2 molecules in the AC2 lipid particles formed by sonication. Collectively these results demonstrate that AC2 forms micelles. NOESY experiments showed that the AC2 molecules forming the micelle structures have hindered motion compared to conventional short-chain phosphatidylcholine micelles. 31P NMR spectroscopy and TEM showed that the AC2 micelles extensively fuse into giant bilayer liposomes (single-layered) when the temperature is reduced from above to below the main phase transition temperature of AC2. This micelle-to-liposome transition is an irreversible process; increasing the temperature above the Tm does not cause the formation of micelles. Thus, a main finding is that AC2 micelles formed by sonication are not thermodynamically stable because they fuse into large unilamellar vesicles that are stable to further changes in temperature. These unusual membrane properties of sonicated AC2 dispersions may be important for the antiviral activity and metabolism of the phospholipids.

Extracellular fluid translocation in perfused rabbit atria: implication in control of atrial natriuretic peptide secretion.

1. Transmural transport of 22Na+, 51Cr-EDTA, [3H]inulin and [14C]Dextran (57 kDa) was measured in perfused rabbit atria. The radiolabelled extracellular space (ECS) markers and [14C]Dextran were introduced into the pericardial space or atrial lumen. Atrial volume changes were induced by steps up and down in atrial pressure. 2. Basal rates of transmural transport of radiolabelled ECS markers across the atrial wall were relatively stable up to 70 min. Atrial stretch and release resulted in a rapid but transient, and reversible increase in the ECS fluid (ECF) translocation. The increased translocation of the ECF into the atrial lumen occurred within 15 s of the reduction of atrial distension and returned to the baseline level within 60 s. 3. Transmural transport of [3H]inulin across the atrial wall was bidirectional. 4. The clearance of radiolabelled ECS markers was molecular-size dependent. The transmural clearance of [3H]inulin was dependent on the distension-reduction volume changes induced by atrial stretch and release. Little transport of [14C]Dextran across the atrial wall was observed. 5. The ECF translocation across the atrial wall was not influenced by changes in external Ca2+ but was suppressed by low temperature. 6. Dynamic changes in the ECS of the atrium were observed in response to atrial distension and reduction. The ECS of the atrium increased on distension and decreased on reduction of atrial distension. 7. Reduction in atrial distension resulted in an increase in the secretion of immunoreactive atrial natriuretic peptide (ANP) which coincided with an increase in the translocation of the ECF. The secretion of immunoreactive ANP was a function of the translocation of the ECF. 8. It is suggested that atrial stretch and release may play a role in driving fluid flow within the interstitium and fluid translocation out of the interstitium. This fluid movement presumably leads to convective transport of released ANP into the atrial lumen.

Single-pass gill extraction and tissue distribution of atrial natriuretic peptide in trout.

A new method is described in the present experiments to quantify atrial natriuretic peptide (125I-ANP) extraction from plasma in a single transit through the gill vasculature of an unanesthetized trout. Tissue distribution of 125I relative to an inert extracellular space marker, 58Co-EDTA, was also measured 1 h after injection of 125I-ANP. Single-pass extraction of 125I-ANP by the gill was 60% in control fish; it fell to 18% in fish previously injected with saline and became negative (relative to 58Co-EDTA) after treatment with the clearance (C-type) receptor inhibitor SC 46542. Approximately 90% of a 125I-ANP bolus injected into control trout is removed from the circulation within 2 min. 125I concentration in gill tissue was nearly seven times greater than that predicted by the 58Co-EDTA space. 125I was also concentrated in brain (2X), gallbladder, gastrointestinal tract, and eye (all 1X). SC 46542 decreased gill 125I binding from seven times to one times the 58Co-EDTA space and increased 125I accumulation in heart, kidney, fat, and skeletal muscle. Extraction of 125I-ANP by the isolated perfused gill was 53%, similar to that observed in vivo. These results show that the gill is a major site for ANP removal from trout plasma and that C-type receptors are predominant in this process.

Characterization of water distribution in cell pellets using nonlabeled sodium thiosulfate as an interstitial space marker.

A procedure for determination of the intracellular water content of cells using a single, nonlabeled solute as an interstitial space marker is proposed. Sodium thiosulfate, which can be accurately assayed by a tritrimetric method, is found to be a good compound for this purpose. Cells are recovered both by filtration and centrifugation; the two techniques gave the same value for internal water, i.e., 650 mg of H2O/g of wet matter for Corynebacterium melassecola and 390 mg of H2O/g of wet matter for Penicillium roquefortii spores. The methodology of data handling, based on a regression technique, is also described. It allows one to obtain very reliable results and should be useful for any marker.

Paracellular permeability pathways in the human placenta: A quantitative and morphological study of maternal-fetal transfer of horseradish peroxidase

Physiological data indicate that both a transcellular and a paracellular pathway are available for transfer across the human placenta but the morphological correlate of the latter is uncertain. We measured the permeability of the dually perfused human placental cotyledon to the predominantly cationic protein horseradish peroxidase (MW 40,000), to the neutral polymer 14C-dextran (MW 50-70,000) and to the extracellular space marker creatinine (MW 113). Following fixation and cytochemistry, we used brightfield microscopy to localize peroxidase reaction product within the same tissue. Steady state unidirectional maternofetal clearance (Kmf) for the peroxidase (0.90 +/- 0.27 microliters/min/g, n = 9) was not significantly different from that for 14C-dextran (0.95 +/- 0.07 microliters/min/g, n = 3) suggesting that charge does not markedly influence peroxidase permeability. The Kmf for creatinine was 13.1 +/- 2.5 microliters/min/g (n = 9); these permeability data are similar to those reported for the placenta in vivo. Microscopically, peroxidase reaction product was localized to the microvillous surface of the syncytiotrophoblast of most villi and to the trophoblastic basement membrane and connective tissue cells of the villous core in a few villi. The reaction product was also associated with fibrin-containing deposits attached to the villous core at sites of discontinuity of the syncytial epithelium. The staining pattern within the deposits was consistent with a diffusion gradient of the peroxidase. These fibrin-containing deposits at discontinuities in the syncytiotrophoblast may provide one paracellular route for peroxidase diffusion from the intervillous space into the villous core.

Transport, uptake, and metabolism of blood-borne vasopressin by the blood-brain barrier

Transport, binding, and metabolism of [phenylalanyl-3,4,5-3H(N)]arginine vasopressin (AVP) by the blood-brain barrier (BBB) was studied in adult guinea-pigs by means of a novel vascular brain perfusion (VBP)/capillary depletion technique and HPLC. A time-dependent, progressive brain uptake of 3H-radioactivity was measured over the 10 min period of VBP both in brain homogenates and in brain tissue depleted of cerebral microvessels. The unidirectional blood-to-brain transport constant, KIN, estimated by multiple-time tissue uptake analysis of the homogenate and postcapillary supernatant, indicated that the BBB transfer rat ffor [3H]AVP (KIN = 2.37±0.25 μl min−1 per gram brain homogenate) was almost 10 times higher than for simultaneously perfused [14C]sucrose, a cerebrosvascular space marker. In contrast to homogenate and postcapillary supernatant, the [3H]radioactivity determined in the vascular pellet after dextran density centrifugation of the brain homogenate was very low and only somewhat higher than for [14C]sucrose. HPLC analysis of the perfused brain tissue revealed time-dependent degradation of the blood-borne neuropeptide. The percentage of intact [3H]AVP as determined in the postcapillary supernatant progressively declined during brain perfusion, from 49% at 1 min to 11.9% at 10 min. The major detectable labeled metabolite was [3H]phenylalanine, the labeled amino acid residue of [3H]AVP. The aminopeptidase inhibitor bestatin (0.5 mM), perfused simultaneously with [3H]AVP by the VBP technique, did not alter tissue uptake of [3H]AVP, indicating that there was no significant hydrolysis of peptide by the luminal BBB surface. The results suggest that rapid in vivo metabolism of AVP occurs after BBB transport in the brain parenchyma with no evidence of significant capillary sequestration, or degradation of AVP by the BBB.

Ion concentrations, fluxes and electrical properties of the embryonic chicken lens

The membrane properties of embryonic chicken lenses were characterized using isotopic and electrical techniques. The lenses had a relatively high water content (80%) and large extracellular space (12·5%). Isotopic uptake measurements indicated that the lens cytoplasm contained 118 m m K + and 26 m m Cl −. A value for intracellular Na + of 14 m m was obtained using Na +-sensitive microelectrodes. A doubleexponential model was used to fit the efflux of 86Rb +, 22Na +, 36Cl − and [ 3H]mannitol (an extracellular space marker) from the lens. When perfused with artificial aqueous humor (AAH) solution, embryonic lenses exhibited membrane potentials of between −20 and −40 mV. The more negative values were generally observed in lenses from older embryos. A ouabain-sensitive component, contributing −7 mV to the membrane potential, was also identified. The relatively depolarized membrane potentials suggested that the lens membranes were only weakly selective for K + over Na +. To test this further, lenses were perfused with AAH containing varying concentrations of K +. The resulting changes in potential were interpreted in terms of the Goldman model. The best fit of the Goldman potential equation indicated that, in the presence of ouabain, the chicken lens membranes had a relative permeability to K +, Na + and Cl − of 1·0, 0·36, 0·51 respectively. Replacing most or all of the Na + in the AAH caused only a small change in the membrane potential rather than the large hyperpolarization towards the K + equilibrium potential predicted by the Goldman model. Including the K + ionophore valinomycin in the low Na +-AAH solutions caused a large increase in 86Rb + efflux but did not result in additional hyperpolarization. This suggested that the insensitivity of the membrane potential to reduced extracellular Na + was not due to voltage or pH inactivation of lens K + channels.

An in situ perfused guinea-pig eye model for blood-ocular transport studies: Application to amino acids

A technique for in situ vascular eye perfusion (VEP) in the guinea-pig has been developed for measurements of the blood-ocular transport kinetics of substrates under controlled conditions of arterial inflow. The blood-aqueous and blood-vitreous barriers remained intact to the vascular space marker [ 3H]dextran (MW 70 kDa) with the perfusion pressure maintained between 80 and 100 mmHg. Several 3H-, 14C- or 35S-labeled amino acids, and 3H- or 14C-labeled sucrose (extracellular space marker) were used to validate the VEP model for transport kinetic studies. Multiple time-point graphic analysis was used to estimate the compartmental unidirectional blood-ocular transport constants, K in, within the 20 min period of the VEP experiment. Blood-to-aqueous humor K in values for [ 3H]serine, [ 14C] N-methyl-α-aminoisobutyric acid (MeAIB) and [ 3H] or [ 14C]sucrose were 3·57 ± 0·38, 1·21 ± 0·13 and 1·13 ± 0·17 μl min −1 g −1, respectively. The respective blood-to-lens K in values for labeled serine, MeAIB and sucrose were 1·71 ± 0·19, 0·09 ± 0·03 and 0·03 ± 0·002 μl min −1 g −1. The uptake of newly secreted amino acids from the aqueous humor in the lens followed the order: [ 35S]methionine > [ 3H]serine ≥ [ 35S]cysteine ≥ [ 3H]alanine > [ 14C]cycloleucine ≥ [ 14C]MeAIB ≥ [ 3H] or [ 14C]sucrose. The data indicate lack of a rapid blood-to-lens uptake of two model amino acids defining the A and L amino acid carriers. A significant lenticular uptake of blood-borne substrates for the ASC amino acid transport system and of methionine was demonstrated. The utility of the VEP model to study carrier-mediated component in overall unidirectional transfer across the blood-ocular barriers was tested with serine. A strong self-inhibition of [ 3H]serine uptake into the aqueous humor (82%) and lens (77%) was obtained by unlabeled amino acid at 4·7 m m. It is concluded that the VEP technique provides a stable well-controlled environment in the choriocapillary circulation of the perfused eye suitable for examining the in situ ocular transport kinetics of different substrates.

Morphology and function of capillary networks in subregions of the rat tuber cinereum.

The differentiated cytology, cytochemistry, and functions within subdivisions of the tuber cinereum prompted this morphometric and physiological investigation of capillaries in the medium eminence and arcuate nucleus of albino rats. Morphometric studies established that the external zone of the median eminence had 3-5 times the number and surface area of true and sinusoidal capillaries than the internal or subependymal median eminence zones, or either of two subdivisions examined in the arcuate nucleus. Type-I true capillaries, around which Virchow-Robin spaces comprise 1% of arcuate tissue area, were situated proximally to the median eminence border. This finding is consistent with a premise that confluent pericapillary spaces enable infiltration of arcuate neurons by factors from capillary blood from the median eminence or Virchow-Robin spaces. Physiologically, the rate of penetration across the median eminence capillaries by blood-borne [14C]alpha-amino-isobutyric acid (a neutral amino acid used as a capillary permeability tracer) was 142 times greater than for capillaries in the distal arcuate nucleus within 12 s of tracer administration. A new finding was that the proximal arcuate nucleus had a permeability x surface area product of 69 microliters g-1 min-1, 34 times greater than that in more distal aspects of the tuber where blood-brain barrier properties exist. We also found that the microcirculatory transit time of a plasma space marker, [14C]sucrose, was considerably longer (1.2 s) in the median eminence and proximal arcuate nucleus than in the distal arcuate or ventromedial nucleus (0.4 s). By virtue of its high capillary permeability and extensive blood-tissue surface area, including the wide Virchow-Robin spaces, the median eminence external zone could be a gateway for flooding other tuberal compartments with blood-borne factors. This effect may be compounded by capillary bed specializations in the proximal arcuate nucleus where Type-I true capillaries, Type-III sinusoids, and pericapillary spaces are confluent with those in the median eminence. The results indicate that the proximal arcuate parenchyma could be exposed to circulating neuroactive substances on a moment-to-moment basis.

Myocardial Na+ during Ischemia and Accumulation of Ca2+ after Reperfusion: A Study with Monesin and Dichlorobenzamil

The intracellular cation contents were determined in isolated perfused rat heart using cobaltic EDTA as a marker of the extracellular space. In hearts in which Na+ accumulation was induced with monensin, a Na+ ionophore, during 20 min-ischemia which otherwise did not result in accumulation of Na+, the levels of Na+ and Ca2+ were significantly higher after reperfusion with a significant decrease in K+. While the recovery of the cardiac mechanical function (CMF) was complete after reperfusion in control hearts, the recovery was incomplete in monensin-hearts. Dichlorobenzamil (DCB), the most specific inhibitor of Na+-Ca2+exchanger, infused for lOmin before induction of ischemia in a dose of 10−5 M, which produced a definite suppression of CMF (over 80%), inhibited the accumulation of Ca2+ and Na+ and the loss of K+ and ATP after 40 min-ischemia and reperfusion. The same dose of DCB given for 3 min before induction of ischemia and after reperfusion, which produced a less than 20% inhibition of CMF, failed to prevent the Ca2+ accumulation after 40 min-ischemia and reperfusion. These findings are at variance with the idea that the accumulation of Na+ during ischemia and the consequent augmented operation of Na+-Ca2+ exchange is responsible for accumulation of Ca2+ after reperfusion.

Artifacts of gendered space: American yard decoration

This article explores the prescribed decorations used to adorn the Anglo‐American home as contemporary visual forms of the historic paradigm that metaphorically equates female with nature and the domestic sphere in complementary opposition to male, culture, and the public domain. Yard ornaments are powerful multivocal symbols that reflect cultural beliefs about ideal gender and the relationship of gendered domains to the processes of biological and social reproduction. Because yard ornaments act as markers of the cultural space that mediates the public and private spheres, they are also fruitfully discussed as liminal objects that serve as forms of social commentary on Anglo‐American culture.

Regulation of T84 cell monolayer permeability by insulin-like growth factors.

When grown on permeable supports, the T84 human colonic epithelial cell line forms polarized monolayer cultures with high-resistance tight junctions between adjacent cells. Addition of either insulin-like growth factor (IGF) I or II to the basolateral but not the apical membrane side of established monolayers caused a dose-dependent decrease in transepithelial resistance over a 4-day period. IGF-I was more potent than IGF-II, with half-maximally effective concentrations of 0.7 and 2.2 nM, respectively. Both IGF-I and -II caused a parallel increase in the transepithelial flux rates for Na+ and the extracellular space marker, mannitol, demonstrating that the decrease in electrical resistance was due to increased permeability through the tight junction-regulated paracellular pathway. Simultaneous addition of cycloheximide prevented the decline in electrical resistance, implying that protein synthesis is necessary for the effect of IGF on paracellular permeability. Treatment of monolayers with IGF produced a subtle condensation of the perijunctional actin ring as visualized using rhodamine-labeled phalloidin. These results demonstrate that IGF-I and -II regulate the paracellular permeability of T84 cell monolayers through a receptor-mediated process that probably involves changes in protein synthesis and cytoskeletal structure.