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Cell swelling and volume regulation.

The extracellular space in the brain is typically 20% of the tissue volume and is reduced to at least half its size under conditions of neural insult. Whether there is a minimum size to the extracellular space was discussed. A general model for cell volume regulation was presented, followed by a discussion on how many of the generally involved mechanisms are identified in neural cells and (or) in astrocytes. There seems to be clear evidence suggesting that parallel K+ and Cl- channels mediate regulatory volume decrease in primary cultures of astrocytes, and a stretch-activated cation channel has been reported. The role of the different channels was discussed. A taurine leak pathway is clearly activated after cell swelling both in astrocytes and in neurones. The relations between the effect of glutamate and cell swelling were discussed. Discussion on the clearance of potassium from the extracellular space was continued. Neither the spatial buffering mechanism nor the role of the Na+/K+ pump was discussed here, but additional possibilities for K+ removal were discussed. At a high extracellular K+ concentration there is a possibility of channel-mediated KCl uptake by the glia cells, and a Na-K-2Cl cotransport system is present in astrocytes and seems to play a role at relatively low extracellular K+ concentrations (6-20 mM). The cotransport system is likely to be regulated, but little is known about its regulation in glia cells.

Animals↗

Sodium bromide by instrumental neutron activation analysis quantifies change in extracellular water space with wound closure in severely burned children.

BACKGROUND: The ability to measure extracellular water (ECW) in critically ill patients can significantly enhance current methods of assessing fluid homeostasis, body composition, and response to nutritional therapy. We measured corrected bromide space to determine change in ECW with wound closure among acutely burned children. METHODS: Fifteen children with burns over 30% of their total body surface area had their ECW determined following hemodynamic stabilization and when wound closure was complete. Plasma samples were obtained at baseline and 4 hours after receiving 25 mg/kg of sodium bromide. Plasma bromide was quantified by instrumental neutron activation analysis. RESULTS: Mean CBS decreased with wound closure (9.1 +/- 4.1 vs 7.9 +/- 3.9 liters; P =.04), indicating a significant decrease in ECW over the course of recovery. A decline in weight also occurred over the same period (32.4 +/- 15.2 vs 29.1 +/- 13.5 kg; P =.007); however, change in corrected bromide space as a proportion of weight was not statistically significant. CONCLUSION: A significant decrease in ECW accompanies the weight loss observed in patients following wound closure. Measurement of bromide dilution space is a convenient method for monitoring ECW that can be done at the bedside.

Acute Disease↗

Evidence for glial control of extracellular pH in the leech central nervous system.

Double-barrelled pH-sensitive microelectrodes were used to measure the intracellular pH (pHi) of neuropil glial cells and the pH of extracellular spaces (pH(e)) within isolated, intact ganglia of the leech Hirudo medicinalis. By application of a weak acid (propionate, 40 mM) or a weak base (ammonium, 20 mM) the total buffer capacity was estimated by changes in glial pHi and in pH(e). The buffering power of glial cells and in the extracellular spaces was increased by up to threefold in the presence of CO2/bicarbonate. The anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3-0.5 mM) reversed this increase in buffering power both in the glial cells and in the extracellular spaces. Inhibitors of the carbonic anhydrases reduced the CO2/bicarbonate-dependent increase in extracellular buffering power. The results suggest that extracellular H+ buffering dependent upon the availability of bicarbonate is linked to DIDS-sensitive bicarbonate transport across the glial membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

Diffusion around a cardiac calcium channel and the role of surface bound calcium.

The diffusion of Ca as it converges to the external mouth of a Ca channel is examined. Diffusional limitation on Ca ions entering Ca channels during current flow, cause local extracellular Ca depletions. Such extracellular Ca depletions have been reported in cardiac muscle. The cardiac sarcolemma has a large number of low-affinity Ca binding sites that can buffer these local Ca depletions. For a hemisphere of extracellular space (of radius less than 0.33 microns) centered on the external mouth of a Ca channel the amount of Ca bound at the membrane surface exceeds that which is free within the associated hemisphere. The ratio of bound Ca/free Ca increases as r decreases, such that the [Ca] nearest the Ca channel is the most strongly buffered by sarcolemmal bound Ca. It is demonstrated that Ca ions coming from these sarcolemmal Ca binding sites contribute quantitatively to the integrated Ca current. The electric field generated by the local depletion of Ca near the channel mouth has little impact on the extent of Ca depletion, but if an additional electric field exists at the mouth of the channel, Ca depletion can be significantly altered. Other low-affinity Ca binding sites in the interstitium may also contribute to the buffering of extracellular Ca. The complex geometry of the extracellular space in cardiac muscle (e.g., transverse tubules and restrictions of extracellular space between cells) increases both the predicted Ca depletions (in the absence of binding) and the bound/free ratio. Thus, the impact of this surface Ca binding is greatly increased. By considering arrays of Ca channels in transverse tubules or in parallel planes (e.g., membranes of neighboring cells), extracellular Ca depletions are predicted which agree with those measured experimentally. Membrane Ca binding may also be expected to buffer increases in [Ca] around the inner mouth of Ca channels. It is demonstrated that in the absence of other intracellular systems most of the Ca entering the cell via Ca channels might be expected to be bound to the inner sarcolemmal surface. It is concluded that surface Ca binding may have a substantial impact on the processes of extracellular Ca depletion (and intracellular Ca accumulation).

Animals↗

Lipoprotein lipase in myocytes and capillary endothelium of heart: immunocytochemical study.

Lipoprotein lipase was immunolocalized by electron microscopy in hearts of young mice; 78% of lipoprotein lipase was in myocytes, 3-6% in extracellular space, and 18% in capillary endothelium. Lipoprotein lipase in myocytes was located primarily in sarcoplasmic reticulum, Golgi sacs, and transport vesicles and also in secretory vesicles at the cell periphery. Lipoprotein lipase in extracellular space was present near the orifice of secretory vesicles of myocytes and in narrow zones spanning the space between myocytes and capillary endothelium. The lowest concentration of lipase associated with endothelial cells was at the basal plasma membrane, whereas the highest concentration was at the surface of luminal projections. Lipoprotein lipase was associated with chylomicrons at the capillary surface but not with chylomicron remnants. Fasting mice for 48 h increased, in heart, lipoprotein lipase activity by 120% and immunolocalized lipase by 270%. The greatest increase (5-fold) occurred at the surface of intraluminal endothelial projections. The findings indicate that lipoprotein lipase in heart is synthesized by myocytes, transferred across extracellular space along cell surfaces and across endothelial cells via vesicles or intracellular channels, and concentrated at the surface of luminal projections of endothelium where the enzyme hydrolyzes triacylglycerol of chylomicrons and very low-density lipoproteins.

Animals↗

Morphologic study of the blood vessels of the superior cervical ganglion of the albino rat.

Blood vessels of the rat superior cervical ganglion were examined by both light and electron microscopy. Direct blood supply to the superior cervical ganglion was derived from a capsular plexus of vessels. Intraganglionic vessels were for the most part capillaries. Some of these capillaries appeared dilated and sinusoidal. Although the ganglion did not seem to be densely vascularized, there was sufficient distribution to accommodate the nerve cell bodies of the ganglion. Individual capillaries served groups of neurons. Occasionally, capillary loops could be observed to surround single neuron perikarya. Ultrastructural studies revealed the presence of two types of capillaries. The majority of the capillaries of the rat superior cervical ganglion demonstrated a continuous, non-fenestrated endothelium. Typical junctional complexes were found on abutting endothelial surfaces. Endothelial flaps and microvilli were also observed on the luminal surface of some of the vessels. Numerous micropinocytotic vesicles were observed on both the luminal and abluminal surfaces of the endothelium. A small number of capillaries demonstrated a fenestrated endothelium. In both types of capillaries there was a basement membrane and an extracellular space containing collagen. Perikaryal cytoplasm was separated from the extracellular space by a thick layer of satellite cell cytoplasm.

Animals↗

Real-time monitoring of the effects of normothermia and hypothermia on extracellular glutamate re-uptake in the rat following global brain ischemia.

Brain hypothermia during ischemia may have a neuroprotective effect on pathological and functional outcomes in vivo. Although a microdialysis study demonstrated that hypothermia decreases glutamate release into the extracellular space, the issue of whether this suppression of the glutamate elevation normally accompanying ischemia is attributable to inhibition of intra-ischemic release or acceleration of post-ischemic re-uptake was not addressed. Recently, we established a real-time method for monitoring glutamate levels in extracellular space, utilizing a dialysis electrode. This method allows detailed analysis of the in vivo dynamics of biphasic glutamate elevation in the extracellular space during the intra-ischemic period and post-ischemic re-uptake. The present results show that post-ischemic hypothermia has little effect on the initial glutamate release, but remarkably enhances post-ischemic glutamate re-uptake.

Animals↗

The effects of ischemia on myocardial vascular flow and permeability.

The effects of brief periods of ischemia on vascular space size, vascular permeability to albumin, tissue water and extracellular space size, and vascular resistance were studied in the perfused isolated rabbit interventricular septum. Vascular space size increased 264% over control after 30 min ischemia and 52% after 60 min ischemia, but vascular resistance increased. Total tissue water did not change after ischemia and extracellular space size did not increase after 30 min ischemia and decreased slightly after 60 min ischemia. These results demonstrate that the initial increase in vascular resistance after ischemia is not initiated by interstitial edema or cellular edema. These events appear to be later phenomena. The initiating cause of the increased vascular resistance appears to be a combination of increased vascular tone and conversion of a portion of vascular flow from low resistance large vessel flow to higher resistance small vessel flow (capillary recruitment).

Animals↗

A simple method for volumetric measurements in isolated cardiac muscle.

A new method for the study of extracellular space and cell volume of cardiac muscle is described. Canine cardiac Purkinje strands and cat papillary muscles were placed within a fluid-filled aperture connecting two sides of an experimental chamber. Direct electrical current was passed through the hole, and changes in the voltage drop across it were correlated with Purkinje strand extracellular space and cell volume. The results of experiments on 21 Purkinje strands and 4 papillary muscles yielded an extracellular space of 51 +/- 2.1% (SEM) and 23.3 +/- 2.1%, respectively. When strands were superfused with hyper- (600 mosM) and hyposmotic (150 mosM) solutions, the preparations were found to attain new steady-state volumes that were 75 +/- 3.1% and 121 +/- 9% of control, respectively. This method can be used for volumetric studies in numerous cardiac muscle preparations and should be applicable to the study of volume abnormalities associated with certain disease states.

Animals↗

Ion diffusion modified by tortuosity and volume fraction in the extracellular microenvironment of the rat cerebellum.

1. The validity of the macroscopic laws of ion diffusion was critically examined within the microenvironment of the extracellular space in the rat cerebellum using ion-selective micropipettes and ionophoretic point sources. 2. The concepts of volume averaging, volume fraction (alpha) and tortuosity (lambda) were defined and shown to be theoretically appropriate for quantifying diffusion in a complex medium such as the brain. 3. Diffusion studies were made with the cations tetramethylammonium and tetraethylammonium and the anions alpha-naphthalene sulphonate and hexafluoro-arsenate, all of which remained essentially extracellular during the measurements. Diffusion parameters were measured for a period of 50s and over distances of the order of 0.1 mm. 4. Measurements of the diffusion coefficients of the ions in agar gel gave values that were very close to those derivable from the literature, thus confirming the validity of the method. 5. Measurements in the cerebellum did not reveal any systematic influences of ionophoretic current strength, electrode separation, anisotropy, inhomogeneity, charge discrimination or uptake, within the limits tested. 6. The pooled data from measurements with all the ions gave alpha = 0.21 +/- 0.02 (mean +/- S.E. of mean) and lambda = 1.55 +/- 0.05 (mean +/- S.E. of mean). 7. These results show that the extracellular space occupies about 20% of the rat cerebellum and that the diffusion coefficient for small monovalent extracellular ions is reduced by a factor of 2.4 (i.e. lambda 2) without regard to charge sign. The over-all effect of this is to increase the apparent strength of any ionic source in the cerebellum by a factor of lambda 2/alpha, about 12-fold in the present case, and to modify the time course of diffusion. 8. These conclusions confirm that the laws of macroscopic diffusion are closely obeyed in the cerebellum for small ions in the extracellular space, provided that volume fraction and tortuosity are explicitly taken into account. It is likely that these conclusions are generally applicable to other brain regions and other diffusing substances.

Animals↗

Role of zinc released by stimulation in rat amygdala.

The movement and role of actively functioning zinc, i.e., vesicular zinc, in the amygdala was studied, based on the data that 65Zn is localized in the limbic system, which may correspond to the regions with high densities of zinc-containing neuron terminals. When release of 65Zn into the extracellular space was examined 2 hr or 25 hr after injection of 65ZnCl2 into the amygdala, 65Zn release was facilitated by stimulation with high K+ 2 hr after injection, but not 25 hr after injection. Even 25 hr after 65Zn injection into the amygdala, approximately 95% of total 65Zn in the brain was detected in the injected area. These results suggest that zinc released into the extracellular space in the amygdala is not readily restored to the presynaptic vesicles. Moreover, to chelate zinc in the extracellular space (and in the synaptic vesicles) in the amygdala, the amygdalae were perfused with 10 microM diethyldithiocarbamate during behavioral tests for odor recognition. The olfactory sensation was temporarily disturbed by the perfusion. These results suggest that vesicular zinc is essential to the function of the amygdala, e.g., olfactory function.

Amygdala↗

In vivo measurement of regional brain and tumor pH using [14C]dimethyloxazolidinedione and quantitative autoradiography. II: Characterization of the extracellular fluid compartment using pH-sensitive microelectrodes and [14C]sucrose.

We measured the extracellular (interstitial) pH (pHe) of RG-2 rat gliomas using H+-sensitive microelectrodes and estimated the volume of tumor extracellular space based on the tissue-plasma ratio of [14C]sucrose. The average RG-2 pHe was 7.63 +/- 0.15 (mean +/- SD, n = 6), whereas the average pHe of contralateral brain tissue was 7.34 +/- 0.10 (n = 3) and arterial pH was 7.36 +/- 0.02. RG-2 extracellular space water volume was estimated to be 0.3 ml water/g tissue. In separate experiments in normal, nontumored rats, intracellular pH (pHi) was calculated for nine gray and white matter regions based on measurements of tissue and plasma [14C]dimethyloxazolidinedione concentration. pHi values ranged from 6.80 to 6.94, and no consistent gray-white differences were observed. Our data suggest that tumor pHi is not more acidic than that of normal brain tissue and that the observed alkalinity of primary brain tumors is due to the presence of a large alkaline extracellular space.

Animals↗

Ultrastructural changes in isolated perfused proximal tubules during osmotic water flow.

Steady-state effects of osmotic gradients on extracellular spaces and cell volumes were studied by ultrastructural morphometry in isolated perfused Ambystoma proximal tubules. Solute clamping, high-resolution pressure and flow control of lumen and bath solutions were all ascertained before and during fixation. Isosmotic removal of organic substrates in the lumen reversibly abolished transport, as confirmed by transepithelial potential decrease from -4.7 +/- 0.5 to -0.5 +/- 0.2 mV (n = 8) but had no effect on ultrastructural parameters. The walls of the extracellular spaces are therefore not deformed by spontaneous solute-coupled water transport. A hyperosmolar lumen generated a streaming potential of -1.56 +/- 0.15 mV (n = 8), reduced cell volume to 65%, reduced lateral intercellular space (LIS) volume to 20%, and LIS volume density to 29% of control without significant effects on the volume of the basal extracellular labyrinth (BEL). A hyperosmolar bath generated a streaming potential of +1.96 +/- 0.30 mV (n = 7), reduced cell volume to 68%, and increased LIS volume density to 236% of control. BEL volume was 55% larger during lumen-to-bath flow than during bath-to-lumen flow. Because cell volume reduction is very similar for both directions of osmotic water flow, the oppositely directed volume changes in the extracellular spaces are secondary to transepithelial water flow. The greater change in volume of LIS compared with BEL indicates that the outermost parts of the LIS are more resistive to transepithelial water flow than the slitlike communications of the BEL with the peritubular space.

Ambystoma↗

Gap junction formation between reaggregated Novikoff hepatoma cells.

We have combined freeze-fracture and electrophysiological methods in a study of gap junction formation between reaggregated Novikoff hepatoma cells. Cell clumps are dissociated with EDTA, and the resulting single cells are allowed to reaggregate (5-180 min) in loose pellets in the presence of calcium at 37 degrees . The earliest electron microscopic evidence for the genesis of new junctions is the appearance of flattened regions of the plasma membrane with a relative paucity of small intramembranous particles. These regions contain instead loosely organized groupings of 9- to 11-nm intramembranous particles, which are seen on the A face of the fractured plasma membrane, while corresponding pits occur on the membrane B face. We have termed the specialized membrane regions "formation plaques." They are seen as early as 5 min after reaggregation and are quite numerous by 30 min. Larger plaques are observed at later times. Plaques seen at 30 min are consistently matched with other plaques on apposed cells, although the extracellular space separating these structures still exceeds 10 nm. By 60 min, some matched plaques display a reduced extracellular space, resembling that of normal gap junctions. Between 30 and 60 min, aggregates of closely packed particles on A faces and hexagonally arranged pits on B faces frequently appear in the formation plaques. The aggregates, which are indistinguishable from small gap junctions, appear to enlarge over the subsequent 2-hr period as the number of unaggregated 9- to 11-nm particles declines. Microelectrode studies demonstrate progressive increases in the percent of interfaces containing lowresistance junctions and in the degree of elctrical coupling in preparations incubated up to 2 hr. Coupling is first detected at about the same time as particle aggregates (or formation plaques with reduced extracellular spaces), and increases as aggregate sizes increase.

Carcinoma, Hepatocellular↗