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PubMed1983

Cation distributions in the hypertrophic myocardium (aortic constriction) of the rat.

Calcium, magnesium, sodium, potassium, and water distributions were determined in rat ventricular muscle during the development of myocardial hypertrophy. Hypertrophy was produced by constriction of the ascending aorta with a silver band. Sham-operated controls were treated similarly, except that the aorta was not constricted. Cation and water distributions were examined at intervals of 1 hour, 1 day, 1 week. Myocardial extracellular space was determined by distribution of [35S] sulphate. In separate experiments, extracellular space was determined in different regions of the normal rat ventricle using [3H] inulin as the extracellular marker. Although some changes were observed in tissue calcium content and the plasma concentrations of several cations, at no time were the cellular concentrations of any cation significantly altered. Myocardial water content and distribution remained nearly constant after constriction of the aorta. Results do not support hypotheses that the heart responds to increased afterload with an accumulation or loss of myocytic calcium sufficiently large to be detectable with standard quantitative methods.

Animals↗
PubMed2003

Gram-negative bacterial ATP-binding cassette protein exporter family and diverse secretory proteins.

Protein translocation to the extracellular space is essential for the invasion, colonization, and survival of pathogenic gram-negative bacteria within a host organism. In addition to the N-terminal signal sequence-dependent secretion system, which is specific for protein transport to the periplasmic space, there are five major systems (type I, II, III, IV, and V) that are known to be involved in protein secretion into the extracellular space. Of the systems, the type I pathway, which is composed of three membrane components including an ATP-binding cassette (ABC) protein, translocates proteins into the extracellular space from the cytosol by directly using the energy generated from ATP hydrolysis, and therefore, the system is a member of the ABC transporter family and is also known as the ABC exporter. To date, ABC exporters have been discovered to be involved in the secretion of a wide variety of exoproteins including RTX (repeats-in-toxin) toxins, cell surface layer proteins, proteases, lipases, bacteriocins, heme-acquisition proteins, and nodulation-related proteins such as the exoglucanases of gram-negative bacteria. A secretory protein and its associated specific ABC exporter are encoded in the same gene cluster in most cases, and ABC exporters show substrate specificity for secretion. Consequently, ABC exporters are present based primarily on the number of secretory protein genes. A secretion signal is situated in the C-terminal region of secretory proteins, however, the characteristics of the secretion signal are not fully understood. Secretory substrates and their linked ABC exporters are reviewed in the following paper.

Journal Article↗
PubMed1990

Immunocytochemical studies of oedema protein clearance in the rat.

Twenty microliters of rat albumin solution was infused into the caudate nucleus of anaesthetized rats and the distribution of the albumin was followed using immunocytochemical methods with LM and EM at 15 min, 24 hr, and 48 hr post-infusion. Fifteen min post-infusion, the albumin was distributed in the extracellular space of the white matter and in the overlying deep cortical layers. At 24 hr post-infusion, the albumin was detected in the extracellular space around the glia limitans. At the surface of the ventricular wall of the 48 hr post-infusion animals, most of the albumin had been cleared from the extracellular space (ECS) of the subependymal white matter and the ependymal clefts, although a large amount of albumin had been observed in these areas at 15 min after infusion. At the temporobasal area of the cortex, there was continuity of the labelled perivascular space of the venous vessel from the deep oedematous area to the cortical surface not only immediately after infusion but also during the chronic phase. In conclusion, oedema fluid and protein migrate not only to the ventricle but upward toward the cortical surfaces to reach the subarachnoid spaces for eventual clearance into CSF. This seemingly occurs in the absence of significant pressure gradients.

Animals↗
PubMed2006

The extracellular electrical resistivity in cell adhesion.

The interaction of cells in a tissue depends on the nature of the extracellular matrix. The electrical properties of the narrow extracellular space are unknown. Here we consider cell adhesion mediated by extracellular matrix protein on a solid substrate as a model system. We culture human embryonic kidney (HEK293) cells on silica coated with fibronectin and determine the electrical resistivity in the cell-solid junction rhoJ=rJdJ by combining measurements of the sheet resistance rJ and of the distance dJ between membrane and substrate. The sheet resistance is obtained from phase fluorometry of the voltage-sensitive dye ANNINE-5 by alternating-current stimulation from the substrate. The distance is measured by fluorescence interference contrast microscopy. We change the resistivity of the bath in a range from 66 Omega cm to 750 Omega cm and find that the sheet resistance rJ is proportionally enhanced, but that the distance is invariant around dJ=75 nm. In all cases, the resulting resistivity rhoJ is indistinguishable from the resistivity of the bath. A similar result is obtained for rat neurons cultured on polylysine. On that basis, we propose a "bulk resistivity in cell adhesion" model for cell-solid junctions. The observations suggest that the electrical interaction between cells in a tissue is determined by an extracellular space with the electrical properties of bulk electrolyte.

Animals↗
PubMed2000

Perspectives on spreading depression.

Spreading depression (SD) consists of a transient suppression of all neuronal activity that spreads slowly across regions of gray matter. The paper is divided into three parts. Martins-Ferreira describes 30 years of research on SD in the isolated retina. Much of this work has relied on the prominent intrinsic optical signals that accompany SD in the retina. By inducing SD to propagate in circles with a velocity of 3.7 mm min(-1), it is possible to investigate the finely balanced electrochemical equilibrium that maintains the traveling wave. SD is accompanied by a slow negative extracellular voltage and ion movements that are greatest in the inner plexiform layer of the retina. Nedergaard discusses the role of astrocytes in SD propagation. Astrocytes mediate slowly moving waves of intracellular Ca(2+) increase, for which gap junctions are essential. SD is accompanied by entry of Ca(2+) into cells and fails when gap junctions are blocked. SD, however, is blocked by glutamate receptor antagonists but glial Ca(2+) waves are not. Astrocytic Ca(2+) waves are probably involved in the initiation of SD but other factors, including K(+), glutamate and purinergic receptors, are necessary for sustained propagation. Nicholson describes studies on the different preparations that helped clarify the role of extracellular space in SD. It has long been known that extracellular K(+) reaches levels of 50 mM or more during SD. Studies with ion-selective microelectrodes showed that extracellular Na(+) and Cl(-) fall by as much as 100 mM during SD, and water leaves the extracellular space. Further work showed that extracellular Ca(2+) falls 10-fold during SD and significant changes in extracellular pH and ascorbate occur. These studies imply that large perturbations of the extracellular milieu occur during SD and are an essential part of the interlocking cascade of events that produce this still mysterious phenomenon.

Animals↗
PubMed1987

Measurement of intra-embryonic pH during the early stages of development in the chick embryo.

Measurements have been made of the pH in the extracellular space, adjacent to the neural tube, in 73 isolated chick embryos in vitro at stages from 4-22 somites. A pH of 7.8-8.4 was observed in the segmented region, while caudally, in the segmental plate, the pH was consistently lower falling by as much as 0.5 pH units at the regressing primitive streak. Variations were noted in the pH of embryos of the same age but the regional variation in pH was a consistent finding in all of the embryos examined. The buffering capacity of the extracellular space was found to be 12.9 mequiv/pH unit/l in the segmented region and 13.9 mequiv/pH unit/l in the segmental plate. Thus it is unlikely that the regional variations in pH result from local variations in the buffering power of the extracellular space. Varying the K+, Cl-, Mg2+ or HCO3- ion concentrations in the bathing medium caused little change in the intra-embryonic pH, while reducing the concentrations of Na+ or Ca2+ caused a small acidification. This suggests that the ectoderm and endoderm form an effective barrier between the embryo and the external environment. Exposure of the embryo to KCN reduced the intra-embryonic pH suggesting that the alkaline environment is maintained by active processes.

Age Factors↗
PubMed1983

Kinetic constants for uptake and metabolism of 3H-(-)noradrenaline in rabbit aorta. Possible falsification of the constants by diffusion barriers within the vessel wall.

1. The neuronal uptake of 3H-(-)noradrenaline into aortic rings from reserpine-pretreated animals was a saturable process with a Km of 2.3 mumol X l-1 and a Vmax of 0.5 nmol X g-1 X min-1. Similar constants were obtained when the neuronal deamination of noradrenaline was taken as an index for neuronal uptake. When the tissue was incubated in the usual way, i.e., when the amine was allowed to enter the aortic rings via both the intimal and the adventitial surface, then there was no initial delay (tlag) for the neuronal uptake of noradrenaline (MAO and COMT inhibited). On the other hand, when the amine entry was restricted to the intimal surface, there was a tlag of 2 min, probably due to the slow equilibration of the extracellular space of the media with the incubation medium. Furthermore, for low amine concentrations the rate of uptake in the latter situation was about 10 times lower than that in the former one. Thus, the rate of uptake clearly depended on the way the amine entered the tissue. 2. The corticosterone-sensitive extraneural uptake of 3H-(-)noradrenaline determined in nerve-free aortic rings was characterised by a high Km (490 mumol X l-1) and Vmax (35 nmol X g-1 X min-1). For uptake2 a tlag of 1 min was observed. 3. The analysis of the extraneuronal O-methylating system in nerve-free aortic rings yielded a Km of 3.6 mumol X l-1 and a Vmax of 0.6 nmol X g-1 X min-1. The tlag for the O-methylation of noradrenaline was in the same order of magnitude as that for uptake2. At low amine concentrations the corticosterone-sensitive accumulation of noradrenaline was prevented by COMT, but not by MAO. The latter enzyme reduced the steady-state accumulation of noradrenaline by about 50%. When the amine entry was restricted to one surface only, the results indicated that the extraneuronal O-methylation of noradrenaline generated a steep concentration gradient of the parent amine within the extracellular space of the aorta. 4. All saturable processes fitted the Michaelis-Menten equation. However, kinetic constants determined in incubated organs may be falsified by poor diffusion of the substrate through the extracellular space.

Animals↗
PubMed1995

Detachment of desmosomes in a microcystic meningioma.

This report demonstrates the detachment of desmosomes in the microcystic area of a frontal convexity meningioma removed from a 69-year-old woman. Well-developed interdigitations of the tumor cell processes with numerous desmosomes and with narrow extracellular spaces were characteristic features of the solid area of the meningioma. By contrast, the microcystic area of the tumor had markedly distended extracellular spaces. Various stages in the separation of desmosomal attachments were seen in this area. The observed configurations ranged from the widening of opposing junctions to the formation of large cavities where hemidesmosome-like structures were evident. The latter lacked basal lamina, and are considered to represent a transition leading to the loss of desmosome, and thus involved in the enlargement of the extracellular space in microcystic meningiomas.

Aged↗
PubMed1969

Calcium exchange and contraction strength of guinea pig atrium in normal and hypertonic media.

A Krebs-Henseleit (KH) medium made hypertonic by adding nonpermeant molecules substantially increased the isometric peak tension at steady-state contractions below 3 per sec in guinea pig atrium at 27 degrees C. Action potential durations were decreased. KH plus 100 mM raffinose or sucrose resulted in similar and nearly maximal changes which were essentially reversible upon return to normal KH. When one active contracting atrium was used to passively stretch a second atrium, the difference in Ca ion exchange (1 min exchange with the extracellular space) between active and stretched atria significantly increased at 1 per sec and at 2 per sec in going from normal to 100 mM hypertonic KH. The calculated mean Ca ion cellular exchange per beat per 100 g of cells (a) doubled in changing from normal to 100 mM hypertonic KH, and (b) decreased slightly in changing from contractions of 1 per sec to 2 per sec in normal KH. These data are consistent with the hypothesis (a) that Ca ion entry per beat from the extracellular space is proportional to membrane depolarized time with a constant medium and a steady-state condition, and the hypothesis (b) that 100 mM hypertonicity doubles the Ca ion entry rate during depolarization. These data enable rejection of the hypothesis that the peak tension is proportional to the Ca ion entry per beat from the extracellular space under steady-state conditions, and suggest that any additional Ca ion involved in the larger contractions at higher frequencies comes from an increase in Ca ion available from intracellular stores.

Action Potentials↗
PubMed1984

[Kinetics of THAM (TRIS) distribution in intra- and extracellular compartments].

The distribution of 14C labelled THAM (tris-hydroxymethylaminomethane) was determined between intra- and extracellular space of nephrectomised Sprague-Dawley rats as a function of time at constant plasma pH of 7.4. The following results were obtained: An equilibrium in the distribution of THAM between ECS and ICS will not occur before 6-12 hours after administration. This indicates that THAM permeates very slowly into the intracellular compartment, which is in contrast to the general assumption that it quickly diffuses into the intracellular space to restore the intracellular acidosis. THAM disappears from the extracellular space in a multiexponential fashion, indicating that it equilibrates with the different body tissues at largely variable rates. The equilibrium which occurs between both body compartments 6-12 hours after THAM application does not agree with the values which are expected for transfer of only the nonionised substance. At plasma pH 7.4 and a "mean whole body pHi" of 6.88, THAM is distributed with a distribution ratio of 4 (ICS/ECS), a value quite different from the value of 11 which would be expected for exclusive nonionic diffusion. Thus THAM is also transferred across the cell membrane in ionised form. These results indicate that the influx of THAM into the intracellular space is too slow (when compared to the renal elimination kinetics) to influence intracellular pH significantly by direct buffer action. Moreover, only a fraction of THAM enters the intracellular space in the nonionised form, thus reducing (to an even greater extent) the direct effect of THAM on the intracellular acid-base equilibrium.

Animals↗
PubMed1988

Uterine vascular permeability, blood flow and extracellular fluid space during implantation in rats.

Vascular permeability to plasma proteins in uterine implantation and non-implantation sites (i.e. dye sites and non-dye sites) was assessed quantitatively by a method which accounts for steady-state volumes of distribution. Extracellular fluid volume and uterine blood flow were also determined. On both the evening of Day 5 and the morning of Day 6, vascular permeability to 125I-labelled human serum albumin, extracellular fluid volume and blood flow were significantly increased in implantation sites compared to non-implantation sites. Vascular permeability in implantation sites was increased significantly between Days 5 and 6, whereas that in non-implantation sites was unchanged. This increase in vascular permeability between Days 5 and 6 was not accompanied by further increases in extracellular fluid volume and blood flow. This result shows a dissociation between vascular permeability and extracellular fluid volume immediately after the onset of implantation and raises important questions as to whether the rat uterus undergoes a truly oedematous response at implantation as has been generally accepted.

Animals↗
PubMedDate not supplied

Caveolae in smooth muscles: nanocontacts.

Smooth muscle cell (SMC) caveolae have been investigated by quantitative and qualitative analysis of transmission electron microscopy (TEM) images of rat stomach, bladder and myometrium, guinea pig taenia coli, human ileum, and rat aortic SMCs. Ultrathin (below 30 nm) serial sections were used for examination of caveolar morphology and their connections with SMC organelles. Average caveolar diameter was smaller in vascular SMCs (70 nm, n=50) than in visceral SMCs (77 nm, n=100), but with the same morphology. Most of the caveolae, featured as flask-shaped plasma membrane (PM) invaginations, opened to the extracellular space through a 20 nm stoma (21 +/- 3 nm) having a 7 nm thick diaphragm. A small percentage of caveolae (3%), gathered as grape-like clusters, did not open directly to the extracellular space, but to irregular PM pockets having a 20-30 nm opening to the extracellular space. In visceral SMCs, caveolae were disposed in 4-6 rows, parallel to myofilaments, whilst aortic SMCs caveolae were arranged as clusters. This caveolar organization in rows or clusters minimizes the occupied volume, providing more space for the contractile compartment. The morphometric analysis of relative volumes (% of cell volume) showed that caveolae were more conspicuous in visceral than in vascular SMCs (myometrium - 2.40%; bladder - 3.66%, stomach - 2.61%, aorta - 1.43%). We also observed a higher number of caveolae per length unit of cellular membrane in most visceral SMCs compared to vascular SMCs (myometrium - 1.06/microm, bladder - 0.74/microm, aorta - 0.57/microm, stomach - 0.48/microm). Caveolae increase the cellular perimeter up to 15% and enlarge the surface area of the plasma membrane about 80% in SMCs. Threedimensional reconstructions (15micro(3)) showed that most caveolae, in both visceral and vascular SMCs, have nanocontacts with SR (87%), other with mitochondria (10%) and 3% apparently have no contact with these organelles. Usually, 15 nm wide junctional spaces exist between caveolae and SR, some of them with nanostructural links between each other or with mitochondria: direct contacts (space <2 nm or none) and molecular links, so called 'feet' (about 12 nm electron dense structures between organellar membranes). Direct contacts possibly allow molecular translocation between the two membranes. Electron-dense 'feet'-like structures suggest a molecular link between these organelles responsible for intracellular Ca(2+) homeostasis (excitation-contraction coupling or pharmaco-mechanical coupling). Close appositions (approximately 15 nm) have also been observed between caveolae and perinuclear SR cisternae, suggesting that caveolae might be directly implicated in excitation-transcription coupling.

Animals↗
PubMed1995

Absorbed energy distribution from radiofrequency electromagnetic radiation in a mammalian cell model: effect of membrane-bound water.

The spatial distributions of induced 27 or 2450 MHz radiofrequency (RF) electric fields (E-fields) and specific absorption rates (SARs) in a three-component spherical cell model (cytoplasm, membrane, extracellular space) were determined by Mie scattering theory. The results were compared to results for the same cell model but with 0.5 nm thick of bound water on the inner (cytoplasmic) and outer (extracellular) membrane surfaces (i.e., five-component cell model). The results provide insight regarding direct frequency-dependent RF radiation effects at the cellular level. Induced E-fields and SARs were calculated for two bound-water characteristic frequencies (400 or 1000 MHz) and ionic conductivities (1-1000 mS/m). In order to estimate the dependence of the results on bound water within the membrane per se, the model was revised to include bound water within the inner and outer membrane surfaces. The results were as follows: 1) on the x-axis, the y- and z-components of the induced E-field were of insignificant magnitude compared to the x-component for an incident E-field parallel to the x-axis; 2) the ratio of transmembrane E-fields induced by 2450 MHz vs. 27 MHz RF [i.e., Ex (2450 MHz)/Ex (27 MHz)] was 0.1; 3) for the three-component cell model, the corresponding SAR ratios [SAR (2450 MHz)/SAR (27MHz)] in the cytoplasm and extracellular space were 1.66 and 5.0, respectively; 4) the SAR rations [SAR (2450 MHz)/SAR (27 MHz)] for the cytoplasm and extracellular space for the five-component cell model were 1.66 and 5.0, respectively; 5) the ratio of the E-fields induced in the cytoplasmic and extracellular layers of bound water in the five-component cell model [E (2450 MHz)/ E (27Mhz)] were 0.62 and 0.63, respectively; 6) the SAR ratios [SAR (2450 MHz)/SAR (27 MHz)] for the cytoplasmic and extracellular bound-water layers were 66 and 65.3, respectively; and 7) variation of bound-water characteristic frequency, ionic conductivity, or bound-water incorporation inside the membrane surfaces, per se, did not significantly affect the E-field or SAR ratios. These results indicate that frequency-dependent nonuniformities may occur in the distribution of induced RF E-fields and SARs at the cellular level.

Animals↗
PubMed2004

Fluid and electrolyte therapy and chronic lung disease.

PURPOSE OF REVIEW: To evaluate the role of fluid and electrolyte therapy in the pathogenesis of chronic lung disease. RECENT FINDINGS: There have been no new studies since 2000, and there are minimal data addressing this issue specifically in infants at highest risk of chronic lung disease (ie, extremely low birth weight infants). Most observational studies demonstrate a significant association between increased fluid intake or differences in measures of changes in total body water balance in the first week of life and chronic lung disease. However, a metaanalysis of three randomized, controlled clinical trials did not reveal a significant increase in the risk of chronic lung disease with higher fluid intakes. The results of two randomized, controlled trials of different sodium intakes were conflicting regarding whether increased intake led to an increased prevalence of chronic lung disease. SUMMARY: The evidence is insufficient to conclude that fluid and electrolyte therapy plays a role in the pathogenesis of chronic lung disease. Even if fluid and electrolyte therapy does play a small role, the data are insufficient to help with decisions about what fluid and electrolyte intake in an individual infant in a specific clinical setting might reduce the risk of chronic lung disease. However, net negative water and sodium balances and the resultant contraction of the extracellular space are probably physiologic in preterm infants in the first week of life and evidence suggests that a positive water and sodium balance and expansion of the extracellular space during this period increase morbidity. It is not clear what degree of contraction is appropriate or whether varying degrees of contraction of the extracellular space alter morbidity.

Chronic Disease↗