Regulation of cell volume via microvillar ion channels

A novel mechanism of cellular volume regulation is presented, which ensues from the recently introduced concept of transport and ion channel regulation via microvillar structures (Lange K, 1999, J Cell Physiol 180:19-35). According to this notion, the activity of ion channels and transporter proteins located on microvilli of differentiated cells is regulated by changes in the structural organization of the bundle of actin filaments in the microvillar shaft region. Cells with microvillar surfaces represent two-compartment systems consisting of the cytoplasm on the one side and the sum of the microvillar tip (or, entrance) compartments on the other side. The two compartments are separated by the microvillar actin filament bundle acting as diffusion barrier ions and other solutes. The specific organization of ion and water channels on the surface of microvillar cell types enables this two-compartment system to respond to hypo- and hyperosmotic conditions by activation of ionic fluxes along electrochemical gradients. Hypotonic exposure results in swelling of the cytoplasmic compartment accompanied by a corresponding reduction in the length of the microvillar diffusion barrier, allowing osmolyte efflux and regulatory volume decrease (RVD). Hypertonic conditions, which cause shortening of the diffusion barrier via swelling of the entrance compartment, allow osmolyte influx for regulatory volume increase (RVI). Swelling of either the cytoplasmic or the entrance compartment, by using membrane portions of the microvillar shafts for surface enlargement, activates ion fluxes between the cytoplasm and the entrance compartment by shortening of microvilli. The pool of available membrane lipids used for cell swelling, which is proportional to length and number of microvilli per cell, represents the sensor system that directly translates surface enlargements into activation of ion channels. Thus, the use of additional membrane components for osmotic swelling or other types of surface-expanding shape changes (such as the volume-invariant cell spreading or stretching) directly regulates influx and efflux activities of microvillar ion channels. The proposed mechanism of ion flux regulation also applies to the physiological main functions of epithelial cells and the auxiliary action of swelling-induced ATP release. Furthermore, the microvillar entrance compartment, as a finely dispersed ion-accessible peripheral space, represents a cellular sensor for environmental ionic/osmotic conditions able to detect concentration gradients with high lateral resolution. Volume regulation via microvillar surfaces is only one special aspect of the general property of mechanosensitivity of microvillar ionic pathways.     

A study of rapid mitochondrial structural changes in vitro by spray- freeze-etching

The spray-freeze-etching technique has been used to study energy-linked mitochondrial structural changes in rat liver mitochondria incubated in vitro. The technique involves spraying the suspension of mitochondria into liquid propane at -190 degrees C, and does not require the use of cryoprotectants or chemical fixatives. The results confirmed that freshly isolated mitochondria have a condensed matrix and that this expands at the expense of the outer compartment to give the orthodox configuration when the mitochondria are incubated in a K+ medium in the presence of substrate and phosphate. Addition of adenosine diphosphate (ADP) caused a rapid shrinkage of the matrix compartment, and the time-course and extent of this shrinkage has been measured quantitatively by coupling a rapid sampling device to the spray-freezing apparatus. These data show that for orthodox mitochondria the onset of phosphorylation is accompanied by a reduction of 30% in the matrix volume in 20's, and there is no evidence that the decrease in matrical volume affects the phosphorylation efficiency. These results suggest that natural ionophores in the mitochondrial inner membrane make it permeable enough to permit a rapid readjustment of matrix volume after the addition of ADP, and that the associated ion movement does not cause uncoupling of oxidative phosphorylation.     

NUCLEAR EXTRUSION AND INTRACISTERNAL INCLUSIONS IN THE RABBIT BLASTOCYST

Observations have been made on the role of a divalent cation (calcium ion) during OsO₄ fixation of nuclei of frog erythrocytes, mainly after isolation from cells. The volume of the nucleus depends partly on the molecular interaction of charged macromolecules, is controlled by the ionic strength of the medium, and hence may be used as a guide in attempts to preserve structure. When the isolation and fixation media contain 0.01 M calcium at pH 6.3 the volume changes, in the light microscope, during processing are small. When the fixative does not contain these ions, reversible volume changes occur during fixation and dehydration. The chromatin of nuclei processed with minimal volume change appears, in the electron microscope, to contain fine dots and lines about 20 to 40 A in diameter, relatively close together. The chromatin structure of nuclei in which volume changes have occurred consists of dense irregularly shaped patches, relatively far apart, and ranging in diameter from about 200 A down to the limits of visibility (20 to 30 A). It is suggested that the latter structure is a precipitation artefact.     

Acid-base disturbance during hemorrhage in rats: significant role of strong inorganic ions.

The present study tests the hypothesis that changes in the strong inorganic ion concentrations contribute significantly to the acid-base disturbance that develops during hemorrhage in the arterial plasma of rats in addition to lactate concentration ([Lac-]) increase. The physicochemical origins for this acid-base disorder were studied during acute, graded hemorrhage (10, 20, and 30% loss of blood volume) in three groups of rats: conscious, anesthetized with ketamine, and anesthetized with urethan. The results support the hypothesis examined: strong-ion difference (SID) decreased in the arterial plasma of all groups studied because of an early imbalance in the main strong inorganic ions during initial hemorrhagic phase. Moreover, changes in plasma [Lac-] contributed to SID decrease in a later hemorrhagic phase (after 10% hemorrhage in urethan-anesthetized, after 20% hemorrhage in ketamine-anesthetized, and after 30% hemorrhage in conscious group). Inorganic ion changes were due to both dilution of the vascular compartment and ion exchange with extravascular space and red blood cells, as compensation for blood volume depletion and hypocapnia. Nevertheless, anesthetized rats were less able than conscious rats to preserve normal arterial pH during hemorrhage, mainly because of an impaired peripheral tissue condition and incomplete ventilatory compensation.     

Evaluation of bone marrow lesion volume as a knee osteoarthritis biomarker - longitudinal relationships with pain and structural changes: data from the Osteoarthritis Initiative

Introduction

Bone marrow lesion (BML) size may be an important imaging biomarker for osteoarthritis-related clinical trials and reducing BML size may be an important therapeutic goal. However, data on the interrelationships between BML size, pain, and structural progression are inconsistent and rarely examined in the same cohort. Therefore, we evaluated the cross-sectional and longitudinal associations of BML volume with knee pain and joint space narrowing (JSN).

Methods

A BML volume assessment was performed on magnetic resonance images of the knee collected at the 24- and 48-month Osteoarthritis Initiative visits from a convenience sample of 404 participants in the progression cohort. During the same visits, knee pain was assessed with WOMAC pain scores and knee radiographs were acquired and scored for JSN. BML volume was summed to generate a total knee volume and an index tibiofemoral compartment volume (compartment with greater baseline JSN). Primary analyses included multiple linear regressions (outcome = pain, predictor = total knee BML volume) and logistic regressions (outcome = JSN, predictor = index tibiofemoral compartment BML volume).

Results

This sample was 49% female with a mean age of 63 (9.2 standard deviation (SD)) years, and 71% had radiographic osteoarthritis in the study knee. Larger baseline BMLs were associated with greater baseline knee pain (P = 0.01), the presence of JSN at baseline (odds ratio (OR) = 1.50, 95% confidence interval (CI) = 1.23 to 1.83), and JSN progression (OR = 1.27, 95%CI = 1.11 to 1.46). Changes in total knee BML volume had a positive association with changes in knee pain severity (P = 0.004) and this association may be driven by knees that were progressing from no or small baseline BMLs to larger BMLs. In contrast, we found no linear positive relationship between BML volume change and JSN progression. Instead, regression of medial tibiofemoral BML volume was associated with JSN progression compared to knees with no or minimal changes in BML volume (OR = 3.36, 95%CI = 1.55 to 7.28). However, follow-up analyses indicated that the association between JSN progression and BML volume change may primarily be influenced by baseline BML volume.

Conclusion

Large baseline BMLs are associated with greater baseline knee pain, the presence of JSN at baseline, and disease progression. Additionally, BML regression is associated with decreased knee pain but not a reduced risk of concurrent JSN progression.     

Subcellular changes in capillary endothelial cells during repair of hyperoxic lung injury

We studied the changes in subcellular ultrastructure associated with the hypertrophy of capillary endothelial cells during repair of hyperoxic (100% O2) lung injury in rats. We used stereologic-morphometric measurements at different magnifications to determine the absolute volume of each subcellular compartment per average capillary endothelial cell. The increases in this value during the first 3 days of postexposure repair were 118% for cytoplasm, 786% for polyribosomes, 310% for rough endoplasmic reticulum, and 79% for mitochondria; the volume of pinocytotic vesicles did not change. By day 7 of repair, only the polyribosomes and rough endoplasmic reticulum were still increased; by day 14 all values were normal. We conclude that the capillary endothelial cell hypertrophy that develops during repair of hyperoxic lung injury is associated with large and heterogeneous increases in subcellular organelles and is not merely due to increases in the cytosol or to cellular edema. These increases seem to be an integral part of the repair process and may be important in the development of tolerance to subsequent oxygen exposure.     

Quantitation of gold labeling and estimation of labeling efficiency with a stereological counting method.

The amount of immunolabeling over a cell compartment of an average cell was estimated by use of an adaptation of the double disector method introduced by Gundersen. The first and last sections of a stack of ultra-thin sections formed a disector in which cell number could be estimated and related to a defined reference volume to give the cell density. Another stack section, selected at random, was immunolabeled and the number of gold particles associated with unit volume reference space (gold "density") estimated in quadrats placed systematically across the section. The ratio of gold density to cell density was used to estimate the number of gold particles lying over a chosen compartment of an average cell, N(gold)/N(cell). Such estimates required neither cell volume nor section thickness measurement and were reproducible. By combining the number of gold particles per cell with estimates of the number of protein antigens per cell, the number of gold particles associated with each antigen could be found (labeling efficiency).     

THE GLUTAMATE AND GLUTAMINE CONTENT OF RAT BRAIN AFTER PORTOCAVAL ANASTOMOSIS

Abstract— Rats have been subjected to portocaval anastomosis and the ammonium ion in plasma and the glutamate and glutamine levels in plasma, red cells and brain have been estimated up to 6 weeks after operation. The glutamine, but not the glutamate, levels in brain were consistently raised, being about 2.5 times greater than normal and the level can be correlated with the level of plasma ammonium ion. Consideration is given to the possibility that the glutamine may be in the greatly enlarged neuroglial compartment in this abnormal metabolic state.     

Changes in the extents of viable and necrotic tissue, interstitial fluid pressure, and proliferation kinetics in clone A human colon tumour xenografts as a function of tumour size

Abstract. Total, viable and necrotic tumour tissue, tumour cell yields, and colony forming efficiencies were measured in clone A human colon tumour xenografts as neoplasms grew from about 100 mm³ to about 6000 mm³. The volumes of the total, viable and necrotic compartments were fit using the Verhulst equation to obtain estimates of growth rates and maximal sizes of the various compartments (carrying capacities). Additionally, at four discrete tumour volumes (250, 850, 2500 and 5500 mm³), hypoxic percentages, proportions of parenchymal tumour and host cells, interstitial fluid pressures, and proliferation kinetics including measurements of apoptosis were determined. There were interesting relationships between the shapes of the curves for total, viable and necrotic tissue to some of the other endpoints measured. Specifically, the volumetric growth curves for the total and viable tumour tissue compartments were identical to a volume of approximately 1000 mm³, but diverged at larger sizes, with the viable cell compartment exhibiting a smaller carrying capacity. The shape of the growth curve for the necrotic compartment exactly mimicked that for the total volume compartment, but was delayed in time by about 21 days. Similarity in shape to that of the overall tumour volume/necrotic volume curves was also seen for the curve for the increase in interstitial fluid pressure, and for the increase in the size of the host cell compartment. In contrast, the growth of the hypoxic compartment and of the parenchymal tumour cell compartment were similar in shape to that of the viable compartment. These data indicate that these compartments are functionally linked. Marked changes in cell kinetic parameters occurred as tumour size increased from 250–5500 mm³. The labelling index and growth fractions decreased from 0.256–0.125, and 0.77–0.40 respectively, and the cell loss factor increased from 0.52–0.74. The volumetric and potential doubling times increased from 4.3–17.6 and 2.1–4.6 days respectively. The cell kinetic changes could not be clearly related to the changes in shape of either the overall tumour volume or the viable tumour volume.     

Effects of long-duration bed rest on structural compartments of m. soleus in man

Magnetic resonance imaging (MRI), histomorphometry and electron microscopy of muscle demonstrate that long-term exposure to actual or simulated weightlessness (including head down bed rest) leads to decreased volume of antigravity muscles in mammals. In muscles interbundle space is occupied by the connective tissue. Rat studies show that hindlimb unloading induces muscle fiber atrophy along with increase in muscle non-fiber connective tissue compartment. Beside that, usually 20% of the muscle fiber volume is comprised by non-contractile (non-myofibrillar) compartment. The aim of the present study was to compare changes in muscle volume, and in muscle fiber size with alterations in myofibrillar apparatus, and in connective tissue compartment in human m. soleus under conditions of 120 day long head down bed rest (HDBR).     

Alzheimer disease periventricular white matter lesions exhibit specific proteomic profile alterations

The white matter (WM) represents approximately half the cerebrum volume and is profoundly affected in Alzheimer’s disease (AD). However, both the WM responses to AD as well as potential influences of this compartment to dementia pathogenesis remain comparatively neglected. Neuroimaging studies have revealed WM alterations are commonly associated with AD and renewed interest in examining the pathologic basis and importance of these changes.     

Role of t-tubules in the control of trans-sarcolemmal ion flux and intracellular Ca2+ in a model of the rat cardiac ventricular myocyte

The t-tubules of mammalian ventricular myocytes are invaginations of the surface membrane that form a complex network within the cell, with restricted diffusion to the bulk extracellular space. The trans-sarcolemmal flux of many ions, including Ca(2+), occurs predominantly across the t-tubule membrane and thus into and out of this restricted diffusion space. It seems possible, therefore, that ion concentration changes may occur in the t-tubule lumen, which would alter ion flux across the t-tubule membrane. We have used a computer model of the ventricular myocyte, incorporating a t-tubule compartment and experimentally determined values for diffusion between the t-tubule lumen and bulk extracellular space, and ion fluxes across the t-tubule membrane, to investigate this possibility. The results show that influx and efflux of different ion species across the t-tubule membrane are similar, but not equal. Changes of ion concentration can therefore occur close to the t-tubular membrane, thereby altering trans-sarcolemmal ion flux and thus cell function, although such changes are reduced by diffusion to the bulk extracellular space. Slowing diffusion results in larger changes in luminal ion concentrations. These results provide a deeper understanding of the role of the t-tubules in normal cell function, and are a basis for understanding the changes that occur in heart failure as a result of changes in t-tubule structure and ion fluxes.     

Simplified models for gas exchange in the human lungs

This paper presents a hierarchy of models with increasing complexity for gas exchange in the human lungs. The models span from a single compartment, inflexible lung to a single compartment, flexible lung with pulmonary gas exchange. It is shown how the models are related to well-known models in the literature. A long-term purpose of this work is to study nonlinear phenomena seen in the cardio-respiratory system (for example, synchronization between ventilation rate and heart rate, and Cheyne-Stokes respiration). The models developed in this paper can be regarded as the controlled system (plant) and provide a mathematical framework to link between "molecular-level", and "systems-level" models. It is shown how changes in molecular level affect the alveolar partial pressure. Two assumptions that have previously been made are re-examined: (1) the hidden assumption that the air flow through the mouth is equal to the rate of volume change in the lungs, and, (2) the assumption that the process of oxygen binding to hemoglobin is near equilibrium. Conditions under which these assumptions are valid are studied. All the parameters in the models, except two, are physiologically realistic. Numerical results are consistent with published experimental observations.     

Ion movements during energy-linked mitochondrial structural changes

The structure of isolated rat liver mitochondria has been observed in the electron microscope following incubation of the mitochondria in vitro under a variety of conditions. The results show that ultrastructural changes are only associated with the energization and deenergization of isolated mitochondria if the composition of the incubation medium permits ion movements in or out of the matrix. The mechanism of energy coupling does not appear to depend on these major mitochondrial structural changes. The addition of low levels of valinomycin greatly increases the rate at which the matrix compartment swells and shrinks on energization and deenergization even at low K⁺ concentrations.     

Haemolymph osmoregulation and the fate of sodium and chloride during dehydration in terrestrial isopods

Osmoregulation of the haemolymph during dehydration was investigated in a selection of temperate oniscidean isopods. Inulin tracer studies show that the haemolymph contributes approximately 69% of water losses in Porcellio scaber, significantly more than predicted from the volume of this compartment (42% of total water). Haemolymph osmolality increases linearly as a function of haemolymph dehydration but at a significantly lower rate than predicted from the change in haemolymph fluid volume. Similar results for Oniscus asellus show that both species display efficient osmoregulation until lethal dehydration. Osmoregulation is associated with significant hyporegulation of haemolymph sodium and chloride. These findings indicate that: (1) cell water is conserved at the expense of the haemolymph; and (2) haemolymph dehydration is associated with the removal of Na(+) and Cl(-) contributing to net osmoregulation. During dehydration, accumulations of both Na(+) and Cl(-) are seen in the hindgut, with significant accumulations of electrolytes also seen in the luminal fluid of the hepatopancreas. Low fluid volumes in the foregut and hindgut suggest macromolecular association as the most plausible mechanism of ion sequestration. Evidence refutes ion excretion and haemocyte sequestration as osmoregulatory mechanisms. Sequestration of Na(+) as urate salts, as shown for Periplaneta and generally assumed for other insects, is insignificant in isopods.     

Osmotically-induced alterations in volume and ultrastructure of mitochondria isolated from rat liver and bovine heart

Detailed studies correlating changes in mitochondrial optical density, packed volume, and ultrastructure associated with osmotically-induced swelling were performed. Various swelling states were established by incubating mitochondria (isolated in 0.25 M sucrose) at 0°C for 5 min in series of KCl and sucrose solutions ranging in tonicity from 250 to 3 milliosmols. Reversibility of swelling was determined by examining mitochondria exposed to 250 milliosmols media after they had been induced to swell. Swelling induced by lowering the ambient tonicity to approximately 130 (liver mitochondria) and 90 (heart mitochondria) milliosmols involves primarily swelling of the inner compartment within the intact outer membrane. Decreasing the ambient tonicity beyond this level results in rupture of the outer membrane and expansion of the inner compartment through the break. The maximum extent of swelling, corresponding with complete unfolding of the cristae and an increase in over-all mitochondrial volume of approximately 6-fold (liver mitochondria) and 11-fold (heart mitochondria), is reached at approximately 15 (liver mitochondria) and 3 (heart mitochondria) milliosmols. Exposure of liver mitochondria to media of lower tonicity results in irreversibility of inner compartment swelling and escape of matrix material. These changes appear to result from increased inner membrane permeability, possibly due to stretching.     

Changes in muscle activity in response to different impact forces affect soft tissue compartment mechanical properties

Electromyographic (EMG) activity is associated with several tasks prior to landing in walking and running including positioning the leg, developing joint stiffness and possibly control of soft tissue compartment vibrations. The concept of muscle tuning suggests one reason for changes in muscle activity pattern in response to small changes in impact conditions, if the frequency content of the impact is close to the natural frequency of the soft tissue compartments, is to minimize the magnitude of soft tissue compartment vibrations. The mechanical properties of the soft tissue compartments depend in part on muscle activations and thus it was hypothesized that changes in the muscle activation pattern associated with different impact conditions would result in a change in the acceleration transmissibility to the soft tissue compartments. A pendulum apparatus was used to systematically administer impacts to the heel of shod male participants. Wall reaction forces, EMG of selected leg muscles, soft tissue compartment and shoe heel cup accelerations were quantified for two different impact conditions. The transmissibility of the impact acceleration to the soft tissue compartments was determined for each subject/soft tissue compartment/shoe combination. For this controlled impact situation it was shown that changes in the damping properties of the soft tissue compartments were related to changes in the EMG intensity and/or mean frequency of related muscles in response to a change in the impact interface conditions. These results provide support for the muscle tuning idea--that one reason for the changes in muscle activity in response to small changes in the impact conditions may be to minimize vibrations of the soft tissue compartments that are initiated at heel-strike.     

Vacuolar reorganization in the motor cells of Albizzia during leaf movement

During the leaf movements of Albizzia julibrissin Durazzini, volume changes in the motor cells of the pulvinule (tertiary pulvinus) are closely correlated with a reversible reorganization of the vacuolar compartment. Motor cells have central vacuoles when expanded, but become multivacuolate during the time the cell volume decreases. The central vacuole reforms — apparently by fusion of small vacuoles — during motor-cell expansion. The volume changes of the vacuolar compartment account for all of the change in the size of the protoplast, while the cytoplasmic volume remains constant during the leaf movements.     

EFFECT OF SHORT-TERM SHADING ON THE CYTOLOGY OF PALISADE TISSUE IN MATURE LEAVES OF THE SUNFLOWER HELIANTHUS ANNUUS

Mature sunflower leaves were exposed to partial shading (35 or 14% of normal sun) or darkness (0% of normal sun) for approximately 8 hr. During this period one-half of each test leaf was shaded; the other half was used as a normal sun control. Palisade cell structure from both halves of each leaf was compared. Shading of leaves had little effect on organelle percent volume values (V_v) with exception of the starch compartment which decreased as shading increased. The surface to volume ratio (S_v) of the chloroplast thylakoids increased while the S_v of the mitochondrial membranes decreased as shading increased. Palisade cell volume did not change in shaded portions of the leaf, except in the fully shaded (dark) tissues where cell volume decreased. Changes in the actual volume of organelle compartments were strongly correlated with changes in cell volume. Thus a general osmotic response may account for some of the volume changes associated with differences in light intensity. Shading increased thylakoid surface areas 10–30% over the full sun controls. The ratio of stromal to granal thylakoid surface area remained constant in both the control and partially shaded samples. However, in darkened samples this ratio decreased as stromal membranes increased more than granal membranes. Changes observed in thylakoid surface areas associated with shading did not support thylakoid models which propose the interconversion of granal membranes to stromal membranes and vice versa.