Hypotonic Treatment Research Articles

Preservation of cytoplasm in the human lymphocyte micronucleus assay.

A major limitation in the quantitative accuracy of the human lymphocyte micronucleus (MN) assay is preservation of the cytoplasm during the cell harvesting. In this short communication, an improved method for cytoplasm preservation in a cytokinesis-blocked, whole-blood microculture (0.3 ml) technique is described. We believe that the timing of the hypotonic treatment, speed of centrifugation, handling of the cell suspension and proper Giemsa staining are important variables in the human peripheral lymphocyte MN assay.

Isolation of single mammalian proximal tubule cells: effects of hypotonic shocks on cell yield and function.

Nord et al. [Am J Physiol 1986; 250:F539-F550] proposed a method to give a high yield of proximal tubule cells by exposing a suspension of rabbit cortical tubules to a hypotonic shock in calcium-free media. The present study describes the effects of both amplitude and duration of the hypotonic treatment on some transport-related characteristics of individual cells as compared to the starting tubule suspension. The averaged cell yield increased by an order of magnitude when the osmolality of the hypotonic solution was varied in four steps from 200 (C200 cells) to 70 mosm/kg H2O (C70 cells) while the proportion of trypan blue-positive cells progressively decreased from 33% for C200 cells to 9.5% for C70 cells. An increase in duration of the hypotonic shock from 0.5 to 6 min did not change the cell yield of C200 cells while it significantly increased that of C70 cells by 61%. Basal and ouabain-sensitive oxygen consumption (QO2) increased by 57 and 155%, respectively, from C70 to C200 cells but was approximately one order of magnitude smaller than the QO2 measured for tubule suspension. Intracellular ATP content averaged 5.5 +/- 0.8 nmol/mg for the starting tubule suspension, 4.6 +/- 0.8 nmol/mg for C70 cells but only 1.3 +/- 0.1 nmol/mg for C200 cells. The maximal velocity for phloridzin-sensitive alpha-methyl glucose transport averaged 13.7 +/- 1.7 nmol min-1 mg-1 for C70 cells and only 6.3 +/- 1.3 nmol min-1 mg-1 for C200 cells which is approximately one order of magnitude smaller than what can be expected from a tubule presenting a good access to luminal membrane. We conclude from these results that, in the process of isolating individual cells from a polarized epithelium, membrane transport rates have decreased by one order of magnitude and this reduction is intensified by a large hypotonic shock. In comparison with C200 cells, the cells obtained with a large hypotonic shock give a high yield, a larger proportion of trypan blue-negative cells and their lower overall transport rate allows the cells to maintain a better electrochemical gradient for Na and a higher intracellular ATP level.

Volume regulation in human fibroblasts: role of Ca2+ and 5-lipoxygenase products in the activation of the Cl- efflux.

Trypsinized human skin fibroblasts in suspension perform regulatory volume decrease (RVD) after cell swelling in hypotonic medium. During RVD, 36Cl- efflux is dramatically increased and the cell membrane is depolarized, indicating the activation of Cl- channels. This activation of Cl- channels depends on extracellular as well as on intracellular Ca2+. The swelling-induced Cl- efflux and the RVD response are inhibited by the 5-lipoxygenase inhibitor ETH 615-139. Finally, following hypotonic treatment, cellular pH decreases. The pH decrease does not involve the Cl-/HCO3- exchange because it is independent of the external Cl- concentration.

Characteristics of endothelin receptors and intracellular signalling in porcine inner medullary collecting duct cells.

In porcine kidneys we investigated the characteristics of endothelin (ET) receptors that are present in papillary tissue but not in glomeruli. Therefore, porcine inner medullary collecting duct (IMCD) cells were separated by Percoll density gradient centrifugation after enzymatic and hypotonic treatment of minced papillary tissue. Studies were performed in fresh cell suspensions and in cells in primary culture. Changes in cytosolic free Ca2+ concentration [Ca2+]i were measured by the use of fura-2. Optimum binding of ET-1 was obtained by incubation for 120 min at 37 degrees C, pH 7.0 when maximal protein content was 40 micrograms. Analysis with the LIGAND program showed an average number of binding sites (Bmax) of 26.0 +/- 30.5 fmol/mg protein and dissociation constant (Kd) of 90.5 +/- 28.6 pmol/L for ET-1 and Bmax of 246.9 fmol/mg protein and Kd of 162.5 pmol/L for ET-3. ET-1, 10(-9) to 10(-6) mol/L, dose dependently raised [Ca2+]i four to tenfold, respectively, from a mean basal level of 41 nmol/L. This rise was significantly attenuated by TMB-8 and by verapamil. Preincubation with Ni2+ almost completely prevented the increment in [Ca2+]i. ET-1 slightly suppressed basal and significantly attenuated arginine vasopressin (AVP)-induced cyclic adenosine monophosphate (cAMP) synthesis. Thus, porcine IMCD cells possess a single class of super high affinity ETB receptors (ETB1). ET-1 raises [Ca2+]i through release from intracellular stores, activation of L-type calcium channels and, probably to a larger extent, through stimulation of other channels, eg, T-type calcium channels or unselective cation channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypotonic cisplatin treatment for carcinomatous pleuritis found at thoracotomy in patients with lung cancer: In vitro experiments and preliminary clinical results

We have developed an intraoperative intrapleural treatment with the use of distilled water combined with cisplatin for carcinomatous pleuritis found at thoracotomy in patients with non-small-cell lung cancer. In the in vitro experiment, three different cell lines were used as a model of malignant pleural effusion. Cell growth was examined after a 3-day culture, which was preceded by exposure of the cells to cisplatin in either phosphate-buffered saline solution or distilled water for 1/2 to 5 minutes. The growth inhibition of tumor cells after hypotonic cisplatin treatment was significantly greater than after treatment with saline solution and cisplatin. Tumor that was obtained by resection of non-small-cell lung cancer was used as a model to demonstrate decreased viability of the tumor after exposure to hypotonic cisplatin. The viability of the tumor in a 6-day culture, preceded by exposure to hypotonic cisplatin (50 micrograms/ml) for 10 minutes, was markedly decreased. Intraoperative intrapleural hypotonic cisplatin was instilled in seven patients with pleural carcinomatosis without side effects and with control of pleural dissemination and pleural effusion for 6 to 29 months.

Mechanisms of regulatory volume decrease in UC-11MG human astrocytoma cells.

Swelling of astrocytes commonly occurs after cerebral ischemia and other brain injuries. Because these cells constitute 20-25% of human brain volume, their swelling is a major factor in the morbidity and mortality associated with cerebral edema. Many cells, including astrocytes, resist or reverse the tendency to swell by activating transport pathways that lead to a regulatory volume decrease. Here we report the results of studies designed to elucidate the mechanisms of the regulatory volume decrease that occurs after astrocytes are swollen by exposure to hypotonic medium. Using UC-11MG cells, a well-characterized, human, astrocytoma-derived line, we observed an increase in membrane permeability to both K+ and Cl- during regulatory volume decrease, consistent with a net loss of these ions. Neither the increase in K+ exit nor the regulatory volume decrease was affected by bumetanide, an inhibitor of anion-cation cotransport. On the other hand, the increased K+ efflux, as well as the regulatory volume decrease, was blocked by Gd3+, suggesting a putative role of stretch-activated cationic channels in the process of volume regulation. Although increases in intracellular free Ca2+ were also observed during hypotonic treatment, they occurred well after the onset of the regulatory volume decrease. Furthermore, the regulatory volume decrease was not affected by blocking the intracellular free Ca2+ increase with dimethyl 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or by removal of extracellular Ca2+. These results indicate that the regulatory volume decrease in UC-11MG cells may involve stretch-activated channels that operate independently of changes in intracellular free Ca2+.

Human spermatozoal tail hypo-osmotic swelling test, motility characteristics in hypotonic saline, and survival of spermatozoa after cryopreservation.

Normozoospermic semen samples (n = 82) were examined to investigate whether the degree of sperm tail swelling in hypo-osmotic medium (fructose and sodium citrate; 150 mOsm/l), and motility characteristics after a 15-min exposure to hypotonic saline (sodium chloride; 150 mOsm/l) could predict the cryosurvival rate (% post-thaw motility/% pre-freeze motility x 100%) of spermatozoa after cryopreservation by the liquid nitrogen vapour freezing method using the TEST-glycerol-egg yolk buffer. The CellSoft automated semen analyser was used to analyse sperm motility in pre-freeze and post-thaw semen samples, and after exposure to hypotonic saline. Sperm tail hypo-osmotic swelling and sperm motility in pre-freeze semen showed no significant correlations (P > 0.05) with the cryosurvival rate. There were significant correlations (P < 0.05) between the cryosurvival rate and the following sperm motility parameters in hypotonic saline: % motility (r = 0.2846), motility index (% motility x curvilinear velocity; r = 0.2809) and % decrease in motility index from the baseline value in semen (r = 0.3378). The % decrease in motility index after hypotonic saline treatment was significantly less (P < 0.05) in the normal (> or = 50% cryosurvival rate; mean +/- SEM 5.9 +/- 3.2%; n = 33) compared with the subnormal (< 50% cryosurvival rate; 27.3 +/- 4.8%; n = 49) cryosurvival groups. This parameter was also determined, by multivariate discriminant analysis, to be capable of classifying each pre-freeze semen sample into either cryosurvival group with 69.5% accuracy.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical properties of ciliary axonemes and membranes as shown by paddle cilia.

Cilia with a distal membrane expansion enclosing a coiled end of the axoneme (paddle cilia or discocilia) have been commonly reported in marine invertebrates. We recently showed that paddle cilia in molluscan veligers are artifacts of non-physiological conditions. Here we investigated the possible mechanisms of formation of paddle cilia under hypotonic conditions; particularly, whether a helical conformational change of doublet microtubules induced by Ca or proton flux is responsible. Typical paddle cilia are induced by hypotonic Ca-free solutions at normal or low pH, showing that axonemal coiling does not require Ca influx or proton efflux. In addition, Triton-demembranated straight axonemes do not coil in high Ca solutions. Most decisively, complete removal of paddle ciliary membranes with detergents, but not mere permeabilization, causes immediate uncoiling and straightening of the axonemes to approximately their original length before hypotonic treatment. These findings and other data show that axonemal coiling in paddles is due to membrane tensile stress acting on an elastic axoneme. Light and electron microscopy of paddles show that axonemes coil uniformly toward the direction of the effective stroke (doublets nos 5-6), even when beating is inhibited by sodium azide or glutaraldehyde before hypotonic treatment. This indicates that axonemes possess an intrinsic asymmetry of stiffness within the beat plane, independent of active microtubule sliding. Paddle cilia thus reveal important mechanical properties of ciliary axonemes and membranes that should be useful for understanding ciliary function.

Chromosomes lacking kinetochore proteins: their meta-anaphase location suggests potential malsegregation.

We have previously reported that a rare chromosome may not carry the kinetochore protein complex--the CENtromere Proteins or CENPs. These chromosomes should not bind to spindle microtubules and, hence, should be found peripheral to the meta-anaphase arrangement exhibited by the chromosomes which do carry CENPs. This communication shows that this actually is the case. When 3T3 mouse cells were not treated with colcemid or hypotonic, the kinetochore-lacking (K-) as well as kinetochore-bearing (K+) chromosomes were found off the spindle zone. When the spindle is disrupted with mild hypotonic treatment or by colcemid, the frequency of K- chromosomes remains unchanged. However, even mild disruption of the spindle with hypotonic treatment increases the frequency of off-lying K+ chromosomes significantly. These data indicate that K- chromosomes do not bind to the spindle and, hence, are a factor in the genesis of aneuploidy. A considerable proportion of K- chromosomes carry the putative centromere DNA indicating that these are not acentric fragments. Since the CREST serum used recognizes all essential kinetochore proteins, the K- centromeres must also lack all essential CENPs.

Morphology of nexin links in relation to interdoublet sliding in the sperm flagellum

In this work, we examine whether the "nexin" linkages of the flagellum can extend in length to accommodate interdoublet sliding. Flagellar bends of large angle were induced in bull spermatozoa by hypotonic treatment. It is argued that this produces large interdoublet displacements that are, nevertheless, still within physiological limits. Such flagella were examined by the rapid-freeze, deep-etch technique and the nexin linkages identified by their position in relation to the inner dynein arms and by their straplike, bipartite, morphology. They were found to bridge perpendicularly (or occasionally at an angle) between the A- and B-tubules of adjacent doublets. The nexin linkages were no more than approximately nm in length, even in regions in which approximately 200 nm of sliding could be inferred. Variable registration between adjacent nexin rows gave some further support to the assumption that sliding had indeed taken place. From this, it is concluded that elastic deformation of the links, such as would accommodate interdoublet sliding, does not occur; some form of displacement must occur between nexin and the adjacent B-tubule.

Mitosis of rotenone-induced endoreduplication in Chinese hamster cells.

Endoreduplication was induced by rotenone with an extremely high frequency (approximately 90% of all the metaphases) in cultured Chinese hamster cells. Endoreduplicated cells were fixed without colchicine or hypotonic treatment, and chromosomal configurations were examined in various mitotic stages. The two sister chromosomes of each diplochromosome at late prophase were widely separated except the centromeric region, but they became gradually paired along the total length as the cell cycle progressed to metaphase. The anaphase cells underwent multipolar division, resulting in three or four aneuploid daughter cells. Indirect immunofluorescence staining using anti-beta tubulin antibody revealed tripolar or tetrapolar spindles and unusual equatorial plates.

Use of fluorescence in situ hybridization to detect chromosome‐specific changes in exfoliated human bladder and oral mucosa cells

Change in chromosome number, numerical aneuploidy, has been consistently linked with cancer development. Since 90% of cancers arise in epithelial tissues, techniques that measure aneuploidy in these tissues would be very useful. Here we describe methods of optimization and suggest use of fluorescent in situ hybridization (FISH) to detect aneuploidy in exfoliated epithelial cells collected from the mouth and bladder. A total of 10,383 urothelial cells and 4,691 buccal cells were scored in order to determine a baseline frequency of aneuploidy in human volunteers using a classical satellite probe for chromosome 9. Protein digestion with pepsin was found to be more efficient at removing the keratinized cell membrane and optimizing probe penetration than acid washes, detergent washes, or hypotonic treatments. A 20 min cellular digestion with 200 micrograms/ml and a 30 min digestion with 300 micrograms/ml of pepsin in 0.01 M HCl optimized probe penetration in urothelial and buccal cells, respectively. Average frequencies for 0, 1, 2, 3, and 4 hybridization regions were 10.3, 10.1, 78.4, 1.0, and 0.3% for urothelial cells and 8.8, 9.8, 79.4, 1.3, and 0.3% for buccal cells, respectively. These results are very similar to those previously described in lymphocytes. The urothelial cells of males had a lower frequency of diploid cells and a higher frequency of cells without hybridization regions than females (P < 0.02). No statistically significant variability was found between individuals or sex groups in buccal cells. Our data show that FISH is a useful tool to detect changes in frequency of aneuploidy in exfoliated epithelial cells and has good potential for monitoring human populations exposed to genotoxic agents.

Revealing nucleolar architecture by low ionic strength treatment

The internal nucleolar architecture of HeLa cells was revealed after a short hypotonic treatment. The response of nucleoli to a gradual reduction in the ionic strength of incubation buffer was assessed by immunofluorescence, confocal microscopy using human autoimmune sera monospecific for antigens present in different nucleolar components, and electron microscopy. The granular component dispersed first, followed by the dense fibrillar component, leaving distinct fibrillar centers remaining. This demonstrates differential sensitivity to low ionic strength treatment in the transcription and the maturation territories of the nucleoli. The changes described develop in only a few minutes and this approach can reveal momentary in situ intranucleolar arrangements. We suggest that the fibrillar centers provide a structural support for RNA-polymerase I complexes and are possibly also attached to a nuclear skeleton. The evidence presented implicates the fibrillar centers as the core elements of nucleoli and that functional nucleoli arise around them.

Calcium -regulated channels and their bearing on physiological activities in Characean cells

Calcium is involved in the regulation of cytoplasmic streaming, membrane excitation, turgor regulation and salt tolerance in the giant internodal cells of the Characeae. To analyse the mechanism of Ca 2+ action, model systems were used, namely, tonoplast-free and plasma m em brane-perm eabilized cells. In the former, the plasma membrane remained intact and its activity could be investigated by m anipulating the cytoplasmic and external media, whereas in the latter, the tonoplast remained intact and its activity could be studied by altering the bathing solution. Studies using these model systems have established the presence of voltage-dependent Ca 2+ -channels in the plasma membrane and Ca 2+ - dependent ion channels in both the plasma membrane and the tonoplast. To further analyse Ca 2+ action on the basis of single channel activities, patch-clam p techniques were applied to plasmolysed protoplasts and isolated cytoplasmic drops. Channel activities were measured using both cell-attached and excised membrane patch modes. A fresh-water member of the Characeae, Nitellopsis , becomes salt-tolerant if millimolar amounts of Ca 2+ are present in the external medium. Under these conditions, excised patches of the plasma membrane exhibit K + -channel activity with unitary conductances of 25-50 pS and a permeability ratio ( P Na / P K ) of 0.28. These K + channels were closed by external Ca 2+ when ATP was present on the cytoplasmic side of the membrane. ATP could be replaced with AMP, which suggests that ATP acted neither as an energy source nor as a substrate for protein phosphorylation, but rather as an effector. In the tonoplast, K + channels having a unitary conductance of 75 pS and a P Na / P K ratio of 0.2 were not activated by Ca 2+ when it was present on the cytoplasmic side of the excised patches, but these channels were activated by Ca 2+ injected into the cytoplasmic drop, which suggested the involvement of an unknown cytoplasmic factor(s) that mediates the Ca 2+ signal. The brackish water Characeae, Lamprothamnium , can regulate elevated turgor induced by hypotonic treatment only when millimolar amounts of Ca 2+ are present in the external medium. In this situation, elevated turgor may first activate Ca 2+ channels and the increased level of Ca 2+ in the cytoplasm may then activate K + and Cl - channels in both the plasma membrane and the tonoplast. In the cytoplasmic-drop-attached mode, single K + channel current-voltage measurements established that the K + channel exhibited a unitary conductance of 50 pS for negative shifts of the voltage, while under positive shifts in the voltage 100 pS channel conductance was observed. The channel with a P Na / P K of 0.02 is highly selective for K + against Na + and this channel is directly activated by Ca 2+ added to the cytoplasmic side of the excised patch. These results suggest that, in Nitellopsis and Lamprothamnium , Ca 2+ regulation of channels in both the plasma membrane and the tonoplast may form the molecular basis for Ca 2+ -regulated physiological functions such as salt tolerance and turgor regulation in characean cells. The mode of Ca 2+ regulation is discussed in light of current findings.

Activation of the Na +/K +/Cl/ − contransport system by reorganization of the actin filaments in Ehrlich ascites tumor cells

Reorganization (disassembly) of the actin filaments in Ehrlich ascites tumor cells, either by hypotonic treatment in the presence of Ca 2+ or by addition of cytochalasin B, results in activation of the Na +/K +/Cl − cotransport system. However, other regulatory processes, some of which may be dependent on an intact filament system, are responsible for the activation of the Na +/K +/Cl − cotransport system after cell shrinkage.

Correlative light and electron microscopy of primary (9+0) cilia: Response of ciliary varicosities to hypotonic treatment in kidney epithelial cells

Primary cilia are centrosomal appendages which in kidney epithelia extend several micrometers from the apical cell surface into the lumen of the kidney tubule. Although considered by some to be “rudimentary appendages” because they are nonmotile, their intimate structural association with the Golgi apparatus and nucleus suggests to others that they mediate interactions between the cell and its environment. Such a sensory role is not without precedence since various photo- and chemoreceptors are highly modified primary cilia. Despite the near-ubiquitous distribution of primary cilia few studies have addressed their function experimentally, and little detailed information is available regarding their behaviorin vivo.

Net efflux of Cl− during hypotonic turgor regulation in a brackish water alga Lamprothamnium*

Abstract. Net efflux of Cl− was measured potentiometrically (Ag/AgCl electrode) during turgor regulation which was induced by hypotonic treatment (hypotonic turgor regulation) in the alga Lamprothamnium succinctum. The efflux of Cl− reached the peak value (11 μmol m −2s−1) several minutes after the hypotonic treatment was started and then declined. The efflux of Cl− and inhibition of the cytoplasmic streaming [reflection of an increase in cytoplasmic concentration of free Ca2+([Ca2+]c)] were blocked under a low external concentration of Ca2+ ([Ca2+]e) (0·01 mol m−3) and resumed after raising [Ca2+]e to the normal value (3·9 mol m−3). The decrease in cell‐osmotic pressure upon hypotonic treatment was inhibited by lowering either turgor pressure or [Ca2h]e. The inhibition was reflected in decreases of both the efflux of Cl− and the membrane conductance. Recovery of the cytoplasmic streaming from the inhibition was also accelerated by the same treatments. It is concluded that an increase in turgor pressure is continuously sensed by the cells and that continuous presence of external Ca2+ is necessary for the hypotonic turgor regulation.

Partitioning of the Chicken Genome by Microcell Hybridization

Chicken fibroblast cells isolated from 7-day-old White Leghorn S-line chicken embryos were used for microcell hybridization. Cells transfected with a pSV2-neo plasmid, which carries the Neor gene and confers resistance to geneticin (G418), were selected and maintained in medium containing G418 (350 μg/mL). Cells were micronucleated by colcemid arrest and hypotonic treatment. Enucleation was carried out by centrifugation in a Percoll gradient in the presence of cytochalasin B. Purified microcells and HeLa S3 cells were mixed and agglutinated by addition of phytohemagglutinin P, followed by polyethylene glycol fusion to generate microcell hybrids. Chicken by human microcell hybrids were selected in RPMI-1640 medium containing 1.4 mg/mL G418. Cloned hybrid cell lines were maintained in the same medium containing 7. mg/mL of G418. The presence of chicken chromosomes in hybrid cells was demonstrated by cytogenetic analysis and high-resolution nonisotopic chromosomal in situ hybridization. Three out of 28 hybrid cell lines analyzed retained single chicken chromosomes.

Mechanisms of dramatic fluctuations of ornithine decarboxylase activity upon tonicity changes in primary cultured rat hepatocytes

We investigated the mechanisms underlying the marked induction of ornithine decarboxylase (ODC) activity by hypotonic treatment and its rapid decay upon reversal to isotonicity in primary cultures of adult rat hepatocytes. Upon hypotonic treatment, ODC synthesis rate increased progressively whereas the amount of ODC mRNA increased only about twofold. In addition, ODC was stabilized severalfold. ODC activity rapidly decreased upon restoration of isotonicity, owing to immediate and nearly complete suppression of ODC synthesis and 3-6-fold stimulation of ODC decay. The stimulation of ODC decay caused by restoration of isotonicity was mostly independent of time and protein synthesis. ODC decay was also stimulated by putrescine, even under hypotonic conditions, depending on time and new protein synthesis. Restoration of isotonicity and putrescine treatment together caused a synergistic stimulation of ODC decay, confirming that these act by different mechanisms.

Stimulation of superoxide anion production in guinea pig polymorphonuclear leukocytes by hypotonic conditions in combination with protein kinase C activators

Conditions for Superoxide anion (O − 2) production were examined in guinea pig polymorphonuclear leukocytes (PMNL). When PMNL were suspended in the hypotonic medium, O − 2 production was significantly enhanced by concurrent treatment with low concentrations of 1-oleoyl-2-acetylglycerol (OAG), a cell-permeable protein kinase C activator. Such hypotonicity or OAG alone had little effect on the production. Other protein kinase C activators also markedly enhanced O − 2 production in combination with hypotonicity, but not in the isotonic medium. Protein kinase C inhibitors, H-7 and staurosporine, dose-dependently inhibited the production. These observations indicate that protein kinase C participates in such synergistic O − 2 production with hypotonicity. Phosphorylation of 46-kDa protein(s), which was commonly enhanced in parallel with an activation of NADPH oxidase in guinea pig PMNL, was increased by treatment with 10 μ m OAG, but the phosphorylation was little altered by hypotonic treatment. Intracellular calcium concentration, arachidonate release, and 1,2-diacylglycerol and phosphoinositide concentrations were slightly altered by hypotonic treatment. A change in phosphatidate (PA) production in PMNL was induced by hypotonic treatment either by itself or in combination with OAG treatment. These results suggest that the combination of cell membrane changes by hypotonic treatment accompanied by the increase in PA and 46-kDa protein phosphorylation by protein kinase C provides the conditions required for a marked increase in O − 2 production. Hypotonicity may be a good tool for studying the mechanism of priming in the activation of NADPH oxidase.