Volume-sensitive K transport in human erythrocytes

Studies have been carried out on human erythrocytes to examine the alterations of K transport induced by swelling or shrinking the cells by osmotic and isosmotic methods. Hypotonic swelling of erythrocytes (relative cell volume, 1.20) resulted in a striking, four- to fivefold augmentation in the ouabain-resistant K influx over the value obtained at a normal cell volume. Shrinking the cells in hypertonic media resulted in a small but statistically significant reduction in K influx. Three different methods of varying cell volume gave similar results. These include the addition of sucrose and of NaCl to hypotonic media and the isosmotic (nystatin) method. The major fraction of the K influx in swollen cells is specific in its requirement for Cl or Br and is not supported by thiocyanate, iodide, nitrate, methylsulfate, or acetate. Bumetanide (0.1 mM), MK-196 (0.2 mM), and piretanide (1 mM) are poorly effective in suppressing K uptake in swollen cells, but at higher concentrations, bumetanide (1 mM) inhibits 80% of the Cl-dependent K influx in swollen cells. The bumetanide concentration required to inhibit 50% of the Cl-dependent K influx is 0.17 mM. The volume-sensitive K influx is independent of both extracellular and intracellular Na, so that the (Na + K + 2Cl) cotransport pathway is not a likely mediator of the volume-sensitive K transport. A variety of inhibitors of the Ca-activated K channel are ineffective in suppressing swelling-induced K influx. Like K uptake, the efflux of K is also enhanced by cell swelling. Swelling-activated K efflux is Cl dependent, is independent of extracellular and intracellular Na, and is observed with both hypotonic and isosmotic methods of cell swelling. The activation of K efflux by cell swelling is observed in K-free media, which suggests that the volume-sensitive K transport pathway is capable of net K efflux. The addition of external K to hypotonic media resulted in an increase in K efflux compared with the efflux in K-free media, and this increase was probably due to K/K exchange. Thus, hypotonic or isosmotic swelling of human erythrocytes results in the activation of a ouabain-resistant, Cl-dependent, Na-independent transport pathway that is capable of mediating both net K efflux and K/K exchange.     

Ionic basis of volume regulation in mammalian cells following osmotic shock

Previous studies with mammalian cultured cells have shown that volume regulation in hypotonic medium requires active Na transport. In the present study, determinations of intracellular Na and K content were made in cultured mouse lymphoblasts during the process of swelling and subsequent shrinking (volume regulation) in hypotonic medium. Na and K content were measured in cells in which the shrinking phase was inhibited by the cardiac glycoside, ouabain. In osmotically-shocked cells, an initial permeability increase to K, and not Na, was observed, which allowed K to diffuse out rapidly, down its gradient. Na, meanwhile, rapidly flowed inward with water entry during the swelling process, and was later lost with the same kinetics as the cell shrinkage. This loss of Na was prevented in the presence of ouabain. The results imply that volume regulation is achieved by pumping Na gained during swelling out of the cells, while any K taken up by the pump is rapidly lost through a more permeable membrane. The loss of osmotically active Na, presumably with accompanying anions, allows water to passively diffuse down its osmotic gradient, reducing cell volume subsequent to the initial passive swelling, during which K was rapidly lost.     

Cyto B dependent and ouabain insensitive regulatory volume decrease in bicellular mouse embryo

Mouse single-cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to determine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicellular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three-dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco's experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van't Hoff law defines swelling phase with the effective hydraulic conductivity of 0.3 micron x min(-1) x atm(-1). Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment.     

Osmotic loading of in situ chondrocytes in their native environment

Changes in the osmotic environment cause changes in volume of isolated cells and cells in tissue explants, and the osmotic environment becomes hypotonic in cartilage diseases such as osteoarthritis (OA). However, it is not known how cells respond to a hypotonic osmotic challenge when situated in the fully intact articular cartilage. A confocal laser scanning microscope was used to image chondrocytes of intact rabbit patellae in an isotonic (300 mOsm) and hypotonic (172 mOsm) immersion medium. Cell volumes were calculated before and 5, 15, 60, 120 and 240 minutes after the change in saline concentration. Local tissue strains and swelling of the entire tissue were estimated from the relative movements of cells and displacements of single cells, respectively. Cell volumes increased rapidly (< or = 5 minutes, p<0.05) by approximately 22%, after which they remained constant for an hour (p>0.05). However, two and four hours post the hypotonic challenge, cell volumes were statistically greater (p<0.05) than those at all earlier time points, and swelling of the entire tissue continued throughout the four hour loading period. The results of our study suggest that osmotic loading induced volume changes of in situ chondrocytes in their native environment occur quickly and continue for hours. Understanding the behaviour of cells in their native environment provides novel insigth into the cell mechanics in ostearthritic joints and so may help understand the onset and progression of this disease.     

A Method to Simultaneously Detect Changes in Intracellular Ca<Superscript>2+</Superscript> Concentration and Cell Volume

A method to simultaneously assess the changes in intracellular calcium concentration and cell volume in single cells was developed using the Ca²⁺-sensitive fluorescent probe Fura-2 and a three-dimensional image-surface reconstruction technique, respectively. Studies with this method showed that Fura-2 loading had no significant effect on the kinetics of A549 human epithelial cell swelling in a hypotonic solution, as well as the volume restoration kinetics. Significant changes in intracellular Ca²⁺ concentration were not observed in the examined volume modulation range. The results suggest that Ca²⁺-mediated signaling pathways are not involved in the autoregulation of the cell volume in A549 cells exposed to hypotonic conditions.     

ULTRASTRUCTURAL STUDIES OF VASOPRESSIN EFFECT ON ISOLATED PERFUSED RENAL COLLECTING TUBULES OF THE RABBIT

Isolated cortical collecting tubules from rabbit kidney were studied during perfusion with solutions made either isotonic or hypotonic to the external bathing medium. Examination of living tubules revealed a reversible increase in thickness of the cellular layer, prominence of lateral cell membranes, and formation of intracellular vacuoles during periods of vasopressin-induced osmotic water transport. Examination in the electron microscope revealed that vasopressin induced no changes in cell structure in collecting tubules in the absence of an osmotic difference and significant bulk water flow across the tubule wall. In contrast, tubules fixed during vasopressin-induced periods of high osmotic water transport showed prominent dilatation of lateral intercellular spaces, bulging of apical cell membranes into the tubular lumen, and formation of intracellular vacuoles. It is concluded that the ultrastructural changes are secondary to transepithelial bulk water flow and not to a direct effect of vasopressin on the cells, and that vasopressin induces osmotic flow by increasing water permeability of the luminal cell membrane. The lateral intercellular spaces may be part of the pathway for osmotically induced transepithelial bulk water flow.     

Osmotic Stress Alters the Intracellular Distribution of Non-erythroidal Spectrin (Fodrin) in Bovine Aortic Endothelial Cells

Cell swelling is known to result in unfolding of membrane invaginations and restructuring of F-actin. The effect of cell swelling on the intracellular distributions of other cytoskeletal proteins that constitute the submembrane cortical cytoskeleton is virtually unknown. This study focuses on the effects of cell swelling on non-erythroidal spectrin (fodrin, also known as spectrin II), a predominant component of the membrane cytoskeleton. The intracellular distribution of spectrin in vascular endothelial cells was studied by optical sectioning using a 3-D deconvolution microscopy system. Our results show that once bovine aortic endothelial cells (BAECs) reach confluency, the non-erythroidal spectrin is localized in the submembrane regions of the cells. Analysis of the intensity profiles of the non-erythroidal spectrin under isotonic and hypotonic conditions show that: (a) the width of the submembrane spectrin staining increases gradually with time within the first 5 minutes after the osmotic shock; (b) significant recovery is observed after 10 minutes even if the cells are maintained in hypotonic medium, and (c) spectrin distribution is altered by disrupting F-actin with latrunculin A but not by stabilizing F-actin with jasplakinolide. We suggest that cell swelling results in partial translocation of the submembrane spectrin to the cytosol and that it may play a major role in initiation of swelling-induced cellular events.     

Evidence for sustained ATP release from liver cells that is not mediated by vesicular exocytosis

Extracellular ATP regulates many important cellular functions in the liver by stimulating purinergic receptors. Recent studies have shown that rapid exocytosis of ATP-enriched vesicles contributes to ATP release from liver cells. However, this rapid ATP release is transient, and ceases in ~30 s after the exposure to hypotonic solution. The purpose of these studies was to assess the role of vesicular exocytosis in sustained ATP release. An exposure to hypotonic solution evoked sustained ATP release that persisted for more than 15 min after the exposure. Using FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide) fluorescence to measure exocytosis, we found that hypotonic solution stimulated a transient increase in FM1-43 fluorescence that lasted ~2 min. Notably, the rate of FM1-43 fluorescence and the magnitude of ATP release were not correlated, indicating that vesicular exocytosis may not mediate sustained ATP release from liver cells. Interestingly, mefloquine potently inhibited sustained ATP release, but did not inhibit an increase in FM1-43 fluorescence evoked by hypotonic solution. Consistent with these findings, when exocytosis of ATP-enriched vesicles was specifically stimulated by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), mefloquine failed to inhibit ATP release evoked by NPPB. Thus, mefloquine can pharmacologically dissociate sustained ATP release and vesicular exocytosis. These results suggest that a distinct mefloquine-sensitive membrane ATP transport may contribute to sustained ATP release from liver cells. This novel mechanism of membrane ATP transport may play an important role in the regulation of purinergic signaling in liver cells.     

Thermal denaturation of the erythrocyte cytoskeleton alters the morphological changes associated with osmotic swelling

1. 1. The shape changes during osmotic swelling of human erythrocytes in a hypotonic medium at room temperature, at 45°C and at the denaturation temperature (49.5°C) of the cytoskeletal protein, spectrin, have been monitored by video microscopy.

2. 2. At room temperature the great majority of cells (which were discoid prior to injection of hypotonic medium) swelled to a spherical shape through an intermediate ellipsoidal form.

3. 3.At 49.5°C (where cells had cupped shapes prior to injection) the transition to the spherical form often involved a stomatocytic rather than ellipsoidal intermediate shape.

4. 4. The cupped form of the cells prior to injection did not account for the high incidence of cells swelling through a stomatocytic intermediate shape at 49.5°C.

5. 5. A theoretical treatment by Svetina and Zeks (1983) attributes the nature of the osmotic swelling transition shape to the difference in area between the outer and inner faces of the membrane. Our results would be consistent with the theoretical predictions if it is assumed that an increase in the area of the inner face of the membrane follows thermal denaturation of the cytoskeleton of cells in hypotonic medium.

Cyto B dependent and ouabain insensitive Regulatory Volume Decrease in bicellular mouse embryo

Mouse single-cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to determine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicellular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three-dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco’s experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van’ t Hoff law defines swelling phase with the effective hydraulic conductivity of 0.3 micron · min⁻¹ · atm⁻¹. Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment.     

The role of intercellular channels in the transepithelial transfer of water and sodium in the frog urinary bladder

Summary Epithelial cells of frog urinary bladders fixed in different physiological states were examined by electron microscopy. It was shown: (1) that when bladders incubated with a hypotonic mucosal medium are water-permeabilized with oxytoxin, arginine-vasotocin, cyclic 3′,5′-AMP and theophylline, this leads to a cellular swelling and the opening of intercellular channels; (2) that these effects are not observed when the transepithelial net water flow is suppressed by abolishing the external osmotic driving force; and (3) that modifications in the rate of active sodium transport do not change the morphological appearance of intercellular channels. These results are expecially discussed with respect to the localization to the intracellular site of the final effect of antidiuretic hormone on water permeability, and to the role of intercellular channels in the transepithelial transfer of water and sodium.     

Loss of cell volume regulation during metabolic inhibition in renal epithelial cells (A6): role of intracellular pH

In renalischemia, tubular obstruction induced by swelling of epithelialcells might be an important mechanism for reduction of the glomerularfiltration rate. We investigated ischemic cell swelling byexamining volume regulation of A6 cells during metabolic inhibition(MI) induced by cyanide and 2-deoxyglucose. Changes in cell volume weremonitored by recording cell thickness (T_c). Intracellular pH (pH_c) measurements were performed with thepH-sensitive probe 5-chloromethyl-fluoresceine diacetate.T_c measurements showed that MI increases cellvolume. Cell swelling during MI is proportional to the rate ofNa⁺ transport and is not followed by a volume regulatoryresponse. Furthermore, MI prevents the regulatory volume decrease (RVD) elicited by a hyposmotic shock. MI induces a pronounced intracellular acidification that is conserved during a subsequent hypotonic shock. Atransient acidification induced by a NH₄Cl prepulse causes a marked delay of the RVD in response to a hypotonic shock. On theother hand, acute lowering of external pH to 5, simultaneously with thehypotonic shock, allowed the onset of RVD. However, this RVD wascompletely arrested ~10 min after the initiation of the hyposmoticchallenge. The inhibition of RVD appears to be related to thepronounced acidification that occurred within this time period. Incontrast, when external pH was lowered 20 min before the hyposmoticshock, RVD was absent. These data suggest that internal acidificationinhibits cellular volume regulation in A6 cells. Therefore, theintracellular acidification associated with MI might at least partlyaccount for the failure of volume regulation in swollen epithelial cells.

     

Effects of Purinergic Stimulation,CFTR and Osmotic Stress on Amiloride-sensitive Na<Superscript>+</Superscript> Transport in Epithelia and <Emphasis Type="Italic">Xenopus</Emphasis> Oocytes

Both stimulation of purinergic receptors by ATP and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibit amiloride-sensitive Na+ transport and activate Cl- secretion. These changes in ion transport may well affect cell volume. We therefore examined whether cell shrinkage or cell swelling do affect amiloride-sensitive Na+ transport in epithelial tissues or Xenopus oocytes and whether osmotic stress interferes with regulation of Na+ transport by ATP or CFTR. Stimulation of purinergic receptors by ATP/UTP or activation of CFTR by IBMX and forskolin inhibited amiloride-sensitive transport in mouse trachea and colon, respectively, by a mechanism that was Cl- dependent. When exposed to a hypertonic but not hypotonic bath solution, amiloride-sensitive Na+ transport was inhibited in mouse trachea and colon, independent of the extracellular Cl- concentration. Both inhibition of Na+ transport by hypertonic bath solution and ATP were additive. When coexpressed in Xenopus oocytes, activation of CFTR by IBMX and forskolin inhibited the epithelial Na+ channel (ENaC) in a Cl- dependent fashion. However, both hypertonic and hypotonic bath solutions showed only minor effects on amiloride-sensitive conductance, independent of the bath Cl- concentration. Moreover, CFTR-induced inhibition of ENaC could be detected in oocytes even after exposure to hypertonic or hypotonic bath solutions. We conclude that amiloride-sensitive Na+ absorption in mouse airways and colon is inhibited by cell shrinkage by a mechanism that does not interfere with purinergic and CFTR-mediated inhibition of ENaC.     

Evidence for Extracellular ATP as a Stress Signal in a Single-Celled Organism

ATP is omnipresent in biology and acts as an extracellular signaling molecule in mammals. Information regarding the signaling function of extracellular ATP in single-celled eukaryotes is lacking. Here, we explore the role of extracellular ATP in cell volume recovery during osmotic swelling in the amoeba Dictyostelium. Release of micromolar ATP could be detected during cell swelling and regulatory cell volume decrease (RVD) phases during hypotonic challenge. Scavenging ATP with apyrase caused profound cell swelling and loss of RVD. Apyrase-induced swelling could be rescued by 100 μM βγ-imidoATP. N-Ethylmalemide (NEM), an inhibitor of vesicular exocytosis, caused heightened cell swelling, loss of RVD, and inhibition of ATP release. Amoebas with impaired contractile vacuole (CV) fusion (drainin knockout [KO] cells) displayed increased swelling but intact ATP release. One hundred micromolar Gd³⁺ caused cell swelling while blocking any recovery by βγ-imidoATP. ATP release was 4-fold higher in the presence of Gd³⁺. Cell swelling was associated with an increase in intracellular nitric oxide (NO), with NO-scavenging agents causing cell swelling. Swelling-induced NO production was inhibited by both apyrase and Gd³⁺, while NO donors rescued apyrase- and Gd³⁺-induced swelling. These data suggest extracellular ATP released during cell swelling is an important signal that elicits RVD. Though the cell surface receptor for ATP in Dictyostelium remains elusive, we suggest ATP operates through a Gd³⁺-sensitive receptor that is coupled with intracellular NO production.     

Cytological effects of prefixation treatment

The effects produced by prefixation treatments on cells in metaphase from 10-day mouse fetuses and from several embryonic stages of the frog were investigated. The technical value of some of these pretreatments is noted. Pretreatment with isotonic solutions (both ionic and non-ionic in the case of the mouse, ionic only in the frog) generally produced a similar effect, viz., chromosomal swelling with little effect on the spindle. A notable exception is provided by frog embryos preceding the neurula stage; spindle disorganization without chromosomal swelling was produced by pretreatment in isotonic modified Niu-Twitty solution, containing no divalent cations. Pretreatment with hypotonic solutions (both ionic and non-ionic in the case of the mouse, ionic only in the frog) generally produced several major effects, viz., despiralization of chromosomes, chromatid separation, and spindle disorganization. The conclusion is drawn from the mouse data that, in order to produce these effects, pretreating solutions must be of low osmotic pressure. Low ionic strength alone (e.g., isotonic sucrose solutions) is not sufficient. As differentiation of frog embryos progressed, pretreatments either of longer duration or with solutions of increasing degrees of hypotonicity were required to produce comparable intensities of the same effects. Many of the effects on metaphases produced by hypotonic pretreatment of frog embryos were reversible by subsequent exposure to isotonic solutions. The significance of results presented here is discussed briefly with respect to some current considerations of the macromolecular structure of chromosomes.     

The influenza virus-induced fusion of erythrocyte ghosts does not depend on osmotic forces

The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or resealed ghosts of human erythrocytes was investigated under isotonic and hypotonic conditions using a recently developed fluorescence assay (Hoekstra, D., De Boer, T., Klappe, K., Wilschut, J. (1984) Biochemistry 23, 5675-5681). The method is based on the relief of fluorescence selfquenching of the fluorescent amphiphile octadecyl rhodamine B chloride (R18) incorporated into the ghost membrane as occurs when labeled membranes fuse with unlabeled membranes. No effect neither of the external osmotic pressure nor of cell swelling on virally mediated ghost fusion was established. Influenza virus fused unsealed ghosts as effectively as resealed ghosts. It is concluded that neither osmotic forces nor osmotic swelling of cells is necessary for virus-induced cell fusion. This is supported by microscopic observations of virus-induced fusion of intact erythrocytes in hypotonic and hypertonic media. A disruption of the spectrin-actin network did not cause an enhanced cell fusion at acidic pH of about 5 or any fusion at pH 7.4.     

The effectiveness of the microsurgical removal of the nuclei in early mouse embryos depending on the time before cleavage division

The efficiency of microsurgical enucleation of zygote and two-cell mice embryos was found to increase sharply 1-1.5 h before cleavage in "late" embryos, and 5-6 h before cleavage in "early" embryos. In "late" embryos, the share of successful enucleations constitutes 80-90%, and in "early" ones, 10-30%. This phenomenon is not connected with cytoskeleton restructuring during the cell cycle, but is determined by the increase in intracellular osmotic pressure at late stages of cell cycle. This process results in the increased elasticity of the embryo and, consequently, the penetration of surgical instruments and removal of karyoplast occurs more efficiently. The artificial increase in the intracellular pressure by addition of distilled water to culture medium also increases the efficiency of microsurgical enucleation.     

Release of ATP from retinal pigment epithelial cells involves both CFTR and vesicular transport

The retinal pigment epithelium (RPE) faces the photoreceptor outer segments and regulates the composition of the interstitial subretinal space. ATP enhances fluid movement from the subretinal space across the RPE. RPE cells can themselves release ATP, but the mechanisms and polarity of this release are unknown. The RPE expresses the cystic fibrosis transmembrane conductance regulator (CFTR), and CFTR is associated with ATP release in other epithelial cells. However, an increasing number of reports have suggested that the exocytotic pathway contributes to release. In the present study, we examined the involvement of CFTR and the vesicular pathway in ATP release from RPE cells. Release from cultured human ARPE-19 cells and across the apical membrane of fresh bovine RPE cells in an eyecup was studied. A cAMP cocktail to activate CFTR triggered ATP release from fresh and cultured RPE cells. Release from both RPE preparations was largely prevented by the broad-acting blocker glibenclamide and the specific thiazolidinone CFTR inhibitor CFTR-172. The block by CFTR-172 was enhanced by preincubation and prevented ATP release with 3.5 µM IC₅₀. The rise in intracellular Ca²⁺ accompanying hypotonic challenge was prevented by CFTR-172. The vesicular transport inhibitor brefeldin A prevented ATP release after stimulation with both hypotonic and cAMP conditions, suggesting vesicular insertion was also involved. These results show an intimate involvement of CFTR in ATP release from RPE cells which can autostimulate receptors on the apical membrane to modify Ca²⁺ signaling. The requirement for both CFTR and vesicular transport pathways suggests vesicular insertion of CFTR may underlie the release of ATP. cystic fibrosis transmembrane conductance regulator; recycling endosomes; brefeldin A; autostimulation; retinal detachment     

Mechanisms of ATP release by human trabecular meshwork cells, the enabling step in purinergic regulation of aqueous humor outflow

Our guiding hypothesis is that ecto-enzymatic conversion of extracellular ATP to adenosine activates A(1) adenosine receptors, reducing resistance to aqueous humor outflow and intraocular pressure. The initial step in this purinergic regulation is ATP release from outflow-pathway cells by mechanisms unknown. We measured similar ATP release from human explant-derived primary trabecular meshwork (TM) cells (HTM) and a human TM cell line (TM5). Responses to 21 inhibitors indicated that pannexin-1 (PX1) and connexin (Cx) hemichannels and P2X(7) receptors (P2RX(7) ) were comparably important in modulating ATP release induced by hypotonic swelling, whereas vesicular release was insignificant. Consistent with prior studies of PX1 activity in certain other cells, ATP release was lowered by the reducing agent dithiothreitol. Overexpressing PX1 in HEK293T cells promoted, while partial knockdown (KD) in both HEK293T and TM5 cells inhibited hypotonicity-activated ATP release. Additionally, KD reduced the pharmacologically defined contribution of PX1 and enhanced those of Cx and P2RX(7) . ATP release was also triggered by raising intracellular Ca(2+) activity with ionomycin after a prolonged lag time and was unaffected by the PX1 blocker probenecid, but nearly abolished by P2RX(7) antagonists. We conclude that swelling-stimulated ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels and P2X(7) receptors, but not by vesicular release. PX1 appears not to be stimulated by intracellular Ca(2+) in TM cells, but can be modulated by oxidation-reduction state. The P2RX(7) -dependent component of swelling-activated release may be mediated by PX1 hemichannels or reflect apoptotic magnification of ATP release, either through itself and/or hemichannels.     

Cytological Effects of Prefixation Treatment

The effects produced by prefixation treatments on cells in metaphase from 10-day mouse fetuses and from several embryonic stages of the frog were investigated. The technical value of some of these pretreatments is noted. Pretreatment with isotonic solutions (both ionic and non-ionic in the case of the mouse, ionic only in the frog) generally produced a similar effect, viz., chromosomal swelling with little effect on the spindle. A notable exception is provided by frog embryos preceding the neurula stage; spindle disorganization without chromosomal swelling was produced by pretreatment in isotonic modified Niu-Twitty solution, containing no divalent cations. Pretreatment with hypotonic solutions (both ionic and non-ionic in the case of the mouse, ionic only in the frog) generally produced several major effects, viz., despiralization of chromosomes, chromatid separation, and spindle disorganization. The conclusion is drawn from the mouse data that, in order to produce these effects, pretreating solutions must be of low osmotic pressure. Low ionic strength alone (e.g., isotonic sucrose solutions) is not sufficient. As differentiation of frog embryos progressed, pretreatments either of longer duration or with solutions of increasing degrees of hypotonicity were required to produce comparable intensities of the same effects. Many of the effects on metaphases produced by hypotonic pretreatment of frog embryos were reversible by subsequent exposure to isotonic solutions. The significance of results presented here is discussed briefly with respect to some current considerations of the macromolecular structure of chromosomes.