Intracellular electrolyte concentrations in the frog skin epithelium: Effect of vasopressin and dependence on the Na concentration in the bathing media

Summary The intracellular electrolyte concentrations of the frog skin epithelium have been determined in thin freeze-dried cryosections using the technique of electron microprobe analysis. Stimulation of the transepithelial Na transport by arginine vasopressin (AVP) resulted in a marked increase in the Na concentration and a reciprocal drop in the K concentration in all epithelial cell layers. The effects of AVP were cancelled by addition of amiloride. It is concluded from these results that the primary mechanism by which AVP stimulates transepithelial Na transport is an increase in the Na permeability of the apical membrane. However, also some evidence has been obtained for an additional stimulatory effect of AVP on the Na pump. In mitochondria-rich cells and in gland cells no significant concentration changes were detected, supporting the view that these cells do not share in transepithelial Na transport. Furthermore, the dependence of the intracellular electrolyte concentrations upon the Na concentration in the outer and inner bathing solution was evaluated. Both in control and AVP-stimulated skins the intracellular Na concentration showed saturation already at low external Na concentrations, indicating that the self-inhibition of transepithelial Na transport is due to a reduction of the permeability of the apical membrane. After lowering the Na concentration in the internal bath frequently a Na increase in the outermost and a drop in the deeper epithelial layers was observed. It is concluded that partial uncoupling of the transport syncytium occurs, which may explain the inhibition of the transepithelial Na transport and blunting of the AVP response under this condition.     

Intracellular ion concentrations in the frog cornea epithelium during stimulation and inhibition of Cl secretion

Summary The intracellular electrolyte concentrations in the isolated cornea of the American bullfrog were determined in thin freeze-dried cryosections using energy-dispersive X-ray microanalysis. Stimulation of Cl secretion by isoproterenol resulted in a significant increase in the intracellular Na concentration but did not change the intracellular Cl concentration. Similar results were obtained when Cl secretion was stimulated by the Ca ionophore A23187. Inhibition of Cl secretion by ouabain produced a large increase in the intracellular Na concentration and an equivalent fall in the K concentration. Again, no increase or decrease in the intracellular Cl concentration was detectable. Clamping of the transepithelial potential to ±50 mV resulted in parallel changes in the transepithelial current and intracellular Na concentration, but, with the exception of the outermost cell layer, in no changes of the Cl concentration. Only when Cl secretion was inhibited by bumetanide or furosemide, together with a decrease in the Na concentration, was a large fall in the Cl concentration observed. Application of loop diuretics also produced significant increases in the P concentration and dry weight, consistent with some shrinkage of the epithelial cells. The results suggest the existence of a potent regulatory mechanism which maintains a constant intracellular Cl concentration and, thereby, a constant epithelial cell volume. Through the operation of this system any variation in the apical Cl efflux is compensated for by an equal change in the rate of Cl uptake across the basolateral membrane. Cl uptake is sensitive to loop diuretics, directly coupled to an uptake of Na, and dependent on the Na and K concentration gradients across the basolateral membrane. Isoproterenol and A23187 seem to increase the Cl permeability of the apical membrane and thus stimulate Cl efflux. Ouabain inhibits Cl secretion by abolishing the driving Na concentration gradient for Cl uptake across the basolateral membrane.     

Differential effects of aldosterone and ADH on intracellular electrolytes in the toad urinary bladder epithelium

Summary Quantitative electron microprobe analysis was employed to compare the effects of aldosterone and ADH on the intracellular electrolyte concentrations in the toad urinary bladder epithelium. The measurements were performed on thin freeze-dried cryosections utilizing energy dispersive x-ray microanalysis. After aldosterone, a statistically significant increase in the intracellular Na concentration was detectable in 8 out of 9 experiments. The mean Na concentration of granular cells increased from 8.9±1.3 to 13.2±2.2 mmol/kg wet wt. A significantly larger Na increase was observed after an equivalent stimulation of transepithelial Na transport by ADH. On average, the Na concentration in granular cells increased from 12.0±2.3 to 31.4±9.3 mmol/kg wet wt (5 experiments). We conclude from these results that aldosterone, in addition to its stimulatory effect on the apical Na influx, also exerts a stimulatory effect on the Na pump. Based on a significant reduction in the Cl concentration of granular cells, we discuss the possibility that the stimulation of the pump is mediated by an aldosterone-induced alkalinization.Similar though less pronounced concentration changes were observed in basal cells, suggesting that this cell type also participates in transepithelial Na transport. Measurements in mitochondria-rich cells provided no consistent results.     

Effect of ouabain,amiloride, and antidiuretic hormone on the sodium-transport pool in isolated epithelia from frog skin (Rana temporaria)

Summary When tracer Na⁺ is added to the solution bathing the apical side of isolated epithelia the observed transepithelial tracer influx increases with time until a steady state is reached. The build-up of the tracer flux follows a single exponential course. The halftime for this build-up under control conditions was 0.92 ±0.06 min, and in the presence of ouabain 4.51±0.7 min. It is shown that the calculated Na⁺-transport pool is located in the cells. The Na⁺-transport pool under control conditions was 35.6 ±3.4 nmol/cm², which corresponds to an intracellular Na⁺ concentration of 7.9mm. Activation of the active Na⁺ transport by addition of antidiuretic hormone resulted in a highly significant increase in the Na⁺ transport pool, and inhibition of the transcellular Na⁺ transport with amiloride resulted in a decrease in the Na⁺-transport pool.Furthermore, the active Na⁺ transport increased along anS-shaped curve with increasing intracellular Na⁺ concentration (Na⁺-transport pool). The Na⁺ pump was found to be half saturated at an intracellular Na⁺ concentration of 12.5mm.     

The volume of mitochondria-rich cells of frog skin epithelium

Summary The pathway for movement of chloride ions across frog skin is not well understood. Mitochondria-rich (MR) cells have been proposed as the route for chloride across the skin. To test this hypothesis we studied the MR cells of the skin of the frog,Rana pipiens, by quantitative light microscopic determination of cell volume. MR cell volume was influenced by changes in the chloride concentration or osmolality of the outside bathing solution. MR cells shrank about 23% when all chloride was removed from the outside (mucosal) bathing solution. MR cells were also shown to be responsive to changes in the osmolality of either the mucosal or serosal bath. Osmotically-induced swelling caused by dilution of the serosal bath resulted in volume regulatory decrease. These results are consistent with the hypothesis that MR cells constitute the pathway for chloride movement across frog skin.     

Localization of chloride conductance to mitochondria-rich cells in frog skin epithelium

Summary Cell volume determinations and electrophysiological measurements have been made in an attempt to determine if mitochondria-rich (MR) cells are localized pathways for conductive movements of Cl across frog skin epithelium. Determinations of cell volume with video microscope techniques during transepithelial passage of current showed that most MR cells swell when the tissue is voltage clamped to serosa-positive voltages. Voltage-induced cell swelling was eliminated when Cl was removed from the mucosal bath solution. Using a modified vibrating probe technique, it was possible to electrically localize a conductance specifically to some MR cells in some tissues. These data are evidence supporting the idea that MR cells are pathways for conductive movements of Cl through frog skin epithelium.     

Influence of lithium upon the intracellular potential of frog skin epithelium

Summary The effect of Li upon the intracellular potential of frog skin (Rana esculenta) was investigated. In the range between 1 and 25mm Li in the epithelial bathing solution, a semilogarithmic linear relationship between [Li] and intracellular potential under short circuit conditions was obtained. The intracellular potential at all [Li] is quantitatively sufficient to explain the previously reported accumulation of Li in the intracellular space of the frog skin epithelium (Leblanc, G. 1972.Pfluegers Arch. 337:1) on the basis of a passive entrance step at the outer border. A reduction of the intracellular potential by Li is also observed in the presence of 6mm Na in the epithelial bathing solution. Consequences regarding the mechanism of uptake of Na across the outer border of the frog skin are discussed.     

Intracellular solute gradients during osmotic water flow: An electron-microprobe analysis

Summary In an attempt to quantify possible intracellular water activity gradients during ADH-induced osmotic water flow, we employed energy dispersive X-ray microanalysis to thin, freezedried cryosections obtained from fresh, shock-frozen tissue of the toad urinary bladder. The sum of all detectable small ions (Na + K + Cl) in the cellular water space was taken as an index of the intracellular osmolarity. Presuming that all ions are osmotically active, they comprise about 90% of the cellular solutes. When the cells were exposed to dilute serosal medium, the reduction in the sum of the ions agreed well with the expected reduction in osmolarity. After inducing water flow by addition of ADH and dilution of the mucosal medium, all epithelial cells showed a fall in osmolarity. The change was more pronounced in granular cells than in basal or mitochondria-rich cells, consistent with the notion that granular cells represent the main transport pathway. Most significantly, intracellular osmolarity gradients, largely caused by an uneven distribution of K and Na, were detectable in granular cells. The gradients were not observed after ADH or mucosal dilution alone, or when the direction of transepithelial water flow was reversed. We conclude from these results that there is a significant cytoplasmic resistance to water flow which may lead to intracellular gradients of water activity. Concentration gradients of diffusible cations can be explained by a flow-induced Donnan-type distribution of fixed negative charges. With regard to transepithelial Na transport, the data suggest that ADH stimulates transport by increasing the Na permeability of the apical membranes of granular cells specifically.     

Localization of a Band 3-related protein in the mitochondria-rich cells of amphibian skin epithelium

Based on immunoblotting procedure, the isolated epithelium of amphibian skin was found to contain a 180 kDa protein which cross-reacts with a polyclonal antiserum raised against human erythrocyte Band 3. Immunoperoxidase and immunofluorescence staining techniques indicated that the Band 3-related protein was localized in the mitochondria-rich cells (MRC) of this epithelium, with characteristic apical labelling pattern. Our findings show that the putative apical anion exchanger of the MRC is immunologically related to the band 3 multigenic family, which catalyzes Cl-HCO₃ ^? transmembranous exchange. It thus suggests a molecular basis for the role played by these cells in the transepithelial Cl pathway and acid-base regulation.     

Amiloride and its analogs as tools in the study of ion transport

Amiloride inhibits most plasma membrane Na+ transport systems. We have reviewed the pharmacology of inhibition of these transporters by amiloride and its analogs. Thorough studies of the Na+ channel, the Na+/H+ exchanger, and the Na+/Ca2+ exchanger, clearly show that appropriate modification of the structure of amiloride will generate analogs with increased affinity and specificity for a particular transport system. Introduction of hydrophobic substituents on the terminal nitrogen of the guanidino moiety enhances activity against the Na+ channel; whereas addition of hydrophobic (or hydrophilic) groups on the 5-amino moiety enhances activity against the Na+/H+ exchanger. Activity against the Na+/Ca2+ exchanger and Ca2+ channel is increased with hydrophobic substituents at either of these sites. Appropriate modification of amiloride has produced analogs that are several hundred-fold more active than amiloride against specific transporters. The availability of radioactive and photoactive amiloride analogs, anti-amiloride antibodies, and analogs coupled to support matrices should prove useful in future studies of amiloride-sensitive transport systems. The use of amiloride and its analogs in the study of ion transport requires a knowledge of the pharmacology of inhibition of transport proteins, as well as effects on enzymes, receptors, and other cellular processes, such as DNA, RNA, and protein synthesis, and cellular metabolism. One must consider whether the effects seen on various cellular processes are direct or due to a cascade of events triggered by an effect on an ion transport system.     

Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.     

Sodium flux in the apical membrane of the toad skin: Aspects of its regulation and the importance of the ionic strength of the outer solution upon the reversibility of amiloride inhibition

Summary Injection of small pulses of concentrate solutions of salts or drugs into the outer bathing fluid led to sudden increases of its solute concentration. Vigorous stirring of the outer bathing solution was used to minimize the thickness of the unstirred layer adjacent to the outer skin surface. Pulses of 1m NaCl injected into the outer compartment induced sharp increases of the SCC following a time course variable with the magnitude of the pulse and the particular condition of each skin. Comparison of the spontaneous decline of the SCC with the decline induced by a small dose of amiloride, where an increase inR was observed, indicates that the spontaneous decline cannot be explained simply as a reduction of the Na permeability of the apical membrane by self-inhibition of feedback inhibition of the apical membrane Na channels. Reduction of the driving force for Na movement into the epithelial cells must play an important role in the process. Reversibility of the amiloride inhibition of the SCC was highly dependent upon the ionic strength of the solution used to rinse and wash out the inhibitor from the outer skin surface. With H₂O, the amiloride molecules washed out slowly as compared to NaCl or KCl solutions. Na or K have the same ability to dislodge the amiloride molecules from their binding sites. This effect is apparently of a purely electrostatic nature.     

The role of nitric oxide in the regulation of ion channels in airway epithelium: Implications for diseases of the lung

The human respiratory tract is covered with airway surface liquid (ASL) that is essential for lung defense and normal airway function. The quantity and composition of ASL is regulated by active ion transport across the airway epithelium. Abnormal electrolyte transport produces changes in ASL volume and composition, inhibits mucociliary clearance and leads to chronic infection of airway surfaces, as is evident in cystic fibrosis. Agonists that induce intracellular increases in cAMP or Ca²⁺ are generally associated with electrolyte secretion. While these mechanisms have been studied in detail for many years, modulation of ion channels by nitric oxide (NO) has emerged only recently as a significant determinant of ion channel function. NO is a physiological regulator of transepithelial ion movement and alterations of its generation and action may play an important role in the pathogenesis of lung disorders characterized by hypersecretion of ASL. This review presents the current understanding of regulation of airway epithelial ion channels by NO and attempts to highlight the importance of this regulation for lung defense.     

Hypoxia‐induced increase in intracellular calcium concentration in endothelial cells: Role of the Na+‐glucose cotransporter

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet‐activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca²⁺]_i) which then activates the phospholipase A₂ (PLA₂). The link between the decrease in ATP and the increase in [Ca²⁺]_i was not known and is investigated in this work. We first showed that the presence of extracellular Na⁺ was necessary to observe the hypoxia‐induced increase in [Ca²⁺]_i and the activation of PLA₂. This increase was not due to the release of Ca²⁺ from intracellular stores, since thapsigargin did not inhibit this process. The Na⁺/Ca²⁺ exchanger was involved since dichlorobenzamil inhibited the [Ca²⁺]_i and the PLA₂ activation. The glycolysis was activated, but the intracellular pH (pH_i) in hypoxic cells did not differ from control cells. Finally, the hypoxia‐induced increase in [Ca²⁺]_i and PLA₂ activation were inhibited by phlorizin, an inhibitor of the Na⁺‐glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na⁺ through the activated Na⁺‐glucose cotransport followed by the activation of the Na⁺/Ca²⁺ exchanger, resulting in a net influx of Ca²⁺. J. Cell. Biochem. 84: 115–131, 2002. © 2001 Wiley‐Liss, Inc.     

A method for the in situ measurement of the electrical quantities in frog skin

A method allowing the measurement of the electrical quantities related to the physiological functions of the frog skin in situ is presented. The method allows the performance of several experiments on the same pithed animal, which remains alive for a number of days. The preparation is very stable, and the electric potential difference and short-circuit current values are higher than in isolated skin. The theory of measurement and the possible systematic errors are discussed. The possibilities of the method are evaluated on comparing the pH and temperature dependence of the electrical quantities in situ with previous measurements on isolated skin.     

Studies on the kinetics of Na+/H+ exchange in OK cells: Introduction of a new device for the analysis of polarized transport in cultured epithelia

Summary The present study describes a new perfusion technique—based on the use of a routine spectrofluorometer—which enables fluorometric evaluation of polarity, regulation and kinetics of Na⁺/H⁺ exchange at the level of an intact monolayer. Na⁺/ H⁺ exchange was evaluated in bicarbonate-free solutions in OK (opossum kidney) cells, a renal epithelial cell line. Na⁺/H⁺ exchange activity was measured by monitoring changes in intracellular pH (pH _i ) after an acid load, using the pH-sensitive dye 27-bis (carboxyethyl) 5–6-carboxy-fluorescein (BCECF). Initial experiments indicated that OK cells grown on a permeable support had access to apical and basolateral perfusion media. They also demonstrate that OK cells express an apical pH _i , recovery mechanism, which is Na⁺ dependent, ethylisopropylamiloride (EIPA) sensitive and regulated by PTH. Compared to resting conditions (pH _i =7.68; pH _o =7.4) where Na⁺/H⁺ exchange is not detectable, transport rate increased as pH _i decreased. A positive cooperativity characterized the interaction of internal H⁺ with the exchanger, and suggests multiple H⁺ binding sites. In contrast, extracellular [Na⁺] increased transport with simple Michaelis-Menten kinetics. The apparent affinity of the exchanger for Na⁺ was 19mM at an intracellular pH of 7.1 and 60mM at an intracellular pH of 6.6. Inhibition of Na⁺/H⁺ exchange activity by EIPA was competitive with respect to extracellular [Na⁺] and theK _i was 3.4 M. In conclusion, the technique used in the present study is well suited for determination of mechanisms involved in control of epithelial cell pH _i and processes associated with their polarized expression and regulation.     

Tissue kinetics,ion transport,and recruitment of mitochondria-rich cells in the skin of the toad (Bufo bufo) in response to exposure to distilled water

Mitochondria-rich cells (MRC) of the amphibian epidermis are responsible for active chloride uptake at low external salinity, and new MRCs are recruited in response to exposure to distilled (deionized) water. The time-course of this recruitment, the tissue kinetics and ion transport have been studied in toads (Bufo bufo) immediately before, and after 2,7, and 14 days exposure to distilled water. General epidermal structure was not affected. However, the numbers of MRCs per mm² (D_MRC) increased throughout the experiment as revealed by staining of epidermal sheets with AgNO₃ (Ag) or methylene blue (MB). Part of the increased D_MRC was accounted for by an increase in MRC subpopulation(s) that stained neither with Ag nor MB. The cell birth rate (K_b) decreased and cell loss by moulting (K_d) increased without any significant change in epidermal cell pool size, indicating a reduced apoptotic rate. The increase in D_MRC was accompanied by a 3-fold increase in Cl^- current (I_Cl). At day-2 there was a transient reduction in the I_Cl per MRC. H⁺ secretion was progressively reduced during prolonged exposure to distilled water. Thus, at day-2 MRCs appeared incompletely differentiated as indicated by decreased I_Cl and H⁺ flux per MRC, and by the increased proportion of MRCs unstained by Ag or MB. Full Cl^- (but not H⁺) transport capacity, was restored at day-7. We conclude that increased D_MRC following exposure to low external Cl^-, rather than being due to an increased K_b, is the combined effect of a decreased apoptotic rate and an increased rate of differentiation, where morphological differentiation precedes functional differentiation.Parts of this study have been presented at the 32th International Congress of Physiological Sciences, 1–6 August, 1993, Glasgow, Scotland, and the 19th meeting of the European Study Group for Cell Proliferation, 5–9 October, 1993, Bruges, Belgium     

Evidence for activation of an active electrogenic proton pump in Ehrlich ascites tumor cells during glycolysis

Summary The addition of glucose to a suspension of Ehrlich ascites tumor cells results in rapid acidification of the extracellular medium due to lactic acid production. The nature of the H⁺ efflux mechanism has been studied by measuring the time course of the acidification, the rate of proton efflux, the direction and relative magnitude of the H⁺ concentration gradient, and the voltage across the membrane. Using the pH-sensitive dye acridine orange, we have established that after addition of 10mm glucose an outward-directed H⁺ concentration gradient develops. As the rate of glycolysis slows, the continued extrusion of H⁺ reverses the direction of the H⁺ concentration gradient. Changes in absorbance of the voltagesensitive dye diethyloxadicarbocyanine iodide (DOCC), and changes in the distribution of the lipid permeant cation tetraphenyl phosphonium, showed a dramatic and persistent hyperpolarization of the membrane voltage after glucose addition. The hyperpolarization was prevented by the protonophore tetrachlorosalicylanalide (TCS) and by valinomycin, but not by the neutral-exchange ionophore nigericin. Inhibitors of lactate efflux were found to reduce the rate of acidification after glucose addition but they had no effect on the magnitude of the resulting hyperpolarization. On the basis of these and other data we suggest that an active electrogenic pump mechanism for H⁺ efflux may be activated by glucose and that this mechanism operates independently of the lactate carrier system.     

Analysis of the monovalent ion fluxes in U937 cells under the balanced ion distribution: recognition of ion transporters responsible for changes in cell ion and water balance during apoptosis

Unidirectional (22)Na, Li(+) and Rb(+) fluxes and net fluxes of Na(+) and K(+) were measured in U937 human leukemic cells before and after induction of apoptosis by staurosporine (1 microM, 4 h) to answer the question which ion transporter(s) are responsible for changes in cell ion and water balance at apoptosis. The original version of the mathematical model of cell ion and water balance was used for analysis of the unidirectional ion fluxes under the balanced distribution of major monovalent ions across the cell membrane. The values of all major components of the Na(+) and K(+) efflux and influx, i.e. fluxes via the Na(+),K(+)-ATPase pump, Na(+) channels, K(+) channels, Na/Na exchanger and Na-Cl symport were determined. It is concluded that apoptotic cell shrinkage and changes in Na(+) and K(+) fluxes typical of apoptosis in U937 cells induced by staurosporine are caused by a complex decrease in the pump activity, Na-Cl symport and integral Na(+) channel permeability.     

Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells

Fluorescently labeled smooth muscle myosin II is often used to study myosin II dynamics in non-muscle cells. In order to provide more specific tools for tracking non-muscle myosin II in living cytoplasm, fluorescent analogues of non-muscle myosin IIA and IIB were prepared and characterized. In addition, smooth and non-muscle myosin II were labeled with both cy5 and rhodamine so that comparative, dynamic studies may be performed. Non-muscle myosin IIA was purified from bovine platelets, non-muscle myosin IIB from bovine brain, and smooth muscle myosin II from turkey gizzards. After being fluorescently labeled with tetramethylrhodamine-5-iodoacetamide or with a succinimidyl ester of cy5, they retained the following properties: (1) reversible assembly into thick filaments, (2) actin-activatable MgATPase, (3) phosphorylation by myosin light chain kinase, (4) increased MgATPase upon light-chain phosphorylation, (5) interconversion between 6S and 10S conformations, and (6) distribution into endogenous myosin II-containing structures when microinjected into cultured cells. These fluorescent analogues can be used to visualize isoform-specific dynamics of myosin II in living cells. J. Cell. Biochem. 68:389–401, 1998. © 1998 Wiley-Liss, Inc.