Three classes of potassium channels in large monopolar cells of the blowfly Calliphora vicina

Summary We have used single electrode voltage clamp in the intact animal and whole-cell recording from dissociated cell bodies to investigate the properties of potassium conductances in large monopolar cells (LMCs) of the first optic ganglion of the blowfly Calliphora vicina. Two classes of voltage gated potassium conductances were found: a delayed rectifier current (Kd) with slow inactivation (_inac = 1–3 sec), and an A current (Ka) showing both faster inactivation (_inac = 21 ms) and also more rapid activation. The reversal potential of both currents is ca. -90 mV with 2 mM [K_o] and 140 mM [K_i], and follows the Nernst slope with increasing [K_o]. The voltage operating range of Ka is unusually negative, with the mid point of the steady-state inactivation curve (V₅₀) at- 101 mV. V₅₀ for Kd is - 84 mV. Although no inward currents were detected, for technical reasons their presence cannot be excluded.In inside-out patches from LMC soma membranes the single channels underlying the currents both have a conductance of ca. 20 pS in symmetrical 140 mM K solutions and channel densities may be as high as 10/m². Less frequently, inside-out patches contained a large conductance (110 pS) calcium-activated potassium channel which existed almost exclusively in a rapidly flickering mode. Open probability increased with depolarization and Ca concentrations greater than 40 nM.In whole-cell recordings, dissociated LMC cell bodies fall into two classes with respect to their voltage sensitive currents: 37 % of cells only showed Kd; the remainder (63%) were dominated by Ka with a variable (0–30%) contribution from Kd. In the intact animal, intracellular recordings from LMCs, combined with dye-marking, indicate that cells expressing only Kd are type L3 cells, whilst L1 and L2 express predominantly Ka. Since L1 and L2 have resting potentials of ca. - 40 mV and maximum hyperpolarizations reaching -90 mV only transiently, inactivation of Ka is unlikely to be removed under most physiological conditions. In contrast, L3 cells have a more negative resting potential (–60 mV) and Kd should play a significant role in signal-shaping, in particular contributing to the falling phase of a prominent spike-like transient in response to dimming.Abbreviations Ka A current - Kd delayed rectifier - LMC large monopolar cell - L1-L3 classes thereof - TTX tetrodotoxin     

The effect of temperature onshibire ts cell clones in the compound eye ofDrosophila melanogaster

Summary We have analysed the effect of temperature on both developing and adult eye cell clones homozygous forshi ^ST139, a temperature-sensitive mutant ofDrosophila melanogaster. The mutant gene, autonomous in its cellular expression, causes structural modifications of ommatidial cells when adult clones of cells are exposed to the restrictive temperature (29°C) for several days. However, the mutant phenotype reverses to normal within 4 days at the permissive temperature (20°C). The results of pulse, shift-up and shift-down experiments show that the temperaturesensitive period for developing compound eye cells is from the late second instar up to the early pupa. Cytodifferentiation of compound eye cells is blocked by restrictive temperature treatment during this period, whereas cell proliferation does not seem to be directly affected. These results are discussed with regard to the other known aspects of the phenotype observed in mutant individuals.     

Development of neural lineages derived from the sine oculis positive eye field of Drosophila

The Anlage of the Drosophila visual system, called eye field, comprises a domain in the dorso-medial neurectoderm of the embryonic head and is defined by the expression of the early eye gene sine oculis (so). Beside the eye and optic lobe, the eye field gives rise to several neuroblasts that contribute their lineages to the central brain. Since so expression is only very short lived, the later development of these neuroblasts has so far been elusive. Using the P-element replacement technique [Genetics, 151 (1999) 1093] we generated a so-Gal4 line driving the reporter gene LacZ that perdures in the eye field derived cells throughout embryogenesis and into the larval period. This allowed us to reconstruct the morphogenetic movements of the eye field derived lineages, as well as the projection pattern of their neurons. The eye field produces a dorsal (Pc1/2) and a ventral (Pp3) group of three to four neuroblasts each. In addition, the target neurons of the larval eye, the optic lobe pioneers (OLPs) are derived from the eye field. The embryonically born (primary) neurons of the Pp3 lineages spread out at the inner surface of the optic lobe. Together with the OLPs, their axons project to the dorsal neuropile of the protocerebrum. Pp3 neuroblasts reassume expression of so-Gal4 in the larval period and produce secondary neurons whose axonal projection coincides with the pattern formed by the primary Pp3 neurons. Several other small clusters of neurons that originate from outside the eye field, but have axonal connections to the dorsal protocerebrum, also express so and are labeled by so-Gal4 driven LacZ. We discuss the dynamic pattern of the so-positive lineages as a tool to reconstruct the morphogenesis of the larval brain.     

Different photoreceptors within the same retina express unique combinations of potassium channels

Single electrode current and voltage clamp recordings in Calliphora, and whole-cell voltage clamp recordings in Drosophila were used to characterise the voltage-gated K channels in both major classes of photoreceptors, R7/8 (long visual fibres, LVFs) and R1-6 (short visual fibres, SVFs). R7/8 were identified by their unique spectral properties, ca. 3–4 fold higher input resistances and 3–4 fold lower cell capacitance. In Calliphora SVFs possess both fast and slow activating delayed rectifier potassium conductances. Drosophila SVFs possess a slowly inactivating delayed rectifier (I_Ks), a very rapidly inactivating A channel encoded by the Shaker gene (I_A), and, in a minority of cells, a third K conductance with intermediate kinetics (I_Kf). In both specs the LVFs lack the slowest component, but exhibit the faster K conductance(s) with properties indistinguishable from those in the SVFs. These findings add to established evidence demonstrating the significant role played by potassium channels in tuning the photoreceptor membrane. The results also suggest that R1-6 photoreceptors and R7/8 form inputs to visual subsystems tuned to different temporal frequencies.Abbreviations LVF long visual fibre - SVF short visual fibre - R1-6 retinular cells 1 to 6 inclusive - R7/8 retinular cell 7 and 8 - I _A rapidly inactivating A type potassium conductance; channel coded by Shaker gene - I _Kf rapidly activating, slowly inactivating delayed rectifier-like potassium conductance - I _Ks slowly activating, slowly inactivating delayed rectifier-like potassium conductance - I _KDs slowly activating delayed rectifier potassium conductance - I _KDf rapidly activating delayed rectifier potassium conductance     

A comparative study of embryonic gene expression inDrosophila andEphestia

Summary The ontogeny of allozyme patterns has been studied in embryos ofDrosophilamelanogaster, which are doubly heterozygous for alleles specifying the slow and fast forms of alcohol dehydrogenase (ADH) and -glycerophosphate dehydrogenase (GPDH). The ontogeny of esterase-2 was studied in embryos and young larvae of the flour mothEphestia kühniella, which are heterozygous for two of the three existing esterase-2 alleles. In freshly laidDrosophila eggs only the maternal enzyme forms are present and during the first 15 hours of development the staining of these forms becomes progressively fainter. After 16 and 17 h, the paternal and hybrid bands of ADH and GPDH respectively become obvious. Before hatching, the intensity distribution in the three-banded pattern of reciprocal hybrids is asymmetric in favour of the persisting maternal enzyme form. InEphestia embryos, however, there is no persistence of the maternal esterases. In all reciprocal heterozygotes a three-banded pattern suddenly appears 96 h after egg deposition, indicating synchronous activation of both parental alleles. The relative intensity distribution in the hybrid patterns approaches that of the mature larvae stepwise and in an allele-specific manner. This result and the fact that the various heterozygous types exhibit unequal total activities suggest that the Esterase-2 alleles have different activities, which are fixed late in embryogenesis.     

Outwardly rectifying chloride channels and CF: A divorce and remarriage

Outwardly rectifying Cl^– channels were originally thought to be the central element in cystic fibrosis. The role of these channels in CF was questioned to such an extent that doubts were rasied about the validity of the original experiments. Recent data reestablishes a role for outwardly rectifying Cl^– channels (ORCC) in CF and suggests that the protein encoded by the CF gene, the cystic fibrosis transmembrane regulator (CFTR), can effect the regulation of more than one channel in the airway. This minireview deals with the rise, fall, and resurrection of the role of outwardly rectifying Cl^– channels in CF.     

Ultrastructure of the compound eye and first optic neuropile of the photoreceptor mutant ora JK84 of Drosophila

Summary The developmental mutant of Drosophila (ora ^JK84) is characterized by nonfunctional photoreceptor cells (R1–6), while the R7/R8 cells are normal. A fundamental question is: Does the near absence of photosensitive membranes inhibit development of the Rl-6 axons and their synapses at the other end of the cell? The retina and first optic neuropile (lamina ganglionaris) were examined with freeze-fracture technique and high voltage electron microscopy. R1–6 have reduced rhabdomere caps; rhabdomeric microvilli have about 50% of the normal diameter and 20% of the normal length. Affected cells exhibit prominent vacuoles which appear to communicate with some highly convoluted microvillar membranes. Almost no P-face particles (putative rhodopsin molecules) are present in the R1–6 rhabdomeres, and particle densities are lower in R7 than previously reported. Near the rhabdomere caps, microvilli of R1–6 are fairly normal, but at more proximal levels they are greatly diminished in length and changed in orientation, while at still more proximal levels they are lost. R1–6, R7, and R8 axons from each ommatidium are bundled into normal pseudocartridges beneath the basement membrane. No abnormalities are found in the lamina ganglionaris, and all synaptic associations as well as the presumed virgin synapses (of R1–6) appear normal. No glial anomalies are present, and R7/R8 axons project through the lamina in the usual fashion. These fine structural findings are correlated with known electrophysiological, biochemical, and behavioral correlates of both sets of photoreceptors (R1–6, and R7/R8).This study was supported substantially by the UW-HVEM Laboratory, in addition to a Faculty Development Award, a UMC Biomedical Research Support Grant N.I.H. RR07053 to W.S.S., and a Hatch Grant, Project 2100 to S.D.C. Freeze fracture was done at the Wisconsin Regional Primate Research Center, N.I.H. Grant RR00167. We thank Professor Hans Ris, Dr. J. Pawley, Dr. D. Neuberger, and Ms. M. Bushlow, HVEM Laboratory, Dept. of Zoology, UW. We also thank Mrs. K. Srivastava, Mr. M.B. Garment, Mr. G. Gaard, and Mr. D. Liu for technical assistance.     

Two visual pigment opsins,one expressed in the dorsal region and another in the dorsal and ventral regions,of the compound eye of a dragonfly,Sympetrum frequens

This paper describes the primary structure of two visual pigment opsins (DfRh1 and DfRh2) in the regionalized compound eye of a dragonfly,Sympetrum frequens. The amino acid sequences were deduced from the nucleotide sequences of cDNAs isolated from a cDNA library of the dragonfly retina. The two opsins both consist of 379 amino acids with 81.3% identity. Analysis of hydropathy indicated that the sequences have seven transmembrane domains like those of previously described opsins. Expression analysis using RT-PCR revealed that DfRh1 was present only in the dorsal region whereas DfRh2 was detected in both the dorsal and the ventral regions of the eye.     

Voltage-gated calcium and Ca2+-activated chloride channels and Ca2+ transients: voltage-clamp studies of perfused and intact cells of Chara

The voltage-clamp technique was used to study Ca²⁺ and Cl^– transient currents in the plasmalemma of tonoplast-free and intact Chara corallina cells. In tonoplast-free cells [perfused medium with ethylene glycol bis(2-aminoethyl ether)tetraacetic acid] long-term inward and outward currents through Ca channels consisted of two components: with and without time-dependent inactivation. The voltage dependence of the Ca channel activation ratio was found to be sigmoid-shaped, with about –140-mV activation threshold, reaching a plateau at V>50 mV. As the voltage increased, the characteristic activation time decreased from approximately 10³ ms in the threshold region to approximately 10 ms in the positive region. The positive pulse-activated channels can then be completely deactivated, which is recorded by the Ca²⁺ tail currents, at below-threshold negative voltages with millisecond-range time constants. This tail current is used for fast and brief Ca²⁺ injection into tonoplast-free and intact cells, to activate the chloride channels by Ca²⁺ . When cells are perfused with EDTA-containing medium in the presence of excess Mg²⁺, this method of injection allows the free submembrane Ca²⁺ concentration, [Ca²⁺]_c, to be raised rapidly to several tens of micromoles per liter. Then a chloride component is recorded in the inward tail current, with the amplitude proportional to . When Ca²⁺ is thus injected into an intact cell, it induces an inward current in the voltage-clamped plasmalemma, having activation–inactivation kinetics qualitatively resembling that in EDTA-perfused cells, but a considerably higher amplitude and duration (approximately 10 A m^–2 and _inact~0.5 s at –200 mV). Analysis of our data and theoretical considerations indicate that the [Ca²⁺]_c rise during cell excitation is caused mainly by Ca²⁺ entry through plasmalemma Ca channels rather than by Ca²⁺ release from intracellular stores.     

Proton-stimulated opening of stomata in relation to chloride uptake by guard cells

The concentration of potassium chloride required in the incubation medium to open stomata in isolated epidermal tissues of Commelina communis L. and Vicia faba L. could be lowered from 100 mM to 10 mM if the proton concentration of the ambient solution was increased from pH 5.6 to pH 3.5. This acidification effect was formerly attributed to the destruction of epidermal and subsidiary cells resulting in a relief of back pressure upon guard cells. While guard cells remain viable at pH 3.5, as demonstrated by their susceptibility to inhibition by uptake of glucose or to uncoupling by DNP, incipient destruction of the cells surrounding them could first be observed 30 min after the onset of the incubation experiment. By this time, however, the stomata had already opened; the time course of stomatal opening at pH 3.5 did not show any lag phase corresponding to the time required for damaging epidermal cells and showed no difference to that at pH 5.6 Thus, the acid-stimulated opening of stomata appears to be a biphasic phenomenon consisting of a physiologic effect onto which the physical effect of the relief of back pressure is superimposed over longer periods of incubation. To interpret the physiologic role of an increased proton concentration in the ambient solution of isolated epidermal strips, it is suggested that guard cells take up protons and chloride ions in an electroneutral symport. While protons are extruded again to generate the negative membrane potential required for potassium influx, chloride ions are retained to maintain electroneutrality.     

Appendage morphogenesis in Drosophila: a developmental study of the rotund (rn) gene

Summary The phenotype of rotund (rn) null alleles is described, and compared to wild type. The mutants are expressed zygotically and cause position specific defects in certain imaginal discs (antenna, legs, wing, haltere and proboscis) and their corresponding adult derivatives. In the discs, specific folds are absent in rn mutants compared to wild type. Clonal analysis shows that the rn ⁺ gene is partially autonomous in its expression in cells destined to form certain distal parts of the adult appendages. The results are consistent with the idea that the rn ⁺ gene is required for normal morphogenesis of specific distal parts of the adult appendages.     

Opposing interactions between homothorax and Lobe define the ventral eye margin of Drosophila eye

Patterning in multi-cellular organisms involves progressive restriction of cell fates by generation of boundaries to divide an organ primordium into smaller fields. We have employed the Drosophila eye model to understand the genetic circuitry responsible for defining the boundary between the eye and the head cuticle on the ventral margin. The default state of the early eye is ventral and depends on the function of Lobe (L) and the Notch ligand Serrate (Ser). We identified homothorax (hth) as a strong enhancer of the L mutant phenotype of loss of ventral eye. Hth is a MEIS class gene with a highly conserved Meis-Hth (MH) domain and a homeodomain (HD). Hth is known to bind Extradenticle (Exd) via its MH domain for its nuclear translocation. Loss-of-function of hth, a negative regulator of eye, results in ectopic ventral eye enlargements. This phenotype is complementary to the L mutant phenotype of loss-of-ventral eye. However, if L and hth interact during ventral eye development remains unknown. Here we show that (i) L acts antagonistically to hth, (ii) Hth is upregulated in the L mutant background, and (iii) MH domain of Hth is required for its genetic interaction with L, while its homeodomain is not, (iv) in L mutant background ventral eye suppression function of Hth involves novel MH domain-dependent factor(s), and (v) nuclear localization of Exd is not sufficient to mediate the Hth function in the L mutant background. Further, Exd is not a critical rate-limiting factor for the Hth function. Thus, optimum levels of L and Hth are required to define the boundary between the developing eye and head cuticle on the ventral margin.     

Cytoplasmic calcium affects the gating of potassium channels in the plasma membrane ofChara corallina: a whole-cell study using calcium-channel effectors

The action of a wide range of drugs effective on Ca²⁺ channels in animal tissues has been measured on Ca²⁺ channels open during the action potential of the giant-celled green alga,Chara corallina. Of the organic effectors used, only the 1,4-dihydropyridines were found to inhibit reversibly Ca²⁺ influx, including, unexpectedly, Bay K 8644 and both isomers of 202–791. Methoxyverapamil (D-600), diltiazem, and the diphenylbutylpiperidines, fluspirilene and pimozide were found not to affect the Ca²⁺ influx. Conversely, bepridil greatly and irreversibly stimulated Ca²⁺ influx, and with time, stopped cytoplasmic streaming (which is sensitive to increases in cytoplasmic Ca²⁺). By apparently altering the cytoplasmic Ca²⁺ levels with various drugs, it was found that (with the exception of the inorganic cation, La³⁺) treatments likely to lead to an increase in cytoplasmic Ca²⁺ levels caused an increase in the rate of closure of the K⁺ channels. Similarly, treatments likely to lead to a decrease in cytoplasmic Ca²⁺ decreased the rate of K⁺ channel closure. The main effect of bepridil on the K⁺ channels was to increase the rate of voltage-dependent channel closure. The same effect was obtained upon increasing the external concentration of Ca²⁺, but it is likely that this was due to effects on the external face of the K⁺ channel. Addition of any of the 1,4-dihydropyridines had the opposite effect on the K⁺ channels, slowing the rate of channel closure. They sometimes also reduced K⁺ conductance, but this could well be a direct effect on the K⁺ channel; high concentrations (50 to 100 μM) of bepridil also reduced K⁺ conductance. No effect of photon irradiance or of abscisic acid could be consistently shown on the K⁺ channels. These results indicate a control of the gating of K⁺ channels by cytoplasmic Ca²⁺, with increased free Ca²⁺ levels leading to an increased rate of K⁺-channel closure. As well as inhibiting Ca²⁺ channels, it is suggested that La³⁺ acts on a Ca²⁺-binding site of the K⁺ channel, mimicking the effect of Ca²⁺ and increasing the rate of channel closure.     

A patch-clamp study of the ionic selectivity of the large conductance,ca-activated chloride channel in muscle vesicles prepared fromAscaris suum

Summary Plasma membrane vesicles prepared from the bag re gion of the somatic muscle cell of the parasiteAscaris suum contain a large conductance, voltage-sensitive, calcium-activated chloride channel. The ability of this channel to conduct a variety of anions has been investigated using the patch-clamp technique on isolated inside-out patches of muscle membrane. Symmetrical Cl solutions (140 mm) produced single-channel I/V plots with reversal potentials of 0 mV, substitution of bath Cl by 140 mM NO₃, Br and I caused depolarizing shifts in the reversal potentials. Replacement of the internal Cl by F (140 mM) caused a large hyperpolarizing shift in the reversal potential. The channel dis played a permeability sequence of I > Br = NO₃> Cl > F which differed from the corresponding conductance sequence Cl > NO₃ = Br = I > F. The ionic environment within the channel pore has been investigated using Reuter and Stevens (1980) plots to describe the selectivity and “fluidity” of the channel pore. In addition, the approach of Wright and Diamond (1977) was employed to estimate the number of cationic binding sites within the channel pore. The channel is relatively fluid but the number of cationic binding sites varies inversely with the ionic radius of the anion from 2.15 for F to 0.89 for the large planar anion NO₃     

Components of fitness in a compound chromosome strain of Drosophila melanogaster

Summary The relative net fitness of a compound chromosome strain of Drosophila melanogaster was about 0.05, compared with the chromosomally normal strain from which it was derived. Based on meiotic considerations alone, the expected relative fitness was about 0.25. There were no significant differences in fertility between the compound and normal strains; the compound strain produced about 28% as many offspring as the normal strain and developed faster than the normal strain in two replicates, and slower in one replicate. The low relative fitness of the compound strain was apparently due to assortative mating, in which normal females discriminated strongly against compound males. Implications for pest control projects are dicussed.     

Cell proliferation, survival, and death in the Drosophila eye

The Drosophila retina has a precise repeating structure based on the unit eye, or ommatidium. This review summarizes studies of the cell proliferation and survival episodes that affect the number of cells available to make each ommatidium. Late in larval development, as differentiation and patterning begin, the retinal epithelium exhibits striking regulation of the cell cycle including a transient G1 arrest of all cells, followed by a ‘Second Mitotic Wave’ cell cycle that is regulated at the G2/M transition by local intercellular signals. Reiterated episodes of cell death also contribute to precise regulation of retinal cell number. The EGF receptor homolog has multiple roles in retinal proliferation and survival.     

The development of ommatidial patterning in metamorphosed eye imaginal discs implants ofDrosophila melanogaster

Summary The development of the rhabdomeric pattern in the compound eye ofDrosophila has been studied using combined transplantation and electron microscope techniques. In a first series of experiments eye imaginal discs of increasing age were implanted into larvae ready to pupate, thus losing variable amounts of the normal time for development. A sequence of differentiative abilities was found in the metamorphosed test pieces. As far as the photoreceptor cells are concerned, the most prominent steps of this sequence are: ability to form groups with other similar elements, anatomical polarization of microvilli, establishment of the rhabdomeric pattern and formation of an equator line. The stability of determination of the equator line was tested in a second experimental series. Fragment of different topographical origin within the mature eye anlage were brought to metamorphosis by implantation into larvae ready to pupate. It was found that an equator line differentiates only in those pieces which according to the published anlage maps contain the prospective equator region prior to metamorphosis. The mitotic abilities of implanted eye imaginal discs were investigated by means of in vitro³H-thymidine pulse-labelling and light microscope autoradiography of the differentiated test pieces. During the third larval stage the eye anlage is traversed by two consecutive mitotic waves, each one of them producing different categories of receptor cells. The first, anterior wave predominantly produces cells oriented toward the poles of the eye within the ommatidia, while the second, posterior wave gives rise to elements exclusively in an equatorial position. The dynamics of this proliferation are discussed in relation to the findings in the implantation experiments. Silver-grain counts support the possibility that at least two successive cell divisions occur in the eye anlage between labeling with tritiated thymidine and beginning of morphological differentiation. The relevance of this finding for the understanding of the concept of acquisition of competence is discussed.     

Reconstitution of sodium channels in large liposomes formed by the addition of acidic phospholipids and freeze-thaw sonication

Phosphatidylcholine (PC) alone or with phosphatidylethanolamine (PE) are sufficient for the reconstitution of Na+ channels in planar lipid bilayers. However, when Na+ channels were first reconstituted into liposomes using the freeze-thaw-sonication method, addition of acidic phospholipids, such as phosphatidylserine (PS), to the neutral phospholipids was necessary to obtain a significant toxin-modulated 22Na uptake. To further investigate the acidic phospholipid effect on reconstitution into liposomes, Na+ channels purified from Electrophorus electricus electrocytes were reconstituted into liposomes of different composition by freeze-thaw sonication and the effect of batrachotoxin and tetrodotoxin on the 22Na flux was measured. The results revealed that, under our experimental conditions, the presence of an acidic phospholipid was also necessary to obtain a significant neurotoxin-modulated 22Na influx. Though neurotoxin-modulated 22Na fluxes have been reported in proteoliposomes made with purified Na+ channels and PC alone, the 22Na fluxes were smaller than those found using lipid mixtures containing acidic phospholipids. Electron microscopy of negatively stained proteoliposomes prepared with PC, PC/PS (1:1 molar ratio), and PS revealed that the acidic phospholipid increases the size of the reconstituted proteoliposomes. The increment in size caused by the acidic phospholipid, due to the associated increase in internal volume for 22Na uptake and in area for Na+ channel incorporation, appears to be responsible for the large neurotoxin-modulated 22Na fluxes observed.     

Fluorescent labelling of Na+,K+-ATPase in intact cells by use of a fluorescent derivative of ouabain: Salinity and teleost chloride cells

Summary Anthroylouabain, a fluorescent derivative of ouabain, was used to localize Na⁺,K⁺-ATPase in transport epithelia of two species of teleosts. Exposure of the opercular membrane of seawater-adapted tilapia (Oreochromis mossambicus) and the jaw skin of the long-jawed mudsucker (Gillichthys mirabilis) to a 2 M anthroylouabain solution resulted in the appearance of cells stained bright blue. These were deemed to be chloride cells by their large size, distinct morphology and co-localization of DASPEI fluorescence, a mitochondrial stain. Addition of ouabain (1 mM final concentration) greatly decreased anthroylouabain fluorescent staining of chloride cells of seawater-adapted fish. Exposure of opercular membranes from freshwater tilapia to 2 M anthroylouabain did not result in significant staining. Anthroylouabain is therefore a useful vital stain for localizing Na⁺,K⁺-ATPase in chloride cells of seawater-adapted teleosts, and may be useful for fluorescent labelling of ouabain-sensitive Na⁺,K⁺-ATPase in other tissues and species.