Coexistence of catecholamine and methionine enkephalin-Arg6-Gly7-Leu8 in neurons of the rat ventrolateral medulla oblongata. Application of combined peptide immunocytochemistry and histofluorescence method in the same vibratome section

An overlapping distribution of catecholamine-containing cells and proenkephaline-A derived peptide-containing neurons have been identified in the rat medulla oblongata. However, it is not evident whether the coexistence of these bioactive substances occurs in the same neurons or not. Therefore, we examined the coexistence of catecholamine and methionine-enkephalin-Arg6-Gly7-Leu8 (MEAGL), a proenkephaline-A derived peptide, using a combination of histofluorescence and peroxidase-anti-peroxidase (PAP) immunohistochemical (modified formaldehyde-glutalaldehyde (Faglu)) methods on the same tissue sections. We found one third of A1/C1 catecholamine fluorescent cells show MEAGL-like immunoreactivity.     

The membrane of the catecholamine storage vesicles of the adrenal medulla

Summary Ultrastructure and biochemical properties—catecholamine uptake and release and ATPase activity—of membrane preparation obtained from bovine adrenal medulla vesicles were studied.Destruction of vesicular organisation by sonication of catecholamine loaded membranes leads to the liberation of catecholamine, demonstrating that the latter is stored within the vesicular space rather than bound to the membrane.Four bands were obtained by sucrose gradient centrifugation, exhibiting distinct differences in ATPase activity and ability to take up catecholamine. The maior portion of material—fraction III—equilibrates in a position corresponding to 0.8 M sucrose. This fraction, although containing only 1/6 of catecholamine of the fraction I, displays the highest uptake of ¹⁴C-noradrenaline and a 4 fold higher ATPase activity than fraction I.Fraction III and IV contain several open membrane fragments and collapsed structures and a small proportion of disrupted mitochondria. Correlative estimations of succinate dehydrogenase- and ATPase-activity indicate that differences in ATPase activity of the fractions are only to a minor extent due to mitochondrial impurities.Negatively stained images reveal the great plasticity of the membrane of catecholamine storage vesicles, which is probably the basis of their ability to undergo the structural changes during the secretory cycle in the intact cell.     

Microfluorimetric quantification of catecholamine fluorescence in rat sympathetic ganglia

Summary The formaldehyde-induced fluorescence (FIF) technique was used to generate catecholamine fluorphores in the perikarya of the sympathetic neurons in the superior cervical ganglion of adult rats. During microfluorimetric quantification, the photodecomposition was eliminated by a rapid measuring procedure with a small excitation field and by using only visible light between the measurements.The catecholamine fluorescence, induced in protein microdroplets with increasing noradrenaline concentrations, was linear up to 2×10^–2 M which exceeds the noradrenaline content of even the most intensively fluorescent neurons. Thus, the differences in fluorescence intensities directly reflect the physiological state of each neuron with respect to their catecholamine content. The mean histograms reveal the changes which can only occur in certain neurons, and which can disappear if the mean only is assessed. The microfluorimetric method was sensitive enough to detect even minute changes induced by reserpine treatment in the catecholamine content of the sympathetic ganglion cells.     

Characteristics of chloride-dependent effects of acetylcholine and neutral aminoacids on neurons of the snail (Achatina)

The response was investigated of neurons composing the cerebral ganglia inAchatina fulica (the Giant African snail) to application of acetylcholine (ACh), gamma-aminobutyric acid (GABA), and glycine (Gly). Chloride-dependent currents induced by these transmitters in 1 1/2-month old siblings were inhibited by dibutyryl-cAMP and strychnine. Inhibition of ACh response produced 10^–8 M GABA was mimicked by application of dibutyryl-cAMP and isobutylmethylxanthine. Complete cross-desensitization was characteristic of both GABA- and Bly-induced response, but this effect did not occur when ACh and GABA (or Gly) were applied. A conclusion was reached on the basis of the pharmacological relationship between GABA- and Gly-induced response that these amino acids act on a single receptor — channel complex in the neurons of infant snails, whereas ACh-, GABA-, and Gly-induced chloride currents were not so related in cells of 4 year-oldAchatina.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 35–43, January–February, 1991.     

Mimosine Arrests the Cell Cycle after Cells Enter S-Phase

-Mimosine (β-N-[3-hydroxy-4-pyridone]-α-aminopropionic acid)—a rare amino acid derived fromMimosaandLeucaenaplants—arrests cells reversibly late during G1 phase or at the beginning of S-phase. If mimosine were to arrest cells immediately before S-phase, it would provide a superb tool for the investigation of the initiation of DNA synthesis. Therefore, we reexamined the point of action of mimosine. Mitotic HeLa cells were released into 200 μMmimosine and grown for 10 h to block them, before the cells were permeabilized and the amino acid removed by washing them thoroughly. On addition of the appropriate triphosphates, DNA synthesis—measured by the incorporation of [³²P]dTTP—began immediately; as it is known that such permeabilized cells cannot initiate DNA synthesis but can only resume elongating previously initiated chains, mimosine must arrest after DNA synthesis has begun. Moreover, cells grown in mimosine assembled functional replication factories—detected by immunolabeling after incorporation of biotin–dUTP—that were typical of those found early during S-phase. Disappointingly, it seems that mimosine—like aphidocolin—blocks only after cells enter S-phase.     

Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons

Pheromones are substances released from animals that, when detected by the vomeronasal organ of other individuals of the same species, affect their physiology and behavior. Pheromone binding to receptors on microvilli on the dendritic knobs of vomeronasal sensory neurons activates a second messenger cascade to produce an increase in intracellular Ca²⁺ concentration. Here, we used whole-cell and inside-out patch-clamp analysis to provide a functional characterization of currents activated by Ca²⁺ in isolated mouse vomeronasal sensory neurons in the absence of intracellular K⁺. In whole-cell recordings, the average current in 1.5 µM Ca²⁺ and symmetrical Cl⁻ was −382 pA at −100 mV. Ion substitution experiments and partial blockade by commonly used Cl⁻ channel blockers indicated that Ca²⁺ activates mainly anionic currents in these neurons. Recordings from inside-out patches from dendritic knobs of mouse vomeronasal sensory neurons confirmed the presence of Ca²⁺-activated Cl⁻ channels in the knobs and/or microvilli. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A/anoctamin1 or TMEM16B/anoctamin2 Ca²⁺-activated Cl⁻ channels, which are coexpressed in microvilli of mouse vomeronasal sensory neurons, and found a closer resemblance to those of TMEM16A. We used the Cre–loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons. Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons. Ca²⁺-activated currents were abolished in vomeronasal sensory neurons of TMEM16A conditional knockout mice, demonstrating that TMEM16A is an essential component of Ca²⁺-activated Cl⁻ currents in mouse vomeronasal sensory neurons.     

Distribution of MAP1A,MAP1B,and MAP2A&;B during layer formation in the optic tectum of developing chick embryos

The expression patterns of three microtubule-associated proteins (MAP1A, MAP1B, and MAP2A&B) were investigated in the developing optic tectum. Expression of MAP1B and middle-molecular-weight peptide of neurofilament (NF-M) was first observed in the same mesencephalic cells on day 3 of incubation, indicating that neuroblasts had been produced. At day 5, MAP1A and MAP2A&B expression appeared in the cellular layer containing the first neuroblasts that differentiate into large multipolar cells. The NF-M⁺ neurites in the striatum album centrale (SAC) and the striatum opticum (SO) were MAP1B⁺ up to day 19, but the intensity of MAP1B immunoreactivity decreased with development. All three MAPs were expressed in large multipolar neurons in the developing stratum griseum centrale from the beginning of maturation. Stratum griseum et fibrosum centrale cellular layers, containing radially arranged piriform neurons, were MAP1A^–/MAP2A&B^– on day 11 but became MAP1A⁺/MAP2A&B⁺ during later stages. These results suggest that the timing of MAP expression in neuronal maturation of large multipolar cells differs from that of piriform cells. The expression of MAPs has revealed specific cellular events in the developing optic tectum. Based on our observations, the development of the optic tectum can be divided into four periods.     

Formation of Polar Bundles of Pili and the Behavior of Azospirillum brasilenseCells in a Semiliquid Agar

This paper describes the formation of single polar bundles of pili on Azospirillum brasilensecells, the twitching motility of cell aggregates, and a new type of social behavior—the dispersal of bacterial cells in semiliquid agar associated with the formation of granular inclusions (the so-called Gri⁺phenotype)—which is an alternative to swarming (the Swa phenotype). The wild-type A. brasilensecells occurring in a semiliquid agar may show either the Swa⁺Gri^–, or Swa^–Gri^–, or Swa^–Gri⁺phenotype. The formation of single polar flagella (Fla) or polar bundles of pili may reflect two alternative states of A. brasilensecells. The components of the Fla system may be involved in the regulation of the phenotypic variation of azospirilla.     

Lithium Induces Apoptosis in Immature Cerebellar Granule Cells but Promotes Survival of Mature Neurons

Lithium (Li⁺) has been used in the treatment of manic—depressive disorders for several decades. More recently, Li⁺ has been shown to affect the signaling pathway of various neurotransmitters and growth/neurotrophic factors. We examined the effect of Li⁺ on the survival of cerebellar granule neurons in culture. Treatment of immature granule cells with Li⁺ resulted in programmed cell death (apoptosis). The death process is accompanied by DNA fragmentation, a hallmark of apoptosis. Following maturation in vitro, granule neurons are dependent on elevated concentrations of extracellular potassium ([K⁺]_o) for survival. Lowering of [K⁺]_o to physiological levels induces apoptosis. Surprisingly, Li⁺ prevents death of mature neurons caused by low [K⁺]_o. Moreover, the concentration range at which Li⁺ exerts its protective effect is the same as that at which it induces apoptosis in immature neurons. Thus, a single agent under similar extracellular conditions has opposing effects on survival, depending on the developmental status of the neuron.     

Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2

The N‐Myc Downstream‐Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4 ^−/−) CRC models and an indirect co‐culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4 ^−/− ENS cell secretome, which is enriched for Nidogen‐1 (Nid1) and Fibulin‐2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS‐derived Nidogen‐1 and Fibulin‐2 enhance colorectal carcinogenesis.     

Different secretory actions of pancreastatin in bovine and human parathyroid cells

Chromogranin A is an acidic protein that is costored and cosecreted with parathyroid hormone (PTH) from parathyroid cells. Pancreastatin (PST), is derived from chromogranin A, and inhibits secretion from several endocrine/neuroendocrine tissues. Effects of different pancreastatin peptides were investigated on dispersed cells from bovine and human parathyroid glands. Bovine PST(1–47) and bovine PST(32–47) inhibited PTH release from bovine cells in a dose-dependent manner. The former peptide was more potent and suppressed the secretion at 1–100 nM. This inhibition was evident in 0.5 and 1.25 mM, but not in 3.0 mM external Ca²⁺. Both peptides failed to alter the concentration of cytoplasmic Ca²⁺([Ca²⁺]_i) of bovine cells. Human PST(1–52) and PST(34–52) did not affect PTH release or [Ca²⁺]_i of parathyroid cells from patients with hyperparathyroidism, nor [Ca²⁺]_i of normal human parathyroid cells. Furthermore, bovine PST(1–47) and bovine PST(32–47) failed to alter the secretion of abnormal human parathyroid cells. The study indicates that PST exerts secretory inhibition on bovine but not human parathyroid cells, and that this action does not involve alterations of [Ca²⁺]_i.     

RAT BRAIN SYNAPTIC VESICLES: UPTAKE SPECIFICITIES OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN IN PREPARATIONS FROM WHOLE BRAIN AND BRAIN REGIONS1

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [³H]norepinephrine and [³H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The K_m for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6-hydroxydopamine, but not of 5,6-dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat-ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6-hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP-Mg²⁺ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with K_is similar to those for inhibition of norepinephrine uptake. Whereas 5,6-dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6-hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.     

Zur Frage eines Photoinaktivierungs-Effektes bei der Polaritätsinduktion inEquisetumsporen undFucus zygoten

Summary Polarity was induced in spores ofEquisetum variegatum and zygotes ofFucus serratus by monochromatic light; the cells were lit partially or by combinations of different types of irradiation.1. Partially lit cells yield the same energy-effect curve which has been found after irradiation with polarized or with unilateral unpolarized light in previous investigations. The energy requirement of the first primary process depends upon the type of irradiation. — 2. The first decline of the curve is caused by photoinactivation of the photoreceptor molecules responsible for the first primary process; this is indicated by the apparent coexistence of active and inactivated pigment in a cell. —3. The cells require approximately 20 minutes at the temperature of 20°C to restore or synthesizede novo the previously inactivated pigment.

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Mit 5 Textabbildungen     

A new serologically defined locus,Qa-1, in theTla-region of the mouse

A new cell-surface antigen, specified by a gene betweenH-2D andTla is described. The provisional notationQa-1 is suggested for the locus determining this newly recognized cell surface component. Qa-1 is distinguished from known TL antigens by the following two criteria. Its expression is not confined to thymocytes — it occurs on lymph node cells (LNC) also; and the phenotypes of the new congenic recombinant strains B6.K1 and B6.K2, derived fromH-2D/Tla crossovers, are Qa-1⁺ Qa-2^–TL^– and Qa-l⁺Qa-2⁺TL^–. Qa-1 antigen is defined by reaction of the standard TL typing serum, (B6 × A -Tla ^b)F₁ anti-A strain leukemia ASL1, with lymph node cells (LNC) in the cytotoxicity assay. Qa-1 antigen evidently is expressed, at least, on a subpopulation of T cells as well as on thymocytes. The gene order isH-2D, Qa-1, Qa-2, Tla.Abbreviations used in this paper LNC lymph node cells pooled from inguinal, axillary, brachial, and mesentric nodes - BA⁺ (C57BL/6-Tla^axA)F1 - BA^– (C57BL/6 × A -Tla ^b)F₁ - PBS phosphate buffered saline pH 7.2 - Thy thymocytes - RMIg Rabbit anti-mouse immunoglobulin Please address proofs and communications concerning this paper to Dr. Thomas Stanton, Sloan Kettering Institute, 1275 York Ave., New York, N.Y. 10021     

Effect of serotonin on neuronal background activity in a hemisegment isolated from the infant rat spinal cord

Neuronal background activity was investigated in a hemisegment of the lumbar section of the spinal cord before and after addition of serotonin (5-HT — 1 × 10^–8–10^–4 M) in 14- to 22-day-old rats. Reversible changes in background firing rate were recorded in 50% and 70.6% of dorsal and ventral horn interneurons respectively. Excitatory response predominated; in the dorsal horn, 62.4% of all cells responding to 5-HT showed an excitatory response, 8.4% an inhibitory reaction, and 29.2% a two-stage response. In the ventral horn, an excitatory and two-stage response were recorded in 91.6% and 8.4% of cells respectively. Application of 5-HT induced an increase in firing rate and depolarization in the ventral horn. Findings from this study would point to a primarily excitatory effect of 5-HT on background in segmental neurons.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 335–343, May–June, 1989.     

Methylglyoxal Evokes Pain by Stimulating TRPA1

Diabetic neuropathy is a severe complication of long-standing diabetes and one of the major etiologies of neuropathic pain. Diabetes is associated with an increased formation of reactive oxygen species and the electrophilic dicarbonyl compound methylglyoxal (MG). Here we show that MG stimulates heterologously expressed TRPA1 in CHO cells and natively expressed TRPA1 in MDCK cells and DRG neurons. MG evokes [Ca²⁺]_i-responses in TRPA1 expressing DRG neurons but is without effect in neurons cultured from Trpa1^−/− mice. Consistent with a direct, intracellular action, we show that methylglyoxal is significantly more potent as a TRPA1 agonist when applied to the intracellular face of excised membrane patches than to intact cells. Local intraplantar administration of MG evokes a pain response in Trpa1^+/+ but not in Trpa1^−/− mice. Furthermore, persistently increased MG levels achieved by two weeks pharmacological inhibition of glyoxalase-1 (GLO-1), the rate-limiting enzyme responsible for detoxification of MG, evokes a progressive and marked thermal (cold and heat) and mechanical hypersensitivity in wildtype but not in Trpa1^−/− mice. Our results thus demonstrate that TRPA1 is required both for the acute pain response evoked by topical MG and for the long-lasting pronociceptive effects associated with elevated MG in vivo. In contrast to our observations in DRG neurons, MG evokes indistinguishable [Ca²⁺]_i-responses in pancreatic β-cells cultured from Trpa1^+/+ and Trpa1^−/− mice. In vivo, the TRPA1 antagonist {"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681"}}HC030031 impairs glucose clearance in the glucose tolerance test both in Trpa1^+/+ and Trpa1^−/− mice, indicating a non-TRPA1 mediated effect and suggesting that results obtained with this compound should be interpreted with caution. Our results show that TRPA1 is the principal target for MG in sensory neurons but not in pancreatic β-cells and that activation of TRPA1 by MG produces a painful neuropathy with the behavioral hallmarks of diabetic neuropathy.     

The genetic map of the mitochondrial genome in yeast

Summary An approach for the screening of mit ^- mutants, the isolation and preliminary classification of a series of such mutants is reported. Loss and retention of 8 mit ^- and 6 drug ^r markers in mitDNA was analyzed in populations of rho^- clones derived from four yeast strains. The populations studied constitute a representative fraction of the rho^- petites formed during growth at 35° C under the influence of mutation tsp-25 which is in common to the four strains. The majority of the rho^- clones retained several of the markers studied. Depending on the marker regarded retention frequencies between 15% (oxi3) and 45% (oli1, cob) were observed. Loss of one and retention of the other of a pair of markers was determined in all rho^- clones of the four populations. The frequencies of marker separation by rho^- deletion thus obtained are assumed to reflect the distance between markers on the mitochondrial genome: the higher the frequency of separation the longer the distance between two markers. Based on these frequencies a unique order of markers on a circular map was determined. Positions of markers on a scale from 0 to 100 were found to be: cap/ery (0) — olil (16) — cob1-1354 (21) — ana101 (22) — cob2-1625 (24) — oli2 (35) — pho1 (40) — oxi3-2501 (44) — oxi3-3771 (47) — par (65) — oxi2 (79) — oxil (87) tms8 (93) —cap (100). The relevance of this map as to the faithful representation of the topology of gene loci on mitDNA is discussed. Correlation of retention frequencies of markers to their map positions reveals a pronounced polarity: mitDNA segments carrying the cob-oli1 segment prevail whereas segments retaining oxi3 are the least frequent.     

Corticofugal postsynaptic influences on neurons of Clarke's column

We carried out intracellular recording from neurons of Clarke's column of the spinal cord of a cat. It is demonstrated that relay neurons of the dorsal spino-cerebellar tract — which are activated by proprioceptive fibers from one muscle only — are not subject to corticofugal postsynaptic control, in contrast to neurons on which excitory and inhibitory influences converge from various groups of afferents. The corticofugal effects in such neurons coincide in direction with effects evoked by flexor reflex afferents. Properties are described of neurons that are situated in the vicinity of Clarke's column but are not relay neurons of the DSCT. The hypothesis is expressed that these neurons are identical to the "border cells" — short-axon interneurons on which axonic collaterals of relay neurons terminate.A. A. Bogomolets' Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 1, No. 1, pp. 15–24, July–August, 1969.     

Cyanine dye fluorescence used to measure membrane potential changes due to the assembly of complement proteins C5b-9

Summary The fluorescent potentiometric indicator diS–C₃-(5) has been used to investigate changes in membrane potential due to assembly of the C5b-9 membrane attack complex of the complement system. EAC1-7 human red blood cells and resealed erythrocyte ghosts—bearing membrane-assembled C5b67 complexes—were generated by immune activation in C8-deficient human serum. Studies performed with these cellular intermediates revealed that the membrane potential of EAC1-7 red cells and ghosts is unchanged from control red cells (–7 mV) and ghosts (0 mV), respectively. Addition of complement proteins C8 and C9 to EAC1-7 red cells results in a dose-dependent depolarization of membrane potential which precedes hemolysis. This prelytic depolarization of membrane potential—and the consequent onset of hemolysis—is accelerated by raising external [K⁺], suggesting that the diffusional equilibration of transmembrane cation gradients is rate limiting to the cytolytic event. In the case of EAC1-7 resealed ghosts suspended at either high external [K⁺] or [Na⁺], no change in membrane potential (from 0 mV) could be detected after C8/C9 additions. When the membrane potential of the EAC1-7 ghost was displaced from 0 mV by selectively increasing the K⁺ conductance with valinomycin, a dose-dependent depolarization of the membrane was observed upon addition of C8 and C9. In these experiments, lytic breakdown of the ghost membranes was <5%. Conclusions derived from this study include: (i) measured prelytic depolarization of the red cell Donnan potential directly confirms the colloid-osmotic theory of immune cytolysis. (ii) The diffusional transmenbrane equilibration of Na⁺ and K⁺ through the C5b-9 pore results in a dose-dependent depolarization of the membrane potential (E _m ) which appears to be rate-limiting to cytolytic rupture of the target erythrocyte. (iii) Enhanced immune hemolysis observed in high K⁺ media cannot be attributed to cation-selective conductance across the C5b-9 pore, and is probably related to the nearequilibrium condition of potassium-containing red cells when suspended at high external K⁺. These experiments demonstrate that carbocyanine dye fluorescent indicators can be used to monitor electrochemical changes arising from immune damage to the plasma membrane under both cytolytic and noncytolytic conditions. Potential application of this method to the detection of sublytic pathophysiological changes in the plasma membrane of complement-damaged cells are discussed.