Epidermal Tissue Homeostasis

Abstract. Toad epidermis is a suitable model for studies on tissue homeostasis because cell pool size, influx into and efflux from the cell pool can be easily determined. the cell pool size was obtained by cell counting on photomicrographs, the influx (cell birth rate) was assessed by the metaphase-arrest technique, and the efflux (cell loss by moulting) assessed by counting the number of cells in the corneal layer and recording of intermoult periods. the importance of the methods for assessing these parameters per square unit of skin surface is emphasized.
These parameters were studied in eight groups of ten adult male toads sacrificed at various hours of the day. There were minor variations in the cell birth rate, fluctuating around a mean of 26 cells/mm²/hr (obtained at the metaphase collection period from 11.00-14.00 hours). By summation of the cell productions during the eight metaphase collection periods of 3 hr, and extrapolation to an intermoult period (time between two moults), a calculated cell production of about 6340 cells/mm² in 10.3 days was obtained, whereas the cell loss at each moult was only 2370 cells/mm². Thus the cell production rate exceeds the rate of cell loss through moults by a factor of 2.7. Arguments are presented that the 'surplus' of cells produced cannot be permanently accommodated within the living epidermis. Consequently a cell deletion rate beyond that by moulting of about 4000 cells/mm² in 10.3 days or 16 cells/mm²/hr can be calculated.
These results are discussed in relation to current concepts of tissue homeostatic mechanism(s). the results are consistent with the hypothesis that controlled cell deletion may be a tissue homeostatic mechanism complementary to controlled cell divisions.     

Epidermal tissue homeostasis. II. Cell pool size, cell birth rate and cell loss in toads deprived of the pars distalis of the pituitary gland

Following removal of the pars distalis of the pituitary gland in toads, epidermal efflux from the stratum corneum recruitment cell pool (i.e. production of corneal layers) is greatly increased. In this investigation the cell birth rate is studied by means of the metaphase arrest technique, as a function of time after pars distalis ablation. The method allows assessment of the total cell production over 14 days after the operation, to be compared with the total efflux and changes in the epidermal cell pool size. Whereas in intact toads the rate of cell production exceeds that of cell loss by moulting by a factor of 2.7, the 'surplus' of cells neither being used for formation of corneal layers nor permanently accommodated within the living epidermis, a 'balance sheet' of efflux and influx indicates that following pars distalis ablation all cells produced are also used for the (excessive) formation of corneal cell layers. The observations lend further support to the hypothesis that controlled cell deletion is a tissue homeostatic mechanism complementary to controlled cell divisions.     

Time-dependent effects of removal of the pars distalis of the pituitary gland on toad epidermal cell and tissue kinetic parameters

Abstract. Amphibian moulting and its hormonal control has been extensively studied, but the possible influence of hormones on epidermal proliferation has been less investigated. In the present contribution to studies on the control of epidermal homeostasis, the proliferative pattern of the toad epidermis following ablation of the pars distalis of the pituitary gland is analysed by an investigation of the changes in the epidermal cell number, metaphase index and [³H]thymidine incorporation at various times after the operation. During the first 24 hr after pars distalis ablation, labelling index (LI) increased concurrently with an initial decrease in the metaphase index (MI), followed by an increase. During the same period of time the mean grain count (MGC) and the grain distribution pattern also changed. From 24 hr to 7 days after operation, MI, LI, and MGC were significantly lower than those of controls, but increased to control levels at 14 days after the operation. Phase durations and their possible changes were not measured directly, but data showed that the S-phase duration (Ts) and the mitotic duration (TM) must have changed in relation to each other during the experiment. Assuming that the MGC is a rough estimate of the DNA-synthesis rate, the compatibility of a postulated change in phase duration with the observed MGC was analysed. This analysis revealed that TS and TM could have decreased up to 18 hr after the operation, whereas these phase durations, after 24 hr and during the rest of the experiment, increased compared to those of controls. Even with these possible changes in phase durations, and in the absence of direct assessment of cell division rates, the observed cell kinetic parameters appeared incompatible with an increased rate of proliferation. This was surprising since the efflux of cells from the living, subcorneal epidermis to the stratum corneum was significantly increased after pars distalis ablation, without a concurrent decrease in the stratum corneum recruitment cell pool (SCRP-number of subcorneal epidermal cells per mm surface). Possible reasons for the discrepancy between the expected increase in proliferation following pars distalis ablation, and the failure to demonstrate this, are discussed.     

Epidermal tissue homeostasis. III. Effect of hydrocortisone on cell pool size, cell birth rate and cell loss in normal toads and in toads deprived of the pars distalis of the pituitary gland

It has previously been shown that in toad epidermis the cell birth rate (Kb) exceeds the rate of cell loss through moulting (Kd) and that the 'surplus' of cells seems to be removed in a controlled manner. Assuming that the epidermis is non-expanding, a Kb/Kd ratio greater than 1 indicates that cell deletion additional to desquamation takes place. In normal toads this ratio is 2-3. Following implantation of hydrocortisone pellets into intact toads (release rate, 18 micrograms/g toad/d), the Kb/Kd ratio, over a period of 14 d of hormone treatment, had increased to about 7, due mainly to an increased Kb and to a lesser extent to a decreased Kd. No change in the epidermal cell pool size had taken place. It was previously shown that, following removal of the pars distalis of the pituitary gland, the Kb/Kd ratio decreased with time, due to a decreasing Kb and an increasing Kd, eventually leading to a decreased epidermal cell pool size. In this paper it is shown that, in pars distalisectomized toads with hydrocortisone pellets implanted, the Kb/Kd ratio is restored to control levels by a restoration of the Kb as well as the Kd. The results differ from those of previous studies in which ACTH or adrenocorticosteroids were administered discontinuously (by injection). Thus, by experimental manipulation, different Kb/Kd ratios can be obtained: low (less than 1, pars distalis ablation), medium (2-3, normal toads) and high (7, hydrocortisone implantation). The potentiality of this unique situation in analysing the important question of how the 'surplus' cells are deleted is discussed.     

Circadian rhythms in mouse epidermal basal cell proliferation. Variations in compartment size, flux and phase duration.

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G2 phase DNA content were measured by flow cytometry of isolated basal cells, and the [3H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G2 phase and traversed the G2 phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G2 phase and mitosis. Circadian variations in the duration of the S phase, the G2 phase and mitosis were clearly demonstrated.     

Trichostrongylus colubriformis: epithelial cell kinetics in the small intestine of infected rabbits

Epithelial cell kinetic parameters were compared in intestines of control and Trichostrongylus colubriformis infected rabbits using a microdissection and metaphase accumulation technique in regions of gut with heavy (proximal site) and small (distal site) burdens of worms. In control animals, the cell production rates were respectively 4.3 cells/crypt/hr in the proximal region and 3.7 cells/crypt/hr in the distal one; and the influx of cells onto villi were respectively 67.5 cells/hr and 37.4 cells/hr. In the parasitized rabbits, in the main site of infection, a fourfold increase was recorded in the cell proliferation rate and in the influx of cells onto villi. In the region distal to the main site of infection, the same parameters were twice the control values, although only a low number of T. colubriformis were recovered from this part of gut. These large modifications in the epithelial renewal probably underlies the morphological and enzymological changes previously described in both parts of the T. colubriformis infected gut.     

Rapid, futile K+ cycling and pool-size dynamics define low-affinity potassium transport in barley

Using the short-lived radiotracer 42K+, we present a comprehensive subcellular flux analysis of low-affinity K+ transport in plants. We overturn the paradigm of cytosolic K+ pool-size homeostasis and demonstrate that low-affinity K+ transport is characterized by futile cycling of K+ at the plasma membrane. Using two methods of compartmental analysis in intact seedlings of barley (Hordeum vulgare L. cv Klondike), we present data for steady-state unidirectional influx, efflux, net flux, cytosolic pool size, and exchange kinetics, and show that, with increasing external [K+] ([K+]ext), both influx and efflux increase dramatically, and that the ratio of efflux to influx exceeds 70% at [K+]ext > or = 20 mm. Increasing [K+]ext, furthermore, leads to a shortening of the half-time for cytosolic K+ exchange, to values 2 to 3 times lower than are characteristic of high-affinity transport. Cytosolic K+ concentrations are shown to vary between 40 and 200 mm, depending on [K+]ext, on nitrogen treatment (NO3- or NH4+), and on the dominant mode of transport (high- or low-affinity transport), illustrating the dynamic nature of the cytosolic K+ pool, rather than its homeostatic maintenance. Based on measurements of trans-plasma membrane electrical potential, estimates of cytosolic K+ pool size, and the magnitude of unidirectional K+ fluxes, we describe efflux as the most energetically demanding of the cellular K+ fluxes that constitute low-affinity transport.     

Increased permeability and subsequent resealing of the host cell membrane early after infection of Escherichia coli with bacteriophage T1.

The addition of T1 to cells growing at 37 degrees C in a minimal medium at 0.4 mM Mg2+ rapidly induced an irreversible loss of K+ and Mg2+ and uptake of Na+ by the cells. Both the ATP pool of the cells and the transmembrane proton motive force were reduced. These cells did not lyse from within, since viral DNA replication and the maturation of the 36,000-molecular-weight phage head protein were inhibited. By contrast, cells lysed when infected at 5.4 mM Mg2+. In these cells, T1 initially induced K+ efflux and Na+ influx and lowered the cytoplasmic ATP concentration. After a few minutes, the cation gradients and ATP pool were restored to levels close to that of control cells. At 5.4 mM Mg2+, the shutoff of host protein synthesis was delayed and coincided with the restoration of the ATP pool. In an ATP synthase-negative mutant, infection with T1 did not affect the cytoplasmic ATP concentration but inhibited host protein synthesis with the same rate as it did in wild-type cells.     

The Effect of Ouabain and External Potassium on the Ion Transport of Rabbit Red Cells

Na efflux of rabbit RBC is approximately 10 mmoles/kg wet weight. hr. One-half of this consists of a ouabain-insensitive exchange diffusion component. Ouabain inhibits 2.5 mmoles/kg.hr of Na efflux. K influx is 3.0 mmoles/kg.hr; 2.2 mmoles/kg.hr are inhibited by ouabain. In contrast with human RBC, ouabain inhibition of Na efflux and K influx of rabbit RBC is easily reversible. After 2 hr, ouabain inhibition of Na efflux is completely compensated for by increased internal Na concentration and Na efflux returns to initial levels. Removal of ouabain at this stage results in stimulation of the efflux by 4.3 mmoles/kg.hr. Na influx is initially not affected by ouabain but is increased by 2.4 mmoles/kg.hr after 2 hr incubation with the drug. Removal of K from normal Ringer does not affect Na efflux and increases Na influx by 1.6 mmoles/kg.hr. Addition of ouabain to K-free Ringer inhibits Na efflux and influx to the same extent (1.6 mmoles/kg.hr). Removal of Na from K-free Ringer has an inhibitory effect on efflux similar to that of ouabain. These findings suggest that the fraction of Na efflux inhibited by removal of external K is completely replaced by a new, ouabain-sensitive exchange diffusion of Na ions.     

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G₂ phase DNA content were measured by flow cytometry of isolated basal cells, and the [³H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G₂ phase and traversed the G₂ phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G₂ phase and mitosis. Circadian variations in the duration of the S phase, the G₂ phase and mitosis were clearly demonstrated.     

Laboratory observations of moulting,growth and maturation in Antarctic krill (Euphausia superba Dana)

Summary Observations of intermoult period, growth and maturation were made on krill which were transported from Antarctic waters and maintained in the laboratory in Australia over a three year period. The mean intermoult period (IP) for each of 10 specimens, with initial body lengths of 24.7=46.8 mm, kept at -0.5° C varied from 22.0 to 29.8 days (overall mean = 26.6 days). These measurements of IP are significantly longer than those obtained in some previous studies. Differences in experimental temperatures, light, body sizes and growth patterns of the specimens between studies are unlikely to be causes of these dissimilar results. The pattern of changes in body length (BL) varies from one individual to the next. The greatest increase in BL over a series of 4–5 moults ranged from 0.024 to 0.070 mm/day, which is equivalent to 0.0020 to 0.0086/day in body weight, assuming exponential growth. This maximum growth rate is about half the rate predicted from the growth scheme of Mauchline (1980) for wild krill. Comparison of growth data for other euphausiids suggests that Mauchline's scheme produces anomalous growth rate. The slower growth rate observed in the present study would extend the estimated life span of krill from 3–4 years, as calculated by Mauchline (1980), to 4–7 years. If krill undergo body shrinkage during the Antarctic winter the estimated life span might be even longer. Examination of the external sexual characters of moults showed both progression and regression of maturity stage in association with changes in BL.     

Cellular cholesterol efflux. Role of cell membrane kinetic pools and interaction with apolipoproteins AI, AII, and Cs.

Efflux of [14C]cholesterol from various cells was monitored in the presence of discoidal complexes of egg phosphatidylcholine and purified apolipoproteins, containing either apoAI, AII, or Cs. Particles containing apoAI were more efficient acceptors than those containing apoAII or Cs when the donor cells were J774 macrophages. No differences were observed when the same acceptor preparations were exposed to Fu5AH rat hepatoma or rabbit aortic smooth muscle cells. The differential efficiency of apolipoproteins in stimulating cholesterol removal from J774 cells was maintained in a plasma membrane-enriched fraction isolated from the same cells. Nonlinear regression analysis of kinetic data obtained from J774 cells exposed to apoAI complexes indicated that cholesterol efflux was best fitted to a curve describing the release from two kinetic compartments. Approximately 10% of cholesterol was transferred from a rapidly exchangeable pool with a t1/2 ranging between 1.5 and 3 h, and the remaining fraction was released from a slower pool with a t1/2 of about 20 h. Modulation of cholesterol efflux from J774 cells by either varying the concentration or the apolipoprotein composition of the acceptors influenced the size of the pools and the t1/2 of the slow pool. Kinetics of cholesterol efflux from membranes isolated from J774 cells also best fit a two-compartment model and modification of the apolipoprotein composition of the acceptor induced a pattern of changes in pool size and half-time similar to that described for whole cells. In the three cell lines studied, we consistently resolved a slow pool with a half-time ranging between 15 and 20 h. In smooth muscle cells only the slow pool was evident, whereas in Fu5AH a very large fast pool was also resolved. In contrast to J774 cells, apolipoprotein composition of the acceptor did not influence the pools in these two cell lines. These results led us to propose a new model regarding the influence of multiple kinetic pools of cholesterol on the regulation of cholesterol desorption from the cell membrane.     

Epidermal tissue homeostasis: Apoptosis and cell emigration as mechanisms of controlled cell deletion in the epidermis,of the toad,Bufo bufo

Summary In normal, non-expanding toad epidermis more cells are produced than needed to replace cells lost by moulting. By implication, cell deletion additional to moulting must take place. This paper deals with the mechanisms by which the surplus of cells is deleted, taking advantage of the fact that the ratio between cell birth rate (K _b) and the rate of desquamation (K _d), which in normal toads is 2 to 3, can be manipulated. In toads deprived of the pars distalis of the pituitary gland it is decreased to 0.2 to 0.3, and in toads with hydrocortisone pellets implanted into the subcutaneous lymph space it is increased to 7 to 10. Thus, structures candidates for the morphological manifestation of the deletion process should occur rarely in toads in which the pars distalis has been removed and frequently in toads with hydrocortisone pellets implanted. Categorization and enumeration of such structures by light microscopy in the epidermis from operated, normal, and hormone-treated toads were performed. The incidence of structures referred to as dark cells and omega-figures were found to correlate relatively well with the K _b/K_d-ratio. A subsequent ultrastructural analysis — on a cell-by-cell basis — of dark cells showed these to reflect various stages of apoptosis. The duration of the apoptotic process was calculated to be approximately 7 h. Light- and electron microscopy of omega-figures combined with histochemical observations of PSA-lectin binding were interpreted as reflecting a release of cells from the basal epidermis and their final elimination within the dermis. It is concluded (i) that apoptosis is an important mechanism of controlled cell deletion, (ii) that emigration to, and elimination in, the dermis is a possible deletion mechanism, and (iii) that necrosis is unlikely to play a role in controlled cell deletion.Supported in part by the Danish National Science Research Council (grant no. 11-6498) (PB)Part of this work was presented at the XVth Meeting of the European Study Group for Cell Proliferation, Sundvollen, Norway, 16–20 September 1987     

Untersuchungen zur Häutungsphysiologie der dekapoden Krebse am Beispiel der StrandkrabbeCarcinus maenas

Under constant laboratory conditions, juvenile shore crabs moult at fixed intervals which depend upon their body size. During one moult every crab exhibits increases of the same relative amounts, independent of its absolute size. Basing on the predictable duration of the intermoult period, the morphological changes in the structure of the cuticle and the development of limb-buds, the intermoult period could be divided into 21 different stages. After studying the moulting rhythm in constant milieu, the influence of the following exogenous and endogenous factors upon the moulting rhythm and growth of normal and of eye-stalkless individuals was investigated: temperature, photoperiod, loss of pereiopods, feeding, and presence of larger specimens. From these investigations it became evident that the moulting rhythm is regulated by growth. The crabs are able to moult only after achieving a minimum of tissue growth. So long as this minimum growth is not achieved, a moult-inhibiting hormone is secreted and moulting is prevented. If the moult-inhibiting hormone is absent, moulting hormone is secreted and initiates a moult. Under dangerous conditions, the crabs are able to delay the next moult. Under unfavourable conditions they consume less food than normal. Therefore, the amount of tissue growth which is the necessary prerequisite for moulting is delayed, and continued release of moult-inhibiting hormone prevents the moult. Under conditions favourable for moulting, or demanding moult (e. g. after loss of many pereiopods) the crabs accelerate the moult. Temperature influences the moulting rhythm by indirect effects on the metabolic rate. During further investigations, the variation of the following parameters were determined quantitatively: content of moulting hormone in whole crabs; content of aminoacids, protein, glucose, Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ in the hemolymph; pH and osmotic pressure in the hemolymph; and Ca⁺⁺ content in skeleton and whole crabs. All parameters mentioned — excepting pH and K⁺ content of the hemolymph — vary characteristically during the intermoult period. The titre of moulting hormone has 4 different maxima. Of all parameters, only the content of animoacids and protein in the hemolymph vary in the same way as the titre of the hormone. From these results the following conclusions are drawn: The moulting hormone not only initiates the moulting process, but controls it at several stages. Only protein metabolism seems to be under direct control of the moulting hormone which stimulates protein-synthesis. Chitin formation, regeneration, apolysis and ecdysis are indirectly controlled by the moulting hormone through protein metabolism. As in most of the other processes mentioned, the calcification of the new cuticle is not under the direct influence of the moulting hormone. The conclusion ofDigby (1966) that calcification in crabs is an electrochemical process, is confirmed.     

Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ -permeable channels

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+ -induced K+ efflux through outwardly directed, K+ -permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+ -sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.     

CDK4 regulation by TNFR1 and JNK is required for NF-kappaB-mediated epidermal growth control

Nuclear factor kappaB (NF-kappaB) mediates homeostatic growth inhibition in the epidermis, and a loss of NF-kappaB function promotes proliferation and oncogenesis. To identify mechanisms responsible for these effects, we impaired NF-kappaB action in the epidermis by three different genetic approaches, including conditional NF-kappaB blockade. In each case, epidermal hyperplasia was accompanied by an increase in both protein levels and tissue distribution of the G1 cell cycle kinase, CDK4. CDK4 up-regulation required intact TNFR1 and c-Jun NH2-terminal kinase (JNK) function. Cdk4 gene deletion concomitant with conditional NF-kappaB blockade demonstrated that CDK4 is required for growth deregulation. Therefore, epidermal homeostasis depends on antagonist regulation of CDK4 expression by NF-kappaB and TNFR1/JNK.     

Different epidermal cell kinetic effects of hydroxyurea when injected at two different times of the day

Circadian rhythms in epidermal basal cell-cycle progression in hairless mouse skin have been repeatedly demonstrated. A dose of 10 mg/animal hydroxyurea (HU), given to inhibit DNA synthesis was injected intraperitoneally to two groups of hairless mice. One group was injected at 10.00 hours MET, when the cell-cycle progression and cell division rate are relatively high, and another group was injected at 20.00 hours, when the same variables are at minimum values. Various cell kinetic methods--[3H]TdR autoradiography, DNA flow cytometry and the stathmokinetic method (Colcemid)--were used to study HU-induced alterations in cell kinetics. Hydroxyurea (HU) immediately reduced the labelling index (LI) to less than 10% of controls when injected at both times of the day, and higher then normal values were observed 8 hr later. A subsequent decrease towards normal values was steeper in the 20.00 hours injected group. The proportion of cells with S-phase DNA content was transiently reduced in both series, but the reduction was less pronounced and control values were reached earlier in the series injected at 10.00 hours. The observed alterations in LI and fraction of cells in S phase were followed by comparable alterations in the fraction of cells in G2 and in the mitotic rate. Hence the changes in G2 and mitotic rate are easily explained as consequences of the previous perturbations in the S phase. The time-dependent differences in the cell kinetic perturbations caused by HU in the S phase may be explained by a circadian-phase-dependent action of HU on the influx and efflux of cells to and from the S phase, respectively. At 10.00 hours the efflux of cells from S is most heavily inhibited; at 20.00 hours the influx is predominantly blocked. Hence, when physiological flux is high HU mainly blocks the efflux from S, but when flux normally is low, HU mainly blocks the entrance to S. Within 20 hours after the HU injection, the cell kinetic variables had approached the unperturbed circadian pattern.     

Potassium fluxes and ouabain binding in growing,density-inhibited and rous sarcoma virus-transformed chicken embryo cells

Potassium fluxes, ouabain binding, and Na⁺ and K⁺ intracellular concentrations were determined for cultures of growing normal, density-inhibited and Rous sarcoma virus-transformed chicken embryo fibroblasts. No significant differences in K⁺ influx or ouabain binding were detected between growing normal cells and Rous sarcoma virus-transformed cells; however, ouabain binding and ouabain-sensitive K⁺ influx were 1.5- to 1.8-fold lower in density-inhibited cells. Thus, potassium influx in this system can be classified as a growth-related, but not transformation-specific change. As determined by both flame photometry and radioisotopic (⁴²K) equilibration, growing normal and density-inhibited cells had similar potassium contents, whereas transformed cells exhibited 1.4-fold higher potassium levels. Sodium ion levels, as measured by flame photometry, were also 2- to 4.5-fold higher in transformed than normal or density-inhibited cells. Complementary studies of potassium efflux showed a 1.3- to 1.5-fold higher rate (based on the percentage of pool exiting the cell) in growing normal versus density-inhibited or transformed fibroblasts. Because of the larger potassium pool in transformed cells, efflux based on absolute number of potassium ions is similar in normal and transformed chicken embryo fibroblasts.     

Role of vacuolar ion pool in Saccharomyces carlsbergensis: potassium efflux from vacuoles is coupled with manganese or magnesium influx.

Saccharomyces carlsbergensis cells accumulated Mn2+ (or Mg2+) ions in the presence of glucose, fructose, or mannose, but not of deoxyglucose, 3-O-methylglucose, and sorbose. Accumulation of one equivalent of Mn/2+ was coupled with the efflux of two equivalents of K+ from the cells. Mg/2+ did not exit during Mn2+ uptake. Preliminary treatment of cells with various proton conductors or glucose led to the loss of K+ and to the proportional inhibition of Mn2+ uptake. Polyene antibiotic candicidin together with glucose elicited rapid efflux of K+ and completely inhibited Mn2+ accumulation. Exogenous K+ (more than 1 mM), 100 microM N,N'-dicyclohexylcarbodiimide, and 30 mM sodium arsenate inhibited both K+ efflux and Mn2+ influx. K+ efflux from S. carlsbergensis cells affected the vacuolar pool of K+ both during the accumulation of Mn2+ or Mg2+ and during glucose uptake.     

Regenerative proliferation of mouse epidermal cells following adhesive tape stripping. Micro-flow fluorometry of isolated epidermal basal cells combined with 3H-TdR incorporation and a stathmokinetic method (colcemid).

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) Laerum, 1969) and brought into a monodisperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G1, S and (G1 + M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. 3H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the G1 phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.