Confinement hinders motility by inducing RhoA-mediated nuclear influx, volume expansion, and blebbing

Cells migrate in vivo through complex confining microenvironments, which induce significant nuclear deformation that may lead to nuclear blebbing and nuclear envelope rupture. While actomyosin contractility has been implicated in regulating nuclear envelope integrity, the exact mechanism remains unknown. Here, we argue that confinement-induced activation of RhoA/myosin-II contractility, coupled with LINC complex-dependent nuclear anchoring at the cell posterior, locally increases cytoplasmic pressure and promotes passive influx of cytoplasmic constituents into the nucleus without altering nuclear efflux. Elevated nuclear influx is accompanied by nuclear volume expansion, blebbing, and rupture, ultimately resulting in reduced cell motility. Moreover, inhibition of nuclear efflux is sufficient to increase nuclear volume and blebbing on two-dimensional surfaces, and acts synergistically with RhoA/myosin-II contractility to further augment blebbing in confinement. Cumulatively, confinement regulates nuclear size, nuclear integrity, and cell motility by perturbing nuclear flux homeostasis via a RhoA-dependent pathway.

Effects of Ethanol and Other Alkanols on Transport of Acetic Acid in Saccharomyces cerevisiae

ABSTRACT In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of V max versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S. cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.

Transport of L-proline and alpha-methyl-D-glucoside by chicken proximal cecum during development

We examined the characteristics of amino acid and sugar absorption by the proximal cecum (PC) of chickens during posthatch development. Rates of absorption of L-proline (Pro) and alpha-methyl-D-glucoside (MG) were measured at 2 days, 5 wk, and 13 wk after hatch with an in vitro everted-sleeve method. For each age, pieces of PC and midjejunum were incubated in solutions containing 0.1-50 mM Pro or MG, and the active and passive components of Pro and MG absorption were determined. Five conclusions may be stated. 1) There are two carrier-mediated transport systems for Pro in the PC: a higher capacity Na(+)-dependent system (Vmax between 1.6 and 3.2 nmol.mg-1.min-1), and a lower capacity Na(+)-independent system (Vmax 0.3-0.8 nmol.mg-1.min-1). 2) Whereas both Pro transport systems are present in the PC at 5 and 13 wk, only the Na(+)-dependent system was found at 2 days. Although rates of transport per milligram tissue by the Na(+)-dependent system fell during development, when rates were normalized to nominal surface area, Vmax was significantly higher in the 5-wk-old group than in the other groups. 3) MG transport is by a Na(+)-dependent system. Vmax values (nmol.mg-1.min-1) were 0.32 (2 days), less than 0.43 (5 wk), and = 0.55 (13 wk). These differences were not affected by normalization to surface area. 4) Because at physiological concentrations passive influx of Pro and MG would be negligible, absorption of amino acids and sugars by the PC would be dependent on the presence of carrier-mediated systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Early Postischemic 45Ca Accumulation in Rat Dentate Hilus

Several studies have found postischemic regional accumulation of calcium to be time-dependent and coincident with the progression of ischemic cell change. In the most vulnerable cells in the hippocampus one would therefore expect to find a primary and specific early uptake of calcium after ischemia. Autoradiograms of 45Ca and 3H-inulin distribution were investigated before and 1 h after 20 min ischemia in the rat hippocampus. Two different methodological approaches were used for administration of 45Ca: (a) administration via microdialysis probes, (b) intraventricular injection. During control conditions the 45Ca autoradiograms showed variations in distribution volume in accordance with 3H-inulin determination of extracellular space size. One hour after ischemia a massive accumulation of 45Ca was found in the dentate hilus. No change in the distribution pattern of 3H-inulin could be demonstrated 1 h after ischemia. We suggest that 45Ca accumulation in dentate hilus 1 h after ischemia is a result of increased Ca2+ uptake before irreversible cell damage occurs and is not due to passive influx of calcium across a leaky plasma membrane.

Complement complex C5b-8 induces PGI2 formation in cultured endothelial cells

The effects of the terminal complement sequence on prostacyclin (PGI2) generation in antibody-sensitized pulmonary arterial endothelial cells were examined. Whereas C5b-7 complement complexes induced no PGI2 formation, addition of purified complement component C8 resulted in a time- and dose-dependent burst of PGI2 release in the absence of overt cell damage. Formation of the complete terminal complement complex C5b-9 enhanced PGI2 release but was accompanied by cytolysis. Extracellular Ca2+ was required for C5b-8-dependent PGI2 formation. Three different blockers of physiological calcium channels failed to suppress the observed stimulatory effect. In contrast, W7 [N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide] and trifluoperazine, inhibitors of calmodulin activity, all reduced the C5b-8-dependent PGI2 generation. None of the inhibitors used impaired Ca2+ flux into the cells. One minute after addition of C8 to endothelial cells carrying C5b-7 complexes, a six- to seven-fold enhanced passive influx of 45Ca2+ into the cells was noted. An enhanced passive influx was also observed for 51Cr O4(2-), [3H] aminobutyric acid, and [3H]sucrose, but not for [3H]inulin and [3H]dextran. These data together suggest that complement C5b-8 complexes may serve as Ca2+ bypass gates in endothelial cells, the ensuing influx of Ca2+ leading to subsequent activation of the arachidonic acid pathway.

Effects of cadmium and zinc on calcium uptake in human red blood cells

Cadmium and zinc increased the accumulation of calcium in human red blood cells by increasing passive influx without enhancing the permeability to other ions. The effect of cadmium and zinc appeared specific to these metals, because barium, magnesium, cobalt, strontium, manganese, and nickel had no effect. Changes in calcium uptake by extracellular sodium, potassium, and pH were not altered by zinc and cadmium. Inhibition of calcium uptake by quinine, oligomycin, and iodoacetate was not affected by cadmium or zinc. These results suggest that cadmium and zinc increase calcium movement through normal influx pathways. Cadmium and zinc acted synergistically apparently by different mechanisms. Zinc and cadmium differentially affected calcium uptake in different extracellular calcium concentrations. The cadmium effect was increased by low concentrations of 2-mercaptoethanol and above pH 8.0, while the zinc effect was less sensitive to these factors. These findings suggest that the cadmium effect involves a disulfide bond between cysteinyl residues and the zinc effect involves a different site.

Effect of sodium on calcium-dependent force in unstimulated rat cardiac muscle

It has previously been demonstrated that 1) changes in superfusate calcium concentration [Ca2+]o within the low millimolar range result in changes in "resting" force and in the light-scattering properties of unstimulated rat cardiac muscle, and 2) if [Ca2+]o is increased from zero to millimolar concentrations, i.e., reperfusion with Ca2+ after a Ca2+-free period, a large influx of Ca2+ occurs and is associated with a substantial increase in resting force. The present study determined whether the Ca2+ influx in either case was influenced by intracellular sodium (Na+i). In unstimulated isometric rat right ventricular papillary muscles equilibrated at 29 degrees C, [Ca2+]o was increased from 1 to 4 mM or from 0 to 2 mM under conditions that vary Na+i, and the resulting change in intracellular calcium concentration [( Ca2+]i) was monitored by changes in both resting force and the frequency of intensity fluctuations in laser light scattered by the muscle. In each case, lowering Na+i by equilibration in lowered extracellular sodium concentration [( Na+]o) or enhancing [Na+]i by equilibration in the absence of extracellular potassium or in the presence of ouabain markedly lowered and enhanced respectively the apparent Ca2+ influx in response to the step increase in [Ca2+]o. Thus, in unstimulated rat cardiac muscle, Na+i modulates the Ca2+ influx resulting from a step increase in [Ca2+]o both under physiological conditions and following a period of Ca2+-free superfusion, i.e., the "Ca2+ paradox." A passive influx of Ca2+ down its electrochemical gradient would not depend on Na+i, and the voltage-time dependent slow Ca2+o channel is inactivated under the experimental conditions employed. The results are best explained by a sarcolemmal Na+-Ca2+ exchange mechanism and suggest that the reversal potential of this electrogenic exchanger is exceeded during a step increase in [Ca2+]o even at the transmembrane potential of resting muscle.

Increased calcium permeability of cold-stored erythrocytes

The calcium, sodium, and magnesium permeability of erythrocytes from blood stored at 4 degrees C in various anticoagulant media has been studied and compared to that of fresh erythrocytes. Passive influx of CA2+ was measured at 37 degrees C in cells pretreated to abolish Ca2+ pumping and was up to fivefold greater for cold-stored erythrocytes than for fresh cells. The Ca2+ leakiness developed gradually after day 2 and reached a maximum by day 7 of cold storage in ACD, CPD, CPD- adenine, or heparin anticoagulants. The total calcium content of cold- stored erythrocytes in ACD was not significantly different from that of fresh erythrocytes. However, when cold-stored erythrocytes were reincubated at 37 degrees C in media containing 1.5 mM ionized calcium and substrates to regenerate ATP, a net gain of Ca2+ occurred that was greater for stored than for fresh erythrocytes. Cold storage of blood for up to 6 wk in any anticoagulant did not alter either sodium or magnesium permeability. Red cell ATP was also measured and fell steadily during cold storage in ACD or CPD, but more increase in Ca2+ permeability preceded any significant change in red cell ATP, it is likely that a selective calcium leak develops independently of the fall in ATP concentration that occurs on cold storage.