Osmolarity Of Incubation Medium Research Articles

Hypertonic shrinking but not hypotonic swelling increases sodium concentration in rat brain synaptosomes

Neurotransmitter release is dependent on both calcium and sodium influx. Hypotonic swelling and hypertonic shrinking of neurons evokes calcium-independent exocytosis of neurotransmitters into the synaptic cleft. To date, there are not too much data available on relationship between extracellular osmolarity and sodium concentration in presynaptic endings. In the present study we investigated the effects of hypotonic swelling and hypertonic shrinking on sodium levels, as measured using fluorescent dyes SBFI-AM and Sodium Green in rat brain synaptosomes. Reduction of incubation medium osmolarity from 310 to 230 mOsm did not raise the intrasynaptosomal sodium concentration. An increase of osmolarity from 310 to 810 mOsm is accompanied by a dose-dependent elevation of sodium concentration from 8.1 ± 0.5 to 46.5 ± 2.8 mM, respectively. This effect was insensitive to several channel inhibitors such as: tetrodotoxin, an inhibitor of voltage-gated sodium channels, bumetanide, an inhibitor of Na +/K +/2Cl − cotransport, gadolinium, an inhibitor of nonselective mechanosensitive channels, ruthenium red, an inhibitor of transient receptor potential channel and amiloride, an inhibitor of epithelial sodium channel/degenerin. Additionally, using the fluorescent dye BCECF-AM, we have shown that hypertonic shrinking caused a dose-dependent acidification of intrasynaptosomal cytosol, which suggests that the Na +/H + exchanger is not involved in the effect of increased osmolarity on cytosolic sodium levels. The increase in intrasynaptosomal sodium concentrations following increases in osmolarity is probably due to sodium influx through another sodium channels.

Calcium regulates the mode of exocytosis induced by hypotonic shock in isolated neuronal presynaptic endings

A decrease in the osmolarity of incubation medium is accompanied by calcium influx in neuronal presynaptic endings. We studied the influence of Ca 2+ on exocytosis induced by hypotonic shock using the hydrophilic fluorescent dye acridine orange and the hydrophobic fluorescent dye FM2–10. It was shown using acridine orange that lowering of osmolarity to 230 mOsm/l induces exocytosis both in calcium-containing and calcium-free medium. By contrast, we were able to demonstrate calcium-dependence of exocytosis using styryl dye FM2–10. Lowering of osmolarity leads to increase of [ 3H] d-aspartate and [ 3H]GABA release in calcium-free medium. Addition of calcium inhibits hypotonic-induced neurotransmitter release. Decreasing of NaCl concentration to 92 mM in isotonic medium is able to induce d-aspartate and GABA release. Thus, our data suggest that hypotonic swelling induces calcium-independent exocytosis possibly by a “kiss and run” mechanism. Calcium influx mediated by stretch channels is able to provoke full fusion between plasma membrane and synaptic vesicles. [ 3H] d-aspartate and [ 3H]GABA released by hypotonic shock is determined by sodium lowering rather than by osmolarity decreasing itself.

Riboflavin transport by rabbit renal basolateral membrane vesicles

The present study examined riboflavin (RF) uptake by isolated rabbit renal basolateral membrane (BLM). RF uptake was linear during the initial 10 seconds and leveled off thereafter with longer incubation. Studies on RF uptake as a function of incubation medium osmolarity indicated that the BLM RF uptake was the results of transport (∼45%) into the intravesicular space as well as binding (∼55%) to membrane surfaces. The RF binding to BLM was Na +-dependent so that replacement of Na + by other cations eliminated the binding component of RF uptake. The process of BLM RF uptake was saturable as a function of substrate concentration and was significantly inhibited by cis-addition of its structural analogs, lumiflavin and lumichrome, indicating the involvement of a carrier-mediated process. The BLM RF uptake was affected by changes in extravesicular pH so that, as compared to pH 7.5, RF uptake was lower at pH 6.5 and higher at pH 8.5. The effect of extravesicular pH persisted when the transmembrane H + gradient was dissipated by FCCP, indicating the direct effect of pH on BLM RF uptake. The BLM RF uptake was not affected by alterations of the transmembrane electrical potential, induced by either the presence of anions with different membrane permeability (Cl −= NO − 3 > SO − 4 >gluconate −) or using nigericin (10 μg/mg protein) with an outwardly or inwardly directed transmembrane K + gradient. The BLM RF uptake was, however, inhibited by probenecid and p-aminohippurate, and was enhanced by trans-RF. In summary, these results demonstrate the existence of a Na +-dependent BLM binding of RF and a membrane-associated carrier system for RF uptake by renal BLM.

Riboflavin transport by rabbit renal brush border membrane vesicles

The present study examined riboflavin (RF) uptake by isolated rabbit renal brush border membrane (BBM). RF uptake was linear for up to 30 s and leveled off thereafter reaching an equilibrium with longer incubation. Studies on RF uptake as a function of incubation medium osmolarity indicated that the uptake was the results of transport (61.4%) into the intravesicular space as well as binding (38.6%) to membrane surfaces. The process of RF uptake was saturable as a function of substrate concentration with an apparent K mof 25.7 ± 7.6 μM and V maxof 75.6 ± 14.7 pmol/mg protein/10 s. cis-Addition of unlabeled RF and its structural analogues, lumiflavin and lumichrome, inhibited the uptake of [ 3H]RF significantly, indicating the involvement of a carrier-mediated process in RF uptake by renal BBM. RF uptake by renal BBM was partly Na +-dependent so that when Na + was replaced by potassium, choline, lithium or tetramethylammonium, the RF uptake was reduced to ca. 60% of the control. This Na +-dependency was unlikely to be due to Na +-cotransport mechanism because RF uptake occurred without the characteristic ‘overshoot’ phenomenon as for other Na +-cotransport systems and the elimination of transmembrane Na +-gradient by preloading Na + to the intravesicular space did not affect RF uptake. In contrast, removal of Na + eliminated the binding component of RF uptake, suggesting the requirement of Na + for RF binding to BBM. The RF uptake was not affected when extravesicular pH was varied within the physiological pH range of 6.5 to 8.5. No effect on BBM [ 3H]RF uptake was found when the transmembrane electrical potential was altered by either the presence of anions with different membrane permeability ( Cl − = NO 3 − SO 4 −gluconate −) or by using nigericin (10 μg/mg protein) with an outwardly or inwardly directed transmembrane K + gradient. The uptake of RF by BBM vesicles was, however, inhibited by probenecid and organic anion transport inhibitors, 4,4-diiso-thiocyanatostilbene-2,2-disulfonic acid (DIDS, 1 mM) and 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS, 1 mM). In summary, these results demonstrate the existence of a membrane-associated, and organic anion inhibitor-sensitive, carrier system for RF uptake by renal BBM.

Riboflavin uptake by rat liver basolateral membrane vesicles

The present study examined riboflavin (RF) uptake by purified rat liver basolateral membrane vesicle (BLMV). Uptake of RF was found to be Na +-and pH-independent in nature. Studies on RF uptake by BLMV as a function of incubation medium osmolarity have indicated that the uptake is the result of transport (66.5%) into the intravesicular space as well as binding (33.5%) to membrane surfaces. The process of RF uptake by BLMV was saturable as a function of substrate concentration with an apparent K m of 3.55 ± 0.70 μM and V maxof 39.89 ± 3.24 pmol/mg protein/5 s, respectively. cis-Addition of unlabeled RF and its structural analogs lumaflavin and lumichrome inhibited the uptake of [ 3H]RF while trans-addition of unlabeled RF stimulated the efflux of [ 3H]RF from preloaded vesicles. No effect on RF uptake was found by the membrane transport inhibitors probenecid, 4,4-diisothiocyanotostilbene-2,2-disulfonic acid (DIDS) and 4-acetamido-4-isothiocyanatostilbene-2,2′-disulfonic acid (SITS). Induction of a transient positive intravesicular space led to a slight stimulation of RF uptake, while induction of a negative intravesicular space led to a slight inhibition in RF uptake. These results demonstrate the existence of a membrane-associated carrier system for RF uptake by liver BLMV. This system appears to be Na +- and pH-independent and is influenced to a certain degree by changes in transmembrane electrical potential.

Riboflavin uptake in microvillous and basal membrane vesicles isolated from full-term human placentas

Riboflavin uptake was characterized using membrane vesicles isolated from the apical (maternal-facing) and basal (fetal-facing) membranes of the syncytiotrophoblast from full-term human placentas. Equilibrium [3H]riboflavin uptake was insensitive to variations in incubation medium osmolarity in contrast to [3H]alanine uptake into an osmotically sensitive space. Osmotic insensitivity suggested riboflavin binding to a membrane component. The dissociation constant of riboflavin binding was similar in microvillous (Kd = 2 microM) and basal membrane vesicles (Kd = 1 microM). Binding capacity was significantly higher in microvillous membranes (Bmax = 11.9 pmol/mg protein). The relatively high affinity binding to the membrane vesicles may represent a first step in riboflavin transport.

An Na+-dependent and an Na+-independent system for glutamine transport in rat liver basolateral membrane vesicles

In the present study the transport of glutamine across rat liver basolateral membrane was examined with special emphasis on the existence of an Na+-independent system and on the characteristics of the Na+-dependent system with respect to stoichiometry of glutamine to Na+. Well-validated and purified liver basolateral membrane vesicles were used in the study. Results of studies on the effect of incubation medium osmolarity and incubation temperature indicated that glutamine uptake by liver basolateral membrane vesicles is largely the result of transport of the substrate into the intravesicular compartment with little binding to basolateral membrane vesicles. Transport of glutamine with time was Na+ gradient dependent (out > in) with a distinct “overshoot” phenomenon. Replacing Na+ with an equivalent concentration of K+, NH4+, choline, or mannitol caused significant inhibition of the initial rate of glutamine transport; on the other hand, Li+ could partially substitute for Na+. The initial rate of transport of glutamine as a function of concentration (0.05–12 mmol/L) was saturable both in the presence and in the absence of an inwardly directed Na+ gradient. Apparent Km values of 2.95 and 3.35 mmol/L and Vmax values of 11,565 and 6663 pmol · mg protein−1 · 10 s−1 were calculated in the presence and absence of a Na+ gradient, respectively. Both in the presence and absence of an Na+ gradient (out > in), transport of [3H]glutamine was significantly inhibited by the addition to the incubation medium of unlabeled glutamine as well as histidine, asparagine, and serine. Transport of glutamine by the Na+-dependent process was significantly inhibited or stimulated, respectively, by inducing a relatively positive or negative intravesicular space. On the other hand, glutamine transport by the Na+-independent process was not affected by changes in transmembrane electrical potential. Using the “activation method,” the stoichiometry of glutamine Na+ transport was found to be 1:1. These results show that glutamine transport in rat liver basolateral membrane vesicles is carrier mediated both in the presence and absence of an Na+ gradient. Furthermore, the Na+-dependent process is electrogenic in nature (net positive) and cotransports one glutamine molecule with one Na+. Transport of glutamine by the Na+-independent system, on the other hand, is electroneutral in nature.

Ion transport in rat liver mitochondria: the effect of the incubation medium osmolarity

A decrease in the incubation medium osmolarity from 320 to 120 mosM reverses the pH dependence of K + efflux from rat liver mitochondria. The K + efflux is no longer inhibited by oligomycin and a free radical scavenger butylhydroxytoluene. At 320 mosM, the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) accelerates the K + efflux, while EGTA inhibits it. At 120 mosM these CCCP and EGTA effects are reversed. In either case the K + efflux is inhibited by Mg 2+. The decrease in osmolarity changes the ruthenium red-insensitive Ca 2+ efflux in the same manner. It has thus been shown that the modification of the mitochondrial structure by changing the incubation medium osmolarity results in a qualitative alteration of the systems regulating the K + and Ca 2+ effluxes. Mitochondria Ion transport ATPase Osmolarity Free radical reaction

Cupric ion stimulation of mitochondrial protein synthesis and dependence on the osmolarity of incubation medium.

AbstractThe effect of cupric ion on amino acid incorporation into protein by isolatec rat liver mitochondria was investigated. In confirmation of previous observations, the incorporation was stimulated as much as 80% by the addition of 2 to 10 μM CuCl2 when the mitochondria were incubated in the medium of Buchanan et al. The stimulation was maximal at 6 μM Cu2+. By contrast, cupric ion at comparable concentrations showed no significant effect on the incorporation when the mitochondria were incubated in the medium of Haldar and Freeman. Experiments involving omission or addition of the components which made the two media different showed that the stimulatory effect of cupric ion depended upon the presence of 50 mM Tris-HCl. Additional experiments with Tris-HCl and sucrose demonstrated that the extent of stimulation depended on the osmolarity of the incubation medium and that cupric ion stimulated the incorporation by preventing the inhibition caused by the hyperosmolarity of the medium.