R.E. Isaacks

Osmotic regulation of myo-inositol uptake in primary astrocyte cultures

Uptake ofmyo-inositol by astrocytes in hypertonic medium (440 mosm/kg H2O) was increased near 3-fold after incubation for 24 hours, which continued for 72 hours, as compared with the uptake by cells cultured in isotonic medium (38 nmoles/mg protein).myo-Inositol uptake by astrocytes cultured in hypotonic medium (180 mosm/kg H2O) for periods up to 72 hours was reduced by 74% to 8 to 10 nmoles/mg protein. Astrocytes incubated in either hypotonic or hypertonic medium for 24 hours and then placed in isotonic medium reversed the initial down- or up-regulation of uptake. Activation of chronic RVD and RVI correlates with regulation ofmyo-inositol uptake. A 30 to 40 mosm/kg H2O deviation from physiological osmolality can influencemyo-inositol homeostasis. The intracellular content ofmyo-inositol in astrocytes in isotonic medium was 25.6 ± 1.3 μg/mg protein (28 mM). This level ofmyo-inositol is sufficient for this compound to function as an osmoregulator in primary astrocytes and it is likely to contribute to the maintenance of brain volume.

Studies on avian erythrocyte metabolism. Effect of organic phosphates on oxygen affinity of embryonic and adult-type hemoglobins of the chick embryo.

The effects of 2,3 diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), and inositol hexaphosphate (IHP) on the oxygen affinity of whole “stripped” hemoglobin (WSH), hemoglobin H (Hb-H), hemoglobin A (Hb-A) and hemoglobin D (Hb-D) isolated from 18-day chick embryo blood have been determined. The effect of the three organic phosphates upon the oxygen dissociation curves is similar and the following order of decreasing oxygen affinity of the organic phosphates was observed for each hemoglobin: 2,3-DPG < ATP < IHP. 2,3-DPG appears to have a slightly greater effect upon the P50 of Hb-H than upon that of either of the two adult-type hemoglobins. However, this effect seems insufficient to suggest a preferential interaction of 2,3-DPG with Hb-H which would account for either the large amounts of 2,3-DPG in the erythrocytes of embryos or the higher oxygen affinity of the whole blood. The effects of the organic phosphates upon the Hill constant of the purified hemoglobins are variable. It is concluded that since the distribution of hemoglobins H, A, and D in the erythrocytes during the developmental period from 18-day embryos to 6-day chicks remains fairly constant, the previously described progressive decrease in oxygen affinity of the whole blood during this period results from changes in the total amount and distribution of the intraerythrocytic organic phosphates.2

Studies on avian erythrocyte metabolism--II. Relationship between the major phosphorylated metabolic intermediates and oxygen affinity of whole blood in chick embryos and chicks.

Abstract 1. The changes in organic phosphates of chicken erythrocytes (RBC) have been determined in relation to the changes in oxygen affinity of whole blood during growth of the embryo and chick. 2. 2,3-diphosphoglyceric acid is the major organic phosphate (42–47%) in the RBC from 15-days of incubation until hatch. The amount of 2,3-DPG during this period is ∼4–6 μmoles/cm3 RBC, decreases abruptly at hatching, and disappears from the RBC within 8 days after hatching. 3. Adenosine triphosphate (ATP) is the major organic phosphate (45–50%) during the first 5 days post-hatching. 4. The P50 of the whole blood during the last week of incubation and the first 5 days post-hatching correlates best with the amount of ATP in the cells. 5. The effect of inositol pentaphosphate on increasing P50 of the whole blood is more gradual and appears to become of major influence in the chick after 4–5 days post-hatching.

Effect of osmolality and anion channel inhibitors on myo-inositol efflux in cultured astrocytes.

Recent studies have shown that swelling‐activated myo‐inositol efflux from rat C6 glioma cells is mediated by a single transport mechanism and most likely by a volume‐sensitive anion channel. In those studies, cells were acclimated in hypertonic medium and then swollen by returning the cells to isotonic medium. In the present study, myo‐inositol efflux was determined in primary cultures of astrocytes by first incubating the cells in isotonic radiolabelled medium for 2 hr and then placing the cells in either unlabelled isotonic, hypertonic, or hypotonic medium and measuring release with time. Computer analyses of efflux data indicated a two‐component system of myo‐inositol efflux. The rate constants for the initial fast component for isotonic and hypotonic cells were 0.0398 ± 0.005 and 0.0631 ± 0.0288 min‐1, respectively. The efflux rates of the slow component, while quite small, were severalfold greater with increasing hypotonic media as compared to the cells in isotonic medium. Several anion membrane transport inhibitors were tested to explore the swelling activated efflux mechanism of myo‐inositol. Furosemide (0.5 mM), 1,9 dideoxyforskolin (0.1 mM), NPPB (0.1 mM), niflumic acid (0.5 mM), and SITS (0.5 mM) blocked the fast component of myo‐inositol efflux by 17, 49, 55, 75, and 93%, respectively. Our results suggest that the fast component of myo‐inositol efflux in primary cultures of astrocytes is mediated by anion transporters or channels and that myo‐inositol flux contributes to cell volume regulation in cultures of primary astrocytes. J. Neurosci. Res. 57:866–871, 1999.

Studies on avian erythrocyte metabolism--I. Procedure for separation and quantitation of the major phosphorylated metabolic intermediates by anion exchange chromatography.

Abstract 1. A method for fractionation of the phosphorylated metabolic intermediates in extracts of chicken red blood cells by anion exchange chromatography using a linear ammonium formate gradient, pH 3·45, is described. 2. The method will resolve myoinositol pentaphosphate (IPP) and/or 2,3-diphosphoglyceric acid (2,3-DPG) as well as the other major phosphorylated intermediates of red cells. 3. The amounts of AMP, Pi, ADP, ATP, and IPP in terms of μmoles of Pi per cm 3 of red blood cells (RBC) of the adult chicken were found to be 0·3, 1·2, 1·0, 3·1, and 17·3, respectively.

Inositol pentaphosphate in erythrocytes of a freshwater fish, Piraracú Arapaima gigas.

Abstract Inositol pentaphosphate (IPP), a characteristic component of avian erythrocytes (RBC), has been found for the first time in teleost. IPP is present in the erythrocytes of a freshwater air-breathing fish, Piraracu, at a level of 1.8 μmoles per ml RBC, representing 39.0 per cent of the total cell phosphate. Guanosine triphosphate (GTP) represents 22.0 per cent of the cell phosphate.

Some studies on blood of the Florida manatee, Trichechus manatus latirostris.

Abstract 1. 1. Hematological data on the Florida manatee, Trichechus manatus latirostris have been collected. 2. 2. The red blood cells (129.5μ3) of the manatee are larger than those of the dolphin or man and contain 6.84 and 0.27μmoles/cm3 of 2,3-diphosphoglyceric acid and adenosine triphosphate, respectively. 3. 3. Serum electrolytes, blood chemistries and serum enzyme activities are very similar to those of man except for unusually low levels of glutathione reductase activity (15 μmoles/min per 100cm3 RBC). 4. 4. The whole blood P50 values for the manatee and baby manatee are 16.5 and 20.6mm Hg, respectively. 5. 5. The diploid chromosome number of this species is 48.

Effect of ammonia and methionine sulfoximine on myo-inositol transport in cultured astrocytes

Ammonia causes astrocyte swelling which is abrogated by methionine sulfoximine (MSO). Since myo-inositol is an important osmolyte, we investigated the effects of ammonia and MSO on myo-inositol flux in cultured astrocytes for periods up to 72 hours. Uptake of myo-inositol was significantly decreased by 26.7 (P < 0.05) and 39.3 (P lt; 0.006) percent after 48 hours of exposure to 5 or 10 mM ammonia, respectively. The maximum rate of uptake was 14.0 ± 0.5 nmol/hour/mg protein which was reduced to 7.45 ± 0.27 and 7.02 ± 0.57 nmoles/hour/mg protein by 5 or 10 mM ammonia, respectively. The Kms by Michaelis-Menten equation for the control, and in the presence of 5, or 10 mM ammonia were 32.5 ± 4.52, 44.4 ± 5.82, and 39.3 ± 7.0 μM, respectively. Kms by Hanes-Woolf plot for the control, 5, or 10 mM ammonia were 25, 45, and 40 μM, respectively. Treatment of astrocytes with either 5 or 10 mM NH4Cl for 6 hours caused a decrease in myo-inositol content by 66% and 58%, respectively. MSO (3 mM) partially diminished the ammonia-induced inhibition of myo-inositol uptake and decreased myo-inositol content by 31% after 24 hours. Additionally, ammonia increased myo-inositol efflux briefly through the fast efflux component but had little effect on myo-inositol efflux through the slow efflux component of astrocytes exposed to ammonia for up to 72 hours. Predominantly decreased myo-inositol influx coupled with brief efflux through the fast component may represent an adaptive response to diminish the extent of ammonia-induced astrocyte swelling.

Effect of osmolality and myo-inositol deprivation on the transport properties of myo-inositol in primary astrocyte cultures

Abstractmyo-Inositol uptake measured in primary astrocyte cultures was saturable in the presence of Na+ with a Km of 13–18 μM and a Vmax of 9.4 nmoles/mg protein/hour in myo-inositol-fed cells, indicating a high affinity transport system. In myo-inositol-deprived cells, Km was about 53 μM with a Vmax of 13.2 nmoles/mg protein/hour. Decreasing osmolality decreased the Vmax to about 1.9 nmoles/mg protein/hour whereas increasing osmolality increased Vmax about 5-fold, while Kms were essentially unchanged in myo-inositol fed cells. In cells deprived of myo-inositol, Vmax decreased in hypotonic medium and increased in hypertonic medium almost 10-fold, but with more than a doubling of the Km regardless of the osmolality. Glucose (25 mM) inhibited myo-inositol uptake 51% whereas the other hexoses used inhibited uptake much less. Our findings indicate that myo-inositol uptake in astrocytes occurs through an efficient carrier-mediated Na+-dependent co-transport system that is different from that of glucose and its kinetic properties are affected by myo-inositol availability and osmotic stress.

Relative oxidation of glutamate and glucose by vertebrate erythrocytes

1. Lungfish erythrocytes (RBC), unlike those in other species of fishes, oxidize endogenous glutamate at a higher rate than they oxidize glucose. This pattern closely resembles that found in invertebrates. 2. The exogenous glutamate oxidation rate of RBC from most species of fish as well as other groups of vertebrates is approximately equal to or less than that observed for glucose. 3. These findings suggest that the nucleated RBC of most vertebrates appear to rely less on the TCA cycle for energy production and more on the glycolytic metabolism of carbohydrates. 4. The RBC of lungfish are also distinguished from RBC of other vertebrates by their relatively higher permeability to exogenous substrates. For example, chicken RBC have a glucose accumulation rate which is approximately one third that observed for lungfish RBC at twice the medium glucose concentration. 5. The unique characteristics of lungfish RBC may be related to their adaptation to the high concentrations of urea produced during estivation.