February. Raven Press, into Plasma Membrane Vesicles Derived from Loui - PDF document

February. Raven Press, into Plasma Membr
February. Raven Press, into Plasma Membrane Vesicles Derived from Louisiana State University Neil, Orleans, the transport both GABA into rat synaptosomes. They showed that DABA is competitive inhibitor inhibitor they showed that was par- tially inhibited They pointed out that but not but not rat cerebellum. emphasized the similarity of of and ["IGABA. Several investigators prepared membrane vesicles which gradients. Kanner membranes active the pres- the characteristics characteristics and [3H]GABA transport into these there are stantive differences raise the DABA is transport system. were prepared from male method of synaptosomes were the plasma was prepared aliquots [about the procedure of Lowry Transport measurements were carried Unless otherwise noted, buffered with After centrifu- min). the membranes were resus- pended (about solution of same composition measure trans- was transferred solution consisting chloride buffered with cedure generates Na* Na+lnsldr) and external transport also contained ["HIGABA (26,600 (26,600 (6850 c.p.m.ipmol) to give a final concen- tration of After incubation specified time ambient temperature transport was M-NaCI in diameter Whatman GFiA were washed twice of 0.20 After drying measured by liquid scintillation using Budget-Solve (Research Products measure background was added before the membrane fraction; and washing were then measurement was the meas

urement threefold variation rates and ex
urement threefold variation rates and extent from membrane preparation a given membrane preparation, varied only three membrane preparations Transport Measurement experiments were method pre- uptake (Roskoski, Transport was 0.9% NaCl by filtration through GFiA filters were washed twice and DABA were obtained from monensin were Other drugs and compounds were purchased from General Characteristics for about uptake exhibits the same experimental condi- tions, there is appreciable uptake (Fig. 1). substrate con- transport can with higher is not concentration because of GABA differs from that into vesicles (Fig. Uptake plateaus latter, possesses to maintain ion occurs rather were next performed for transport. Both DABA uptake. Trans- port into membrane vesicles and synaptosomes was mea- labeled substrate membrane vesicles using a substrate. The values terminations. Similar results obtained with four dif- transport are Na+-dependent choline for Na+ decreased also dependent on external was examined. In GABA, DABA phosphate under conditions less than with NaCl. This SCN- increases the more readily more negative intravesicular electromotive The thiocyanate both substances suggests that their substantiate this hypothesis, were also performed with valinomycin, enhances the transport concentration gradient (from inside to outside) produces a more negative vesicular electromotive postulated electr

ogenic nature increases the rate externa
ogenic nature increases the rate external solution 4.14 2.54 synaptosome membrane vesicles were and Methods. specified salt choline chloride, and pM-valinomycin. Incubations were membrane preparations. Addition DABA Gramicidin D The synaptosome membrane fraction was reconstituted and Methods. chloride and were dissolved portions were added the transport the means duplicate determinations To further demonstrate the role the energy for determined. Monensin, gradient (Harold Nigericin, which also abolishes similarly de- an ionophore specificity (Mueller the rate These studies support the that the gradients provide the driving into the membrane vesi- were performed to determine specificity of the internal cation and Rb+ optimal for internal Tris Li+ also sup- ports substantial uptake (Table uptake into the membrane vesicles. DABA and Cation DABA were reconstituted Materials and reactions were terminated and Methods. membrane prepara- This finding, different spe- the external anion, that there are substantive kinetic analysis two components into membrane vesicles (Fig. into membrane vesicles were obtained when was measured In both and the results are consistent tive inhibitory patterns. The synaptosome preparation more physiological ion In performing similar the inhibi- the first place, the apparent into synaptosomes (Fig. equation: slope slope )(Plowman, 1972). Similar results were obtain

ed in three other membrane of GABA tran
ed in three other membrane of GABA transport in rat synaptosomes. Transport into synaptosomes was described previously (Roskoski, that the [3H]GABA was varied from 1 bations were carried at 24". The data the mean duplicate determinations. Similar results obtained with synaptosome preparations. and membrane vesicles (not shown) data are transport system. cromolar concentrations concentrations were used, the extrapolated wide range with single single into membrane vesicles, moreover, and temperature-dependent finding was weak inhibitor ranging from found with seems to a system than the the effect of the various classes structural classes including leucine, acid, phenylalanine, and glycine are without effect. was the only amino acid that substantially decreased decreased uptake. Net Transport of GABA into Membrane Vesicles In addition to characterizing the requirements transport into membrane vesi- was performed to determine whether the would mediate net previously performed substrate transport into membrane vesicles, mea- not chemical have been performed. that exchange intravesicular and external reconstituted membrane vesicles was measured equivalent amount radioactive uptake (Table transport into synaptosomes. Transport was measured the concentration [3H]DABA was were performed at 24". were obtained with three ferent synaptosome preparations. GABA TRANSPORT Membrane vesicles were pre

pared and reconstituted, and was measure
pared and reconstituted, and was measured Materials and were performed performed DABA in medium containing chloride plus the specified Means of duplicate determinations given. Similar results dissipates the inhibits chemical from the ex- ternal medium. Reconstitution potassium phos- phate and chosen to use of membrane vesicles There are several systems. Transport perature-dependent. Transport dissipate the also electrogenic, valinomycin. On hand, there several differences the properties these two is required for but not different from These characteristics can membrane vesicles with with DABA uptake (pmoUmg protein) None 20 pM 0.67 mv 2.0 mv 20 mv 40 mv 1.20 1.18 1.04 0.94 0.88 0.63 Membrane vesicles reconstituted and Materials and the external transport concentration. Incubations were per- per- concentration of 0.14 p~. Similar results were obtained different membrane prep- prep- 'HIDABA uptake Addition (1 mM) (pmoVmg protein) None Alanine Leucine Lysine Phen ylalanine Glycine DABA P- Alanine 3.1 2 0.15 2.0 2 0.12'1 3.3 2 0.14 3.2 t 0.14 2.9 5 0.13 3.2 -c 0.16 1.0 * 0.11" 3.1 t 0.14 Membranes were prepared and reconstituted, and transport was measured Materials and Methods. Portions of unlabeled amino acid were added to the external trans- port solution quadruplicate determinations determinations 'HIDABA concentration with two brane preparations. specified artificially imposed ion gra

dients, and exhibits an uptake into reco
dients, and exhibits an uptake into reconstituted The latter with pre- Simon and Martin feeble inhibitor Chemical Radioactive Transport transport transport solution (nmoUmg) vesicles were Materials and the same solution with specified solution solution (10.4 c.p.m.!pmol). After 3 min at ambient temperature, were centrifuged an Eppendorf Microfuge. After was gently the tube decrease the extravesicular extravesicular After adding the solution was heated any metabolizing and the sonication with a Kontes After centrifugation taken for the fluorometric determination described (Roskoski. fication of the procedure and Aprison were taken spectrometry. The represent the means duplicate samples. results were obtained two other membrane preparations. system (Table This argues that that and further raises the possibility, first hypothesized into elements that take up GABA. These results, synaptosomes and membrane vesicles, et al., range. They present studies, transport studies and the present slices and membrane the former barriers exist the exogeneously applied the slices, for affects the that transport which may the preparation the present experiments, the chloride and potassium provides the best evidence for the between brain slices preparations seem the respective experiments, and those of Simon raise the DABA may into cellular which lack high-affinity GABA transport system, and the need for

additional GABA uptake work was and Apri
additional GABA uptake work was and Aprison mination of enzymic methods. M., Altendorf and Hirata B., Denner elevated (K+), transmitters from cortical synaptosomes: transmitter uptake mechanisms synaptic transmission. L. L. GABA uptake system: Comparison homogenates and the effects some inhibitors. y-aminobutyric acid membrane vesicles Differential labeling and nerve terminals microinjection of of and ['HIGABA into single folia the cerebellum. Qutirt. Biol. Krnjevic K. and Raiteri high affin- for GABA and synaptosomes. nerve endings can simulate High affinity uptake sys- glycine, glutamic aspartic acids rat central nervous tissues. and Randall phenol reagent. the sodium-dependent transport acid by experimental bimolecular macrocyclic antibiotics. Biochem. Biophys. McGraw-Hill Book New York. and Johnston alkali ions Riddall D. transmitter uptake into slices of rat Effect of slice Net uptake and GABA by high transport systems. uptake after depolarization-induced acetylcholine release Net uptake by a rat cortical synaptosomal transport plasma membrane vesicles from human platelets. and Roskoski of y-amino- butyric acid synaptosomal transport the uptake gamma amino synaptosomal fraction rat brain. Biochem. Biophys. 157, 348-355. and exchange and Roberts carrier-mediated transfer ['4C]-y-aminobutyric acid Biochem. Pharmacol. 14, 273-388. tive neural uptake and release release acid by rat ce