Protonmotive force and catecholamine transport in isolated chromaffin granules.

The effect of the transmembrane potential (delta psi) and the proton concentration gradient (delta pH) across the chromaffin granule membrane upon the rate and extent of catecholamine accumulation was studied in isolated bovine chromaffin granules. Freshly isolated chromaffin granules had an intragranular pH of 5.5 as measured by [14C]methylamine distribution. The addition of ATP to a suspension of granules resulted in the generation of a membrane potential, positive inside, as measured by [14C]thiocyanate (SCN-) distribution. The addition of carboxyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a proton translocator, resulted in a reversal of the potential to negative values (measured by [3H]tetramethylphenylphosphonium (TPMP+)) approaching -90 mV. Changing the external pH of a granular suspension incubated with FCCP produced a linear perturbation in the measured potential from positive to negative values, which can be explained by the distribution of protons according to their electrochemical gradient. When ammonia (1 to 50 mM) was added to highly buffered suspensions of chromaffin granules there was a dose-dependent decrease in the transmembrane proton gradient (delta pH) and an increase in the membrane potential (delta psi). On the other hand, thiocyanate or FCCP, at varying concentration, produced a dose-related collapse of the membrane potential and had no effect upon the transmembrane proton gradient. The addition of larger concentrations of catecholamines caused a decrease in the transmembrane proton gradient and an increase in the membrane potential. Time-resolved influx of catecholamines into the granules was studied radiochemically using low external catecholamine concentrations. The accumulation of epinephrine or norepinephrine was over one order of magnitude greater in the presence of ATP than in its absence. The rate and extent of amine accumulation was found to be related to the magnitude of the membrane potential at fixed transmembrane proton concentration (delta pH) values. Likewise, the accumulation was related to the magnitude of the delta pH at fixed membrane potential values. These results suggest that the existence of both a transmembrane proton gradient and a membrane potential are required for optimal catecholamine accumulation to occur.     

Biological amine transport in chromaffin ghosts. Coupling to the transmembrane proton and potential gradients.

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in chromaffin ghosts. The chromaffin ghosts were formed by hypo-osmotic lysis of isolated bovine chromaffin granules and extensive dialysis in order to remove intragranular binding components and dissipate the endogenous electrochemical gradients. Upon ATP addition to suspensions of chromaffin ghosts, a transmembrane proton gradient alone, a transmembrane gradient alone, or both, could be established, depending upon the compositions of the media in which the ghosts were formed and resuspended. When chloride was present in the medium, addition of ATP resulted in the generation of a transmembrane proton gradient, acidic inside of 1 pH unit (measured by [14C]methylamine distribution), and no transmembrane potential (measured by [14C]-thiocyanate distribution). When ATP was added to chromaffin ghosts suspended in a medium in which chloride was substituted by isethionate, a transmembrane potential, inside positive, of 45 mV and no transmembrane proton gradient, was measured. In each medium, the addition of agents known to affect proton or potential gradients, respectively, exerted a predictable mechanism of action. Accumulation of [14C]epinephrine or [14C]5-hydroxytryptamine was over 1 order of magnitude greater in the presence of the transmembrane proton gradient or the transmembrane potential than in the absence of any gradient and, moreover, was related to the magnitude of the proton or potential gradient in a dose-dependent manner. When ghosts were added to a medium containing chloride and isethionate, both a delta pH and delta psi could be generated upon addition of ATP. In this preparation, the maximal rate of amine accumulation was observed. The results indicate that amine accumulation into chromaffin ghosts can occur in the presence of either a transmembrane proton gradient, or a transmembrane potential gradient, and that the maximal rate of accumulation may exist when both components of the protonmotive force are present.     

Transmembrane proton-motive potential of Spiroplasma floricola

In Spiroplasma floricola, the transmembrane proton-motive potential delta p was studied. It is composed of a transmembrane electric potential difference, delta psi, and a transmembrane proton gradient, delta pH, according to delta p = delta psi - (Z.delta pH). Using a potential-sensitive carbocyanine dye and 5,5'-dimethyl[2-14C]oxazolidine-2,4-dione as probes, delta psi and delta pH were measured at different [H+] of the medium, and delta p was calculated to be remarkably constant at -123 mV +/- 16% over a wide range of external pH values. Inhibition experiments indicated that it is generated by a membrane-bound, electrogenic, proton-translocating ATPase.     

5-Hydroxytryptamine transport in cells and secretory granules from a transplantable rat insulinoma.

Mechanisms of transport of 5-hydroxytryptamine in the pancreatic B-cell were investigated by using cell suspensions and secretory granules prepared from a transplantable rat insulinoma. (1) Cells incubated with 5-hydroxy[G-3H]tryptamine at concentrations ranging from 0.1 microM to 5 mM accumulated the radioisotope principally by a simple diffusion process. The incorporated radioactivity was recovered principally as the parent molecule and was recovered predominantly in soluble protein and secretory-granule fractions prepared from the tissue. (2) Isolated granules incubated in buffered iso-osmotic medium without ATP accumulated the amine to concentrations up to 38-fold that of the medium. This process was insensitive to reserpine and occurred over a wide range of 5-hydroxytryptamine concentrations (0.075 microM-25 mM). Above 5 mM, 5-hydroxytryptamine accumulation decreased in parallel with the breakdown of the delta pH across the granule membrane. Uptake was favoured by alkaline media and was reduced by the addition of (NH4)2SO4. In both cases a close correlation was observed between uptake and the transmembrane delta pH, a finding that suggested that 5-hydroxytryptamine permeated the membrane as the free base and equilibrated across the membrane with the delta pH. Binding of 5-hydroxytryptamine to granule constituents also played a part in this process. ATP caused a further doubling of granule 5-hydroxytryptamine uptake by a process that was sensitive to reserpine (0.5 microM). Inhibitor studies suggested that amine transport in this instance was linked to the activity of the granule membrane proton-translocating ATPase. (3) It was concluded that the uptake of amines driven by proton gradients across the insulin-granule membrane could account for the accumulation in vivo of amines in the B-cell.     

Participation of a transmembrane proton gradient in 5-hydroxytryptamine transport by platelet dense granules and dense-granule ghosts.

Dense granules, the storage organelles for 5-hydroxytryptamine in blood platelets, have been isolated from porcine platelets and are shown to transport 5-hydroxytryptamine in response to a transmembrane proton gradient (delta pH). Transport in the absence of delta pH is minimal, and it is shown that a rapid increase in transport takes place as delta pH increases. Direct measurements with [14C]methylamine show a delta pH of 1.1 units (acid inside) for intact granules. Osmotically active ghosts of dense granules from which 95% of the endogenous 5-hydroxytryptamine content has been released have also been prepared. Ghosts swell in the presence of ATP and Mg2+, and this swelling is shown to be due to the entry of protons via a process linked to ATP hydrolysis. Proton entry is also apparently linked to anion penetration in ghosts. Steady-state 5-hydroxytryptamine transport in ghosts is stimulated approx. 3-fold on the addition of ATP to the incubation medium, and the stimulation of 5-hydroxytryptamine transport in ghosts correlates with the formation of a transmembrane delta pH. Ghosts generate a delta pH of 1.1-1.3 pH units (acid inside) in the presence of 5 mM-ATP/2.5 mM-MgSO4. delta pH is generated within 3 min at 37 degrees C and is dissipated by the ionophore nigericin and by NH4Cl. It is shown that an Mg2+-stimulated ATPase activity is present on the ghost membrane, and inhibition of the ATPase leads to a corresponding decrease in 5-hydroxytryptamine transport. The results presented support the idea that 5-hydroxytryptamine transport into platelet dense granules is dependent on the presence of a transmembrane delta pH and, together with previous findings by others, suggest a generalized mechanism for biogenic amine transport into subcellular storage organelles.     

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions.

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions

The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using [14C]dimethyloxazolidinedione and [14C]benzoic acid as transmembrane pH gradient (delta pH) probes and [14C]triphenylmethylphosphonium as a membrane potential (delta psi) indicator. The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5. The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5. Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5. The transmembrane electrical potential decreased as the external pH decreased. At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5. The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5. The ability to maintain a high internal pH at a low extracellular pH suggests that C. acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents.     

Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane.

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5'-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.     

Nisin dissipates the proton motive force of the obligate anaerobe Clostridium sporogenes PA 3679.

The influence of nisin on the proton motive force (delta p) generated by glucose-energized cells of the obligate putrefactive anaerobe Clostridium sporogenes PA 3679 was determined. The components of delta p, the transmembrane potential (delta psi) and the pH gradient (delta pH), were determined from the distributions of the lipophilic cation [3H]TPP+ ([3H]tetraphenylphosphonium bromide) and [14C]salicylic acid, respectively. The cells maintained a constant delta p of -111 mV, consisting of a delta pH of 0.4 to 1.0 pH units at an external pH of 5 to 7 and a delta psi of -60 to -88 mV. Nisin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N'-dicyclohexylcarbodiimide (DCCD) at pH 6.0 elicited the complete release of preaccumulated [3H]tetraphenylphosphonium bromide and [14C]salicylic acid, with a concomitant depletion of delta psi and delta pH. Nisin and DCCD caused rapid drops in intracellular ATP levels from 1.2 to 0.01 and 0.06 nmol/mg of cells (dry weight), respectively. Cells exposed to nisin and DCCD lost the ability to form colonies, thus suggesting that delta psi and delta pH are necessary for cell viability. The data suggest that depletion of delta p and exhaustion of cellular ATP reserves are the basis for nisin inhibition of C. sporogenes PA 3679.     

Nisin dissipates the proton motive force of the obligate anaerobe Clostridium sporogenes PA 3679.

The influence of nisin on the proton motive force (delta p) generated by glucose-energized cells of the obligate putrefactive anaerobe Clostridium sporogenes PA 3679 was determined. The components of delta p, the transmembrane potential (delta psi) and the pH gradient (delta pH), were determined from the distributions of the lipophilic cation [3H]TPP+ ([3H]tetraphenylphosphonium bromide) and [14C]salicylic acid, respectively. The cells maintained a constant delta p of -111 mV, consisting of a delta pH of 0.4 to 1.0 pH units at an external pH of 5 to 7 and a delta psi of -60 to -88 mV. Nisin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N'-dicyclohexylcarbodiimide (DCCD) at pH 6.0 elicited the complete release of preaccumulated [3H]tetraphenylphosphonium bromide and [14C]salicylic acid, with a concomitant depletion of delta psi and delta pH. Nisin and DCCD caused rapid drops in intracellular ATP levels from 1.2 to 0.01 and 0.06 nmol/mg of cells (dry weight), respectively. Cells exposed to nisin and DCCD lost the ability to form colonies, thus suggesting that delta psi and delta pH are necessary for cell viability. The data suggest that depletion of delta p and exhaustion of cellular ATP reserves are the basis for nisin inhibition of C. sporogenes PA 3679.     

Proton pump-generated electrochemical gradients in rat liver multivesicular bodies. Quantitation and effects of chloride

Endocytic vesicles possess an electrogenic proton pump, and measurements of ATPase activity suggest that Cl- may stimulate proton pump activity. This study was undertaken to measure the steady-state pH, potential (delta psi), and total proton electrochemical gradients established by the rat liver multivesicular body (MVB) proton pump and to examine the effects of Cl- (0.5-140 mM) on these gradients. Radiolabeled [( 14C] methylamine and 36Cl-) and fluorescent (fluorescein isothiocyanate-conjugated low density lipoproteins) probes were used to assess internal pH (pHi) and delta psi. In the absence of ATP, pHi averaged 7.37 +/- 0.05 (extracellular pH 7.31 +/- 0.02), and delta psi ranged from -32 to -71 mV; but neither pHi nor delta psi varied consistently with [Cl-]. In the presence of ATP, pHi decreased progressively with increasing [Cl-] to a plateau value of about 5.89 at greater than or equal to 25 mM Cl-, and MVB exhibited an interior positive delta psi that was maximal at the lowest Cl- concentration (+65.5 mV) and decreased as medium Cl- increased. The total ATP-dependent proton electrochemical gradient (proton-motive force (delta p] averaged 118.0 +/- 4.3 mV and did not change in any consistent manner as [Cl-] varied almost 300-fold. However, initial rates of MVB acidification increased with increasing [Cl-]. These studies indicate that: (a) in the absence of ATP, isolated MVB exhibited a negative delta psi, probably a Donnan potential; (b) in the presence of ATP and at a [Cl-] similar to that in hepatocyte cytoplasm (25 mM), MVB pHi was 5.89, and delta psi was +9.6 mV; and (c) over the range of [Cl-] tested, the magnitudes of delta pH and delta psi were inversely related, apparently related to Cl- availability, but the ATP-dependent delta p did not vary. Therefore, it is concluded that Cl- increases the initial rate of vesicle acidification in MVB and also affects the relative chemical and electrical contributions of the steady-state proton pump-determined delta p. Cl-, however, does not alter steady-state delta p.     

The protonmotive force and beta-galactoside transport in Bacillus acidocaldarius.

The acidophilic and thermophilic bacterium, Bacillus acidocaldarius maintains a cytoplasmic pH between 5.85 and 6.31 over a range of external pH from 2.0 to 4.5. Consistently, the pH optimum of beta-galactosidase, as assayed in cell extracts, is between pH 6.0 and 6.5. An electrical potential (delta-psi), interior positive, is also maintained across the membrane. A delta-psi of approximately 34 mV was calculated from determinations of thiocyanate uptake by cells at pH 3.5. Addition of the proton conductor carbonyl cyanide m-chlorophenylhydrazone increased the delta-psi. Treatment of cells with valinomycin (in the absence of external potassium ions) or high concentrations of thiocyanate, to abolish the delta psi, resulted in collapse of the transmembrane proton gradient (delta pH). Active transport of methylthio-beta, D-galactoside occurred optimally at pH 3.5. Transport of the galactoside was inhibited by various compounds which could dissipate the transmembrane delta pH and by respiratory inhibitors. A decrease in the delta pH and an increase in the delta psi occurred upon addition of methylthio-beta, D-galactoside to cells of B. acidocaldarius. Thus the transport of this solute appears to involve an electrogenic symport with protons. The transport system is most active at 50 degrees C and shows little activity at 25 degrees C, although the delta pH is the same at the two temperatures. Gramicidin inhibits methylthio-beta, D-galactoside transport equally effectively at 50 degrees C and 25 degrees C, while nigericin inhibits only after a lag at 25 degrees C.     

Spectrophotometric measurements of transmembrane potential and pH gradients in chromaffin granules

The electrical potential (delta psi) and proton gradient (alpha pH) across the membranes of isolated bovine chromaffin granules and ghosts were simultaneously and quantitatively measured by using the membrane- permeable dyes 3,3'dipropyl-2,2'thiadicarbocyanine (diS-C3-(5)) to measure delta psi and 9-aminoacridine or atebrin to measure delta pH. Increases or decreases in the delta psi across the granular membrane could be monitored by fluorescence or transmittance changes of diS-C3- (5). Calibration of the delta psi was achieved by utilization of the endogenous K+ and H+ gradients, and valinomycin or carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP), respectively, with the optical response of diS-C3-(5) varying linearly with the Nernst potential for H+ and K+ over the range -60 to +90 mV. The addition of chromaffin granules to a medium including 9-aminoacridine or atebrin resulted in a rapid quenching of the dye fluorescence, which could be reversed by agents known to cause collapse of pH gradients. From the magnitude of the quenching and the intragranular water space, it was possible to calculate the magnitude of the alpha pH across the chromaffin granule membrane. The time-course of the potential-dependent transmittance response of diS-C3-(5) and the delta pH-dependent fluorescence of the acridine dyes were studied simultaneously and quantitatively by using intact and ghost granules under a wide variety of experimental conditions. These results suggest that membrane-permeable dyes provide an accurate method for the kinetic measurement of delta pH and delta psi in an amine containing subcellular organelle.     

Existence of an adenosine 5'-triphosphate dependent proton translocase in bovine neurosecretory granule membrane

The addition of ATP to bovine neurohypophysial secretory granules suspended in isotonic sucrose medium induces a positive polarization, delta psi, of their interior without affecting their internal pH. In KCl-containing media, ATP failed to generate large delta psi but induced a pH gradient (delta pH; interior acidic). These observations are consistent with the existence in the neurosecretory granule membrane of an ATP-dependent inward electrogenic H+ translocase (H+ pump), capable in KCl-containing media of acidifying the granule matrix by H+-Cl- cotransport. The delta psi and delta pH generated by the H+ pump, defined as the ATP-induced changes sensitive to the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), were blocked by N,N'-dicyclohexylcarbodiimide, an inhibitor of all H+ pumps, and were insensitive to oligomycin, a mitochondrial ATPase inhibitor. In sucrose medium, measurements were complicated by a Donnan equilibrium reflecting the presence in the granule of peptide hormones and neurophysins which resulted in a CCCP-resistant resting delta pH. In KCl-containing media, the Donnan equilibrium was destroyed since the membrane is permeable to cations, but under these conditions a CCCP-resistant K+-diffusion potential was observed. The ATP-induced delta psi was also monitored by the extrinsic fluorescent probe bis(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol. The hypothesis of a granule H+ pump is further supported by the presence of an oligomycin-resistant ATPase in the preparation and the ultrastructural localization of such an activity on the granule membrane. The H+ pump has been found in both newly formed and aged neurosecretory granules. Its possible physiological function is discussed with reference to that of chromaffin granules, with which it has many similarities.     

Quantitative association between electrical potential across the cytoplasmic membrane and early gentamicin uptake and killing in Staphylococcus aureus

The relationship between the magnitude of the transmembrane electrical potential and the uptake of [14C]gentamicin was examined in wild-type Staphylococcus aureus in the logarithmic phase of growth. The electrical potential (delta psi) and the pH gradient across the cell membrane were determined by measuring the equilibrium distribution of [3H]tetraphenyl-phosphonium and [14C]acetylsalicylic acid, respectively. Incubation in the presence of the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) led to an increase in delta psi with no measurable effect on the pH gradient at external pHs ranging from 5.0 to 6.5, and the effect on delta psi was DCCD concentration dependent. In separate experiments, gentamicin uptake and killing were studied in the same cells under identical conditions. At pH 5.0 (delta psi = -140 mV), no gentamicin uptake occurred. In the presence of 40 and 100 microM DCCD, delta psi was increased to -162 and -184 mV, respectively, and gentamicin uptake was observed in a manner that was also dependent on the DCCD concentration. At pH 6.0 (delta psi = -164 mV), gentamicin uptake occurred in the absence of the carbodiimide but was enhanced in a concentration-dependent fashion by 40 and 100 microM DCCD (delta psi = -174 and -216 mV, respectively). In all cases increased gentamicin uptake was associated with an enhanced bactericidal effect. The results indicate that initiation of gentamicin uptake requires a threshold level of delta psi (-155 mV) and that above this level drug uptake is directly dependent on the magnitude of delta psi.     

The internal pH of isolated serotonin containing granules of pig platelets.

The [14C]methylamine distribution method was utilized to measure the internal pH of isolated serotonin containing granules of pig blood platelets under varying conditions. The granules used were isolated by a new protocol which stressed platelet rupture under controlled conditions and preservation of isotonicity throughout the isolation procedure, In a well buffered external medium, pH 6.85, The deltapH was measured as 1.11 with the internal pH being found acidic (pH 5.74). Increasing the external pH produced a corresponding increase in the deltaH. The pH gradient could be collapsed by the addition of ionophores and uncouplers which are known to transport protons across biological membranes. In addition, the deltapH was constant for granules suspended in various ionic media, thus suggesting that the deltaH did not arise secondarily due to the establishment of a Donnan equilibrium. The existence of the acidic intragranular space is discussed with respect to previous ancillary findings. Also, an explication of the possible physiological significance of the deltaH is presented.     

Regulation of the transmembrane potential of isolated chromaffin granules by ATP, ATP analogs, and external pH.

The transmembrane potential of isolated chromaffin granules has been measured using the permeant ions [14C]methylamine and [35S]thiocyanate, as well as the fluorescent probe, 9-aminoacridine. At pH 7.0, the granule membrane had a Nernst proton potential of -45mV, inside negative. This potential was sensitive to the external pH, but was unaffected by K+,Na+, Ca2+, Mg2+, or other cations. The pH of zero potential was 6.25 for both methylamine and thiocyanate. Thiocyanate also had a Nernst potential of similar magnitude and sign to that of methylamine at pH 7.0, and was also sensitive to variation in external pH. Mg2+ATP was found to depolarize the granule membrane by a saturable mechanism with a K 1/2 for ATP of 40 muM. Ca2+ was only 30% as effective as Mg2+ in supporting the ATP effect. The pH optimum for this process was 6.25 and appeared to be accompanied by a marked alkalinization of the granule interior. The specificity for ATP was further tested with structural analogs of ATP and GTP. The rate of change of membrane potential in response to changes in external pH or Mg2+ATP was estimated using the fluorescent probe 9-aminoacridine. Changes came to completion in less than 1 s. This suggested that the ATP effects were not dependent on an enzymatic transformation but on an ATP-induced conformational change in the membrane. We conclude that the chromaffin granule exists in at least two proton permeability states, corresponding to the presence or absence of Mg2+ATP. These states may be related to hormone release from granules and regulation of secretion in vivo.     

p-Chloroamphetamine induces serotonin release through serotonin transporters.

p-Chloroamphetamine (PCA) interacts with serotonin transporters in two membrane vesicle model systems by competing with serotonin for transport and stimulating efflux of accumulated serotonin. In plasma membrane vesicles isolated from human platelets, PCA competes with [3H]imipramine for binding to the serotonin transporter with a KD of 310 nM and competitively inhibits serotonin transport with a KI of 4.8 nM. [3H]Serotonin efflux from plasma membrane vesicles is stimulated by PCA in a Na(+)-dependent and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, PCA competitively inhibits ATP-dependent [3H]serotonin accumulation with a KI of 1.7 microM and, at higher concentrations, stimulates efflux of accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due in part to dissipation of the transmembrane pH difference (delta pH) generated by ATP hydrolysis. Part of PCA's ability to stimulate efflux may be due to its transport by the vesicular amine transporter. Flow dialysis experiments demonstrated uptake of [3H]PCA into chromaffin granule membrane vesicles in response to the delta pH generated in the presence of Mg2+ and ATP. In plasma membrane vesicles, no accumulation was observed using an NaCl gradient as the driving force. We conclude that rapid nonmediated efflux of transported PCA prevents accumulation unless PCA is trapped inside by a low internal pH.     

ATP-dependent uptake of 5-hydroxytryptamine by secretory granules isolated from thyroid parafollicular cells.

The current study was done to test the hypotheses that parafollicular granules contain a vacuolar ATPase (V-ATPase) similar to that found in chromaffin granules, that the transport of H+ into granules mediated by this enzyme drives the granular uptake of 5-hydroxytryptamine (5-HT, serotonin), and that secretagogues stimulate both the acidification of parafollicular granules and their ability to take up 5-HT by opening an anion channel in the granular membrane. Our studies indicate that parafollicular granules contain a V-ATPase that is antigenically similar to that of the V-ATPase of adrenal chromaffin granules; however, the parafollicular granular membrane differs from that of chromaffin granules in permeability to Cl- and K+. The membranes of granules derived from resting parafollicular cells appear to be relatively impermeable to Cl- but permeable to K+. Parafollicular granules (and ghosts derived from them) manifest ATP-dependent transmembrane transport of 5-HT. This transport is more dependent on the pH difference (delta pH) than on the membrane potential component of the proton electrochemical gradient across the granular membrane. Transport of 5-HT is thus inhibited more by exposure of parafollicular granules to agents, such as nigericin, that collapse delta pH than by those, such as valinomycin, that decrease transmembrane difference in potential. ATP-dependent uptake of 5-HT by granules isolated from secretagogue-stimulated parafollicular cells is greater than that into granules isolated from unstimulated cells. Since secretagogues open a Cl- channel in parafollicular granule membranes, which enhances acidification of the granules, the facilitation of 5-HT uptake by secretagogues is probably due to an increase in delta pH.     

Characterization of a H+-ATPase in rat brain synaptic vesicles. Coupling to L-glutamate transport

Synaptic vesicles contain a H+-ATPase that generates a proton electrochemical gradient (delta mu H+) required for the uptake of neurotransmitters into the organelles. In this study, the synaptic vesicle H+-ATPase was examined for structural and functional similarities with other identified ATPases that generate a delta mu H+ across membranes. The synaptic vesicle H+-ATPase displayed immunological similarity with the 115-, 72-, and 39-kDa subunits of a vacuolar-type H+-ATPase purified from chromaffin granules. Functionally, the ATP-dependent H+ pumping across synaptic vesicles and ATP hydrolysis were sensitive to the sulfhydryl-modifying reagents, N-ethylmaleimide and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, at concentrations known to affect vacuolar-type H+-ATPases. In addition, as with vacuolar-type H+-ATPases, the presence of NO3-, SO4(2-), or F- inhibited the generation of a delta mu H+, but addition of vanadate or oligomycin had no effect. The delta mu H+ is a function of the pH gradient (delta pH) and membrane potential (delta psi sv) across the synaptic vesicle. Acidification (delta pH) of the synaptic vesicle interior was enhanced in the presence of permeant anions, such as Cl-, or the K+ ionophore, valinomycin. In the absence of permeant anions, the H+-ATPase generated a delta psi sv that effected the transport of L-glutamate into the synaptic vesicles. Dissipation of delta psi sv by incubation with increased external Cl- or nigericin resulted in the abolition of glutamate uptake, despite the continued maintenance of a delta mu H+ across the synaptic vesicle as a substantial delta pH. The results suggest that the synaptic vesicle H+-ATPase is of a vacuolar type and energizes the uptake of anionic glutamate by virtue of the delta psi sv component of the delta mu H+ it generates.