beta-Amino acid transport across the renal brush-border membrane is coupled to both Na and Cl

Sodium-dependent beta-alanine uptake into dog renal brush-border membrane vesicles was studied. Kinetic analysis indicated a single transport system, highly specific for beta-amino acids, with Km = 35 microM at 100 mM NaCl. Sodium-dependent beta-alanine transport was markedly anion-dependent, being highest in the presence of chloride (Cl greater than Br greater than SCN greater than NO3 approximately I greater than F) and virtually nonexistent in the presence of gluconate and other nonphysiological chloride substitutes. In addition, it was observed that beta-alanine uptake could be driven against a concentration gradient by a chloride gradient. Similar results were found for sodium. Taken together, these observations provide strong evidence that beta-alanine transport across the renal brush-border membrane is coupled to both sodium and chloride. Studies of the dependence of beta-alanine flux on chloride and sodium concentrations indicated that one chloride ion and multiple sodium ions were involved in the beta-alanine transport event. beta-Alanine flux on chloride found to involve the net transfer of positive charge, consistent with these stoichiometric assignments. The hallucinogen harmaline inhibited beta-alanine uptake in a 1:1 fashion, presumably by acting at a single site on the transport molecule. The ability of harmaline to inhibit beta-alanine uptake was decreased when the chloride concentration was lowered but was unchanged when the sodium concentration was decreased. These results indicate that harmaline does not compete with sodium for a binding site on the carrier as has been suggested for other sodium-coupled transport systems, and that instead, chloride may be required for harmaline binding to the beta-alanine transporter.     

Determination of catecholamine permeability coefficients for passive diffusion across phospholipid vesicle membranes

Summary A convenient catecholamine transport assay has been developed which permits continuous, instantaneous monitoring of transmembrane flux. Epinephrine transport has been examined by spectrophotometrically monitoring adrenochrome formation resulting from the passive diffusion of catecholamine into unilamellar phospholipid vesicles containing entrapped potassium ferricyanide. Ferricyanide oxidation of epinephrine under the conditions employed is fast compared to membrane transport, which obviates the need for intravesicular concentration or volume determinations. Epinephrine transport data over a pH 6 to 7 range have been fitted to an integrated rate equation from which a permeability coefficient for neutral epinephrine of 2.7±1.5×10^–6 cm/sec has been obtained.     

Topical epinephrine causes a decrease in density of β-adrenergic receptors and catecholamine-stimulated chloride transport in the rabbit cornea

A single administration, or twice daily administration for 4.5 days, of topical 2% epinephrine to the rabbit eye in vivo causes a 30–40% decrease in the density of β-adrenergic receptors on membranes prepared from the cornea. Such treatment also causes complete loss of the ability of excised corneas to respond to epinephrine in vitro with enhanced active chloride transport. These findings indicate that stimulation with a high concentration of catecholamine depresses the entire pathway from receptor to physiological response.     

Synthesis, in vitro anti-inflammatory and cytotoxic evaluation, and mechanism of action studies of 1-benzoyl-β-carboline and 1-benzoyl-3-carboxy-β-carboline derivatives

In the present study, various 1-substituted and 1,3-disubstituted β-carboline derivatives were synthesized by a modified single-step Pictet-Spengler reaction. The compounds were examined for cytotoxicity and anti-inflammatory activity, as measured by the inhibition of prostaglandin E(2) (PGE(2)) production and nitric oxide (NO) production. While only two compounds (28 and 31) showed marginal cytotoxicity against four human cancer cell lines, most of the tested compounds exhibited potent inhibitory activity of both NO and PGE(2) production. Moreover, compounds 6 and 16 significantly reduced the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2), suggesting that β-carboline analogs can inhibit NO and PGE(2) production at the translational level. In addition, several of the β-carboline derivatives (1, 2, 4-8, 11, 13, 22, 25, 27, 31, and 41-43) displayed significant inhibitory activity of superoxide anion (O(2)(·-)) generation or elastase release compared to the reference compound, with 6 being the most potent. N-Formyl-L-methionyl-phenylalanine (FMLP)-induced phosphorylation of c-JunN-terminal kinase (JNK) and protein kinase B (AKT) were also inhibited by 6, suggesting that it suppresses human neutrophil functions by inhibiting the activation of JNK and AKT signaling pathways. Therefore, the synthetic 1-benzoyl-3-carboxy β-carboline analogs may have great potential to be developed as anti-inflammatory agents.     

Influence of aerobic energy deficit, ouabain and harmaline on the phenylalanine and galactose active transport by snail intestine

The effect of anaerobiosis (N2 bubbling of the medium) or 10(-4) M dinitrophenol on the penetration of 0.5 mM Phe in snail and rat everted intestine, in 2 min and 30 min incubation periods, has been studied. The aerobic energy deficit inhibits the amino acid net entry in both species, but whereas the active transport is annulled in rat, snail intestine is capable of continuing to accumulate Phe against a gradient. The prolonged action (30 min of preincubation) of 1 mM ouabain inhibits 0.1 mM Phe and 0.1 mM galactose entry in snail intestine. Amino acid uptake is far higher than the one obtained in the absence of Na+, in which condition Phe keeps accumulating against a gradient in the tissue water. Galactose active transport, instead, becomes null in the presence of the glucoside or in the absence of Na+. One mM harmaline is able to inhibit the initial entry of galactose into the tissue, while higher than 5 mM concentrations are required to inhibit that of Phe. Results confirm that snail intestine is capable of easily carrying out active transport processes with energy from anaerobic origin. On the other hand Phe transport is less sensitive to the absence of Na+, presence of ouabain or harmaline than that of galactose, so that contrary to what has been observed for the sugar, the active transport of the amino acid is not annulled in any of the three conditions.     

Mitochondrial dysfunction and biotransformation of β-carboline alkaloids,harmine and harmaline,on isolated rat hepatocytes

The cytotoxic effects and biotransformation of harmine and harmaline, which are known β-carboline alkaloids and potent hallucinogens, were studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to harmine caused not only concentration (0–0.50 mM)- and time (0–3 h)-dependent cell death accompanied by the formation of cell blebs and the loss of cellular ATP, reduced glutathione, and protein thiols but also the accumulation of glutathione disulfide. Of the other analogues examined, the cytotoxic effects of harmaline and harmol (a metabolite of harmine) at a concentration of 0.5 mM were less than those of harmine. The loss of mitochondrial membrane potential and generation of oxygen radical species in hepatocytes treated with harmine were greater than those with harmaline and harmol. In the oxygen consumption of mitochondria isolated from rat liver, the ratios of state-3/state-4 respiration of these β-carbolines were decreased in a concentration-dependent manner. In addition, harmine resulted in the induction of the mitochondrial permeability transition (MPT), and the effects of harmol and harmaline were less than those of harmine. At a weakly toxic level of harmine (0.25 mM), it was metabolized to harmol and its monoglucuronide and monosulfate conjugates, and the amounts of sulfate rather than glucuronide predominantly increased with time. In the presence of 2,5-dichloro-4-nitrophenol (50 μM; an inhibitor of sulfotransferase), harmine-induced cytotoxicity was enhanced, accompanied by decrease in the amount of harmol-sulfate conjugate, due to an increase in the amount of unconjugated harmol and the inhibition of harmine loss. Taken collectively, these results indicate that (a) mitochondria are target organelles for harmine, which elicits cytotoxicity through mitochondrial failure related to the induction of the MPT, mitochondrial depolarization, and inhibition of ATP synthesis; and (b) the toxic effects of harmine are greater than those of either its metabolite harmol or its analogue harmaline, suggesting that the onset of harmine-induced cytotoxicity may depend on the initial and/or residual concentrations of harmine rather than on those of its metabolites.     

Neural uptake of catecholamines and their molecular structures: A histopharmacologic study

Using ultrastructural and histofluorescence methods, we investigated the uptake mechanism of catecholamines by the nerve terminals in the cutaneous smooth muscles of stump-tailed macaques (Macaca arctoides). This in vivo approach ultilized the observed cytotoxic effects of 6-hydroxydopamine on these catecholamine-containing terminals and the protective effects of simultaneous treatment with catecholamines (dopamine, norepinephrine, and epinephrine), their 3–0-methylated derivatives (metanephrine and normetanephrine), and catechol acids (3,4-dihydroxymandelic acid and 2, 4, 5-trihy-droxymandelic acid). Both catecholamines and 3–0-methylated derivatives protected these nerve terminals from destruction by 6-hydroxydopamine, but catechol acids did not. However, the 3–0-methylated derivatives were less effective than the catecholamines. The degree of protection afforded by these amines depended largely on their concentration. Only catecholamines intensified the electron density of the intravesicular mass or the fluorescence in the nerve terminals; therefore, 3–0-methylated derivatives may inhibit 6-hydroxydopamine uptake at axoplasmic membrane sites, but not inside the axon. These observations led to the discovery that there are two sites for the catecholamine uptake process. One site is the axoplasmic membrane. The terminals are protected by catecholamines and their 3–0-methylated derivatives from 6-hydroxydopamine uptake and thus destruction. The other site is the intra-axonal compartments. Here competitive binding between the vesicular protein and both 6-hydroxydopamine and the catecholamines plays a main role.     

Inhibition of the Na+/I- symporter by harmaline and 3-amino-1-methyl-5H-pyrido(4,3-b)indole acetate in thyroid cells and membrane vesicles

Novel inhibitors of the Na+/I- symporter were identified using rat-thyroid-derived FRTL-5 cells and sealed vesicles from calf thyroid as model systems. Na(+)-dependent 125I- uptake was inhibited by the hallucinogenic drug harmaline and by a chemically related convulsive agent, 3-amino-1-methyl- 5H-pyrido(4,3-b)indole acetate (TRP-P-2). TRP-P-2 (Ki = 0.25 mM) was tenfold more effective as an inhibitor than harmaline (Ki = 4.0 mM). Inhibition by TRP-P-2 was competitive with respect to Na+ and was fully reversible. Although TRP-P-2 is a relatively low-affinity inhibitor, its affinity for the Na+ site of the Na+/I- symporter is over 100 times higher than that of Na+ (Km = 50 mM). 45Ca(2+)-efflux rates in calf thyroid membrane vesicles were not affected by TRP-P-2, indicating that membrane integrity is not disrupted by the drug. These findings show that TRP-P-2 may be a potentially useful tool for the identification and characterization of the Na+/I- symporter.     

Inhibition of Norepinephrine Transport and Reserpine Binding by Reserpine Derivatives

Reserpine, a competitive inhibitor of catecholamine transport into adrenal medullary chromaffin vesicles, consists of a trimethoxybenzoyl group esterified to an alkaloid ring system. Reserpine inhibits norepinephrine transport with a Ki of approximately 1 nM and binds to chromaffin-vesicle membranes with a KD of about the same value. Methyl reserpate and reserpinediol, derivatives that incorporate the alkaloid ring system, also competitively inhibit norepinephrine transport into chromaffin vesicles with Ki values of 38 +/- 10 nM and 440 +/- 240 nM, respectively. Similar concentrations inhibit [3H]reserpine binding to chromaffin-vesicle membranes. 3,4,5-Trimethoxybenzyl alcohol and 3,4,5-trimethoxybenzoic acid, derivatives of the other part of the reserpine molecule, do not inhibit either norepinephrine transport or [3H]reserpine binding at concentrations up to 100 microM. Moreover, trimethoxybenzyl alcohol does not potentiate the inhibitory action of methyl reserpate. Therefore, the amine binding site of the catecholamine transporter appears to bind the alkaloid ring system of reserpine rather than the trimethoxybenzoyl moiety. The more potent inhibitors are more hydrophobic compounds, suggesting that the reserpine binding site is hydrophobic.     

Linear and circular dichroism of harmine and harmaline interacting with DNA

The interaction of the β-carboline derivatives harmine and harmaline with calf thymus DNA was studied using linear and circular dichroism techniques. Absorption linear dichroism in an electric field indicated that the transition moment (at 336 nm) of harmine lies at angle χ = 82° relative to the helix axis and that the same angle for harmaline (at 365 nm) is only 67°. The 82° angle found for harmine is compatible with the interpretation that the molecule is intercalated between two consecutive base pairs of the DNA. The c.d. results in the u.v. region for the DNA harmine complex support this interpretation, since the increase in the magnitude of all the DNA c.d. bands can also be explained by assuming an intercalation. Since a 67° angle was found for harmaline, an intercalation is in that case unlikely, the molecule is probably in a tilted position in one of the DNA grooves. However, harmaline can induce a conformational change in nucleic acids, as shown by the modification of the c.d. spectra of DNA and, especially, of poly[d(A-T)], the addition of harmaline to this polynucleotide gradually inducing its pre-melting at 4°C. This study shows that the hydrogenation of a double bond of the pyridine ring converting harmine to harmaline greatly alters the interactions of the molecule with DNA.     

Effects of Mg2+ on calcium accumulation by two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium accumulation by two fractions of sarcoplasmic reticulum presumably derived from longitudinal tubules (light vesicles) and terminal cisternae (heavy vesicles) was examined radiochemically in the presence of various free Mg2+ concentrations. Both fractions of sarcoplasmic reticulum exhibited a Mg2+-dependent increase in phosphate-supported calcium uptake velocity, though half-maximal velocity in heavy vesicles occurred at a much higher free Mg2+ concentration than that in light vesicles (i.e., approx. 0.90 mM vs. approx. 0.02 mM Mg2+). Calcium uptake velocity in light vesicles correlated with Ca2+-dependent ATPase activity, suggesting that Mg2+ stimulated the calcium pump. Calcium uptake velocity in heavy vesicles did not correlate with Ca2+-dependent ATPase activity, although a Mg2+-dependent increase in calcium influx was observed. Thus, Mg2+ may increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles. Analyses of calcium sequestration (in the absence of phosphate) showed a similar trend in that elevation of Mg2+ from 0.07 to 5 mM stimulated calcium sequestration in heavy vesicles much more than in light vesicles. This difference between the two fractions of sarcoplasmic reticulum was not explained by phosphoenzyme (EP) level or distribution. Analyses of calcium uptake, Ca2+-dependent ATPase activity, and unidirectional calcium flux in the presence of approx. 0.4 mM Mg2+ suggested that ruthenium red (0.5 microM) can also increase the coupling of ATP hydrolysis to calcium transport in heavy vesicles, with no effect in light vesicles. These functional differences between light and heavy vesicles suggest that calcium transport in terminal cisternae is regulated differently from that in longitudinal tubules.     

The occurrence of 2-methyl-1,2,3,4-tetrahydro-β-carboline and variation in alkaloids in Phalaris arundinacea

2-Methyl-1,2,3,4-tetrahydro-β-carboline was isolated from reed canarygrass (Phalaris arundinacea L.) and the occurrence of 2-methyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline confirmed. Clones of reed canarygrass that contained N,N-dimethyltryptamine or 2-methyl-1,2,3,4-tetrahydro-β-carboline did not contain their respective methoxy or hydroxy derivatives. Five of the 12 clones tested contained either or both of 5-methoxy-N,N-dimethyltryptamine and 2-methyl-6-methoxy-1,2,3,4-tetrahydro-β-carboline. The data suggest that clones that contain gramine are not likely to contain N,N-dimethyltryptamine and/or β-carbolines. Thus, an inverse biosynthetic relationship between gramine and the tryptamine and β-carboline alkaloids seems to exist. However, further work is needed to firmly establish any such relationship between these alkaloids.     

Transport of 1,5-anhydro-D-glucitol into insulinoma cells by a glucose-sensitive transport system

The uptake of 1,5-anhydro-D-glucitol (1,5-AG) occurs by passive mechanisms in cells or tissues that have passive glucose transporters. It is known that serum 1,5-AG concentrations are reduced in patients with diabetes mellitus. To elucidate the metabolism of this substance and its physiological role in pancreatic β-cells, we assayed 1,5-AG transport in the insulinoma-derived cell lines, RINr and MIN6. Both cell lines showed an insulin-insensitive, concentration-dependent uptake of 1,5-AG with a saturation time of approximately 120 min, and most of the 1,5-AG in the cytoplasm was in the free form. A biphasic saturation curve was obtained using a wide range of 1,5-AG concentrations, suggesting that accumulation was mediated by a high affinity and a low affinity transporter. The high affinity transporter had a K_m of 10.4 in RINr cells and 13.0 mM in MIN6 cells, and the low affinity transporter had a K_m of 131 in RINr cells and 211 mM in MIN6 cells. Uptake of 1,5-AG was markedly inhibited by phloretin and cytochalasin B, but was only slightly affected by phloridzin. Uptake of 1,5-AG was markedly inhibited by glucose at physiological concentrations (1.0–10 mM), as well as by galactose and mannose. The 1,5-AG concentration required to inhibit 2-deoxyglucose uptake exceeded that of glucose by >100 times, being much higher than the physiological concentrations of 1,5-AG. These results indicate that the 1,5-AG carrier system in insulinoma cells is distinct from that in either the somatic cells or renal tubular cells. These findings also suggest that a unique 1,5-AG transport system is present in pancreatic β-cells.     

Harmaline distribution in single muscle fibres and the inhibition of sodium efflux

Harmaline, a known inhibitor of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in cell membranes, inhibited 50% of the ²²Na efflux from barnacle muscle fibres at an extracellular concentration of 2.4 mM. Injected harmaline inhibited 50% of the efflux at an estimated intracellular concentration of about $\text{8}\text{mM}\text{·}\text{kg}{}^{\mathrm{?1}}$, assuming complete equilibration with no binding. Total fibre harmaline was measured in separate fibres by ultraviolet spectrophotometry. Fibres in 3 mM harmaline saline accumulated harmaline with a half-time of 17 min and a final total fibre concentration of $\text{6–12}\text{mM}\text{·}\text{kg}{}^{\mathrm{?1}}$. In harmaline-free saline this accumulated harmaline was lost exponentially with a half-time of 35 min; injected harmaline was lost exponentially from fibres with a half-time of 50 min. It is proposed that harmaline crosses the fibre membrane as the uncharged base and that its apparent accumulation against a concentration gradient is mainly due to intracellular binding with an additional contribution from a transmembrane pH gradient. It is concluded that, in fibres exposed to harmaline saline, the intracellular concentration can reach a sufficiently high value, as judged from the results of the injection experiments, to inhibit Na⁺ efflux at an interior-facing site on the fibre membrane. In contrast, harmaline appears to inhibit the Na⁺-dependent uptake of l-glutamate at an extracellular site.     

Topographical similarities between harmaline inhibition sites on Na+-dependent amino acid transport system ASC in human erythrocytes and Na+-independent system asc in horse erythrocytes

Na+-dependent system ASC and Na+-independent system asc are characterized by a common selectivity for neutral amino acids of intermediate size such as L-alanine and by their interactions with dibasic amino acids. For system ASC, the positive charge on the dibasic amino acid side chain is considered to occupy the Na+-binding site on the transporter. We report here the use of harmaline (a Na+-site inhibitor in some systems) as a probe of possible structural homology between these two classes of amino acid transporter. Harmaline was found to inhibit human erythrocyte system ASC noncompetitively with respect to L-alanine concentration, but approximated competitive inhibition with respect to Na+ concentration (apparent Ki = 2.0 and 0.9 mM, respectively). Similarly, harmaline noncompetitively inhibited L-alanine uptake by horse erythrocyte systems asc1 and asc2 (apparent Ki = 2.0 and 1.9 mM, respectively). In contrast, harmaline functioned as a competitive inhibitor of L-lysine uptake by system asc1 (apparent Ki = 2.6 mM). It is concluded that harmaline competes with Na+ for binding to system ASC and that a topographically similar harmaline inhibition site is present on system asc. This site does not however bind Na+, the asc1 transporter exhibiting normal L-alanine and L-lysine influx kinetics in the total absence of extracellular cations.     

Inhibition of Mrp2- and Ycf1p-mediated transport by reducing agents: evidence for GSH transport on rat Mrp2

Mammalian Mrp2 and its yeast orthologue, Ycf1p, mediate the ATP-dependent cellular export of a variety of organic anions. Ycf1p also appears to transport the endogenous tripeptide glutathione (GSH), whereas no ATP-dependent GSH transport has been detected in Mrp2-containing mammalian plasma membrane vesicles. Because GSH uptake measurements in isolated membrane vesicles are normally carried out in the presence of 5-10 mM dithiothreitol (DTT) to maintain the tripeptide in the reduced form, the present study examined the effects of DTT and other sulfhydryl-reducing agents on Ycf1p- and Mrp2-mediated transport activity. Uptake of S-dinitrophenyl glutathione (DNP-SG), a prototypic substrate of both proteins, was measured in Ycf1p-containing Saccharomyces cerevisiae vacuolar membrane vesicles and in Mrp2-containing rat liver canalicular plasma membrane vesicles. Uptake was inhibited in both vesicle systems in a concentration-dependent manner by DTT, dithioerythritol, and β-mercaptoethanol, with concentrations of 10 mM inhibiting by ∼40%. DTT’s inhibition of DNP-SG transport was noncompetitive. In contrast, ATP-dependent transport of [³H]taurocholate, a substrate for yeast Bat1p and mammalian Bsep bile acid transporters, was not significantly affected by DTT. DTT also inhibited the ATP-dependent uptake of GSH by Ycf1p. As the DTT concentration in incubation solutions containing rat liver canalicular plasma membrane vesicles was gradually decreased, ATP-dependent GSH transport was now detected. These results demonstrate that Ycf1p and Mrp2 are inhibited by concentrations of reducing agents that are normally employed in studies of GSH transport. When this inhibition was partially relieved, ATP-dependent GSH transport was detected in rat liver canalicular plasma membranes, indicating that both Mrp2 and Ycf1p are able to transport GSH by an ATP-dependent mechanism.     

Transport of Catecholamines by Native and Reconstituted Rat Heart Synaptic Vesicles

Highly purified “heavy” synaptic vesicles were isolated from rat heart by differential centrifugation. Because of the high intravesicular concentrations of proteins, catecholamine, and ATP, resealed vesicle ghosts were prepared and used to study the detailed kinetics of catecholamine transport. ATP stimulated the uptake of /-norepinephrine and was saturable with a K_m for l-norepinephrine at 3.3 μM and 1.8 mM for ATP. The ghosts also accumulated 5-hydroxytryptamine and l-epinephrine via an ATP-dependent mechanism. Uptake was stereospecific for the l-form. A functional catecholamine transporter could be solubilized by the detergent octyl-glucoside and incorporated into phospholipid vesicles, which, after detergent removal, generated proteoliposomes that accumulated l-norepinephrine. Reserpine- and l-propranolol-sensitive accumulation against a concentration gradient is achieved by artificially creating a pH gradient across the membrane, and lends further support to the idea that at least the initial phase of catecholamine transport is driven by the trans-membrane pH gradient created by the proton-translocating ATPase.     

Restoration of Catecholamine Content of Previously Depleted Adrenal Medulla In Vitro: Importance of Synthesis in Maintaining the Catecholamine Stores

The functional integrity of adrenal chromaffin storage vesicles was studied in the perfused rat adrenal gland subjected to intense exocytosis. Continuous perfusion with 55 mM K+-Krebs solution produced a large and uninterrupted secretion of catecholamines. Total amounts secreted within 45 min were 4.66 micrograms and represented almost 30% of the total tissue catecholamine content. If perfusion with excess K+ was extended to 90 min, the secretion increased further to 5.76 micrograms. Despite such a large secretory response, the catecholamine content of the K+-stimulated adrenal medulla was comparable to that of unstimulated control, suggesting an enhanced resynthesis to maintain the normal levels. Pretreatment of rats with alpha-methyl-p-tyrosine, and including this agent in the perfusion medium during stimulation with K+, caused a marked reduction in catecholamine content. The degree of depletion depended on the extent of stimulation with K+ (45% in 45 min and 60% in 90 min). Although depleted catecholamine stores did not show spontaneous recovery in 2 h, inclusion of tyrosine, L-3,4-dihydroxyphenylalanine or dopamine (but not epinephrine or norepinephrine) completely restored the catecholamine content of previously depleted adrenal medulla. Repletion achieved by tyrosine was time dependent (evident in 30 min and maximum in 2 h) and blocked by alpha-methyl-p-tyrosine but not by calcium deprivation. The ratio of epinephrine to norepinephrine remained constant during various stages of the experiment, suggesting both types of vesicles were equally affected by different treatments. The secretory response (10 Hz for 30 s) was unaffected even though tissue catecholamine stores were significantly depleted (50%). In summary, we have demonstrated that catecholamine content of the isolated perfused adrenal gland can be reduced by stimulation of exocytotic secretion in the presence of tyrosine hydroxylase inhibitor. Since the depleted stores can be fully refilled by synthesis of catecholamines from its precursors, it is suggested that chromaffin vesicles may be reutilized for the purpose of synthesis, storage, and secretion of adrenal medullary hormones.     

Kinetic and quantitative measurements of catecholamine transport in chromaffin ghosts using a glassy carbon electrode

A method for the on-line kinetic measurement of net catecholamine uptake and release in ghosts derived from bovine chromaffin granules is described. Changes in free catecholamine concentration in 1 to 2 ml of media containing chromaffin ghosts were continuously measured through the amperometric detection of their oxidation products through a glassy carbon electrode set at 0.5-V potential vs a reference electrode. Parallel measurements of catecholamine uptake and release in the ghosts under various metabolic conditions show a good quantitative agreement between the values obtained with the electrode and those obtained through high-performance liquid chromatography after separation of the ghosts from the medium. Initial velocities of ATP-dependent uptake of epinephrine, norepinephrine, dopamine, and isoproternol by the ghosts are shown. This method permits, for the first time, quantitation of unidirectional movement of catecholamines in the presence of minute quantities of biological samples. The advantages, limitations, and suitability of this method to measure catecholamine transport in other systems are discussed.