Harmaline Inhibition Research Articles

Cation and harmaline interactions with Na +-independent dibasic amino acid transport system y + in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish ( Eptatretus stouti)

Transport systems y +, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na +-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na + site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na +-site inhibitor) with the same affinity as asc (apparent K i range 1–4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na +-independent, dibasic amino acid transport system y + accepts neutral amino acids when Na + or another acceptable cation is also present. This latter observation implies that the y + translocation site binds Na + and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y +-mediated lysine uptake (5 μM, 20°C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na + functioning as a competitive inhibitor of transport. Apparent K m and K m values for lysine transport at 20°C were 15.2 μM and 183 μmol/l cells per h, respectively, in sucrose medium and 59.4 μM and 228 μmol/l cells per h in Na + medium. Similar results were obtained with y + in erythrocytes of a primitive vertebrate, the Pacific hagfish ( Eptatretus stouti), indicating that Na +-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 μM, the IC 50 value for Na +-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K + and guanidinium +, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y + translocation site, the presence of 150 mM Na + increased the harmaline inhibition constant ( K l) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y +, asc and ASC represent a family of closely related transporters with a common evolutionary origin.

Harmaline inhibition of amino acid transport in human and in horse erythrocytes

Harmaline inhibition of amino acid transport in human and in horse erythrocytes

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 Na +-Ca 2+ exchange mechanism in cardiac sarcolemmal vesicles by harmaline

1. Harmaline was found to inhibit the Na +-Ca 2+ exchange mechanism present in cardiac sarcolemmal vesicles. 2. The inhibition was dose-dependent and was observed in the range 10 −5M-10 −2m harmaline. 3. The effect was demonstrated on both 45Ca 2+-uptake and 45Ca 2+-efflux. 4. The observed K i value for harmaline inhibition of 45Ca 2+-uptake was found to be 2.5 × 10 −4M.

Characteristics of monoamine oxidase activity in mouse brain

Summary Mouse brain homogenates were incubated in vitro and monoamine oxidase (MAO) activity determined fluorometrically using kynuramine as a substrate. Optimum activity was found at pH 9.0, the amount of product formed was linear with respect to both time of incubation and amount of homogenate in the incubation, and the activity of MAO was temperature-dependent, with a Q 10 of 2.1. Deamination of kynuramine by MAO was inhibited by the monoamines dopamine, noradrenaline and serotonin, but not by the diamine histamine, nor by the amino acids lysine and glutamine. The MAO inhibitors pargyline and iproniazid gave single dose-response sigmoid inhibition curves, whereas harmaline produced a double sigmoid inhibition curve, indicating the presence of two forms of MAO (MAO-A and MAO-B) with differing sensitivities to harmaline inhibition. The pargyline inhibition curve did not differ in the presence of harmaline, suggesting that, in vitro , pargyline has no preference for either form of MAO. These results show that the fluorometric kynuramine assay is a simple and sensitive method for MAO measurement. This MAO can metabolise monoamines but not related substrates, is inhibited by typical MAO inhibitors and apparently exists in two forms.

The effect of harmaline and related β-carbolines on the acetylcholine-stimulated contractions of guinea-pig ileum

Five members of the Harmala family of alkaloids, four commercially available and one synthetic homologue, were tested for their actions on the acetylcholine-elicited contractions of the guinea-pig ileum. Under normal Tyrode conditions over comparable concentration ranges harmalol was without effect, harmaline and harmaline inhibited competitive-noncompetitive inhibition whilst the related 2-methylated derivatives effected purely competitive antagonism. Binding studies using [ 3H]QNB have further confirmed the competitive aspect of this antagonism. The competitive-noncompetitive inhibitory property of harmaline on smooth muscle muscarinic receptors has not been previously reported. Harmaline is known to compete with Na + binding sites and significantly complete substitution of Na + by K + in the Tyrode medium was found to abolish the noncompetitive component of harmaline inhibition. Explanations are offered for both the competitive and noncompetitive components of the inhibition produced by the Harmala alkaloids.

Harmaline inhibition of Na-dependent transport in renal microvillus membrane vesicles.

The effects of the hallucinogen harmaline on D-glucose, L-alanine, and Na+ transport were studied in microvillus membrane vesicles isolated from the rabbit renal cortex. Harmaline had no effect on glucose transport in the absence of Na+, but reversibly inhibited sugar flux in the presence of NaCl. Inhibition of Na+-dependent glucose transport was inversely related to the Na+ concentrations. The hallucinogen competitively inhibited the Na+ activation of phlorizin binding to the membranes but did not inhibit phlorizin binding in the absence of Na+. Harmaline inhibited Na+-dependent alanine transport and, at higher drug concentrations, the amino acid flux in the absence of NaCl. Harmaline competitively inhibited the rate of Na+ uptake which, in the absence of glucose and alanine, is known to occur via Na+-H+ exchange. The hallucinogen trans-inhibited the efflux of glucoe and Na+ from membrane vesicles preloaded with the solutes. These findings suggest that harmaline is a direct inhibitor of microvillus membrane transport processes and acts as a competitive inhibitor of Na+ transport sites. Harmaline may therefore be a useful investigative tool for studying mechanisms of Na+-coupled transport in the luminal membrane of the proximal tubular cell.

The effect of harmaline on intestinal sodium transport and on sodium-dependent D-glucose transport in brush-border membrane vesicles from rabbit jejunum.

Harmaline inhibition of sodium uptake and of sodium-dependent D-glucose transport was investigated using brush-border membrane vesicles from frozen rabbit jejunum. Under sodium-gradient conditions, "initial" D-glucose uptake (20 s) was inhibited by harmaline at concentrations above 0.5 mM, but at lower harmaline concentrations D-glucose uptake was stimulated by 10--15%. When a similar potassium gradient was used, harmaline had no effect. At concentrations up to 2 mM, harmaline did not alter the equilibrium uptake of D-glucose or D-mannitol. After pre-equilibration with sodium (25 mM), G-glucose uptake was inhibited at harmaline concentrations ranging from 0.1 to 2 mM. Sodium (10 mM) uptake was also inhibited by harmaline. Increasing the sodium concentration reduced the inhibitory effect of harmaline on tracer sodium uptake as well as on sodium-dependent D-glucose uptake. Similar to phlorizin, harmaline (1 mM) was able to prevent glucose-induced sodium influx across the brush-border membrane. Sodium uptake into brush-border membrane vesicles seems to be inhibited at lower harmaline concentrations than sodium-dependent D-glucose uptake. At high (2 mM) inhibitor concentrations, however, sodium-dependent glucose uptake is more strongly inhibited than sodium uptake. These results suggest that harmaline inhibits both sodium and sodium-dependent transport across intestinal brush-border membranes by interacting with specific sodium-binding sites.

Studies of monoamine oxidases: Inhibition of bovine brain MAO in intact mitochondria by selective inhibitors

The inhibition of mitochondrial monoamine oxidase (MAO) from beef brain cortex by the selective inhibitors, clorgyline, harmaline, Deprenyl and pargyline, was compared using five substrates: serotonin (5-HT), β-phenylethylamine (PEA), tyramine, tryptamine and dopamine. Dose-response studies, consistent with the classification of MAO, types A and B, indicated that serotonin deamination was more sensitive to clorgyline and harmaline inhibition than was phenylethylamine. However, the curves for all substrates were double-sigmoidal, rather than being a single sigmoid curve for 5-HT and PEA. Deprenyl and pargyline did not exhibit any marked selectivity for inhibiting PEA deamination without prior preincubation of enzyme and inhibitor. The rate of inhibition was variable and was dependent upon the substrate, the nature of the inhibitor and the inhibitor concentration. Dual inhibitor studies, using the “type A” inhibitor, clorgyline, and the “type B” inhibitor, Deprenyl, together, resulted in almost complete MAO inhibition, regardless of substrate. Combining the two type A inhibitors, clorgyline and harmaline, or the two type B inhibitors, deprenyl and pargyline, resulted in inhibitions that were equal to or only slightly greater than the inhibition produced by a single inhibitor. These results suggested that there are at least two distinct sites in beef brain MAO from cortical mitochondria which may be interacting. The deamination of all substrates occurs at both sites.