Reserpine Binding Research Articles

Functional molecular mass of binding sites for [3H]dihydrotetrabenazine and [3H]reserpine and of dopamine beta-hydroxylase and cytochrome b561 from chromaffin granule membrane as determined by radiation inactivation.

The monoamine transporter of chromaffin granule membrane has two distinct high-affinity binding sites for tetrabenazine and reserpine, which can be assayed by [3H]dihydrotetrabenazine and [3H]reserpine binding, respectively. The functional molecular mass of the components bearing these sites has been investigated by the radiation inactivation technique. The decline of [3H]dihydrotetrabenazine binding activity with increasing radiation doses followed a single exponential, from which a functional molecular mass of 68 kDa was derived for tetrabenazine binding sites. [3H]Reserpine binding activity declined in a more complex way; however, under conditions where high-affinity reserpine binding sites were specifically assayed, the decline was also exponential, corresponding to a functional molecular mass of 37 kDa for these sites. The figures obtained for high-affinity tetrabenazine and reserpine binding sites are consistent with previous values obtained by photoaffinity of tetrabenazine and serotonin binding sites, respectively. It is thus concluded that the monoamine transporter has an oligomeric structure. By the radiation inactivation technique, cytochrome b561 and dopamine beta-hydroxylase have functional molecular masses of 25 and 123 kDa, respectively. The latter value might be attributed to the dimeric form of the enzyme.

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.

Pharmacologie moléculaire du transporteur des catécholamines des granules chromaffines de la médullo-surrénale bovine

Tetrabenazine (TBZ) and reserpine are two inhibitors of the catecholamine uptake system of the chromaffin granule membrane. They are structural analogs of the substrates dopamine and serotonin and they inhibit the monoamine transporter, which catalyzes a H+/neutral amine antiport. [3H]Dihydrotetrabenazine ([3H]TBZOH) is bound by chromaffin granule membranes on one class of site (T sites, KD = 3 nM); [3H]reserpine is bound on T sites and a second class of site (R1 sites, KD = 0.7 nM). The two sites are involved in monoamine translocation. The substrates displace the ligands with different efficiency: noradrenaline (Km = 10 microM) displaces reserpine efficiently (EC50 = 30 microM), but TBZOH poorly (EC50 = 2000 microM); m-iodobenzylguanidine, which has recently been shown to be a substrate of the monoamine uptake system (Km = 5 microM), displaces TBZOH efficiently (EC50 = 25 microM), but reserpine inefficiently (EC50 = 300 microM). Since both substrates are translocated by the same transporter, this result confirms the existence of two sites with different properties. T sites are characterized by a linear relationship between the reciprocal of the dissociation constants of various drugs displacing [3H]TBZOH and their partition coefficient in octanol/H2O mixtures. This relationship, which indicates a hydrophobic environment of T sites, does not exist for R1 sites. T sites have been identified by covalent labeling with a derivative of TBZ coupled to an arylazido group. The labeled sites are borne by a 65,000 dalton protein. The kinetics of reserpine binding are accelerated in the presence of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and characterization of the catecholamine transporter in bovine chromaffin granules using [3H]reserpine.

Characterization of the catecholamine transporter in chromaffin granule membranes has been hampered by the lack of a radioligand with high specific activity which binds selectively to the carrier with high affinity. We report here the identification of a high affinity binding site for [3H]reserpine on chromaffin granule membranes isolated from bovine adrenal gland which has the characteristics expected of the catecholamine transporter. [3H]Reserpine bound predominately to a high affinity site with a Kd for [3H]reserpine of 9 nM and a binding site density of 7.8 pmol/mg of protein. Comparison of the characteristics of the high affinity reserpine binding site to the characteristics of catecholamine transport indicated that (a) the Ki and rank order of potency for inhibition of [3H]reserpine binding by various biogenic amines was similar to their Ki for inhibition of catecholamine transport (b) both the inhibition of (-)-[3H]norepinephrine transport and inhibition of [3H]reserpine binding showed similar stereo-specificity, and (c) Kd for binding of reserpine to chromaffin granule membranes was similar to the Ki for reserpine inhibition of catecholamine transport. These results demonstrate that the high affinity binding site for [3H]reserpine on chromaffin granule membranes is associated with the catecholamine transporter.

Reserpine labels the catecholamine transporter in synaptic vesicles from bovine caudate nucleus

Tritiated reserpine binds to synaptic vesicles from bovine caudate with high affinity ( K app d = 1.25 nM, B max = 3.3 pmol/mg protein). This interaction is both ATP-dependent and sensitive to the protonophores CCCP and nigericin, suggesting that a proton electrochemical gradient is required for binding. Dopamine, epinephrine, norepinephrine and serotonin all inhibit reserpine binding at concentrations similar to those required for inhibition of dopamine uptake. Treatment with saponin to release vesicle contents results in complete loss of accumulated dopamine but retention of bound reserpine. these results indicate that reserpine binds to the catecholamine transport system of synaptic vesicles with high affinity and specificity.

Lithium attenuates dopamine depleting effects of reserpine and tetrabenazine but not that of alpha methyl-p-tyrosine

Concurrent administration of lithium (Li) significantly attenuates the dopamine (DA) depleting effects of reserpine and tetrabenazine, but does not change alpha-methyl-p-tyrosine (AMPT) induced DA depletion in rat brain. This effect of Li is probably mediated, in part, by inhibiting the magnesium-dependent binding of both reserpine and tetrabenazine to their specific receptor sites. Such interaction between these drugs may attenuate the beneficial effects of tetrabenazine and reserpine on patients with tardive dyskinesia or tardive dystonia who are treated concurrently with lithium for their psychiatric disorder.

Conditions required for reserpine binding to the catecholamine transporter on chromaffin granule ghosts

Conditions required for reserpine binding to the catecholamine transporter on chromaffin granule ghosts

Noradrenaline storage in deoxycorticosterone—saline hypertensive rats

Accumulation of labeled noradrenaline ( 3H-NA) in heart slices provided a measure of the storage ability of peripheral adrenergic nerves in rats made hypertensive by DOCA and salt (NaCl) administration. Measurements of dopamine-β-hydroxylase (DBH) and the irreversible binding of tritiated reserpine ( 3H-R) in the heart and mesenteric arteries were taken as indices of the density of storage vesicles in nerve endings. The turnover of transmitter in peripheral adrenergic nerves was estimated by measuring the disappearance of NA after blockade of its synthesis. The storage of 3H-NA in heart slices, measured by the retention of amine, was not changed during the development and maintenance phases of DOCA hypertension in the rat. DBH activity (31. 1 nmole of synephrine formed/g of heart and 193.7 nmole./g of mesenteric artery) and the binding of 3H-reserpine (4.4 ng/g/heart and 4.7 ng/g of mesenteric artery) were similar to the values observed in tissues of normotensive rats. Turnover of NA was significantly accelerated in the cardiac nerves of DOCA hypertensive rats (T 1 2 6.2 h vs. 11.7 h for controls). These results show that the storage function and the density of synaptic vesicles are not changed in the peripheral adrenergic nerves of DOCA hypertensive rats. The faster disappearance of endogenous NA or exogenous labeled NA observed in vivo in hypertensive rats may be attributed to an increased release due to enhancement of nerve impulse flow, not to an impaired storage ability.

Binding of reserpine to plasma albumin and lipoproteins

The binding of reserpine to plasma albumin and lipoproteins was studied by equilibrium gel filtration. Reserpine was shown to be over 96 per cent bound to plasma proteins. Two discrete peaks of plasma-protein binding were identified, the one in the lipoprotein fraction and the other corresponding to plasma albumin. Binding to the lipoproteins showed large variations between normal subjects (mean extent of binding of 5.4 ± 0.8) and hyperlipoproteinemic patients (mean of 11.2 ± 0.9), and correlated very significantly with the triglycéride and total lipid concentration and, to a lesser extent, with the concentration of cholesterol in the plasma. It is concluded that plasma lipoproteins, as well as albumin, bind reserpine, and that the degree of lipoproteinemia may contribute to variations in plasma-drug binding.

On the formation of adrenergic amine storage granules as measured by reserpine labeling.

Reserpine appears to be bound specifically and irreversibly to amine storage granules in the adrenergic neuron. The presence of reserpine persistently attached to amine granule should reflect the life span of the vesicles, and any additional binding of reserpine should then measure the rate of appearance of newly formed amine granules. Measurement of the persistent binding of tritiated reserpine (3H-Reserpine) in hearts of rats given labeled compound at various times after a saturating dose of unlabeled reserpine showed that the recovery of 3H-reserpine binding capacity occurred rapidly. Four days after reserpine the 3H-reserpine binding was 30% of control rats. The rate of return of binding capacity was then slowed being 65% of normal in 24 days. Calculation of the mean daily increment of binding capacity showed that the rate was an inverse linear function of the binding capacitu existing in the neurone at an early phase (1--8 days) but became constant at a later interval (8--24 days). It is suggested that the return of reserpine binding capacity reflects the appearance of new storage granules and that the rate of storage granule synthesis in the cell body is under feedback control. The signal for enhanced granule formation does not appear to be related to the intensity of nerve traffic not to the concentration of transmitter in the neuron since manaeuvers which alter these parameters did not change the rate of reserpine binding recovery.

Localization and binding of reserpine in the membrane of adrenomedullary amine storage granules.

The subcellular localization of 3H-reserpine persisting in the adrenal gland of the rat 18–60 h after parenteral administration was studied by differential and sucrose gradient centrifugation techniques. This persistent reserpine is associated with the catecholamine storage granule. More specifically, the drug is exclusively localized in the vesicular membrane where it is attached by a firm bond which is not reversed by prolonged dialysis or by incubation with adrenomedullary granules from non-reserpinized rats. Binding experiments conducted in vitro indicate that membranes isolated from bovine adrenomedullary granules or mitochondria bind reserpine at a single binding site with a molarity in these preparations of 9.4×10−9M/mg of protein with a binding constant of 4.7×10−7M. The in vitro binding of 3H-reserpine is reversible upon dialysis and has a low specificity since unlike the binding in vivo, it is not affected by preincubation of membranes with high concentrations of unlabeled reserpine. There was a very low affinity of reserpine for the intragranular constituents of adrenomedullary granules. It is concluded that a rapid specific and irreversible binding of reserpine occurs in vivo in the membranes of amine storage granules which cannot be realized by bovine adrenomedullary granule membranes in vitro.

In vivo persistent binding of 3H-reserpine to rat brain subcellular components

24 hr, but not 1 hr, after injection of 3H-reserpine, the gross subcellular distribution profile of the labelled drug in the brain differed from that determined by adding 3H-reserpine to brain homogenates from untreated rats. Evidence for the occurrence of an ‘in vivo’ persistent binding of reserpine to the ‘microsomal’ fraction is discussed.

Further studies on the nature of persistent reserpine binding: Evidence for reversible and irreversible binding

Tritium-labeled reserpine of high specific activity was injected into rats, and drug levels measured in heart, spleen, adrenal glands, and small intestine. All tissues showed a first-order reserpine decline terminating about 24–30 hr after injection. After this time there was a semi-permanent binding persisting for many days. Experiments in which high doses of unlabeled reserpine were given 18 or 30 hr after the labeled drug reveal that at the 18 hr time a portion of the drug is reversibly bound, while at the 30 hr time the drug is irreversibly bound. It is suggested that the reversibly bound phase is associated with blockade of the intraneuronal amine carrier system, while the irreversible phase is associated with prolonged alteration of the granular amine storage mechanism.

Specific binding of reserpine—: Association with norepinephrine depletion

Small doses of 3H-reserpine (10–100 μg/kg) were injected i.v. into rats and the drug concentration in heart, spleen and liver measured at various intervals. Eighteen hours after a dose of 25 μg per kg, concentrations of the tritiated compound were: 4.98 ng per g heart, 6.39 ng per g spleen, and 1.48 ng per g liver. The biologic half-life of 3H-reserpine in heart and spleen was about 18 hr; that of liver was about 4 hr. Specificity studies showed that the labeled material extracted from rat heart 18 hr after administration of the tritiated compound behaved similarly to authentic reserpine. Pretreatment of rats with tetrabenazine (20 mg/kg) or unlabeled reserpine (0.5 mg/kg) significantly reduced the amount of 3H-reserpine found in heart and spleen 18 hr after administration of the radioactive drug, while administration of either tetrabenazine or unlabeled reserpine after the injection of 3H-reserpine had no significant effect on levels of the labeled drug. The concentration of norepinephrine remaining in heart 18 hr after the administration of small doses of reserpine was inversely related to the concentration of persisting drug and the correlation was highly significant. It is concluded that the persistent depletion of norepinephrine by small doses of reserpine is induced and maintained by the physical presence of minute amounts of very highly bound reserpine. Calculations reveal that each molecule of reserpine persisting in heart is associated with a deficit of about 500 norepinephrine molecules. It is also calculated that about 20 molecules of reserpine persist for each affected catecholamine-containing granule.

The binding of the calcium transport inhibitors reserpine, chlorpromazine and prenylamine to the lipids of the membranes of the sarcoplasmic reticulum.

1. Reserpine, chlorpromazine, prenylamine and imipramine are bound to vesicles isolated from the sarcoplasmic reticulum of rabbit striated muscle cells as well as by the lipids which can be extracted from the vesicular preparations. 2. At concentrations of 5×10−7 M reserpine, chlorpromazine and prenylamine, drug binding can just be detected. At drug concentrations of 10−4 M the drug uptake amounts to 0.1 μmol per mg vesicular protein, 0.2 μmol drug per mg vesicular lipid and ∼0.4 μmol drug per mg phosphatidylcholine. 3. Drug binding is reversible and temperature independent. 4. Drug binding is not diminished when the phospholipids are hydrolysed by phospholipase A, C or D. 5. Drug binding is abolished by hydrogenation of the isolated lipids as well as of the lipids bound to the membranes. 6. In addition calcium uptake the Ca++ induced increase in ATP breakdown and the phosphate transfer reaction are inhibited when the lipids in the membranes are hydrogenated. 7. The normal ATPase as well as the lipid free proteins myokinase and actin are not affected by hydrogenation.