Molecular evidence and functional expression of a novel drug efflux pump (ABCC2) in human corneal epithelium and rabbit cornea and its role in ocular drug efflux

Cornea is considered as a major barrier for ocular drug delivery. Low ocular bioavailability of drugs has been attributed primarily to low permeability across corneal epithelium, thus leading to sub-therapeutic concentrations of drug in the eye and treatment failure. The role of drug efflux proteins, particularly the P-glycoprotein (P-gp) in ocular drug bioavailability has been reported. The objective of this research was to determine whether human corneal epithelium expresses multidrug resistance associated proteins (MRPs) contributing to drug efflux by employing both cultured corneal cells and freshly excised rabbit cornea. SV40-HCEC and rPCEC were selected for in vitro testing. SV40-HCEC and freshly excised rabbit corneas were utilized for transport studies. [³H]-cyclosporine-A and [¹⁴C]-erythromycin, which are known substrates for ABCC2 and MK-571, a specific inhibitor for MRP were applied in this study. RT-PCR indicated a unique and distinct band at ∼272 bp corresponding to ABCC2 in HCEC, SV40-HCEC, rabbit cornea, rPCEC, and MDCKII-MRP2 cells. Also RT-PCR indicated a unique band ∼181 bp for HCEC and SV40-HCEC. Immunoprecipitation followed by Western Blot analysis revealed a specific band at ∼190 kDa in membrane fraction of SV40-HCEC, MDCKII-MRP2 and no band with isotype control. Uptake of [³H]-cyclosporine-A and [¹⁴C]-erythromycin in the presence of MK-571 was significantly enhanced than control in both SV40-HCEC and rPCEC. Similarly a significant elevation in (A → B) permeability of [³H]-cyclosporine-A and [¹⁴C]-erythromycin was observed in the presence of MK-571 in SV40-HCEC. A → B transport of [³H]-cyclosporine-A was elevated in the presence of MK-571 in freshly excised rabbit cornea indicating potential role of this efflux transporter and high clinical significance of this finding.     

Permeability characteristics of novel mydriatic agents using an in vitro cell culture model that utilizes sirc rabbit corneal cells

The purpose of this study was to evaluate the permeability characteristics of a previously reported in vitro corneal model that utilizes SIRC rabbbit corneal cells and to investigate the permeability of three novel esters of phenylephrone chemical delivery systems (CDS) under different pH conditions using this in vitro model. The SIRC rabbit corneal cell line was grown on transwell polycarbonate membranes, and the barrier properties were assessed by measuring transepithelial electrical resistance (TEER) using a voltohmmeter. The permeabilities of esters of phenylephrone CDS across the SIRC cell layers were measured over a pH range 4.0–7.4. The esters tested include phenylacetyl (1), isovaleryl (2), and pivalyl (3). The SIRC rabbit corneal cell line, when grown on permeable filters, formed tight monolayers of high electrical resistance with TEER values increasing from 71.6 ±20.8 Ω·cm² at day 3 in culture to 2233.42 ±15.2 Ω·cm² at day 8 in culture and remained constant through day 14 in culture. The transepithelial permeability coefficients (P_app) at pH 7.4 ranged from 0.58 × 10⁻⁶ cm/s for the hydrophilic marker, mannitol, to 43.5 × 10⁻⁶ cm/s for the most lipophilic molecule, testosterone. The Papp at pH 7.4 for phenylephrine was 4.21 × 10⁻⁶ cm/s. The P_app values and the lag times of the three esters of phenylephrone were pH dependent. The P_app for 1, 2, and 3 at pH 7.4 were 14.76 × 10⁻⁶, 13.19 × 10⁻⁶, and 12.86 × 10⁻⁶ cm/s, respectively and the permeabilities decreased at conditions below pH 7.4. The lag times at pH 7.4 were 0.10, 0.17, and 0.12 h for 1, 2, and 3, respectively, and the values increased at lower pH conditions. The TEER values of SIRC cell line observed at day 8 to day 14 in the present investigation are similar to the resistance value reported for rabbit cornea (2 kΩ·cm²). All the esters showed significantly (p < 0.05) higher permeabilities than phenylephrine at pH 7.4. The rate and extent of transport of the drugs across the cell layers were influenced by the fraction of ionized and un‐ionized species and the intrinsic partition coefficient of the drug. The results indicate that the permeability of ophthalmic drugs through ocular membranes may be predicted by measuring the permeability through the new in vitro cell culture model.     

Purine salvage pathways for the biosynthesis in vitro of adenine nucleotides in the guinea pig vas deferens

The guinea pig vas deferens has been found to possess at least two active anabolic pathways for adenine nucleotide biosynthesis. Our studies in vitro show that [³H]adenosine and [³H]adenine may be important precursors in purine salvage. Both precursors are eventually converted to [³H]AMP prior to final incorporation into [³H]ATP. [³H]adenosine is transported across the cell membrane and then phosphorylated intracellularly to [³H]AMP with the subsequent formation of [³H]ADP and [³H]ATP. [³H]adenine, on the other hand, is probably converted to [³H]AMP by a mechanism that does not involve [³H]adenosine transport, since 6-nitrobenzylthioguanosine (6-NBTG), a nucleoside transport inhibitor, does not inhibit [³H]adenine utilization. Conversion of [³H]adenosine to [³H]inosine or [³H]hypoxanthine prior to incorporation into [³H]ATP is probably only of minor importance since: (1) 6-NBTG reduces [³H]adenosine conversion to phosphorylated adenine compounds by 90 per cent; and (2) the adenosine deaminase inhibitor, 6-thioguanosine, even at high concentrations could only slightly reduce the amount of [³H]ATP formed from [³H]adenosine. Incubation of vas deferens with either [³H]inosine or [³H]hypoxanthine also failed to result in appreciable labeling of adenine nucleotide pools. The relative contributions of tritium labeled nucleosides and bases to [³H]ATP synthesis in vitro in the guinea pig vas deferens are; [³H]adenosine > [³H]adenine ? [³H]inosine > [³H]hypoxanthme.     

Saturable uptake of [3H]-tryptamine in rabbit platelets is inhibited by 5-hydroxytryptamine uptake blockers

Summary [³H]-tryptamine is taken up by rabbit platelets through an active and staurable process which is temperture sensitive, sodium-dependent and inhibited by imipramine and non-tricyclic 5-hydroxytryptamine (5-HT) uptake blockers.There is an excellent correlation between the Ki for the inhibition of [³H]-tryptamine and [³H]-5-HT uptake in rabbit platelets for a series of 5-HT uptake blockers. These results indicate that [³H]-tryptamine is actively transported through the membrane of blood platelets by the same carrier that transports 5-HT.     

Effect of reserpine on adenosine uptake and metabolism,and subcellular transport of platelet adenosine triphosphate in washed rabbit platelets

There is a slow exchange of adenine nucleotides between the metabolically active (cytoplasmic) pool and the releasable amine storage organelle pool of blood platelets. Reserpine is known to inhibit serotonin uptake into platelet storage organelles. Therefore, we have determined whether reserpine also inhibits the uptake of adenine nucleotides from the cytoplasm into the storage organelles of rabbit platelets. Transport of adenine nucleotides from the metabolically active pool into the releasable amine storage granule pool was followed by labeling the metabolically active pool of adenine nucleotides by incubating the platelets with [¹⁴C]adenosine or [¹⁴C]adenine. Practically complete release of amine storage granule constituents was induced at various times in aliquots of the labeled platelet suspensions by treatment with a high concentration of thrombin (0.45 units/ml. The fraction of the total labeled [¹⁴C]ATP released was taken as a measure of ATP transport from the metabolically active pool into the releasable pool. Reserpine (0.2 and 2 μM) decreased the rate of ATP transport into the storage granules by about 50 per cent. Platelets obtained from rabbits that had received 5 mg/kg of reserpine intraperitoneally 18 hr prior to the collection of blood released less ATP and ADP than control platelets from animals that had not received any drugs. This was not due to inhibition of the release reaction by reserpine. Since reserpine reduces the amount of adenine nucleotides in the storage granules, we conclude that if it affects the rate of efflux of adenine nucleotides from the granules at all, this effect must be slight compared with the inhibition of the uptake into the granules. Reserpine was also found to decrease the incorporation of [8-¹⁴C]adenosine into platelet adenine nucleotides by inhibiting adenosine uptake into the platelets noncompetitively (K_i = 2 μM). Inosine uptake was also inhibited by reserpine. The effect of reserpine on adenosine uptake was reversible. In contrast, the effect of reserpine on ATP transfer from the metabolically active pool into the releasable pool was irreversible. This is in keeping with earlier observations that some reserpine binds to platelets reversibly and some binds irreversibly.     

Binding and uptake studies with [3H]CP-93, 129, a radiolabeled selective 5-HT1B receptor ligand

3-(1,2,5,6-Tetrahydro-4-pyridyl)-5-pyrrolo[3,2-b]pyridone, CP-93, 129, is a selective agonist ligand for 5-HT_1B receptors. High affinity binding sites of [³H]CP-93, 129 were found in rat whole brain membranes, which showed K_D and B_max values similar to those for 5-HT_1B sites labeled by [³H]5-HT. Uptake of [³H]CP-93, 129 in crude rat synaptosomes was also observed, which was potently inhibited by 5-HT uptake blockers and 5-HT but not by desipramine (NE uptake blocker) or tametraline (NE and DA uptake blocker). Because of this sensitivity to 5-HT uptake inhibitors and the structural similarity of CP-93, 129 to serotonin, [³H]CP-93, 129 uptake probably occurred in 5-HT neurons.     

3H]Ouabain binding and dissociation in rabbit colon: effect of ions and drugs

²²Na fluxes (J) and [³H]ouabain binding were studied in vitro in the distal portion of the rabbit colon, mounted as a membrane, separating two perspex chambers. J_MS^Na (flux of ²²Na from mucosal to serosal chamber) was 2.1 and J_SM^Na was 1.0 μmoles cm² × hr· J_SM^Na was completely abolished by 10^?3 M ouabain, placed in the serosal side. J_SM^Na was unaffected by ouabain. [³H] Ouabain binding to the serosal surface of the colon was inhibited to the same maximal effect by ‘cold’ ouabain (I₅₀ = 5 × 10^?7 M), by K⁺ placed in the serosal chamber (I₅₀ = 3.5 mM) and by replacement of Na⁺ with choline on the serosal side. Increased tissue concentration of Na⁺ did not affect [³H]ouabain binding, suggesting that extracellular rather than intracellular sodium plays a major role in cardiac glycoside binding. Various cardiac glycosides (digoxin, digitoxin. ouabain) inhibited [³H]ouabain binding to the serosal side of the colon but other steroids (estriol, testosterone, desoxycorticosterone) had not effect.Efflux of [³H]ouabain, bound to the serosal side of the colon, was differentiated into dissociation of nonspecifically bound [³H]ouabain (95 per cent in 60 min) and dissociation of specifically bound [³H]ouabain (20 per cent in 60 min). Dissociation of specifically bound [³H]ouabain was accelerated when Na was replaced by choline (50 per cent in 60 min). The dissociation rate of nonspecifically bound ouabain was unaffected by replacement of Na⁺· [³H]Ouabain binding to mucosal surface was unaffected by ‘cold’ ouabain, by increased K⁺ in the medium or by replacement of Na⁺. It is concluded that there is no specific binding of [³H]ouabain to the mucosal surface. Omission of Ca, Mg or phosphate from the medium did not affect [³H]Ouabain binding to either mucosal or serosal surfaces of rabbit colon.     

Carrier mediated uptake of L-tyrosine and its competitive inhibition by model tyrosine linked compounds in a rabbit corneal cell line (SIRC)--strategy for the design of transporter/receptor targeted prodrugs

The objective of this study was to investigate the presence of amino acid transporters on the corneal epithelium and to enhance corneal drug absorption through prodrug modification targeted to the amino acid transporters. SIRC was used as a model cell line representing the corneal epithelium. Uptake studies were carried out using [3H] L-tyrosine at 37 degrees C. Temperature, energy and pH dependence studies were carried out. The uptake seems to be composed of a major saturable and minor non-saturable component (V(max) =2.9+/-0.62 nmoles/min/mg protein, K(m) =71+/-21 microM, K(d) =2.6+/-0.6 nl/min/mg protein). No significant inhibition of uptake was observed in the presence of metabolic inhibitors or in the absence of sodium. Competitive inhibition studies were performed in the presence of various amino acids and model tyrosine conjugates (p-nitro and p-chloro benzyl ether conjugate of L-tyrosine). Uptake was inhibited by neutral aromatic and large neutral aliphatic amino acids. L-Tyrosine uptake was inhibited by its ether conjugates in a concentration dependent manner suggesting that these compounds may be sharing the same transport mechanism. This study provides biochemical evidence of the presence of a large neutral amino acid transport system on the corneal epithelium, which may be utilized to enhance the corneal drug transport.     

Riboflavin transport mediated by riboflavin transporters (RFVTs/SLC52A) at the rat outer blood-retinal barrier

The previous in vivo study revealed the carrier-mediated transport of riboflavin (vitamin B₂) across the blood-retinal barrier (BRB). In the present study, the blood-to-retina supply of riboflavin across the outer BRB was assessed in RPE-J cells, a rat-derived in vitro cell model of the outer BRB that is formed by the retinal pigment epithelial cells. In the directional uptake analysis on collagen-coated Transwell® inserts, RPE-J cells showed higher basal-to-cell (B-to-C) uptake (22.8 μL/mg protein) of [³H]riboflavin than apical-to-cell (A-to-C) uptake (13.5 μL/mg protein). RPE-J cells showed concentration- and temperature-dependent uptake of [³H]riboflavin with a K_m of 297 nM, suggesting the involvement of carrier-mediated process in the blood-to-retina transport of riboflavin across the outer BRB. In RPE-J cells, [³H]riboflavin uptake was affected under a K⁺-replacement condition while no effect was observed under a choline-replacement condition and at different pH values. Uptake of [³H]riboflavin by RPE-J cells was markedly reduced by riboflavin, flavin adenine dinucleotide (FAD), and lumichrome with no significant effect noted for other vitamins. The obtained results suggested the involvement of riboflavin transporters (SLC52A/RFVT) at the outer BRB, and this is supported by the expression and knockdown analyses of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3).     

Nucleoside transport in primary cultured rabbit tracheal epithelial cells

The present study aimed at elucidating the mechanisms of nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) grown on a permeable filter support. Uptake of ³H-uridine, the model nucleoside substrate, from the apical fluid of primary cultured RTEC was examined with respect to its dependence on Na⁺, substrate concentration, temperature and its sensitivity to inhibitors, other nucleosides and antiviral nucleoside analogs. Apical ³H-uridine uptake in primary cultured RTEC was strongly dependent on an inward Na⁺ gradient and temperature. Ten micromolar nitro-benzyl-mercapto-purine-ribose (NBMPR) (an inhibitor of es-type nucleoside transport in the nanomolar range) did not further inhibit this process. ³H-uridine uptake from apical fluid was inhibited by basolateral ouabain (10 μM) and apical phloridzin (100 μM), indicating that uptake may involve a secondary active transport process. Uridine uptake was saturable with a K_m of 3.4 ± 1.8 μM and the V_max of 24.3 ± 5.2 pmoles/mg protein/30 s. Inhibition studies indicated that nucleoside analogs that have a substitution on the nucleobase competed with uridine uptake from apical fluid, but those with modifications on the ribose sugar including acyclic analogs were ineffective. The pattern of inhibition of apical ³H-uridine, ³H-inosine and ³H-thymidine uptake into RTEC cells by physiological nucleosides was consistent with multiple systems: A pyrimidine-selective transport system (CNT1); a broad nucleoside substrate transport system that excludes inosine (CNT4) and an equilibrative NBMPR-insensitive nucleoside transport system (ei type). These results indicate that the presence of apically located nucleoside transporters in the epithelial cells lining the upper respiratory tract can lead to a high accumulation of nucleosides in the trachea. At least one Na⁺-dependent, secondary, active transport process may mediate the apical absorption of nucleosides or analogous molecules.     

Uptake and displacement of [3H]dihydroalprenolol, [3H]epinephrine and [3H]clonidine in isolated perfused rabbit lung

Uptake and displacement of three adrenergic receptor ligands, [³H]dihydroalprenolol ([³H]DHA), [³h]epinephrine ([³H]EPI) and [³H]clonidine ([³H]CLON), were examined in isolated rabbit lungs by recirculating perfusion. Removal of [³h]DHA was the most extensive (85% uptake; 6.6 $\text{ml}\text{min}$ clearance), [³H]CLON removal was intermediate (50%; 3.8 $\text{ml}\text{min}$), and [³H]EPI removal was the lowest (33%; 1.2 $\text{ml}\text{min}$). Specific displacement of each radioligand from lung was attempted using several competing agents. Both (?)- and (+)-propranolol equally displaced [³H]DHA from lung. Phentolamine, (?)-phenylephrine and (?)-epinephrine were unable to displace 10 nM [³H]EPI from lung, although the latter two agents did produce concentration-dependent increases in perfusion pressure. High concentrations of (?)-epinephrine, which produced near maximal physiological responses, inconsistently displaced 30–40 nM [³H]EPI from lung. [³H]Clonidine was displaced by unlabeled clonidine at concentrations that caused increases in perfusion pressure. Pretreatment of lungs with either 10 μM phentolamine or phenoxybenzamine did not alter the total amount of [³H]CLON displaced by clonidine, suggesting that [³H]CLON was displaced predominantly from non-specific sites, perhaps preventing detection of [³H]CLON displacement from specific (receptor) sites. Alternatively, these results may be interpreted as inhibition of uptake of each radioligand. Thus, both (?)- and (+)-propranolol interfered with [³H]DHA removal, suggesting a common mechanism for uptake and/or retention for these two β-adrenergic receptor antagonists. Inhibition of ³H]EPI removal was observed only at high concentrations of (?)-epinephrine which indicates that pulmonary removal of epinephrine occurs through a low affinity uptake system. [³H] Clonidine removal was effectively inhibited by the same (μM) concentrations of unlabeled clonidine that produced physiological responses. Neither phentolamine nor phenoxybenzamine was able to interfere with pulmonary removal of [³H]CLON. Therefore, uptake and displacement of these adrenergic receptor radioligands showed no correlation with pharmacological effects produced by these agents in isolated perfused rabbit lung. The results are more closely associated with inhibition of removal and/or non-specific retention of the radioligands examined.     

Mouse equilibrative nucleoside transporter 2 (mENT2) transports nucleosides and purine nucleobases differing from human and rat ENT2

Several mammalian nucleoside transporters have been identified at the molecular level. Human and rat equilibrative nucleoside transporter 2 (hENT2 and rENT2, respectively) was previously reported to have the dual ability of transporting both nucleosides and nucleobases. In the present study, we characterized the transport of a variety of nucleosides and nucleobases via recombinant mouse ENT2 (mENT2). Cloned mENT2 mediated the uptake of nucleosides and purine nucleobases, but not pyrimidine nucleobases. The mENT2-mediated uptake of adenosine was significantly inhibited by nucleosides and nucleobases, irrespective of purine and pyrimidine. The K(m) values for the uptake of nucleosides and purine nucleobases mediated by mENT2 varied between 1.24 and 16.3 microM, and the transport clearances of adenosine and hypoxanthine via the transporter were greater than those of other substrates. Therefore, we concluded that mENT2 is nucleoside and purine nucleobase transporter, and pyrimidine nucleobases are blockers for the transporter, differing from hENT2 and rENT2 that were reported to also transport pyrimidine nucleobases.     

Effect of 4-chlorobiphenyl on substrate transport and phospholipid metabolism in mouse L5178Y lymphoma cells

The toxicity of 4-chlorobiphenyl, a constituent of Aroclor 1221, was studied in mouse L5178Y cells, in vitro. 4-Chlorobiphenyl had a varied effect on the uptake of small precursor molecules. Uptake of [³H]l-leucine, [³H]l-serine, [³H]uridine and [³H]thymidine was inhibited, while that of [³H]inositol was stimulated. There was no significant effect on either [¹⁴C]ethanolamine or [¹⁴C]choline uptake. However 4-chlorobiphenyl significantly inhibited incorporation of [¹⁴C]ethanolamine into phosphatidylethanolamine and caused a 2- to 3-fold stimulation in the incorporation of [¹⁴C]choline into phosphatidylcholine. This effect on phosphatidylcholine metabolism depended on the adsorption and continued presence of 4-chlorobiphenyl on the cell plasma membrane. The stimulation of [¹⁴C]choline incorporation was reversed when treated cells were placed in fresh growth medium under conditions where 95 per cent of the 4-chlorobiphenyl was desorbed from the cell surface. The effect of 4-chlorobiphenyl on substrate uptake and phospholipid metabolism appears to depend upon the interaction of the agent with the cell membrane surface.     

Identification of dihydrofolate reductase in rabbit brain

After partial purification, rabbit brain extracts were assayed for dihydrofolate reductase by spectrophometric, radiochemical and methotrexate binding assays. By these assays, the specific activity of rabbit brain dihydrofolate reductase was about 15 per cent that of liver. Both rabbit liver and brain dihydrofolate reductase activities were abolished by methotrexate and had comparable K_m (3 μM), pH maxima (4.8), and cofactor requirements (NADPH). In vivo, a small percentage of [³H]folic acid was reduced to [³H]methyltetrahydrofolate when injected into the left lateral ventricle. Thus, both in vitro and in vivo, the central nervous system has the ability to reduce oxidized folates.     

Transport of acyclovir ester prodrugs through rabbit cornea and SIRC-rabbit corneal epithelial cell line.

The purpose of this study is to assess the permeability of acyclovir (ACV) prodrugs through the rabbit corneal cell line (SIRC) as well as the cornea, and characterize the SIRC cell line for transport and metabolism studies of ester prodrugs. Prodrug derivatization of an acycloguanosine antiviral agent, acyclovir, was employed to improve its permeability across the cornea. New Zealand albino rabbits were used as an animal model for corneal studies. The SIRC cell line grown on polyester membranes was used for transport of these prodrugs. SIRC cells grown on the membrane support for 10 days developed four to six layers of epithelial cells, and this is comparable to the normal rabbit corneal epithelial layer. Transport experiments were conducted across the rabbit cornea and confluent SIRC cells using side-by-side diffusion-cell apparatus. Enzymatic hydrolysis of these compounds was evaluated in SIRC cell lysates. Appropriate reversed phase HPLC method(s) were employed for quantitation of both the prodrug and ACV simultaneously. Corneal permeabilities of some of these prodrugs (Malonyl ACV and Acetyl ACV) were higher relative to ACV. The SIRC cell line permeability values of all the prodrugs were higher compared to that of the intact cornea. The total amount of ACV-prodrugs transported, i.e., unhydrolyzed prodrugs and regenerated ACV, across the SIRC cell line was more relative to ACV. Hydrolytic studies in the SIRC cell line homogenate demonstrated the bioreversion potential of the prodrugs and the presence of enzymes, particularly the cholinesterase in the SIRC cell line. It may be concluded that the SIRC cell line is leakier compared to the cornea. Keeping in mind the limitations, the SIRC cell line after further characterization may be used for transport and metabolism studies of ester prodrugs.     

Effects of phencyclidine on [3H]catecholamine and [3H]serotonin uptake in synaptosomal preparations from rat brain

Phencyclidine inhibited uptake in vitro of [³H]norepinephrine (ic₅₀ 0.52 μM), [³H]dopamine (ic₅₀ 0.73 μM) and [³H]serotonin (ic₅₀ 0.80 μM) in crude synaptosomal preparations from rat brain through a competitive mechanism. Phencyclidine was fairly similar in potency to d-amphetamine and methylphenidate in inhibiting catecholamine uptake but was 8 times more potent than d-amphetamine and 34 times more potent than methylphenidate in inhibiting [³H]serotonin uptake.     

Presynaptic agonist effect of phentolamine in the rabbit vas deferens and rat cerebral cortex

Clonidine inhibited the electrically-induced twitch response of the rabbit and rat isolated vas deferens preparations and also the K⁺-evoked release of [³H]noradrenaline from rat cortical slices. This effect of clonidine was antagonized competitively by yohimbine. Phentolamine inhibited the electrically-induced twitch response of the rabbit, but not the rat, vas deferens and in low concentrations (<·1 μm ) also inhibited the K⁺-evoked release of [³H]NA from rat cortical slices. These inhibitory effects of phentolamine were antagonized by yohimbine in a competitive manner but were not antagonized by indoramin, an α₁-adrenoceptor antagonist. In the rabbit vas deferens, the effects of phentolamine were shown not to be due to the stimulation of β-, H_1-, H_2-, 5-HT- or muscarinic receptors. These results are consistent with the view that phentolamine can act as an agonist at presynaptic α₂-adrenoceptors in the rabbit vas deferens and rat cortex but not in the rat vas deferens.     

Different effects of serotonin (5-HT) uptake blockers in caudate nucleus and hippocampus of the rabbit: Role of monoamine oxidase in dopaminergic terminals

Slices of rabbit hippocampus or caudate nucleus were incubated with [³H]-5-HT (0.1 µM, 60 min) or with [³H]-DA. In hippocampal tissue, the 5-HT uptake blockers chlorimipramine, fluvoxamine, and 6-nitroquipazine (0.1, 1, 10 µM) reduced the percentage content of [³H]-5-HT in a concentration dependent manner. The degree of inhibition of [³H]-5-HT content produced by the 5-HT uptake inhibitors was not affected by the MAO inhibitors pargyline or amezinium (which by themselves enhanced [³H] loading) or the catecholamine uptake inhibitor nomifensine (which by itself did not affect [³H] loading). In caudate nucleus tissue, however, the [³H]-5-HT accumulation was reduced only at the highest concentration of the 5-HT uptake blockers (10 µM). In the additional presence of the MAO inhibitors or nomifensine (which by themselves increased or diminished, respectively, the [³H] labelling) the 5-HT uptake inhibitors became more potent in reducing the percentage [³H]-5-HT accumulation of caudate nucleus slices. These results indicate (1) that a false labelling of [³H]-5-HT into dopaminergic terminals in the caudate nucleus can be prevented by nomifensine, (2) that the 5-HT uptake blockers seem to accumulate within the dopaminergic terminals, where they may display a MAO inhibitory property. The 5-HT uptake blockers were ineffective on the percentage tritium accumulation of caudate nucleus slices incubated with [³H]-DA, regardless of the presence of pargyline or nomifensine. Tritiated DA and deaminated [³H]-metabolites were separated in the superfusate of [³H]-DA-release experiments in caudate nucleus tissue. In the presence of 6-nitroquipazine the percentage efflux of unmetabolized [³H]-DA was significantly enhanced in a concentration and time dependent manner. In comparison to 6-nitroquipazine, fluvoxamine was less potent in that respect. 6-Nitroquipazine inhibited the electrically evoked [³H]-DA and [³H]-ACh release from caudate nucleus slices in a concentration dependent manner. The effects on [³H]-DA release were abolished in the presence of pargyline. The inhibition of [³H]-ACh release was significantly diminished by the D₂-receptor antagonist domperidone. In conclusion, some 5-HT-related drugs may diminish the release of ACh from caudate nucleus slices via an enhanced dopaminergic transmission due to inhibition of MAO within the dopaminergic terminals.     

[3H]2-(2-Benzofuranyl)-2-imidazoline,a highly selective radioligand for I2-imidazoline receptor binding sites Studies in rabbit kidney membranes

2-(2-Benzofuranyl)-2-imidazoline (2-BFI) has recently been characterised as a selective ligand for the I₂-type of imidazoline-receptor binding site(s) (I₂-RBS). The present studies determined the relative levels of specific [³H]2-BFI binding to membrane homogenates of brain and kidney from rat, guinea pig and rabbit and identified the pharmacological characteristics of [³H]2-BFI binding sites in rabbit kidney membranes. Rabbit kidney membranes had the highest relative density of specific [³H]2-BFI binding of all tissues studied (2000?fmol/mg protein). Rabbit brain and guinea pig kidney had moderate levels of specific [³H]2-BFI binding (350–500?fmol/mg protein), while rat kidney and guinea pig and rat brain displayed much lower densities of binding (40–65?fmol/mg protein). Studies of [³H]2-BFI binding kinetics in rabbit kidney homogenates revealed binding to two distinct sites with K _d values of 0.10?±?0.01?nmol/l and 1.00?±?0.36?nmol/l respectively. Equilibrium saturation studies were also consistent with the presence of two binding sites – [³H]2-BFI (0.01–20?nmol/l) bound to sites with affinities of 0.10?± 0.01?nmol/l and 0.92?±?0.13?nmol/l and binding densities of 470?±?80 and 840?±?60?fmol/mg protein (n=3), representing 36 and 64% respectively. Drug inhibition studies revealed that l-adrenaline; α₁-adrenoceptor drugs (prazosin, l-phenylephrine) and α₂-adrenoceptor drugs (rauwolscine, methoxyidazoxan, 2-(2,4-(O-methoxyphenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione (ARC-239) had extremely low affinities for [³H]2-BFI binding sites (IC₅₀?≥?10?μmol/l). Putative I₁-RBS compounds, p-aminoclonidine, moxonidine, imidazole-4-acetic acid and cimetidine, inhibited [³H]2-BFI binding to rabbit renal membranes with low to very low affinities (K _i values 3 to ≥100?μmol/l), suggesting [³H]2-BFI does not label I₁-RBS in rabbit kidney membranes. I₂-RBS compounds – 2-(4,5-dihydroimidaz-2-yl)-quinoline (BU224), 2-(4,5-dihydroimidaz-2-yl)-quinoxaline (BU239), idazoxan and cirazoline – potently inhibited [³H]2-BFI binding (K _i values 0.37–1.6?nmol/l), confirming the labelling of I₂-RBS. Inhibition of [³H]2-BFI binding by certain compounds was consistent with their interaction with two binding site populations – for example (drug, K _i values) guanabenz, 0.65?nmol/l and 0.17?μmol/l; naphazoline, 0.94?nmol/l and 2.8?μmol/l; amiloride, 76?nmol/l and 26?μmol/l rilmenidine, 150?nmol/l and 50?μmol/l; and clonidine, 230?nmol/l and 70?μmol/l. The high affinity of amiloride for a high proportion (85%) of the binding is consistent with the presence of the I_2A-subtype of I-RBS in rabbit kidney. These results demonstrate that [³H]2-BFI is a highly selective and high affinity radioligand for I₂-RBS which should be useful for the further characterisation of these sites in mammalian tissues.