Expression and function of P-glycoprotein and absence of multidrug resistance-related protein in rat and beige mouse peritoneal mast cells.

To clarify the function of the multidrug transporter P-glycoprotein in mast cells we used the green fluorescent compound Bodipy-FL-verapamil, which is a substrate of P-glycoprotein. This compound is also transported by Multidrug Resistance-related Protein (MRP), another membrane transport protein expressed in many tumour resistant cells as well as in normal cells. When rat peritoneal mast cells were incubated with Bodipy-verapamil, a rapid uptake of this compound was observed. Pretreatment with modulators of P-glycoprotein activity, such as verapamil and vinblastine, increased Bodipy-verapamil intracellular concentrations. In addition, Bodipy-verapamil efflux from these cells was rapid and also inhibited by verapamil and vinblastine. In contrast, no effect was observed when cells were treated with agents, such as probenecid and indomethacin, that are known inhibitors of MRP. Methylamine and monensin, substances that modify the pH values in the granules, were able to lower the concentrations of Bodipy-verapamil. Microscopical observations, conducted in both rat and beige mouse mast cells, demonstrated that the fluorochrome accumulated in the cytoplasmic secretory granules. RT-PCR performed on rat peritoneal mast cells revealed the presence of MDR1a and MDR1b mRNAs; on the contrary, MRP mRNA was not expressed. Mast cells were further treated with the fluorescent probe LysoSensor Blue, a weak base that becomes fluorescent when inside acidic organelles. This substance accumulated in mast cell granular structures and its fluorescence was reduced either by treatment with P-glycoprotein modulators or with agents that disrupt pH gradients. In conclusion, these data further confirm the presence of an active P-glycoprotein, but not of MRP, in rat peritoneal mast cells. These findings, coupled with previous ultrastructural data, lend further support to the assumption that this protein is located on the mast cell perigranular membrane. The functional role of P-glycoprotein in these cells is at present unclear, but a possible involvement in the transport of molecules from the granules to the cytosol can be hypothesized. Alternatively, this protein might be indirectly implicated in changes of pH values inside secretory granules.

Kinetics of doxorubicin handling in the LLC-PK1 kidney epithelial cell line is mediated by both vesicle formation and P-glycoprotein drug transport.

The subcellular distribution of doxorubicin was evaluated in living non-fixed LLC-PK1 cells, which maintain the structural and functional characteristics of the kidney proximal tubule epithelium and also express P-glycoprotein. After 10 min incubation, doxorubicin fluorescence was detectable in the nucleus. The intensity of nuclear fluorescence progressively increased, reaching the maximum at the end of the first hour. Then, the nuclear signal started to decrease and, at 2 h, doxorubicin fluorescence disappeared almost completely from the cell nucleus. Cytoplasmic fluorescent vesicles first appeared in the perinuclear region after 10 min doxorubicin exposure and increased in number and size over a period of 2 h. From 2 to 5 h, fluorescent vesicles moved unidirectionally to the cell periphery. Disappearance of doxorubicin punctate fluorescence in LLC-PK1 cells treated with methylamine or monensin demonstrated that drug accumulation occurred inside acidic compartments. In addition, the cytoplasmic pattern of doxorubicin fluorescence was very similar to that observed upon exposure to the acidotropic tracer LysoSensor Blue. Involvement of P-glycoprotein in doxorubicin handling by LLC-PK1 cells was suggested by modified intracellular doxorubicin distribution after cell incubation with verapamil and vinblastine. Moreover, the fluorescent P-glycoprotein substrate Bodipy FL Verapamil was shown to accumulate in LLC-PK1 cells in a manner that is quite similar to that observed for doxorubicin. P-glycoprotein expression was evaluated by immunoblot using the JSB-1 and C219 monoclonal antibodies. Immunofluorescence analysis was performed using the JSB-1 monoclonal antibody. P-glycoprotein immuno-reactivity was found both on the plasma membrane and intracytoplasmically in a perinuclear position. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that MDR1 gene was expressed. This study indicates that a rapid intracellular redistribution accompanies the process of doxorubicin uptake by LLC-PK1 cells. Although these cells are non-tumour cells derived from the normal epithelium of the proximal renal tubule, they display a model of doxorubicin redistribution which is characteristic of doxorubicin-resistant tumour cells.

Mechanisms of inhibitory effects of zinc and cadmium ions on agonist binding to adenosine A1 receptors in rat brain.

The dose-dependent inhibition of zinc and cadmium ions of agonist binding to A1 adenosine receptors in rat brain is prevented by histidine and cysteine, respectively. In the present study, the possible different mechanisms of Zn2+ and Cd2+ inhibitions were examined. The effects of Zn2+ and Cd2+ on equilibrium binding parameters of the agonists N6-cyclohexyl-[2,8-3H]-adenosine ([3H]CHA) or chloro-N6-cyclopentyl-adenosine ([3H]CCPA) and the antagonist cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) were compared with those effects of reagents or binding conditions which altered histidyl or cysteinyl residues of the A1 receptor. Zn2+ pretreatment did not change A1 agonist or antagonist affinity, but did reduce the Bmax. The inhibitory effects of Zn2+ pretreatments were also maintained after several membrane washings. Diethylpyrocarbonate, a histidine-specific alkylating reagent, behaved like zinc ions: pretreatment with A1 agonist protected the histidyl residues of the [3H]CHA binding site against modification by Zn2+, while the modification of the protonation state of the nitrogen of the imidazole group of histidines by changing pH indicated that the interactions of Zn2+ with the histidyl residues were feasible with their unprotonated form. These findings suggest the formation of coordination bonds between Zn2+ and histidines critical for [3H]CHA or [3H]DPCPX binding, which may prevent the ligand interaction with the specific sites without modifying the binding kinetics of radioligand to the non-chelated recognition sites. Cd2+ pretreatment reduced the [3H]CCPA affinity, but did not modify the affinity of the antagonist [3H]DPCPX, the Bmax remaining unaffected. As with cadmium effects, the oxidation of the thiol group of cysteine by dithionitrobenzoic acid (DTNB) reduced [3H]CCPA affinity without changing the number of binding sites. The reducing reagent dithiothreitol, which alone was unable to modify [3H]CCPA binding, overcame the inhibiting effects of both Cd2+ and DTNB. These findings suggest that cadmium ions may oxidize SH groups of cysteines localized on the A1 receptor molecule or a cysteine localized in the region of G(i)alpha subunit involved in the coupling with receptors. This mechanism can justify potential conformational modifications of the receptor molecule producing the decrease in affinity.

Transport of doxorubicin in mast cell granules and the effect of the calcium antagonist verapamil.

P-glycoprotein has been identified in mast cells stabilized in culture as well as in rat peritoneal mast cells, and is primarily concentrated on the granular membrane. This study aimed to define the role of this protein in the transport and accumulation of doxorubicin in mast cell granules and in its histamine releasing effect. The reverting agent verapamil, that is a substrate for P-glycoprotein, inhibited doxorubicin uptake in intact mast cells in a dose and time dependent manner, but had no effect on the exocytotic action of the antineoplastic drug. Doxorubicin was also concentrated in granules with intact membranes and the uptake was dependent on temperature and showed a trend for saturation. Verapamil and vinblastine, another substrate for P-glycoprotein, significantly reduced doxorubicin concentrations in intact granules. Similar results were obtained with the metabolic inhibitors sodium metavanadate, N-ethylmaleimide, and sodium azide, whereas ouabain, an inhibitor of sodium-potassium ATPase, was without effect. Doxorubicin was taken also up in granule remnants, consisting of a proteoglycan matrix without membrane, that are extruded from mast cells upon stimulation. However, the uptake was not dependent on temperature and was not modified by P-glycoprotein substrates or metabolic inhibitors. Rat peritoneal mast cells were examined for the expression of P-glycoprotein at the protein level with C219 monoclonal antibody, using Western blot, confirming that P-glycoprotein was expressed in mast cells. These data suggest the presence of a P-glycoprotein active in the transport of doxorubicin, in mast cell granules.

Increased density of M1 receptors in the hippocampus of juvenile rats chronically deprived of NGF.

Binding studies were used to assess the changes in affinity and/or number of M1 muscarinic receptors in hippocampi from juvenile rats chronically deprived of NGF. NGF deprivation was obtained by implanting into right ventricle at postnatal day 2 (P2) hybrydoma cells secreting high levels of monoclonal antibodies against NGF (alphaD11). Parenteral myeloma cells (P3U) were used as controls. Competition experiments were used to characterise the [3H]-PNZ binding sites in membrane preparations of hippocampi from rats sacrificed at P15. [3H]-PNZ bound M1 receptors both in P3U and alphaD11 group as shown by displacing potency order of antagonists: TLZ=4-DAMP>PNZ>p-F-HHSiD>MTC. The deprivation of NGF for two weeks significantly increased the number of M1 receptors without changing the Ki values of antagonists with exception of methoctramine which showed an increase in affinity in alphaD11 group. Similar changes in binding parameters were already observed after the first week of anti-NGF treatment. In contrast, a treatment for a week with implant at postnatal day 15 failed to produce any changes in M1 binding parameters. These results provide further physiological evidence for developmentally regulated modulatory role of NGF in the cholinergic function in the hippocampus.

Inhibitory and excitatory effects of adenosine antagonists on spontaneous locomotor activity in mice.

The behavioral effect of the adenosine antagonists CPT, PACPX, DPCPX and PD 115,199 on spontaneous locomotor activity was investigated in mice after parenteral administration. CPT, PACPX and PD 115,199 affected locomotor activity in a biphasic way. Doses in the nanomolar/kg range significantly reduced locomotion (PACPX> or =PD 115,199>>CPT). Higher doses were progressively less active until they became ineffective or slightly stimulated locomotion. NECA, a mixed A1/A2 agonist, and CCPA, a highly selective A1 agonist, also induced a biphasic behavior, with low doses stimulating and high doses inhibiting locomotion. The stimulant effect of 1 nmol/kg NECA was antagonized by depressant doses of antagonists, whereas antagonists-induced hypomotility was potentiated by a depressant dose of NECA (20 nmol/kg). It is suggested that the blockade of A1 receptors by antagonists is probably responsible for reducing locomotor activity, whereas the activation of A2 receptors by agonists is likely responsible for reducing locomotion in mice.

An NK1 receptor-dependent component of the slow excitation recorded intracellularly from rat motoneurons following dorsal root stimulation.

Intracellular recording from lumbar motoneurons of the neonatal rat spinal cord in vitro was used to study how recently developed non-peptide antagonists such as SR-140333 and SR-48698, known to block distinct subtypes of tachykinin receptors peripherally, might affect synaptic transmission elicited by electrical stimulation of dorsal root fibres. SR-140333 (1 microM) preferentially antagonized responses mediated by an exogenously applied agonist acting on the NK1 receptor subclass, while SR-48968 (0.5 microM) preferentially reduced responses mediated by an exogenously applied agonist acting on the NK2 receptor subclass. SR-48968 did not affect fast or slow excitatory postsynaptic potentials (EPSPs) or 'wind-up' responses induced by repetitive, low-frequency stimulation (mimicking certain types of nociceptive input); binding studies using this radiolabelled ligand disclosed specific binding activity (21 fmol/mg protein) selectively displaced by an NK2 receptor agonist. SR-140333 reduced the late component of fast and slow EPSPs, and of wind-up. Pharmacological block of ionotropic glutamate receptors abolished all dorsal root-evoked EPSPs. In comparison to glutamate receptor blockers, SR-140333 was a weaker antagonist of slow synaptic responses, though it displayed preferential antagonism towards some components of the wind-up phenomenon. The present results provide evidence obtained with a novel NK1 antagonist that a neuropeptide (presumably substance P), although not directly released by primary afferents onto motoneurons, is a neurotransmitter (acting via NK1 receptors) in the pathway mediating slow synaptic responses of motoneurons, and is presumably involved in signalling nociceptive inputs from the periphery.

Biochemical and pharmacological evidence for the presence of A1 but not A2a adenosine receptors in the brain of the low vertebrate teleost Carassius auratus (goldfish).

In whole brain membranes of goldfish, 3H-chlorocyclopentyladenosine bound to adenosine A1 receptors. The A1 receptors were ubiquitously distributed in the brain with a maximum in the hypothalamus and a minimum in the spinal cord. In superfused goldfish cerebellar slices, cyclohexyladenosine inhibited the cyclic AMP accumulation stimulated by forskolin and the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline, reversed this effect. In the same brain preparation, 30 mM K+ stimulated the release of glutamate, glutamine, glycine and GABA in a Ca(2+)-dependent manner, whereas the aspartate and taurine release was Ca(2+)-independent. Cyclohexyladenosine, in a dose-dependent manner, inhibited the 30 mM K(+)-evoked release of glutamate whereas that of aspartate was unaffected. The CHA inhibition of glutamate-evoked release was reversed by 8-cyclopentyltheophylline. The adenosine A2a receptors were not detectable in whole brain membranes of goldfish either using the specific agonist 3H-CGS 21680 or 3H-5'-N-ethylcarboxamidoadenosine. The presence of A2b seems to be suggested by the NECA stimulation of cyclic AMP accumulation, which was reversed by 8-cyclopentyltheophylline. The results, taken together, indicate that adenosine has a neuromodulatory function in the nervous system of lower vertebrates which is comparable to that described in mammalian brain.

Effects of chronic administration of adenosine antagonists on adenosine A1 and A2a receptors in mouse brain.

In the present study the effects of chronic in vivo treatments with caffeine and more selective antagonists (PACPX and PD115,199) on the binding parameters of 3H-CHA and 3H-NECA to cortical A1 and striatal A2a adenosine receptors of mouse brain are shown. The drugs were injected intraperitoneally once a day for 6 or 20 days. Treatment for 20 days with caffeine (250 mumol/kg), PD115,199 (50-250 nmol/kg) and PACPX (250 nmol/kg) shifted the A1 low affinity receptors into an agonist-specific high affinity state. Moreover, after 20 days of treatment, the antagonists decreased the affinity of 3H-CHA to A1 receptors in the high affinity state. Antagonist treatments for 6 days did not modify the 3H-CHA binding parameters. The A2a striatal receptors were dose- and time-dependently up-regulated by caffeine and PD115,199, whereas PACPX displayed an up-regulation independent of dose or length of treatment. Moreover, PD115,199 decreased the affinity of 3H-NECA to A2a striatal receptors. This effect on affinity was visible after 20 days of treatment with 50 and 250 nmol/kg. This study provides evidence for a sensitivity of A2a receptors greater than that of A1 receptors and for a different regulation of cortical A1 and striatal A2a adenosine receptors of mouse brain after chronic treatment with antagonists.

Strain-related differences in adenosine receptor density and in behavioral sensitivity to adenosine analogs in mice.

The behavioral effects of the adenosine agonists 5'-N-ethylcarboxamidoadenosine (NECA) was investigated in two strains of inbred mice, CD1 and CBA. NECA dose dependently reduced spontaneous locomotor activity with similar potency (ED50 = 36 +/- 1.5 and 36 +/- 1.1 nmol/kg IP for CBA and CD1 mice, respectively) and efficacy (> 90% at 100 nmol/kg) in the two strains. One nmol/kg NECA, an ineffective dose in CBA mice, exerted a significant stimulant action in CD1 mice. In saturation experiments, no differences were found in the density or in the affinity of striatal A2a receptors labeled with [3H]NECA. A strain-related difference was found in the density of striatal A1 receptors labeled with [3H]CCPA. In CBA mice, the Bmax value was 32% less than in CD1 mice (0.646 +/- 0.037 and 0.951 +/- 0.073 pmol bound/mg protein, respectively, p < 0.05). No differences in [3H]CCPA binding parameters were found in cortical and hippocampal membranes obtained from the two strains, whereas a higher density of A1 binding sites was found in the cerebellum of CBA mice. The present results show a close correlation between binding studies and the depressant action of NECA and present evidence for strain-related differences in regional distribution of central adenosine receptors and in behavioral response to purinergic drugs.

Effects of divalent cations on adenosine agonist binding to A1 receptors and non-A1/non-A2 sites in rat cerebral cortex.

In the present study results are reported concerning the effects of several divalent cations on the binding characteristics of [3H]-cyclohexyladenosine on A1 adenosine receptors and of [3H]-N-ethylcarboxamidoadenosine on non-A1/non-A2 sites in membranes from cerebral cortex of the rat. The [3H]-cyclohexyladenosine binding to A1 receptors was dose-dependently increased by Mn2+, Co2+, Ca2+. The binding characteristics of the agonist were differently affected by Ca2+/Mn2+ and Mg2+. Ca2+ and Mn2+ increased the Bmax value without any change in Kd, whereas Mg2+ decreased the Kd value without changing the Bmax. In the presence of Ca2+ and Mg2+ the Kd value was similar to that obtained in the presence of Mg2+, whereas the Bmax value was similar to the apparent number of binding sites calculated in the presence of Ca2+. The cations, Cu2+, Cd2+, Zn2+, decreased the A1 binding with IC50 values of 19.6 microM, 39.2 microM and 103.9 microM, respectively. The binding characteristics of [3H]-N-ethylcarboxamidoadenosine to non-A1/non-A2 sites were affected by Ca2+, Mn2+, Co2+ and Mg2+ in the opposite manner to A1 receptors. They decreased the binding with IC50 values of 20.1 mM, 22.8 mM, 93.0 mM and 18.1 mM, respectively. This occurs through an enhancement in Kd values without changes in the number of binding sites. The findings on A1 receptor and non-A1/non-A2 binding site, taken together, suggest that cations could also exert a modulatory action via specific interactions with divalent cation binding sites on the receptor molecule.

Circadian rhythm of cortical and striatal adenosine receptors.

In order to investigate diurnal variations in binding parameters of A1 and A2 adenosine receptors, Kd and Bmax were calculated in mice that had been housed under controlled light-dark cycles for 4 weeks (light on from 7.00 to 19.00 h). A1 cortical receptors were labelled by N6-cyclohexyl-[3H]adenosine, and A1 and A2 striatal receptors by 5'-N-ethylcarboxamido[8-3H]adenosine. Significant differences were found for Bmax values measured at 3-h intervals across a 24-h period. Cyclic variations of the number of binding sites were shown, with a minimum number of A1 and A2 receptors during the light period and a maximum during the dark period. The amplitude for cortical A1 receptors, between 03.00 and 18.00 h, was 39%, while for striatal A1 receptors, between 03.00 and 15.00 h, was 92%. The amplitude for the A2 striatal receptors, between 03.00 and 15.00 h, was 147%. No substantial rhythm was found in the Kd values. These differences in the amplitude could suggest a different physiological modulation of the two adenosine receptor subtype moiety which could reflect a physiologically-relevant mechanism by which adenosine exerts its modulatory role in the central nervous system.

Anti-AIDS therapy: an update.

This review first examines AIDS as regards its historical aspects and current situation. Attention focuses on some basic etiopathogenic characteristics of this disease. After this point, the main pharmacological aspects are examined and, in particular, attention is paid to the most two interesting targets for pharmacological therapy. Lastly, the dynamic and kinetic profiles of the most promising compounds for therapeutic application are considered.

Circadian rhythm in adenosine A1 receptor of mouse cerebral cortex.

In order to investigate diurnal variation in adenosine A1 receptors binding parameters, Bmax and Kd values of specifically bound N6 - cyclohexyl-[3H]adenosine were determined in the cerebral cortex of mice that had been housed under controlled light-dark cycles for 4 weeks (light on from 7.00 to 19.00 h). Significant differences were found for Bmax values measured at 3-hr intervals across a 24-h period, with low Bmax values during the light period and high Bmax values during the dark period. The amplitude between 03.00 and 18.00 hr was 33%. No substantial rhythm was found in the Kd values. It is suggested that the changes in the density of A1 receptors could reflect a physiologically-relevant mechanism by which adenosine exerts its modulatory role in the central nervous system.

Adenosine receptors in cortical-derived vesicles of the rat: studies on binding sites and accumulation of cyclic AMP.

A vesicular preparation derived from the cerebral cortex of the rat was used to obtain, under the same experimental condition, binding parameters and stimulation data for cyclic AMP. Two analogues of adenosine were employed in the binding studies: [3H]NECA, a mixed A1/A2 agonist and [3H]CHA, a more selective A1 agonist. The [3H]CHA seemed to bind to a single high affinity site (Kd = 1.31 nM, Bmax = 0.327 pmol bound); saturation data for [3H]NECA were resolved for the presence of a high and a low affinity binding site (Kd1 = 3.08 nM, Bmax1 0.115 pmol bound; Kd2 = 204 nM, Bmax2 1.59 pmol bound), but only when calcium ions were omitted from the incubation medium. At 0 degree C, [3H]NECA bound to a single, low affinity site; the presence of calcium ions (1 mM) significantly reduced the affinity of [3H]NECA (Kd 419 nM), with respect to the absence of calcium (Kd 208 nM), without affecting the Bmax value. The influence of calcium ions was also investigated on the binding of [3H]CHA and a reduction of the Bmax value (36%) was found. Regardless of the presence or the absence of calcium ions, NECA stimulated accumulation of cyclic AMP in a dose-dependent way with an EC50 of 2.79 microM; this value did not correlate with the Kd of the low affinity binding site for [3H]NECA. Thus, the purpose of establishing a correlation between binding sites for analogues of adenosine and the site in the cerebral cortex through which the accumulation of cyclic AMP is induced, was not achieved. It is concluded that the stimulatory effect of analogues of adenosine on adenylate cyclase might not be a receptor-mediated effect. The complex influence of calcium ions on affinity and binding capacity of analogues of adenosine is discussed.