Identification of dopamine plasma membrane and vesicular transporters in human peripheral blood lymphocytes.

Plasma membrane dopamine transporter (DAT), vesicular monoamine transporters (VMAT) type-1 and -2 and the expression of the dopaminergic markers dopamine and tyrosine hydroxylase were assessed in membranes and/or in cytospin centrifuged human peripheral blood lymphocytes. The radiolabeled DAT ligand [3H]GBR12935 was bound to peripheral lymphocytes in a manner consistent with the specific binding to a dopamine uptake system, with a dissociation constant similar to that found in striatum, but with a lower density of binding sites. On the other hand, no specific binding occurred in cerebellum used as a test tissue not expressing DAT. Western blot analysis using antibodies raised against amino or carboxy terminus of DAT or against VMAT-1 or VMAT-2 revealed labeling of single bands of approximately 76, 55 or 68 KDa, respectively, displaying similar migration characteristics in lymphocytes and test tissues used for comparison. Immunofluorescence revealed that anti-dopamine, anti-tyrosine hydroxylase, anti-DAT, anti-VMAT-1 and anti-VMAT-2 antibodies labeled the total population of cytospin-centrifuged lymphocytes mounted on microscope slides. Confocal laser microscopy demonstrated that dopamine and VMAT-2 immunoreactivity was developed mainly in cytoplasmic punctiform areas likely corresponding to vesicles and to a lower extent was associated to plasma membrane. Tyrosine hydroxylase immunoreactivity was diffused to cytoplasm and to plasma membrane of lymphocytes, whereas DAT and VMAT-1 immunoreactivity were located almost exclusively in lymphocyte plasma membrane and cytoplasm, respectively. Lymphocyte DAT characterized in this study has probably functional relevance as [3H]dopamine was taken up by intact lymphocytes and uptake was inhibited specifically by compounds known to affect dopamine transport. These findings indicate that human peripheral blood lymphocytes possess DAT plasma membrane and VMAT-1 and VMAT-2 transporters. Increasing evidence indicates that dopamine transporter changes may be related to neuronal injury. In view of this assessment of lymphocyte DAT and VMAT transporters can be considered for identifying pathologies characterized by impaired dopaminergic neurotransmission.

Pharmacological profile of enantiomerically pure chiral muscarinic agonists: desoxymuscarines.

The enantiomers desoxymuscarine 6 were tested in vitro on guinea pig tissues, and their muscarinic potency was evaluated at M2 (heart force and rate) and M3 (ileum and bladder) receptor subtypes together with the enantiomers of the parent compound muscarine 1. The eutomers (+)-1 and (+)-6 and distomers (-)-1 and (-)-6 were also assayed in vivo on pithed rat. Affinity, relative efficacy and enantio-selectivity were also determined for the compounds under study at M2 (heart force and rate) and M3 (ileum and bladder), in order to investigate muscarinic receptor heterogeneity. The results of this study have been discussed in comparison with the data previously reported for the structurally related fluoromuscarine (+)-4 and difluoromuscarines (+)-5 and (-)-5.

Structure-activity relationship at alpha-adrenergic receptors within a series of imidazoline analogues of cirazoline.

Several analogues of cirazoline (2), a selective alpha1-adrenoreceptor agonist, were prepared and their pharmacological profiles studied. Although at the alpha1-adrenoreceptor all the compounds displayed a significant agonist activity, at the alpha2-adrenoreceptor they showed either agonist or antagonist activity depending on the nature of the phenyl substituent. The qualitative structure-activity relationship led us to the conclusion that the oxygen atom in the side-chain is essential for alpha1-agonist activity, while the cyclopropyl ring is not, and may be replaced by several groups. Of the groups studied, isopropoxy appears to be the best. Instead, the same substitution (i.e., isopropoxy for the cyclopropyl ring) at alpha2-adrenoreceptors causes a reversal of activity. On the other hand, the cyclopropyl ring seems to be important for alpha1-selectivity. Compound 20 is the most potent alpha1-agonist of the series, being equiactive with cirazoline on rat vas deferens and in pithed rat.

Synthesis and pharmacological characterization of new chiral derivatives of muscarine and allo-muscarine.

Novel derivatives of natural muscarine and allo-muscarine, i.e. the benzyl ethers (-)-10 and (-)-12 and the benzoate (-)-13, were synthesized in very high enantiomeric excess. Target compounds were tested in vitro on guinea pig tissues, and their muscarinic potency was evaluated at M2 (heart force and rate) and M3 (ileum and bladder) receptor subtypes. The derivatives under study were also assayed in vivo on pithed rat. In addition, muscarinic receptor heterogeneity was investigated by determining the affinity and the relative efficacy of compounds (-)-10, (-)-12 and (-)-13 at M2 (heart force and rate) and M3 (ileum and bladder) receptor subtypes.

Alterations of T cell distribution and functions in prenatally cypermethrin-exposed rats: possible involvement of catecholamines.

The synthetic pyrethroid insecticide, cypermethrin (50 mg/Kg) was given during gestation to pregnant rats by gavage in corn oil. Prenatal cypermethrin-exposure induces a marked and long-lasting increase of adrenaline (A) and noradrenaline (NA) plasma concentrations. The enhancement of plasma catecholamine levels was accompanied by a marked increase of CD5+, CD4+, and CD8+ total T cell numbers in the peripheral blood, while in the spleen a reduction of all T cell subsets was observed. In addition, peripheral blood lymphocytes (PBL) from rats prenatally exposed to cypermethrin showed an enhanced capability to proliferate in response to different doses of Concanavalin A (ConA), or human recombinant interleukin-2 (hrIL-2), whereas an impaired proliferative response was observed in the spleen. The percent increase of NA, but not A plasma concentrations paralleles the immunomodulatory effects induced by cypermethrin neonatal exposure on T cell distribution and mitogen-induced proliferation in the peripheral blood and spleen. Collectively, our results suggest that the changes in mitogen-induced proliferative responses in the peripheral blood and spleen of prenatally cypermethrin-exposed rats may be attributable to pesticide-induced catecholamine release, which causes an increased output of CD5+, CD4+, and CD8+ T cells from the spleen to the peripheral blood, and a consequent lymphocytosis.

Influence of neonatal treatment with the pyrethroid insecticide cypermethrin on the development of dopamine receptors in the rat kidney.

The influence of neonatal treatment with the pyrethroid insecticide cypermethrin ((R,S)alpha-cyano-3-phenoxybenzyl (1R,S)-cis-trans-3-(2,2-dichloro-vinyl)-2,2-dimethylcyclopropane carboxylate) on postnatal development of renal dopamine receptors was investigated by radioligand binding assay techniques. Treatment with cypermethrin was made on rats from the 10th to the 16th day after birth. Dopamine D1- and D2-like receptors were assayed in frozen sections of kidney of 21-, 30-, 60- and 90-day-old rats using as ligands of dopamine D1- and D2-like receptors [3H]([R](+)-(chloro-2,3,4,5,-tetrahydro-5-phenyl-1,4,-benzazepinal hemimaleate) (SCH 23390) and [3H]spiperone, respectively. Treatment with cypermethrin was without effect on the affinity (Kd value) or the density (Bmax value) of dopamine D1- and D2-like receptors of rats of 21 days of age. In older groups, treatment with the compound reduced the affinity and increased the density of dopamine D1-like receptors, whereas it was without effect on the affinity of dopamine D2-like receptors and decreased their density. These findings indicate that neonatal treatment with the pyrethroid insecticide cypermethrin induces long-lasting impairment of renal dopamine D1- and D2-like receptors and that kidney is a target of the toxic action of the compound. Renal dopamine receptor changes caused by cypermethrin are consistent with possible alterations of renal tubular function and of sympathetic neuroeffector modulation. The above data suggest also that, different from the adult, neonatal exposure to pyrethroid insecticides may induce toxic effects.

Cypermethrin-induced alteration of thymocyte distribution and functions in prenatally-exposed rats.

The synthetic pyrethroid insecticide, cypermethrin (50 mg/kg) was given during gestation to pregnant rats by gavage in corn oil. Prenatal cypermethrin exposure induced a significant decrease in the absolute number of all thymocyte subsets during the first 30 days after birth, being the double negative CD4-CD8-, single positive CD4 and CD8 T cells preferentially affected. Later on day 60 and 90 double positive CD4+CD8+ and single positive thymocytes gradually recovered, while the total number of CD4 CD8 cells was increased. Moreover, thymocytes from rats prenatally exposed to cypermethrin showed an impaired ability to proliferate in response to different doses of Concanavalin A (ConA) and human recombinant interleukin-2 (hrIL-2) and to produce and/or release IL-2. Overall, our results indicate that cypermethrin administered during prenatal period can affect multiple steps in thymocyte differentiation pathways resulting in an altered cell subset distribution and an impairment of thymocyte functions.

Prenatal exposure to cypermethrin modulates rat NK cell cytotoxic functions.

The synthetic pyrethroid insecticide, cypermethrin, was given during gestation to pregnant rats by gavage in corn oil. Peripheral blood and spleen cytotoxic activity of dams and their offspring were then evaluated at different times (30, 60, 90, 120 days) after birth. Pups showed a significant increase in peripheral blood natural killer (NK) and antibody-dependent (ADCC) cytotoxic activity paralleled with a similar increase in the percentage of NK-RP1+ cells and decreased activity in the spleen. Pregnant cypermethrin-exposed dams showed no changes in peripheral blood or spleen cytotoxic function during the postnatal period. Overall, these results suggest that immunomodulation of cytotoxic activity observed in the offspring is likely attributable to a specific effect of cypermethrin administered during the prenatal period.

Synthesis and pharmacological characterization of enantiomerically pure muscarinic agonists: difluoromuscarines.

The four homochiral 4-deoxy-4,4-difluoromuscarine stereoisomers (difluoromuscarines) were prepared in very high enantiomeric excess. A convenient sequence based on the use of natural as well as "unnatural" ethyl lactate allowed the synthesis of target compounds, whose absolute configuration is dictated by that of the starting synthon. Quaternary ammonium salts (+)-5, (-)-5, (-)-6, and (+)-6 were tested in vitro on guinea pig tissues, and their muscarinic potency was evaluated at M2 (heart) and M3 (ileum and bladder) muscarinic receptor subtypes. The eutomer (+)-5 and distomer (-)-5 were also tested in vivo on pithed rat, and their muscarinic activity at the M1 receptor subtype was compared with those of racemic muscarine [(+/-)-1] and (2S,4R,5S)-4-deoxy-4-fluoromuscarine [(+)-4]. Further pharmacological parameters such as affinity, relative efficacy, and enantioselectivity have been determined for compounds (+)-5 and (-)-5 at M2 (heart force and rate) and M3 (ileum and bladder) receptors in order to investigate muscarinic receptor heterogeneity. The four homochiral difluoromuscarines behave as muscarinic agonists in all the tests with a potency trend which is different from that previously observed with the 4-deoxy-4-fluoromuscarines and (+/-)-1, thus indicating the intervention of the second fluorine atom on the receptor-ligand interaction. Moreover, the second fluorine atom produces significant differences in the affinity and relative efficacy values of compounds (+)-5 and (-)-5 at M2 and M3 subtypes, which could be attributed to a heterogeneity between the muscarinic receptors mediating heart rate and heart force and those involved in the contraction of ileum and bladder.

Selective blockade of muscarinic M2 receptors in vivo by the new antagonist tripitramine.

The antimuscarinic effects of tripitramine (1, 1, 24--tris[[5, 11-dihydro-6-oxo-6H-pyrido [2,3-b][1,4]- benzodiazepin-11-yl)carbonyl]methyl]-8, 17-dimethyl-1, 8, 17, 24-tetraazatetracosane tetraoxalate), a member of a series of polymethylene tetraamines with in vitro cardioselectivity, were assessed in two in vivo preparations: anaesthetized and pithed rats. The well-known M2 selective antagonist methoctramine was used in a comparative study. Tripitramine (0.0202 mumol/kg i.v.) proved to be a potent antagonist at cardiac M2 receptors that mediate the decrease in heart rate in the pithed rat; the same dose of this antagonist in the anaesthetized rat did not significantly affect the depressor action of methacholine mediated by vascular M3 receptors. In the pithed rat, this dose did not affect the ganglionic M1 receptor-mediated tachycardia and pressor response to muscarine or McN-A-343. These in vivo data are consistent with the in vitro findings and confirm that tripitramine is a more potent and selective muscarinic M2 receptor antagonist than methoctramine.

Activity of N-(2-phenylethyl)-N-n-propyl-2-(3-hydroxyphenyl) ethylamine derivatives as dopamine receptor ligands.

The N-n-propyl-N-(2-phenylethyl)-2-(3-hydroxyphenyl)ethylamine (1, RU 24213) had been previously identified as selective agonist of DA D2 receptor subtype. In this paper we describe the synthesis and in vitro binding affinities of several derivatives of 1 substituted with fluorine, chlorine and methyl or hydroxy groups on the phenyl ring of the N-2-phenylethyl moiety. The results obtained indicate that these substitutions do not improve the D2 binding affinity. The introduction on the phenyl ring of two fluorine or chlorine atoms decreases with D1 affinity, and the dichloro derivatives are highly selective for the D2 receptor. Preliminary behavioural tests confirm that the dichloro derivatives behave as D2 selective agonists.

Acute toxicity of two pyrethroids, permethrin, and cypermethrin in neonatal and adult rats.

The present study aims specifically at obtaining a comparison of the acute toxicity of cypermethrin (CY), a type I pyrethroid, and permethrin (PERM), a type II pyrethroid, administered orally as a single dose to neonatal and adult rats, and at assessing the importance of pyrethroid biotransformation in CY and PERM toxicity through use of drug metabolism inhibitors. Our experiments show that CY is more toxic than PERM to adult and neonatal rats. The sensitivity of neonatal rats both to CY and to PERM toxicity is higher, the younger the animals. CY is much more toxic than PERM in the neonatal rat, compared with the adult. In rats aged 8, 16, and 21 days, pretreatment with piperonyl butoxide (PB), a monooxygenase inhibitor, or with tri-o-tolyl phosphate (TOTP), an esterase inhibitor, does not produce significant variations in the lethal effects of CY and PERM. Instead, in the adult rats, a significant increase in CY (chi 2 = 5.97; p < 0.05) and PERM (chi 2 = 4.37; p < 0.05) mortality occurred in rats pretreated with esterase inhibitors, whereas no increase in CY and PERM toxicity was found in adult animals pretreated with monooxygenase inhibitor. It was concluded that the higher level of sensitivity of the neonate rat to pyrethroid toxicity is probably due to incomplete development of the enzymes which catalyze the metabolism of pyrethroids in the liver of young animals. It is suggested that ester hydrolysis is an important pyrethroids detoxification reaction in the adult rat.

Synthesis and pharmacological evaluation of enantiomerically pure 4-deoxy-4-fluoromuscarines.

Four isomers of [(4-fluoro-5-methyl-tetrahydrofuran-2-yl)methyl]trimethylammonium iodide (4-deoxy-4-fluoro-muscarines) were prepared in enantiomerically and diastereomerically pure form from (S)-(-)-methyl 4-methylphenyl sulfoxide, ethyl fluoroacetate, and allyl bromide. Their absolute configurations were assigned by 1H NMR analyses. The four optically pure compounds were tested in vitro on guinea pig and their muscarinic potency was evaluated at M3 (ileum and bladder) and M2 (heart) muscarinic receptor subtypes. Compound 1a, the most potent isomer of the series, was also tested in vivo on pithed rat and its muscarinic activity at the M1 receptor subtype was compared with that of muscarine. Moreover, affinity and relative efficacy were calculated in vitro for this compound at M2 (heart force and rate) and M3 (ileum and bladder) receptors in order to investigate muscarinic receptor heterogeneity. The 4-deoxy-4-fluoromuscarines display a similar trend of potency as the corresponding muscarines and compound 1a shows differences in the affinity constants among the studied tissues. Replacement of a hydroxyl group for a fluorine atom in the 4 position of muscarine produces 1 order of magnitude increase in affinity for cardiac M2 muscarinic receptors controlling rate, while the affinity at cardiac M2 muscarinic receptors controlling force is unchanged, opening the possibility of a further classification of cardiac muscarinic receptors.

Determination of muscarinic agonist potencies at M1 and M2 muscarinic receptors in a modified pithed rat preparation.

The agonistic potencies of (+/-)muscarine, (+/-)cis-2-methyl-5- [(dimethylamino)methyl]-1,3-oxathiolane methiodide (cis-oxathiolane) and its two enantiomers were determined at muscarinic M1 and M2 receptors in the pithed rat. In non-pretreated animals, i.v. administration of these agents produced bradycardic effects mediated by cardiac M2 receptors followed by increases in heart rate mediated by M1 receptors in sympathetic ganglia. As these responses have been shown to partly overlap, "true" M1 and M2 potencies were determined after selective blockade of M1 and M2 receptors by pirenzepine and methoctramine, respectively. A similar rank order of agonist potencies was obtained at M1 and M2 receptors: (+)cis-oxathiolane greater than (+/-)cis-oxathiolane greater than (+/-)muscarine greater than (-)cis-oxathiolane. At both receptor subtypes, (+)cis-oxathiolane was considerably more potent (ca. 30-fold) than its corresponding (-) enantiomer indicating that the agonist binding sites of the two receptor subtypes may have similar stereochemical properties. While (+/-)muscarine showed similar potencies at M1 and M2 receptors, racemic cis-oxathiolane and its two enantiomers showed a slight selectivity (3-7 fold) for M1 receptors indicating the potential usefulness of these compounds in the development of selective M1 receptor agonists.

Fluoronorepinephrines: further pharmacological evaluation in vitro and in pithed rats.

The effect of 6F-, 5F- and 2F-norepinephrine (6F-, 5F- and 2F-NE) in rat vas deferens, guinea-pig ileum and pithed rats was compared to that of norepinephrine (NE). The rank order of potency on postsynaptic alpha 1-adrenoreceptors, determined from the isometric contraction of vas deferens, was 6F-NE = 5F-NE = NE greater than 2F-NE. A similar pattern was found for presynaptic alpha 2-adrenoreceptor activity in both noradrenergic nerve terminals of vas deferens and cholinergic nerve terminals of the ileum, determined from the inhibition of contraction elicited by electrical field stimulation. The only exception was the 5F isomer which was 7 times less active than NE to activate the alpha 2-adrenoreceptors of vas deferens. Thus, ring fluorination markedly alters both alpha 1- and alpha 2-agonist properties of NE. Moreover, alpha 1/alpha 2 selectivity, at least as far as rat vas deferens is concerned, is not significantly influenced by the introduction of a fluorine atom in the NE molecule. 6F-NE was about 3-4 times more active than NE in pithed rats. In turn, NE was equiactive with 5F-NE. 2F-NE was the least active isomer, being 30- and 100-fold less active than NE and 6F-NE, respectively.

[Synthesis and antiinflammatory activity of various 1,4-benzothiazine derivatives]. / Sintesi ed attività antiinfiammatoria di alcuni derivati 1,4-benzotiazinici.

A new series of dihydro-4H-1,4-benzothiazine derivatives was prepared. These compounds were obtained by reductive N-alkylation reaction with sodium borohydride in acetic acid of the corresponding 4H-1,4-benzothiazine. Some of the latter compounds were synthesized by a new synthetic method employing 2-aminobenzenethiol and alkynes as starting material. Preliminary pharmacological data on the antiinflammatory activity of these compounds by using carrageenin paw edema assay are reported.

Possible influence of tachykinins on body fluid homeostasis in the rat.

The effect on water intake, urine flow and vasopressin release of intracranial injections of substance P, physalaemin and eledoisin was studied in Wistar and Brattleboro, homozygous and heterozygous, rats. The tachykinins strongly inhibited water intake both in Wistar and in Brattleboro, homozygous and heterozygous, rats. Physalaemin and eledoisin reduced urine flow in Wistar and heterozygous, but not in homozygous, Brattleboro rats. Substance P never affected urine elimination. Physalaemin and eledoisin produced a dose-dependent, long lasting release of vasopressin in Wistar rats. Substance P did not affect the release of vasopressin. The results suggest that both substance P and physalaemin could influence brain mechanisms which control water intake, acting as thirst inhibitors, and that physalaemin could also participate in body fluid control by conserving water through vasopressin release.

Drinking stimulation by a new angiotensin, crinia-angiotensin II, in rats and pigeons.

The effects of crinia-angiotensin II on water intake and arterial blood pressure were investigated in conscious rats and pigeons. Injected by intravenous route to rats and pigeons, crinia-angiotensin II produced a hypertensive response practically identical to that induced by intravenous angiotensin II. Injected by intracerebroventricular route crinia-angiotensin II proved to be as active as angiotensin II in eliciting water intake in pigeons, while being less effective in rats. These findings, while demonstrating that naturally occurring angiotensins may be as active as angiotensin II itself in eliciting drinking, suggest that different molecular requirements must be satisfied to activate the angiotensin receptors for drinking in rats and pigeons.

Dual effect of naloxone on drinking behaviour of rats.

Naloxone, administered to rats subcutaneously or by i.c.v. route, produces a dose-dependent inhibition of water intake elicited by angiotensin II or water deprivation. However, doses of the drug which do not affect drinking inhibit the antidipsic effect of subcutaneous morphine. The larger is the dose of morphine, the larger is the antimorphinic effect evoked by naloxone. These data would suggest that, at least in respect to the effects of narcotics on water intake, naloxone is a partial agonist of the nalorphine type, but the slopes of naloxone and of morphine dose-response regression lines are not in keeping with this hypothesis. Thus, the mechanism of the anti-morphic effect elicited by naloxone on drinking behaviour remains to be clarified.