Rafael Villalobos-Molina

Effect of L-dopa and the catechol-O-methyltransferase inhibitor Ro 41-0960 on sulfur amino acid metabolites in rats

L-Dopa is the most effective drug known for the treatment of Parkinsons disease. However, the large doses required to treat this neurodegenerative disorder can significantly affect tissue concentrations of sulfur amino acid metabolites due to peripheral and central O-methylation. These effects include decreases in tissue concentrations of the biochemical methyl donor S-adenosylmethionine (SAM), increases in tissue concentrations of the methylation inhibitor S-adenosylhomocysteine (SAH), and increases in plasma concentrations of homocysteine, recently identified as an independent risk factor for vascular disease. In the present study, the ability of the catechol-O-methyltransferase inhibitor Ro 41-0960 to prevent L-Dopa-induced changes in SAM, SAH, and homocysteine concentrations was determined in rats. Rats were injected intraperitoneally with Ro 41-0960 or vehicle 30 min prior to an intraperitoneal injection of L-Dopa or vehicle. One hour after the second injection, the rats were killed and their brains, livers, spleens, kidneys, and plasma collected. SAM and SAH concentrations were then determined in discrete brain regions and peripheral tissues, and total homocysteine concentrations were determined in plasma. In the rats treated with only L-Dopa, decreased SAM concentrations and increased SAH concentrations were found in all brain regions and peripheral tissues measured, and increased homocysteine concentrations were found in plasma, consistent with previous reports. In rats pretreated with Ro 41-0960, however, these L-Dopa-induced effects on sulfur amino acid metabolite concentrations were attenuated or prevented entirely. It remains to be determined if this sparing effect of Ro 41-0960 on sulfur amino acid metabolites has clinical significance.

α1-Adrenoceptors mediating contraction in arteries of normotensive and spontaneously hypertensive rats are of the α1D or α1A subtypes

Alpha 1-Adrenoceptor subtypes mediating contraction in carotid, aorta, mesenteric and caudal arteries from both Wistar Kyoto (WKY) normotensive and spontaneously hypertensive (SHR) rats were investigated by using the alpha 1A-adrenoceptor agonist methoxamine and antagonized with selective, competitive antagonists WB-4101, 5-methyl urapidil or BMY 7378 (8-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4,5)decane -7,9-dione dihydrochloride). Isometric tension changes were recorded after methoxamine addition to the arterial rings, and the effects of the antagonists determined. All the antagonists shifted to the right the concentration-response curve to methoxamine. pA2 values indicate that all arteries but caudal express the alpha 1D-adrenoceptor subtype, since BMY 7378 values were high in these arteries. Due to the high pA2 values for 5-methyl urapidil and WB-4101 and the low values for BMY 7378 we conclude that the tail artery expresses the alpha 1A and not the alpha 1B subtype. No differences were found between both strains of rats, suggesting that hypertension does not modify the alpha 1-adrenoceptors in conductance arteries.

Functional evidence of α1D-adrenoceptors in the vasculature of young and adult spontaneously hypertensive rats

The role of α1D‐adrenoceptors in the vasculature of spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY), of different ages was assessed in pithed rats by the use of the selective α1D‐adrenoceptor antagonist BMY 7378 (8‐[2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]‐ethyl]‐8‐azaspiro [4.5]decane‐7,9‐dione dihydrochloride). BMY 7378 displaced the pressor effect of phenylephrine in young pre‐hypertensive pithed SHR rats, but produced no effect in young WKY rats (dose ratio of 3.4 and 1.6, respectively), while in adult rats BMY 7378 produced a greater shift in the phenylephrine response curve than in younger animals (dose ratio of 3.2 and 6.2 in WKY and SHR, respectively). The presence of α1D‐adrenoceptors in the vasculature of pre‐hypertensive rats, suggests its role in the pathogenesis/maintenance of increased blood pressure.

α1D- and α1A-adrenoceptors mediate contraction in rat renal artery

To investigate the alpha 1-adrenoceptor subtype(s) mediating contraction in rat renal artery, we have compared the effect of the alpha 1-adrenoceptor antagonists, 5-methylurapidil, BMY 7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl) ethyl) 8-azaspiro (4.5) decane-7,9-dione 2HCl) and chloroethylclonidine on functional responses to noradrenaline. A clear blockade by chloroethylclonidine (10(-4) M) of noradrenaline-induced contraction was observed and, along with this effect. pKB values of 9.12 and 8.40 for BMY 7378 and 9.75 and 10.06 for 5-methylurapidil were obtained, indicating that the renal artery expresses the alpha 1D-adrenoceptor subtype as the one involved in contraction and not only the alpha 1A subtype as has been reported.

Evidence for an age-dependent functional expression of α1D-adrenoceptors in the rat vasculature

Abstract The role of the α 1 -adrenoceptor subtypes, and their possible change with maturation, in α 1 -adrenoceptor-induced pressor responses in the rat has not been established. Thus, the effects of the α 1D -, α 1A/1D - and α 1B/1D -adrenoceptor antagonists, BMY 7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl) 8-azaspiro (4.5) decane-7,9-dione 2HCl), 5-methyl-urapidil and chloroethylclonidine, respectively, on the pressor responses induced by phenylephrine in 1- and 5-month-old pithed rats were investigated. The pressor responses induced by phenylephrine were competitively antagonized by both BMY 7378 and chloroethylclonidine in 5-month-old, but not in young immature animals; in marked contrast, 5-methylurapidil antagonized with similar potency the phenylephrine-induced pressor responses in animals of both ages. The present pharmacological data suggest that functional expression of α 1D -adrenoceptors in the rat resistance vessels increases with age; α 1A -, but not α 1B - or α 1D -adrenoceptors, seem to predominate in immature animals. These findings represent the first evidence that age-related changes in functional α 1 -adrenoceptor subtypes occur in the systemic vasculature in vivo. ©xa01997 Elsevier Science B.V.

Differential response to chloroethylclonidine in blood vessels of normotensive and spontaneously hypertensive rats: role of α1D‐ and α1A‐adrenoceptors in contraction

The effects of chloroethylclonidine on α1‐adrenoceptor‐mediated contraction in endothelium‐denuded caudal arteries and aorta from normotensive Wistar and Wistar Kyoto (WKY), and from spontaneously hypertensive (SHR) rats were evaluated. Chloroethylclonidine elicited concentration‐dependent contractions. Maximal contraction was similar in caudal arteries among strains (∼40% of noradrenaline effect). However, chloroethylclonidine elicited a higher contraction in aorta from SHR than from normotensive rats. In Wistar aorta chloroethylclonidine produced the smallest contractile response. In SHR aorta, BMY 7378 and 5‐methylurapidil blocked chloroethylclonidine‐elicited contraction, while (+)‐cyclazocine did not inhibit it; while in caudal arteries, 5‐methylurapidil blocked chloroethylclonidine action; the other antagonists had no effect. In chloroethylclonidine‐treated aorta noradrenaline elicited biphasic contraction‐response curves, indicating a high affinity (pD2, 8.5–7.5) chloroethylclonidine‐sensitive component and a low affinity (pD2, 6.3–5.2) chloroethylclonidine‐insensitive component. The high affinity component was blocked by chloroethylclonidine; while in caudal arteries noradrenaline elicited monophasic contraction‐response curves with pD2 values (6.5–5.7) similar to the low affinity component in aorta. Chloroethylclonidine inhibition of noradrenaline response was greater in aorta than in caudal arteries. Chloroethylclonidine increased the EC50 values of noradrenaline ∼1000 fold in aorta and ∼10 fold in caudal arteries. In SHR aorta BMY 7378 protected α1D‐adrenoceptors and in caudal arteries 5‐methylurapidil protected α1A‐adrenoceptors from chloroethylclonidine alkylation, allowing noradrenaline to elicit contraction. These results show marked strain‐dependent differences in the ability of chloroethylclonidine to contract aorta; moreover, chloroethylclonidine stimulates α1D‐adrenoceptors in aorta and α1A‐adrenoceptors in caudal arteries. The higher contraction observed in aorta from SHR and WKY suggests an augmented number of α1D‐adrenoceptors in these strains.

Area specific alterations in muscarinic stimulated low Km GTPase activity in aging and Alzheimer's disease: implications for altered signal transduction

Carbachol-stimulated low Km GTPase activity (an index of muscarinic receptor-G protein coupling) was examined in hippocampus, basal ganglia, orbital frontal gyrus and superior frontal gyrus obtained from mature, aged and Alzheimers Disease (AD) groups. Results indicated that carbachol-stimulated low Km GTPase activities in basal ganglia were as follows: mature controls > aged > AD, and there was a trend toward a similar pattern of decline in the hippocampus. No differences were seen in the two cortical areas examined; however, carbachol-stimulated low Km GTPase activity was small in the mature controls. Importantly, there were significant negative correlations between disease duration and carbachol-stimulated low Km GTPase activity in all areas examined except the orbital frontal gyrus. The longer the duration of the disease the lower the carbachol-stimulated low Km GTPase activity. Results suggest that age and disease-related changes in mAChR-G protein interactions in the basal ganglia may contribute to reduced signal transduction (ST). In addition, since decreased carbachol-stimulated low Km GTPase activity has also been observed in the aged rat; thus, investigations of the factors involved in decrements in signal transduction in the aged rat may be useful for understanding these alterations in aged humans or victims of AD.

α1-Adrenoceptor subtype selectivity of tamsulosin: Studies using livers from different species

The subtype selectivity of the alpha 1-adrenoceptor antagonist, tamsulosin, was tested using hepatocytes and liver membranes from guinea pigs and rabbits (expressing alpha 1-adrenoceptors with alpha 1A pharmacology) and rats (alpha 1B-adrenoceptors). Tamsulosin blocked the alpha 1-adrenergic activation of phosphorylase with higher affinity in hepatocytes from guinea pigs and rabbits than in those from rats. [3H]Tamsulosin binding to liver membranes was rapid, reversible and saturable. The Kd values obtained also indicated higher affinity for alpha 1A-adrenoceptors (70 and 140 pM, for liver membranes obtained from guinea pigs and rabbits, respectively) than for those of the alpha 1B-subtype (510 pM). Chloroethylclonidine potently and completely inactivated [3H]tamsulosin binding sites in membranes from rabbit and rat livers, but not those in guinea pig liver membranes. Binding competition and inactivation experiments were performed to further characterize the receptor subtypes present in the livers of these animals. In summary, tamsulosin is a very potent alpha 1-adrenoceptor antagonist that has higher affinity for alpha 1A-adrenoceptors than for those of the alpha 1B-subtype.

α1-adrenoceptor subtypes in aorta (α1A) and liver (α1B)

The effect of several alpha 1 adrenoceptor antagonists on the alpha 1-adrenoceptor-mediated stimulation of phosphatidylinositol labeling was studied comparatively in rat hepatocytes and rabbit aorta. It was observed that 5-methyl urapidil and WB 4101 were much more potent in rabbit aorta than in hepatocytes. The orders of potency were prazosin much greater than 5-methyl urapidil greater than or equal to WB 4101 in liver cells and WB 4101 greater than or equal to 5 methyl urapidil = prazosin in aorta. Treatment with chlorethylclonidine inhibited 70-80% of the stimulation of labeling induced by epinephrine in rat liver, but only 30-40% of that in aorta. Our data suggest the existence of two pharmacologically distinct receptors in these tissues i.e.m alpha 1A-adrenoceptors in aorta and alpha 1B in liver cells.

Chloroethylclonidine is a partial α1A-adrenoceptor agonist in cells expressing recombinant α1-adrenoceptor subtypes

Abstract Chloroethylclonidine increased cytosol [Ca 2+ ] in rat-1 fibroblasts stably expressing α 1a -adrenoceptors. The effect of the imidazoline was dose-dependent with a maximal effect (≈3-fold increase in [Ca 2+ ] i ) at 10 μM and it was blocked by phentolamine and 5-methyl urapidil, indicating that it was mediated through α 1 -adrenoceptors. Noradrenaline (1 μM) induced a much bigger effect (≈6–8-fold) in the same cells. When Chloroethylclonidine was added before noradrenaline a dosedependent inhibition of the effect of the natural catecholamine was observed. Chloroethylclonidine did not modified cytosol [Ca 2+ ] in rat-1 fibroblast expressing α 1b - or α 1d -adrenoceptors. However, the imidazoline acutely inhibited the effect of noradrenaline in these cells. It is concluded that Chloroethylclonidine interacts with α 1a -adrenoceptors as a partial agonist inducing Ca 2+ mobilization in a very short time frame and that it is able to inhibit the action of noradrenaline when co-incubated with the catecholamine in cells expressing any of the three α 1 -adrenoceptor subtypes.