NT3/TrkC Pathway Modulates the Expression of UCP-1 and Adipocyte Size in Human and Rodent Adipose Tissue.

Neurotrophin-3 (NT3), through activation of its tropomyosin-related kinase receptor C (TrkC), modulates neuronal survival and neural stem cell differentiation. It is widely distributed in peripheral tissues (especially vessels and pancreas) and this ubiquitous pattern suggests a role for NT3, outside the nervous system and related to metabolic functions. The presence of the NT3/TrkC pathway in the adipose tissue (AT) has never been investigated. Present work studies in human and murine adipose tissue (AT) the presence of elements of the NT3/TrkC pathway and its role on lipolysis and adipocyte differentiation. qRT-PCR and immunoblot indicate that NT3 (encoded by NTF3) was present in human retroperitoneal AT and decreases with age. NT3 was also present in rat isolated adipocytes and retroperitoneal, interscapular, perivascular, and perirenal AT. Histological analysis evidences that NT3 was mainly present in vessels irrigating AT close associated to sympathetic fibers. Similar mRNA levels of TrkC (encoded by NTRK3) and ß-adrenoceptors were found in all ATs assayed and in isolated adipocytes. NT3, through TrkC activation, exert a mild effect in lipolysis. Addition of NT3 during the differentiation process of human pre-adipocytes resulted in smaller adipocytes and increased uncoupling protein-1 (UCP-1) without changes in ß-adrenoceptors. Similarly, transgenic mice with reduced expression of NT3 (Ntf3 knock-in lacZ reporter mice) or lacking endothelial NT3 expression (Ntf3flox1/flox2;Tie2-Cre+/0) displayed enlarged white and brown adipocytes and lower UCP-1 expression. Conclusions: NT3, mainly released by blood vessels, activates TrkC and regulates adipocyte differentiation and browning. Disruption of NT3/TrkC signaling conducts to hypertrophied white and brown adipocytes with reduced expression of the thermogenesis marker UCP-1.

Changes in adrenoceptors and G-protein-coupled receptor kinase 2 in L-NAME-induced hypertension compared to spontaneous hypertension in rats.

This work compares the expression of adrenoceptors (ARs) and G-protein-coupled receptor kinase (GRK) 2 (RT-PCR and immunoblotting) and functional responses in conductance (aorta) and resistance vessels (mesenteric resistance arteries; MRA) in two different models of rat hypertension: hypertension induced by chronic treatment with L-NAME (N(G)-nitro-L-arginine methyl-ester) (L-NAME-treated rats; LNHR), and genetically induced hypertension (spontaneously hypertensive rats; SHR). Changes found in the aorta, but not in the MRA, were: (1) a loss of contractile capacity, more evidently in α1-AR-mediated contraction, and an impairment of endothelium-dependent vasorelaxation, with both changes occurring independently of the hypertensive model; (2) a diminished sensitivity to α1-AR-induced vasoconstriction along with increased ß2-AR-mediated vasodilation in LNHR, and (3) a lower expression of ARs and GRK2 in LNHR. The two latter changes are the opposite of those previously found in aortas of SHR. In the MRA of LNHR, a diminished sensitivity to isoprenaline, in parallel with a reduced expression of ß1-AR, was observed without changes in GRK2 expression. In the MRA of SHR, the increased GRK2 expression was not accompanied by significant changes in either ß-AR expression or the vasorelaxant potency of isoprenaline. The present results highlight that changes in AR function differ not only between vessels but also between hypertensive models. Moreover, they suggest that changes in GRK2 expression could contribute to regulating ß2-AR function in conductance vessels but not ß1-AR function in resistance vessels.

ß-Adrenoceptors differentially regulate vascular tone and angiogenesis of rat aorta via ERK1/2 and p38.

ß-Adrenoceptors (ß-ARs) modulate ERK1/2 and p38 in different cells, but little is known about the contribution of these signaling pathways to the function of ß-ARs in vascular tissue. Immunoblotting analysis of rat aortic rings, primary endothelial (ECs) and smooth muscle cells (SMCs) isolated from aorta showed that ß-AR stimulation with isoprenaline activated p38 in aortic rings and in both cultured cell types, whereas it had a dual effect on ERK1/2 phosphorylation, decreasing it in ECs while increasing it in SMCs. These effects were reversed by propranolol, which by itself increased p-ERK1/2 in ECs. Isoprenaline ß-AR mediated vasodilation of aortic rings was potentiated by the ERK1/2 inhibitor, U0126, in the presence or absence of endothelium or L-NAME, whereas inhibition of p38 had no impact. Isoprenaline moderately decreased sprouting from aorta rings in the Matrigel angiogenesis assay; conversely propranolol not only prevented isoprenaline inhibition, but stimulated angiogenesis. ERK1/2 inhibition decreased angiogenesis, while a dramatic stimulation was observed by p38 blockade. Our results suggest that ERK1/2 activation after ß-ARs stimulation in the smooth muscle hinders the vasodilator effect of isoprenaline, but in the endothelium ß-ARs decreases ERK1/2 and increases p38 activity reducing therefore angiogenesis.

Antihypertensive mechanism of lactoferrin-derived peptides: angiotensin receptor blocking effect.

Looking for antihypertensive mechanisms beyond ACE inhibition, we assessed whether lactoferrin (LF)-derived peptides can act as receptor blockers to inhibit vasoconstriction induced by angiotensin II or endothelin-1. The lactoferricin B (LfcinB)-derived peptide LfcinB20-25 (RRWQWR), the low molecular weight LF hydrolysate (LFH < 3 kDa), and two peptides identified in LFH < 3 kDa (LIWKL and RPYL) were tested in ex vivo assays of vasoactive responses. The peptide RPYL was tested in radioligand receptor binding assays. Both LFH < 3 kDa and individual peptides inhibited angiotensin II-induced vasoconstriction. RPYL showed the highest ex vivo inhibitory effect and also inhibited binding of [(125)I]-(Sar(1),Ile(8))-angiotensin II to AT1 receptors. By contrast, neither LFH < 3 kDa nor RPYL inhibited endothelin-1 and depolarization-induced vasoconstrictions. In conclusion, LF-derived peptides selectively inhibit angiotensin II-induced vasoconstriction by blocking angiotensin AT1 receptors. Therefore, inhibition of angiotensin II-induced vasocontriction is suggested as a mechanism contributing along with ACE inhibition to the antihypertensive effect of some LF-derived peptides.

Structure-activity relationship of dopaminergic halogenated 1-benzyl-tetrahydroisoquinoline derivatives.

Two series of halogenated 1-benzyl-7-chloro-6-hydroxy-tetrahydroisoquinolines were prepared to explore the influence of each series on the affinity for dopamine receptors. All the compounds displayed a high affinity for D(1)-like and/or D(2)-like dopamine receptors in striatal membranes, although they were unable to inhibit [(3)H]-dopamine uptake in striatal synaptosomes. The halogen placed on the benzylic ring in 1-benzyl-THIQs, compounds of the series 1, 2'-bromobenzyl derivatives with K(i) values into the nanomolar range, and the series 2, 2',4'-dichlorobenzyl-THIQ homologues, proves to be an important factor to modulate affinity at dopamine receptor.

The impact of alpha1-adrenoceptors up-regulation accompanied by the impairment of beta-adrenergic vasodilatation in hypertension.

In human and animal hypertension models, increased activity of G-protein-coupled receptor kinase (GRK) 2 determines a generalized decrease of beta-adrenergic vasodilatation. We analyzed the possibility of differential changes in the expression and functionality of alpha(1A), alpha(1B), alpha(1D), beta(1), beta(2), and beta(3)-ARs also being involved in the process. We combined the quantification of mRNA levels with immunoblotting and functional studies in aortas of young and adult spontaneously hypertensive rats (SHRs) and their controls (Wistar Kyoto). We found the expression and function of beta(1)-adrenoceptors in young prehypertensive SHRs to be higher, whereas a generalized increase in the expression of the six adrenoceptors and GRK2 was observed in aortas of adult hypertensive SHRs. alpha(1D)- and beta(3)-adrenoceptors, the subtypes that are more resistant to GRK2-mediated internalization and mostly expressed in rat aorta, exhibited an increased functional role in hypertensive animals, showing two hemodynamic consequences: 1) an increased sensitivity to the vasoconstrictor stimulus accompanied by a decreased sensitivity to the vasodilator stimulus (alpha(1D)-ARs are the most sensitive to agonists, and beta(3)-ARs are the least sensitive to agonists); and 2) a slower recovery of the basal tone after adrenergic stimulus removal because of the kinetic characteristic of the alpha(1D) subtype. These functional changes might be involved in the greater sympathetic vasoconstrictor tone observed in hypertension.

8-NH2-boldine, an antagonist of alpha1A and alpha1B adrenoceptors without affinity for the alpha1D subtype: structural requirements for aporphines at alpha1-adrenoceptor subtypes.

Structure-activity analysis of 21 aporphine derivatives was performed by examining their affinities for cloned human alpha (1A), alpha (1B) and alpha (1D) adrenoceptors (AR) using membranes prepared from rat-1 fibroblasts stably expressing each alpha (1)-AR subtype. All the compounds tested competed for [ (125)I]-HEAT binding with steep and monophasic curves. The most interesting compound was 8-NH (2)-boldine, which retains the selective affinity for alpha(1A)-AR (pKi = 6.37 +/- 0.21) vs. alpha(1B)-AR (pKi = 5.53 +/- 0.11) exhibited by 1,2,9,10-tetraoxygenated aporphines, but shows low affinity for alpha(1D)-AR (pKi < 2.5). Binding studies on native adrenoceptors present in rat cerebral cortex confirms the results obtained for human cloned alpha (1)-AR subtypes. The compounds selective for the alpha (1A) subtype discriminate two binding sites in rat cerebral cortex confirming a mixed population of alpha (1A)- and alpha (1B)-AR in this tissue. All compounds are more selective as inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. A close relationship was found between affinities obtained for cloned alpha (1A)-AR and inhibitory potencies on noradrenaline-induced contraction or inositol phosphate accumulation in tail artery, confirming that there is a homogeneous functional population of alpha(1A)-AR in this vessel. On the contrary, a poor correlation seems to exist between the affinity of 8-NH (2)-boldine for cloned alpha (1D)-AR and its potency as an inhibitor of noradrenaline-induced contraction or inositol phosphate accumulation in rat aorta, which confirms that a heterogeneous population of alpha (1)-AR mediates the adrenergic response in this vessel.

Comparison of the alpha-adrenoceptor-mediated effects of beta3-adrenoceptor ligands in rat pulmonary artery.

We have recently shown that the beta-adrenoceptor ligands CGP 12177, bupranolol, and SR 59230A (aryloxypropanolamines), but not BRL 37344 and CL 316243 (phenylethanolamines), exhibit significant affinity for alpha1-adrenoceptors and that CGP 12177 displays partial agonist properties at alpha-adrenoceptors in rat pulmonary artery. In this study, bupranolol and SR 59230A were further evaluated for their potential alpha-adrenoceptor mediated effects (i.e., agonist and/or antagonist properties) in rat intralobar pulmonary artery and compared with BRL 37344 and CL 316243. Bupranolol induced a relaxation in phenylephrine-precontracted arteries, but had no effect in prostaglandin F2alpha(PGF2alpha) -precontracted ones. SR 59230A also elicited a relaxation in phenylephrine-precontracted arteries. In PGF2alpha -precontracted arteries, SR 59230A induced a contractile response that was insensitive to the irreversible alpha-adrenoceptor antagonist phenoxybenzamine. BRL 37344 at high concentrations, but not CL 316243, produced slight relaxation in both phenylephrine- and PGF2alpha -precontracted arteries. The contractile response to phenylephrine was antagonized by bupranolol and SR 59230A in a competitive manner (pA2: 6.38 and 7.08 respectively). The concentration-response curve to phenylephrine was also shifted to the right by BRL 37344 (mean pKb: 4.45), but not by CL 316243 (100 microM). This study indicates that the aryloxypropanolamine derivatives bupranolol and SR 59230A exhibit competitive antagonist, but no agonist properties on alpha1-adrenoceptors, SR 59230A also inducing alpha-adrenoceptor-independent contraction. Among the phenylethanolamines, BRL 37344 but not CL 316243, also exerts an antagonist effect on alpha1-adrenoceptors, with a much lower potency than the aryloxypropanolamines studied.

Correlation between mRNA levels and functional role of alpha1-adrenoceptor subtypes in arteries: evidence of alpha1L as a functional isoform of the alpha1A-adrenoceptor.

The mRNA levels for the three alpha1-adrenoceptor subtypes, alpha1A, alpha1B, and alpha1D, were quantified by real-time RT-PCR in arteries from Wistar rats. The alpha1D-adrenoceptor was prominent in both aorta (79.0%) and mesenteric artery (68.7%), alpha1A predominated in tail (61.7%) and small mesenteric artery (73.3%), and both alpha1A- and alpha1D-subtypes were expressed at similar levels in iliac artery. The mRNA levels of the alpha1B-subtype were a minority in all vessels (1.7-11.1%). Concentration-response curves of contraction in response to phenylephrine or relaxation in response to alpha1-adrenoceptor antagonists on maximal sustained contraction induced by phenylephrine were constructed from control vessels and vessels pretreated with 100 micromol/l chloroethylclonidine (CEC) for 30 min. The significant decrease in the phenylephrine potency observed after CEC treatment together with the inhibitory potency displayed by 8-{2-[4-(2-methoxyphenyl)-1-piperazinyl]-8-azaspiro (4,5) decane-7-dionedihydrochloride} (BMY-7378, an alpha1D-adrenoceptor antagonist) confirm the relevant role of alpha1D-adrenoceptors in aorta and iliac and proximal mesenteric arteries. The potency of 5-methylurapidil (an alpha1A-adrenoceptor antagonist) and the changes in the potency of both BMY-7378 and 5-methylurapidil after CEC treatment provided evidence of a mixed population of alpha1A- and alpha1D-adrenoceptors in iliac and distal mesenteric arteries. The low potency of prazosin (pIC50 < 9) as well as the high 5-methylurapidil potency in tail and small mesenteric arteries suggest the main role of alpha1A/alpha1L-adrenoceptors with minor participation of the alpha1D-subtype. The mRNA levels and CEC treatment corroborated this pattern and confirmed that the alpha1L-adrenoceptor could be a functional isoform of the alpha1A-subtype.

Role of alpha-adrenergic receptors in the effect of the beta-adrenergic receptor ligands, CGP 12177, bupranolol, and SR 59230A, on the contraction of rat intrapulmonary artery.

This study investigates the effect of the aryloxypropanolamines 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one (CGP 12177), bupranolol, and 3-(2-ethylphenoxy)-1[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate (SR 59230A) [commonly used as beta(3)- and/or atypical beta-adrenergic receptors (beta-AR) ligands] on the contractile function of rat intralobar pulmonary artery. Affinities of beta-AR ligands for alpha(1)-adrenergic receptors (alpha(1)-AR) were also evaluated using [(3)H]prazosin binding competition experiments performed in rat cortical membranes. In intralobar pulmonary artery, CGP 12177 did not modify the basal tone, but antagonized the contraction induced by the alpha(1)-AR agonist phenylephrine (PHE). In arteries precontracted with PHE, CGP 12177 elicited relaxation, whereas in those precontracted with prostaglandin F(2alpha) (PGF(2alpha)), it further enhanced contraction. CGP 12177 induced an increase in intracellular calcium concentration in pressurized arteries loaded with Fura PE-3 and precontracted with PGF(2alpha). In PGF(2alpha) precontracted arteries, phentolamine (an alpha-AR antagonist) and phenoxybenzamine (an irreversible alpha-AR antagonist) antagonized the contractile responses to PHE and CGP 12177. Both responses were also decreased by bupranolol and SR 59230A. Specific [(3)H]prazosin binding was displaced by CGP 12177, bupranolol, and SR 59230A with pK(i) values of 5.2, 5.7, and 6.6, respectively. In contrast, (+/-)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium (BRL 37344) and disodium 5-[(2R)-2-([(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243) (nonaryloxypropanolamines beta(3)-AR agonists) displayed very low affinity for [(3)H]prazosin binding sites (pK(i) values below 4). These data suggest that CGP 12177 exhibits partial agonist properties for alpha(1)-AR in rat pulmonary artery. They also show that bupranolol and SR 59230A exert an alpha(1)-AR antagonist effect. As a consequence, these aryloxypropanolamine compounds should be used with caution when investigating the role of beta(3)- and atypical beta-AR in the regulation of vascular tone.

Simplified tetrandrine congeners as possible antihypertensive agents with a dual mechanism of action.

A series of O- and/or N-substituted derivatives of (+/-)-coclaurine (1a) were synthesized as simplified structural mimics of the antihypertensive alkaloid tetrandrine (2) and assayed for binding to brain cortical sites labeled with the alpha(1)-adrenergic radioligand [(3)H]prazosin or the calcium channel radioligand [(3)H]diltiazem. The introduction of O-benzyl groups on the coclaurine molecule, which exhibits only adrenergic antagonist activity, led to the appearance of calcium channel blocking activity comparable to that of tetrandrine while retaining adrenolytic activity in the same concentration range. Contraction of aortal rings with noradrenaline or KCl was relaxed more potently by some of these coclaurine derivatives than by tetrandrine, suggesting leads for the development of novel antihypertensive drugs with a dual mechanism of action.

Functional characterization of alpha 1-adrenoceptor subtypes in vascular tissues using different experimental approaches: a comparative study.

1. The alpha(1)-adrenergic responses of rat aorta and tail artery have been analysed measuring the contractility and the inositol phosphate (IP) formation induced by noradrenaline. Three antagonists, prazosin, 5-methylurapidil (alpha(1A) selective) and BMY 7378 (alpha(1D) selective) have been used in different experimental procedures. 2. Noradrenaline possesses a greater potency inducing contraction and IP accumulation in aorta (pEC(50)-contraction=7.32+/-0.04; pEC(50)-IPs=6.03+/-0.08) than in the tail artery (pEC(50)-contraction=5.71+/-0.07; pEC(50)-IPs=5.51+/-0.10). Although the maximum contraction was similar in both tissues (E(max)-tail=619.1+/-55.6 mg; E(max)-aorta-698.2+/-40.8 mg), there were marked differences in the ability of these tissues to generate intracellular second messengers the tail artery being more efficient (E(max)-tail=1060+/-147%; E(max)-aorta=108.1+/-16.9%). 3. Concentration response curves of noradrenaline in presence of antagonist together with concentration inhibition curves for antagonists added before (CICb) or after (CICa) noradrenaline-induced maximal response in Ca(2+)-containing or Ca(2+)-free medium have been performed. A comparative analysis of the different procedures as well as the mathematical approaches used in each case to calculate the antagonist potencies, were completed. 4. The CICa was the simplest method to characterize the predominant alpha(1)-adrenoceptor subtype involved in the functional response of a tissue. 5. In aorta, where constitutively active alpha(1D)-adrenoeptors are present, the use of different experimental procedures evidenced a complex equilibrium between alpha(1D)- and alpha(1A)-adrenoceptor subtypes. 6. The appropriate management of LiCl in IP accumulation studies allowed us to reproduce the different experimental procedures performed in contractile experiments giving more technical possibilities to this methodology.

Alterations by age of calcium handling in rat resistance arteries.

This study investigated the alterations by age of Ca handling in rat small mesenteric arteries. The contractile responses to phenylephrine and caffeine in small mesenteric arteries from young and old rats were studied in Ca -containing and Ca -free medium. In Ca -containing medium the contraction to phenylephrine (100 micro M) but not to caffeine (10 mM) was greater in old than in young rats. Concentration-response curve to phenylephrine was affected to the same extent by nifedipine (1 micro M) in both age groups, whereas ryanodine (20 micro M) decreased the maximal response to phenylephrine only in young rats. These results suggest the participation of intracellular Ca handling on the observed differences by aging. In Ca -free medium, phenylephrine (10 micro M) but not caffeine (10 mM) induced a greater contraction in old than in young animals, corroborating the results obtained in Ca -containing solution. The greater response to phenylephrine observed in old rats cannot be explained by an increase in the inositol 1,4,5-trisphosphate (IP ) formation because the accumulation of inositol phosphates by phenylephrine was not affected by aging. Results obtained in Ca -free medium using caffeine after phenylephrine or vice versa suggest a common intracellular Ca pool. Pretreatment with ryanodine in Ca -free medium almost abolished contractile response to phenylephrine and caffeine in young rats but only partially decreased them in old animals, suggesting an impairment in the Ca -induced Ca release (CICR) mechanism leading to an increase in the stored Ca content. The greater amount of stored Ca could explain the higher contractile response to phenylephrine observed in aged rats. As a consequence of all these changes due to aging, an imbalance between the two Ca release mechanisms from sarcoplasmic reticulum was observed with a major role of Ca induced release by IP at the expense of an impairment of CICR mechanism. This observation will also help explain the results obtained in the presence of extracellular Ca, where phenylephrine induced a greater maximum response in old animals in spite of a decrease in the midrange sensitivity to this agonist.

Pathological role of a constitutively active population of alpha(1D)-adrenoceptors in arteries of spontaneously hypertensive rats.

1. The role of a constitutively active population of alpha(1D)-adrenoceptors was analysed in arteries obtained from spontaneously hypertensive rats (SHR) and controls (WKY) divided into three groups: young prehypertensive, adult hypertensive, and adult animals chronically treated with captopril (50 mg kg(-1) per day orally) in order to prevent the hypertensive state. 2. In adult SHR, a significant increase in BMY 7378 potency (not in prazosin potency) was observed in aorta, mesenteric artery, and the first and second branches of the small mesenteric arteries with respect to WKY rats. This difference was not observed in iliac and tail arteries, which suggests an increased functional role of alpha(1D)-adrenoceptors only in some vessels of SHR. 3. The increase in the resting tone (IRT) observed in absence of agonist, inhibited by BMY 7378, that represents the constitutively active population of alpha(1D)-adrenoceptors, was also significantly greater in aorta and mesenteric artery from adult SHR. 4. In young and captopril treated adult animals, no differences between strains with respect to BMY 7378 potency, or IRT were observed. 5. The increase in the functional role of alpha(1D)-adrenoceptors and their constitutive activity observed in hypertension is prevented by captopril treatment. The pathological consequence of this change is the slower rate of recovery of the basal tone after removal of an adrenergic stimulus, observed in vessels from hypertensive animals that had shown an increase in the functionality of constitutively active alpha(1D)-adrenoceptors. This change was not observed in prehypertensive or captopril treated animals.