Involvement of endothelins in deoxycorticosterone acetate-salt hypertension through the modulation of noradrenergic transmission in the rat posterior hypothalamus.

NEW FINDINGS: What is the central question of this study? Does ex vivo administration of endothelin-1 and endothelin-3 regulate noradrenergic transmission in the posterior hypothalamus of deoxycorticosterone acetate-salt hypertensive rats compared with normotensive rats? What is the main finding and its importance? Endothelin-1 and endothelin-3 enhanced diverse mechanisms leading to increased noradrenergic transmission in the posterior hypothalamus of deoxycorticosterone acetate-salt hypertensive rats. Unveiling the role of brain endothelins in hypertension would probably favour the development of new therapeutic targets for the treatment of essential hypertension, which still represents a challenging disease with high mortality. Brain catecholamines participate in diverse biological functions regulated by the hypothalamus. We have previously reported that endothelin-1 and endothelin-3 (ET-1 and ET-3) modulate catecholaminergic activity in the anterior and posterior hypothalamus of normotensive rats. The aim of the present study was to evaluate the interaction between endothelins and noradrenergic transmission in the posterior hypothalamus of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We assessed the effects of ET-1 and ET-3 on tyrosine hydroxylase activity and expression, neuronal noradrenaline (NA) release, neuronal NA transporter (NAT) activity and expression, monoamine oxidase activity and NA endogenous content and utilization (as a marker of turnover) in the posterior hypothalamus of DOCA-salt hypertensive rats. In addition, levels of ETA and ETB receptors were assayed in normotensive and hypertensive rats. Results showed that tyrosine hydroxylase activity and total and phosphorylated levels, NAT activity and content, NA release, monoamine oxidase activity and NA utilization were increased in DOCA-salt rats. Both ET-1 and ET-3 further enhanced all noradrenergic parameters except for total tyrosine hydroxylase level and NA endogenous content and utilization. The expression of ETA receptors was increased in the posterior hypothalamus of DOCA-salt rats, but ETB receptors showed no changes. These results show that ET-1 and ET-3 upregulate noradrenergic activity in the posterior hypothalamus of DOCA-salt hypertensive rats. Our findings suggest that the interaction between noradrenergic transmission and the endothelinergic system in the posterior hypothalamus may be involved in the development and/or maintenance of hypertension in this animal model.

Regulation of the norepinephrine transporter by endothelins: a potential therapeutic target.

Neuronal norepinephrine (NE) uptake is a crucial step in noradrenergic neurotransmission that regulates NE concentration in the synaptic cleft. It is a key mechanism mediated by the NE transporter (NET) which takes the neurotransmitter into the presynaptic neuron terminal or the adrenal medulla chromaffin cell. The activity of NET is short and long terms modulated by phosphorylation mediated by protein kinases A, C, and G and calcium-calmodulin-dependent protein kinase, whereas the transporter availability at the cell surface is regulated by glycosylation. Several neuropeptides like angiotensins II, III, and 1-7, bradykinin, natriuretic peptides, as well as endothelins (ETs) regulate a wide variety of biological effects, including noradrenergic transmission and in particular neuronal NE uptake. Diverse reports, including studies from our laboratory, show that ETs differentially modulate the activity and expression of NET not only in normal conditions but also in diverse cardiovascular diseases such as congestive heart failure and hypertension. Current literature supports a key role for the interaction between ETs and NE in maintaining neurotransmission homeostasis and further suggests that this interaction may represent a potential therapeutic target for various diseases, particularly hypertension.

Enhanced assymetrical noradrenergic transmission in the olfactory bulb of deoxycorticosterone acetate-salt hypertensive rats.

The ablation of olfactory bulb induces critical changes in dopamine, and monoamine oxidase activity in the brain stem. Growing evidence supports the participation of this telencephalic region in the regulation blood pressure and cardiovascular activity but little is known about its contribution to hypertension. We have previously reported that in the olfactory bulb of normotensive rats endothelins enhance noradrenergic activity by increasing tyrosine hydroxylase activity and norepinephrine release. In the present study we sought to establish the status of noradrenergic activity in the olfactory bulb of deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Different steps in norepinephrine transmission including tyrosine hydroxylase activity, neuronal norepinephrine release and uptake were assessed in the left and right olfactory bulb of DOCA-salt hypertensive rats. Increased tyrosine hydroxylase activity, and decreased neuronal norepinephrine uptake were observed in the olfactory bulb of DOCA-salt hypertensive rats. Furthermore the expression of tyrosine hydroxylase and its phosphorylated forms were also augmented. Intriguingly, asymmetrical responses between the right and left olfactory bulb of normotensive and hypertensive rats were observed. Neuronal norepinephrine release was increased in the right but not in the left olfactory bulb of DOCA-salt hypertensive rats, whereas non asymmetrical differences were observed in normotensive animals. Present findings indicate that the olfactory bulb of hypertensive rats show an asymmetrical increase in norepinephrine activity. The observed changes in noradrenergic transmission may likely contribute to the onset and/or progression of hypertension in this animal model.

Involvement of membrane tubulin in erythrocyte deformability and blood pressure.

OBJECTIVE: To test the hypothesis that erythrocyte deformability is influenced by changes in the content of membrane tubulin (Mem-tub). METHODS AND RESULTS: Human erythrocytes contain tubulin distributed in three pools (membrane, sedimentable, soluble). Erythrocytes from hypertensive humans have a higher proportion of Mem-tub. Increased Mem-tub content in hypertensive patients was correlated with decreased erythrocyte deformability. Treatment of erythrocytes from normotensive individuals with taxol increased Mem-tub content and reduced deformability, whereas treatment of hypertensive patients erythrocytes with nocodazole had the opposite effect. In-vivo experiments with rats were performed to examine the possible relationship between Mem-tub content, erythrocyte deformability, and blood pressure. Spontaneously hypertensive rats (SHRs) showed lower erythrocyte deformability than normotensive Wistar rats. During the development of hypertension in SHR, tubulin in erythrocytes is translocated to the membrane, and this process is correlated with decreased deformability. In-vivo treatment (intraperitoneal injection) of SHR with nocodazole decreased Mem-tub content, increased erythrocyte deformability, and decreased blood pressure, whereas treatment of Wistar rats with taxol had the opposite effects. CONCLUSION: These findings indicate that increased Mem-tub content contributes to reduced erythrocyte deformability in hypertensive animals.

Endothelin-1 and -3 modulate the neuronal norepinephrine transporter through multiple signalling pathways in the rat posterior hypothalamus.

We have previously reported that endothelin-1 and -3 modulate different steps of noradrenergic transmission in the hypothalamus. We showed that endothelins modify neuronal norepinephrine transport activity through the regulation of the kinetic constant and internalization. In the present work we sought to define the endothelin receptors and intracellular mechanisms involved in the down-regulation of neuronal norepinephrine uptake induced by endothelin-1 and -3 in the rat posterior hypothalamic region. Results showed that endothelin-1 reduced norepinephrine uptake through ET(B) receptors, whereas endothelin-3 through a non-conventional or atypical endothelin receptor. In both cases, the effect on norepinephrine uptake was coupled to protein kinase A and C as well as nitric oxide pathways. However, neither protein kinase G nor intracellular or extracellular calcium and calcium/calmodulin-dependent protein kinase II were involved. In addition, the same intracellular mechanisms participated in the reduction of nisoxetine binding (norepinephrine transporter internalization index) induced by both endothelins. Present findings reveal the underlying mechanisms involved in the regulation of the neuronal norepinephrine transporter by endothelins and further support the role of these peptides in the modulation of noradrenergic transmission at the presynaptic nerve endings in the posterior hypothalamus.

Regulation of the neuronal norepinephrine transporter by endothelin-1 and -3 in the rat anterior and posterior hypothalamus.

We previously reported that endothelin-1 and endothelin-3 modulate norepinephrine neuronal release and tyrosine hydroxylase activity and expression in the hypothalamus. In the present study we sought to establish the role of endothelin-1 and -3 in the regulation of norepinephrine uptake in the anterior and posterior hypothalamus. Results showed that in the anterior hypothalamus endothelin-3 increased neuronal norepinephrine uptake whereas endothelin-1 decreased it. Conversely, in the posterior hypothalamic region both endothelins diminished the neuronal uptake of the amine. Endothelins response was concentration dependent and maintained at all studied times. Endothelins also modified the kinetic and internalization of the NE neuronal transporter. In the anterior hypothalamic region endothelin-3 increased the V(max) and the B(max) whereas endothelin-1 decreased them. However, in the posterior hypothalamic region both endothelins diminished the V(max) as well as B(max). Neither endothelin-1 nor endothelin-3 modified neuronal norepinephrine transporter K(d) in the studied hypothalamic regions. These findings support that in the posterior hypothalamic region both endothelins diminished neuronal norepinephrine transporter activity by reducing the amine transporter expression on the plasmatic membrane. Conversely, in the anterior hypothalamic region endothelin-3 enhanced neuronal norepinephrine transporter activity by increasing the expression of the transporter on the presynaptic membrane, whereas endothelin-1 induced the opposite effect. Present results permit us to conclude that both endothelins play an important role in the regulation of norepinephrine neurotransmission at the presynaptic nerve endings in the hypothalamus.

Short-term regulation of tyrosine hydroxylase activity and expression by endothelin-1 and endothelin-3 in the rat posterior hypothalamus.

Brain catecholamines are involved in several biological functions regulated by the hypothalamus. We have previously reported that endothelin-1 and -3 (ET-1 and ET-3) modulate norepinephrine release in the anterior and posterior hypothalamus. As tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis, the aim of the present work was to investigate the effects of ET-1 and ET-3 on TH activity, total enzyme level and the phosphorylated forms of TH in the rat posterior hypothalamus. Results showed that ET-1 and ET-3 diminished TH activity but the response was abolished by both selective ET(A) and ET(B) antagonists (BQ-610 and BQ-788, respectively). In addition ET(A) and ET(B) selective agonists (sarafotoxin S6b and IRL-1620, respectively) failed to affect TH activity. In order to investigate the intracellular signaling coupled to endothelins (ETs) response, nitric oxide (NO), phosphoinositide, cAMP/PKA and CaMK-II pathways were studied. Results showed that N(omega)-nitro-l-arginine methyl ester and 7-nitroindazole (NO synthase and neuronal NO synthase inhibitors, respectively), 1H-[1,2,4]-oxadiazolo[4,3-alpha]quinozalin-1-one and KT-5823 (soluble guanylyl cyclase, and PKG inhibitors, respectively) inhibited ETs effect on TH activity. Further, sodium nitroprusside and 8-bromoguanosine-3',5'-cyclic monophosphate (NO donor and cGMP analog, respectively) mimicked ETs response. ETs-induced reduction of TH activity was not affected by a PKA inhibitor but it was abolished by PLC, PKC and CaMK-II inhibitors as well as by an IP(3) receptor antagonist. On the other hand, both ETs did not modify TH total level but reduced the phosphorylation of serine residues of the enzyme at positions 19, 31 and 40. Present results suggest that ET-1 and ET-3 diminished TH activity through an atypical ET or ET(C) receptor coupled to the NO/cGMP/PKG, phosphoinositide and CaMK-II pathways. Furthermore, TH diminished activity may result from the reduction of the phosphorylated sites of the enzyme without changes in its total level. Taken jointly present and previous results support that ET-1 and ET-3 may play a relevant role in the modulation of catecholaminergic neurotransmission in the posterior hypothalamus of the rat.

Involvement of nitric oxide pathways in short term modulation of tyrosine hydroxylase activity by endothelins 1 and 3 in the rat anterior hypothalamus.

The ability of endothelins 1 and 3 (ET-1 and ET-3) to reduce neuronal norepinephrine release through ETB receptor activation involving nitric oxide (NO) pathways in the rat anterior hypothalamus region (AHR) was previously reported. In the present work, we studied the effects of ET-1 and -3 on tyrosine hydroxylase (TH) activity and the possible involvement of NO pathways. Results showed that ET-1 and -3 (10 nM) diminished TH activity in AHR and this effect was blocked by a selective ETB receptor antagonist (100 nM BQ-788), but not by a ET(A) receptor antagonist (BQ-610). To confirm these results, 1 microM IRL-1620 (ET(B) agonist) reduced TH activity whereas 300 nM sarafotoxin S6b falled to modify it. N(omega)-Nitro-L-arginine methyl ester (10 microM), 7-nitroindazole (10 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-ona (10 microM), KT5823 (2 microM), inhibitors of nitric oxide synthase, neuronal nitric oxide synthase, NO-sensitive-guanylyl cyclase, and protein kinase G, respectively, did not modify the reduction of TH activity produced by ETs. In addition, both 100 microM sodium nitroprusside and 50 microM 8-bromoguanosine-3',5'-cyclic monophosphate (NO donor and guanosine-3',5'-cyclic monophosphate analog, respectively) diminished TH activity. Present results showed that ET-1 and ET-3 diminished TH activity through the activation of ET(B) receptors involving the NO/guanosine-3',5'-cyclic monophosphate/protein kinase G pathway. Taken jointly present and previous results it can be concluded that both ETs play an important role as modulators of norepinephrine neurotransmission in the rat AHR.