Sandra I. Hope

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.

Hypothalamic Noradrenergic Hyperactivity and Detrimental Bone Status in an Animal Model of Nutritional Growth Retardation

We have studied hypothalamic noradrenergic activity in relation with bone status in a nutritional growth retar- dation model (ND). Control rats (C) were fed ad libitum. ND received 80% of the diet consumed by C for 4 weeks and later refed ad libitum for 8 weeks. Food restriction induced detrimental effects on body and femur weight and length (P<0.05) and bone biomechanical properties (P<0.001). Thickness of proliferative and hypertrophic zone (�m) of growth plate cartilage and bone volume (%, mean±SE) were 225.96±5.70 v. 280.70±12.52, 95.16±5.81 v. 134.60±9.30, 17.64±3.23 v. 26.80±2.03, respectively (P<0.05); anterior and posterior hypothalamus norepinephrine uptake and release and tyrosine hydroxylase activity (% of control) were 79.05±3.56, 67.00±10.00, 164.26±16.58 and 80.65±5.92, 147.00±1.00, 152.42±9.30, respectively (P<0.05). Thus, impaired biomechanical bone performance in ND could be due, in part, to the increased hypothalamic noradrenergic activity in response to restriction. Normalization of parameters with refeeding suggests no long-term side-effects in undernourished rats.

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.

Regulation of the Norepinephrine Transporter by Endothelins

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.

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

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.

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.

Altered expression of Aquaporin-2 in one-kidney, one-clip hypertension

Aims: The aim of the present study was to evaluate the regulation of Aquaporin‐2 (AQP2) water channel in the kidney of one‐kidney, one‐clip rats (Goldblatt‐1 model). In addition, some mechanisms that underlie the role of AQP2 in the Goldblatt‐1 model were evaluated. Main methods: Sprague‐Dawley rats were divided in three groups: control two‐kidney, no clip (C, 2 K‐NC); nephrectomized one‐kidney, no clip (N, 1 K‐NC) and Goldblatt one‐kidney, one‐clip (G, 1 K‐1C). AQP2 expression (by westernblot, real time PCR, immunohistochemistry and immunofluorescence), vasopressin V2 receptor expression (by real time PCR), cAMP concentration, NFkB and TonEBP (cytosol to nucleus ratio) were evaluated in the renal medulla. Key findings: AQP2 expression, V2 receptor expression and cAMP concentration were decreased in the renal medulla of 1 K‐1C rats, NFkB translocation was favoured towards the nucleus suggesting its activation while TonEBP translocation was not altered in this model of hypertension. Significance: In this model of hypertension the decrease of AQP2 expression could be a mechanism that counteracts the high blood pressure promoting water excretion and this may be consequence of decreased vasopressin sensitivity and/or the increased activity of NFkB at renomedullary collecting duct level. Given that renovascular hypertension is among the most common causes of secondary hypertension, it is important to elucidate all the relevant mechanisms involved in the generation or in the compensation of the hypertensive state in order to improve the diagnoses and treatment of the patients.