Role of astrocytes and ectonucleotidases in purinergic signaling of the paraventricular nucleus of hypothalamus in salt-induced hypertension.

Author(s)

Renato Willian Martins Sá

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2020-12-14

Examining board members

Vagner Roberto Antunes; Hildebrando Candido Ferreira Neto; Lisete Compagno Michelini; Davi José de Almeida Moraes

• Advisor: Vagner Roberto Antunes

Abstract

In the present study we investigated whether salt-load (SS) stimulates the release of ATP in the PVN environment, and which mechanism would be responsible for this phenomenon. Two hypotheses have been raised: (1) a higher content of ATP in PVN during SS is a result of a failure of ectonucleotidases to metabolize ATP; and (2) SS can stimulate PVN astrocytes to release ATP. Male Wistar rats 290-350 g (n = 12) were used for in vivo studies and Sprague Dawley rats 290-350 g (n = 41) for in vitro. Animals were divided in 2 group: 1) SS: group that received hypertonic saline solution (2% NaCl) in replacement of tap water for 4 or 7 days, and 2) control group. A radiotelemetry system was used to measure the progression of hypertension in SS rats over the seven days of salt loading. mRNA expression of ecto-nucleotide triphosphate diphosphohydrolase (E-NTPD) 1, 2 and 3 and ecto-5\'-nucleotidase (E-5\'-NT) in PVN was analyzed by RT-qPCR. The release of ATP, adenosine and the activity of ectonucleotidases were measured via amperometric biosensors overlaid on the PVN region of hypothalamic slices. The function of PVN astrocytes was assessed by interference with the AVV-sGFAP-dnSNARE-EGFP vector. Mean arterial pressure (MAP) of SS rats increased progressively from day 2 to 7 [MAP: 97 ± 4 mmHg (day 0) vs 129 ± 4 mmHg (day 7), p<0.0001]. The power of the LF component increased progressively from day 0 to day 7 in SS rats [LF: 1.21 ± 0.22 mmHg2 (day 0) vs 5.01 ± 0.96 mmHg2 (day 6), p<0.0001]. E-NTPD3 mRNA expression was higher in the SS group than in control [0.99 ± 0.5 &Delta;&Delta;Ct (control) vs. 3.09 ± 0.5&Delta;&Delta;Ct (SS) p = 0.0247]. SS rats showed an increase in ATP release [1.13 ± 0.8 &mu;M (control) vs. 19.16 ± 6.9 &mu;M (SS), p<0,0001], but not in adenosine [2.15 ± 0.9 &mu;M (control) vs. 1.19 ± 0.6 &mu;M (SS)]. The activity of ectonucleotidases was tested in vitro by quantifying the maximum production of adenosine in response to exogenous application of ATP (50&mu;M). No difference was detected when comparing the responses between the groups. PVN glial fibrillar glial protein (GFAP) immunofluorescence intensity was higher in SS rats than in control [6.9 ± 1.3 a.u. (control) vs 27.2 ± 6.2 a.u. (SS)]. PVN unilateral transfection with dn-SNARE vector decreased the release of ATP in SS rats when compared to the non-transfected side as a control [13.88 ± 0.07 &mu;M (non-transfected side) vs. 6.64 ± 1.64 &mu;M (transfected side). In conclusion, our results showed that SS gradually increases the BP as well as the sympathetic influence on the cardiovascular system and stimulates the glial release of ATP in the PVN by exocytosis. (AU)

• FAPESP's process: 16/03359-8 - Role of glia and ectonucleotidases in the purinergic signaling of the paraventricular nucleus of the hypothalamus in salt-induced hypertension
• Grantee: Renato Willian Martins Sá
• Support Opportunities: Scholarships in Brazil - Doctorate