Study of the interaction between ATP and glutamate in neurons of the paraventricular nucleus of the hypothalamus and its relationship with the sympathoexcitatory response induced by changes in osmolarity.

Neste trabalho investigamos a interação entre ATP-glutamato na modulação de potenciais de ação e atividade sináptica de neurônios PVN-RVLM, além de avaliar se esta interação induziria mudanças na atividade simpática lombar (ANSL) por estímulo osmótico. Utilizamos de técnicas de imunohistoquímica, whole-cell patch clamp e registro eletroneurográfico. Observou-se que o ATP aumenta a frequência de potenciais de ação em neurônios PVN-RVLM, efeito bloqueado por acido quinurênico (KYN) e PPADS. A injeção de ATP no PVN aumenta a ANSL (25 nmol: 72%), um efeito atenuado por PPADS e/ou KYN, e também por CNQX. O ATP não afeta a função sináptica, mas aumenta correntes glutamatérgicas induzidas por aplicação AMPA em 52%, a qual foi bloqueada por PPADS ou por quelação de Ca2+ intracelular. Além disso, o estímulo osmótico ativa neurônios do PVN que expressam receptores P2X2 e potencia as correntes mediadas por AMPA (53%), um efeito bloqueado por PPADS. Finalmente, demonstrou-se que receptores P2 no PVN são importantes na simpatoexcitação induzida por estímulo osmótico agudo.In the present study we investigate the interaction of ATP-glutamate on the firing activity and synaptic function in PVN-RVLM neurons, besides whether that interaction would be translated in changes on sympathetic nerve activity (SNA) induced by osmotic stimulus. Immunohistochemistry, whole-cell patch clamp and electroneurography technical approaches were used. Our data have shown that ATP increases firing rate of PVN-RVLM neurons, an effect blocked by kynurenic acid (KYN) or PPADS. ATP injection into the PVN enhanced SNA (72%), which was attenuated by PPADS and/or KYN, or CNQX. ATP did not affect synaptic function but, glutamatergic currents evoked by AMPA application were augmented with ATP (AMPA area: 52%), blocked by PPADS and chelation of intracellular Ca2+. In addition, we observed that acute osmotic stimulus activates P2X2 expressing neurons in the PVN. Moreover, an osmotic challenge potentiated AMPA responses (53%), an effect blocked by PPADS. Finally, we demonstrated that P2 receptors in the PVN are important for osmotically-driven sympathoexcitation

Role of kidneys in ouabain-induced hypertension rats.

A Na+K+-ATPase (NKA) é uma proteína de membrana que participa de mecanismos de transporte nos túbulos renais para a reabsorção de sódio e outros substratos. Sabe-se que a administração de ouabaína (OUA), um inibidor da NKA, induz hipertensão arterial em ratos. No entanto, o papel dos rins nesse modelo de hipertensão não está bem elucidado. Desta forma, o objetivo do presente estudo foi avaliar as possíveis alterações na função renal induzidas pelo tratamento crônico com OUA por 5 ou 20 semanas. Sendo assim, foi observado que o tratamento com OUA promoveu hipertensão de mesma magnitude nos dois grupos avaliados. Além disso, a administração de OUA induziu o aumento da ingestão de água, do fluxo urinário e da expressão protéica da isoforma 1 da NKA. Porém, não foi capaz de alterar de maneira significativa o ritmo de filtração glomerular, assim como a fração de excreção de Na+ e K+. Pode-se concluir que, o tratamento crônico com OUA induz hipertensão, porém parece que os rins não contribuem de forma importante para o processo hipertensivo neste modelo de hipertensão.Na+K+-ATPase (NKA) is an integral membrane protein that participates in transport mechanisms along renal tubules for sodium reabsorption and other substrates. Its known that ouabain (OUA) administration, a NKA inhibitor, induces hypertension in Wistar rats. However, the role of kidneys in this model of hypertension is not elucidated. So, the aim of this study was to evaluate the possibles alterations in renal function induced by chronic treatment with OUA by 5 or 20 weeks. Chronic treatment with OUA induced hypertension in a similar magnitude in both experimental groups. Moreover, OUA administration was able to increase water intake, urinary flow, and protein expression of 1 isoform of NKA. However, OUA treatment did not alter significantly the glomerular filtration rate, likewise the fractional excretion of Na+ and K+. In summary, chronic OUA treatment induces mild hypertension independent of the period of administration, but the kidneys dont play an important role in the hypertensive process in this model of hypertension

Purinergic and glutamatergic interactions in the hypothalamic paraventricular nucleus modulate sympathetic outflow

P2X receptors are expressed on ventrolateral medulla projecting paraventricular nucleus (PVN) neurons. Here, we investigate the role of adenosine 5′-triphosphate (ATP) in modulating sympathetic nerve activity (SNA) at the level of the PVN. We used an in situ arterially perfused rat preparation to determine the effect of P2 receptor activation and the putative interaction between purinergic and glutamatergic neurotransmitter systems within the PVN on lumbar SNA (LSNA). Unilateral microinjection of ATP into the PVN induced a dose-related increase in the LSNA (1 nmol: 38 ± 6 %, 2.5 nmol: 72 ± 7 %, 5 nmol: 96 ± 13 %). This increase was significantly attenuated by blockade of P2 receptors (pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid, PPADS) and glutamate receptors (kynurenic acid, KYN) or a combination of both. The increase in LSNA elicited by L-glutamate microinjection into the PVN was not affected by a previous injection of PPADS. Selective blockade of non-N-methyl-D-aspartate receptors (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, CNQX), but not N-methyl-D-aspartate receptors (NMDA) receptors (DL-2-amino-5-phosphonopentanoic acid, AP5), attenuated the ATP-induced sympathoexcitatory effects at the PVN level. Taken together, our data show that purinergic neurotransmission within the PVN is involved in the control of SNA via P2 receptor activation. Moreover, we show an interaction between P2 receptors and non-NMDA glutamate receptors in the PVN suggesting that these functional interactions might be important in the regulation of sympathetic outflowSao Paulo Research Foundation (FAPESP, 07/04085-0)Sao Paulo Research Foundation (FAPESP, 10/17997-0)FAPESP, 10/05037-

A Short-Day Photoperiod Delays the Timing of Puberty in Female Mice via Changes in the Kisspeptin System

The reproduction of seasonal breeders is modulated by exposure to light in an interval of 24 h defined as photoperiod. The interruption of reproductive functions in seasonally breeding rodents is accompanied by the suppression of the Kiss1 gene expression, which is known to be essential for reproduction. In non-seasonal male rodents, such as rats and mice, short-day photoperiod (SP) conditions or exogenous melatonin treatment also have anti-gonadotropic effects; however, whether photoperiod is able to modulate the puberty onset or Kiss1 gene expression in mice is unknown. In the present study, we investigated whether photoperiodism influences the sexual maturation of female mice via changes in the kisspeptin system. We observed that SP condition delayed the timing of puberty in female mice, decreased the hypothalamic expression of genes related to the reproductive axis and reduced the number of Kiss1-expressing neurons in the rostral hypothalamus. However, SP also reduced the body weight gain during development and affected the expression of neuropeptides involved in the energy balance regulation. When body weight was recovered via a reduction in litter size, the timing of puberty in mice born and raised in SP was advanced and the effects in hypothalamic mRNA expression were reverted. These results suggest that the SP delays the timing of puberty in female mice via changes in the kisspeptin system, although the effects on hypothalamic–pituitary–gonadal axis are likely secondary to changes in body weight gain

Commissural nucleus of the solitary tract regulates the antihypertensive effects elicited by moxonidine

The rostral ventrolateral medulla (RVLM) contains the presympathetic neurons involved in cardiovascular regulation that has been implicated as one of the most important central sites for the antihypertensive action of moxonidine (an α2-adrenergic and imidazoline agonist). Here, we sought to evaluate the cardiovascular effects produced by moxonidine injected into another important brainstem site, the commissural nucleus of the solitary tract (commNTS). Mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic nerve activity (sSNA) and activity of putative sympathoexcitatory vasomotor neurons of the RVLM were recorded in conscious or urethane-anesthetized, and artificial ventilated male Wistar rats. In conscious or anesthetized rats, moxonidine (2.5 and 5 nmol/50 nl) injected into the commNTS reduced MAP, HR and sSNA. The injection of moxonidine into the commNTS also elicited a reduction of 28% in the activity of sympathoexcitatory vasomotor neurons of the RVLM. To further assess the notion that moxonidine could act in another brainstem area to elicit the antihypertensive effects, a group with electrolytic lesions of the commNTS or sham and with stainless steel guide-cannulas implanted into the 4th V were used. In the sham group, moxonidine (20 nmol/1 μl) injected into 4th V decreased MAP and HR. The hypotension but not the bradycardia produced by moxonidine into the 4th V was reduced in acute (1 day) commNTS-lesioned rats. These data suggest that moxonidine can certainly act in other brainstem regions, such as commNTS to produce its beneficial therapeutic effects, such as hypotension and reduction in sympathetic nerve activity.Sao Paulo State Foundation (FAPESP - 11/11934-9)Sao Paulo State Foundation (FAPESP - 10/09776-3)Sao Paulo State Foundation (FAPESP - 09/54888-7)Sao Paulo State Foundation (FAPESP - 2011/50770-1)Sao Paulo State Foundation (FAPESP - 10/19336-0