Characterization of a functional (pro)renin receptor in rat brain neurons

Abstract

(Pro)renin receptor (PRR), the newest member of the renin-angiotensin system (RAS), is turning out to be an important player in the regulation of the cardiovascular system. It plays a pivotal role in activation of the local RAS and stimulates signalling pathways involved in proliferative and hypertrophic mechanisms. However, the role of PRR in the brain remains unknown. Thus, our objective in this study was to determine whether a functional PRR is present in neurons within the brain. Neuronal co-cultures from the hypothalamus and brainstem areas of neonatal rat brain express PRR mRNA. Immunoreactivity for PRR was primarily localized on the neuronal cell soma and in discrete areas in the neurites. Treatment of neurons with renin, in the presence of 2 microm losartan, caused a time- and dose-dependent stimulation of phosphorylation of extracellular signal related kinase ERK1 (p44) and ERK2 (p42) isoforms of mitogen-activated protein kinase. Optimal stimulation of fourfold was observed within 2 min with 20 nm renin. Electrophysiological recordings showed that treatment of the neurons with renin, in the presence of 2 microm losartan, resulted in a steady and stable decrease in action potential frequency. A 46% decrease in action potential frequency was observed within 5 min of treatment and was attenuated by co-incubation with a PRR blocking peptide. These observations demonstrate that the PRR is present in neurons within the brain and that its activation by renin initiates the MAP kinase signalling pathway and inhibition of neuronal activity.

Figure 1. Pro(renin) receptor expression in the WKY rat brain neuronal cultures

A, PRR mRNA in neuronal and astroglial cultures from WKY rat brains. Neuronal and astroglial cells were established in primary culture as described in the Methods. Cells were collected, RNA isolated and subjected to quantitative real-time RT-PCR as described in the Methods. Data are means + S.E.M., n = 9 for neuronal cultures and 6 for astroglial cultures. ^*P < 0.001 versus astroglial cultures. B, localization of PRR immunoreactivity in neuronal cultures. Neuronal cultures from WKY rat brains were fixed and subjected to the immunocytochemical protocol described in the Methods with the use of anti-PRR and -NeuN antibodies. Cultures incubated without primary antibodies (Ba, b and c) were used as controls. Phase contrast (Ba), green (Bb) and red fluorescence (Bc) examination of control cultures. The PRR and NeuN staining is shown at lower (Bd and e) and higher magnifications (Bg and h), while Bf and i represent merged images. Arrows in Bg indicate localization of PRR immunoreactivity on cell body and neuronal processes (contrast was adjusted to enhance immunostained clusters). Scale bar represents 50 μm in Ba, b and c, 25 μm in Bd, e and f, and 10 μm Bg, h and i.

Figure 2. Effects of renin on ERK1/2 phosphorylation in neuronal cultures

A, dose–response relationship. Neuronal cultures, in triplicate, were incubated with the indicated concentrations of renin in the presence of 2 μM losartan for 2 min at 37°C. Proteins were isolated and subjected to 10% SDS-PAGE separation and Western blotting with the use of ERK1/2 and phosphorylated ERK1/2 antibodies as described in the Methods. Top left panel is a representative Western blot showing phosphorylated (p) ERK in each set of treatment conditions. Top right panel is a representative Western blot showing total ERK in each set of treatment conditions. Bottom panel shows quantification of phosphorylated bands that have been normalized with total ERK1/2 and compared with control, normalized to unity. Data are means + S.E.M. (n = 3), ^*P < 0.05 compared with control. B, time course. Neuronal cultures were incubated with 20 nM recombinant human renin in the presence of 2 μM losartan for the indicated time periods. Phosphorylated levels of ERK1/2 were quantified as described in A. Top left panel is a representative Western blot showing p-ERK in each set of treatment conditions. Top right panel is a representative Western blot showing total ERK in each set of treatment conditions. Bottom panel shows quantification of phosphorylated ERK1/2. Data are means + S.E.M. (n = 3). ^*P < 0.05 compared with control.

Figure 3. Effect of renin on APF in WKY rat brain neurons

Representative traces of bridge-mode recordings in neurons performed after 10 min equilibration between the cell cytosol and pipette solution. A, control cells depicting firing rate in basal conditions and 15 min after establishing basal. B, renin (20 nM)-treated cells showing basal firing, trace at 10 min in the presence of 2 μM losartan and trace with renin and losartan co-treatment (5 min). C, bar graphs comparing APF levels in neuronal recordings, from control neurons (left) compared with treated neurons (right) that were subjected to the indicated treatments. Means + S.E.M.; n = 6, ^*P < 0.01 versus control. D, expansion of the representative traces shown in A and B to compare changes in resting membrane potential from baseline in control neurons (left) and in losartan plus renin-treated neurons (right). E, representative trace recordings (left) and bar graph (right) showing change in resting membrane potential after hyperpolarizing pulse administration in neurons before and after renin treatment during current-clamp recordings. ΔRMP, change in resting membrane potential. Means + S.E.M.; n = 5, ^*P < 0.05 versus control.

Figure 4. Effect of PRR inhibitor peptide on renin-induced decrease in APF

Representative traces of bridge-mode recordings performed after 10 min equilibration with pipette solution in control neurons (A) and PRRB-treated neurons (C). In both cases, neurons in the presence of 2 μM losartan were screened for the inhibitory effect of renin (20 nM). Control neurons were subjected to renin washout with recording buffer for 30 min, while the treatment group had a 10 min washout, followed by a 20 min treatment with 10 μM PRRB to determine whether the inhibitory peptide would block the effect of renin. Bar graphs are shown derived from bridge-mode recordings in neurons, representing APF levels for the indicated treatments, for the control (B) and PRRB-treated groups (D). Means + S.E.M. (n = 9). ^*P < 0.01 versus basal.