Role of phosphatidylinositol 3-kinase in angiotensin II regulation of norepinephrine neuromodulation in brain neurons of the spontaneously hypertensive rat - PubMed (original) (raw)

Role of phosphatidylinositol 3-kinase in angiotensin II regulation of norepinephrine neuromodulation in brain neurons of the spontaneously hypertensive rat

H Yang et al. J Neurosci. 1999.

Abstract

Chronic stimulation of norepinephrine (NE) neuromodulation by angiotensin II (Ang II) involves activation of the Ras-Raf-MAP kinase signal transduction pathway in Wistar Kyoto (WKY) rat brain neurons. This pathway is only partially responsible for this heightened action of Ang II in the spontaneously hypertensive rat (SHR) brain neurons. In this study, we demonstrate that the MAP kinase-independent signaling pathway in the SHR neuron involves activation of PI3-kinase and protein kinase B (PKB/Akt). Ang II stimulated PI3-kinase activity in both WKY and SHR brain neurons and was accompanied by its translocation from the cytoplasmic to the nuclear compartment. Although the magnitude of stimulation by Ang II was comparable, the stimulation was more persistent in the SHR neuron compared with the WKY rat neuron. Inhibition of PI3-kinase had no significant effect in the WKY rat neuron. However, it caused a 40-50% attenuation of the Ang II-induced increase in norepinephrine transporter (NET) and tyrosine hydroxylase (TH) mRNAs and [3H]-NE uptake in the SHR neuron. In contrast, inhibition of MAP kinase completely attenuated Ang II stimulation of NET and TH mRNA levels in the WKY rat neuron, whereas it caused only a 45% decrease in the SHR neuron. However, an additive attenuation was observed when both kinases of the SHR neurons were inhibited. Ang II also stimulated PKB/Akt activity in both WKY and SHR neurons. This stimulation was 30% higher and lasted longer in the SHR neuron compared with the WKY rat neuron. In conclusion, these observations demonstrate an exclusive involvement of PI3-kinase-PKB-dependent signaling pathway in a heightened NE neuromodulatory action of Ang II in the SHR neuron. Thus, this study offers an excellent potential for the development of new therapies for the treatment of centrally mediated hypertension.

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Figures

Fig. 1.

A, Effect of Ang II on PI3-kinase activity in WKY and SHR brain neurons. Time course: neuronal cultures of WKY and SHR were incubated with 100 n

Ang II for the indicated time periods. PI3 kinase activity was measured after immunoprecipitation with the PI3-kinase p85 subunit-specific antibody essentially as described in Materials and Methods. Top, Representative autoradiogram of a TLC plate. Bottom, Quantitation of radioactive spots corresponding to PIP, mean ± SE (n = 3 experiments). * Significantly different from zero time (p < 0.01). # Significantly different from Ang II-treated WKY rat neuron (p < 0.01). B, Effect of Ang receptor antagonists. WKY and SHR neuronal cultures were incubated without (Control) or with (2, 4, 6) 100 n

Ang II for 10 min in the absence or presence of 10 μ

losartan (Los) or PD 123319 (PD). PI3-kinase activity was determined as described in Materials and Methods. Top, Representative autoradiogram. Bottom, Quantitation of radioactive spot, mean ± SE (n = 3). * Significantly different from control (p < 0.0l). C, Ang II concentration. Incubation and assay conditions with indicated concentration of Ang II for 10 min were essentially as described in_A_. Top, Representative autoradiogram.Bottom, Data from three experiments ± SE. * Significantly different from no Ang II (p< 0.05).

Fig. 2.

Effect of Ang II on PI3-kinase activity in hypothalami of adult WKY rats and SHRs. WKY rats and SHRs were cannulated and subjected to Ang II injection and isolation of hypothalamic blocks. Tissues were homogenized and used for the measurement of PI3-kinase activity as described in Materials and Methods. Top, Representative autoradiogram.Bottom, Quantitation of PIP spots from three animals. Data are mean ± SE (n = 3). * Significantly different from zero time (p < 0.01).

Fig. 3.

Effect of Ang II on immunoreactive PI3-kinase in WKY and SHR brain neurons (in vitro) and in hypothalami (in vivo). Lysates from neuronal cultures and hypothalamic blocks of adult WKY and SHR neurons were prepared, and PI3-kinase immunoreactivity was precipitated with the use of p85 PI3-kinase-specific antibody. Western blotting was performed as described in Materials and Methods.

Fig. 4.

Ang II-induced nuclear translocation of PI3-kinase in WKY and SHR neurons. Neuronal cultures of WKY rats and SHRs were incubated without or with 100 n

Ang II for indicated time periods. Cytoplasmic and nuclear fractions were isolated and subjected to Western analysis as described in Materials and Methods: (A) cytoplasmic fractions and (B) nuclear fractions. Whole-cell lysates from control and Ang II-treated cells were also run in separate experiments to determine levels of the enzyme. A, B,Top, Representative autoradiograms;bottom, quantitation of immunoreactive bands. Mean ± SE (n = 3). * Significantly different from control (p < 0.05).

Fig. 5.

Effect of Wortmannin on Ang II stimulation of PI3-kinase activity in WKY and SHR neurons. Neuronal cultures of WKY rats and SHRs were incubated with 100 n

Ang II for 15 min in the absence and presence of 100 n

Wortmannin. PI3-kinase activity was determined as described in Materials and Methods. A, Representative autoradiogram.B, Mean ± SE (n = 3). * Significantly different from untreated cells (p < 0.01). ** Significantly different from Ang II-treated cells (p < 0.01).

Fig. 6.

Neuronal cultures of WKY rats and SHRs were incubated with 100 n

Ang II in the absence or presence of 100 n

Wortmannin for 4 hr at 37°C. Levels of NET mRNA (A), TH mRNA (B), and [3H]-NE uptake (C) were measured essentially as described in Materials and Methods. A, B, Top, Representative autoradiograms; bottom, mean data from three experiments ± SE. * Significantly different from control (p < 0.01). ** Significantly different from Ang II-treated neurons (p < 0.05). # Significantly different from Ang II-treated WKY rat neurons (p < 0.01).

Fig. 7.

Effects of Wortmannin and PD 98059 on NE neuromodulation in WKY and SHR neurons. WKY rat and SHR neurons were treated without or with 100 n

Ang II (AngII) for 4 hr at 37°C in the absence or presence of 100 n

Wortmannin (Wort) or 50 μ

PD 98059 (PD). mRNA levels for NET (A) and TH (B) were measured essentially as described in Materials and Methods.Top, Representative autoradiograms;bottom, mean ± SE (n = 3). * Significantly different from control (p< 0.01). ** Significantly different from Ang II-treated neurons (p < 0.05). # Significantly different from Ang II-treated WKY rat neurons (p < 0.01).

Fig. 8.

Effect of Wortmannin on Ang II stimulation of AP-1 binding activity in the nuclear fractions of WKY and SHR neurons. Nuclear extracts of control, Ang II-treated (100 n

) or Ang II + Wortmannin-treated WKY rat and SHR neurons were subjected to gel shift analysis to determine the levels of AP-1 binding activity essentially as described in Materials and Methods.

Fig. 9.

Effect of Ang II on protein kinase B activity in WKY and SHR neurons. A, Time course: neuronal culture of WKY rat and SHR were incubated with 100 n

Ang II for the indicated time at 37°C. PKB activity in neuronal extract was determined essentially as described in Materials and Methods.Top, Representative autoradiogram;bottom, mean ± SE (n = 3). * Significantly different from zero time (p< 0.05). # Significantly different from the WKY (p < 0.05). B, Ang II concentrations: neuronal cultures of WKY rat and SHR were incubated with the indicated concentrations of Ang II for 10 min at 37°C before determination of PKB activity. Top, Representative autoradiogram; bottom, mean ± SE (n = 3). * Significantly different from control (p < 0.05). # Significantly different from the WKY (p < 0.05). C, Effect of Ang receptor antagonists: experimental conditions were essentially as described in the legend to Figure10_B_. WKY rat and SHR neuronal cultures were incubated without (C) or with 100 n

Ang II in the absence or presence of 10 μ

losartan (Los) or PD123319 (PD).Top, Representative autoradiogram;bottom, mean ± SE (n = 3). * Significantly different from control (p< 0.05). ** Significantly different from Ang II treatment (p < 0.05).

Fig. 10.

Effects of PD 98059 and Wortmannin on PKB activity in WKY and SHR neurons. WKY rat and SHR neurons were treated without or with 100 n

Ang II (AngII) for 10 min at 37°C in the absence or presence of 50 μ

PD 98059 (PD) or 100 n

Wortmannin (Wort). Top, Representative autoradiogram; bottom, mean ± SE (n = 3) * Significantly different from control (p < 0.05). ** Significantly different from Ang II-treated neurons (p < 0.05).

Fig. 11.

Ang II-induced nuclear translocation of PKB in WKY and SHR neurons. Neuronal cultures of WKY and SHR were incubated without or with 100 n

Ang II for the indicated time periods. Cytoplasmic and nuclear fractions were isolated and subjected to Western analysis as described in Materials and Methods.A, Cytoplasmic fractions; B, nuclear fractions. A, B, Top, Representative autoradiograms;bottom, quantitation of immunoreactive bands. Mean ± SE (n = 3). * Significantly different from control (p < 0.01).

Fig. D1.

Summary of signaling mechanisms of AT1 receptor-mediated neuromodulation in WKY and SHR neurons.

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Role of phosphatidylinositol 3-kinase in angiotensin II regulation of norepinephrine neuromodulation in brain neurons of the spontaneously hypertensive rat - PubMed (original) (raw)