Chronic blockade of phosphatidylinositol 3-kinase in the nucleus tractus solitarii is prohypertensive in the spontaneously hypertensive rat

Abstract

Phosphatidylinositol 3-kinase (PI3K) within brain stem neurons has been implicated in hypertension in the spontaneously hypertensive rat (SHR). Previously, we demonstrated elevated expression of PI3K subunits in rostral ventrolateral medulla and paraventricular nucleus of SHRs compared with Wistar-Kyoto rats. Here, we considered expression levels of PI3K in the nucleus tractus solitarii, a pivotal region in reflex regulation of arterial pressure, and determined its functional role for arterial pressure homeostasis in SHRs and Wistar-Kyoto rats. We found elevated mRNA levels of p110beta and p110delta catalytic PI3K subunits in the nucleus tractus solitarii of adult (12 to 14 weeks old) SHRs relative to the age-matched Wistar-Kyoto rats (fold differences relative to beta-actin: 1.7+/-0.2 versus 1.01+/-0.08 for p110beta, n=6, P<0.05; 1.62+/-0.15 versus 1.02+/-0.1 for p110delta, n=6, P<0.05). After chronic blockade of PI3K signaling in the nucleus tractus solitarii by lentiviral-mediated expression of a mutant form of p85alpha, systolic pressure increased from 175+/-3 mm Hg to 191+/-6 mm Hg (P<0.01) in SHRs but not in Wistar-Kyoto rats. In addition, heart rate increased (from 331+/-6 to 342+/-6 bpm; P<0.05) and spontaneous baroreflex gain decreased (from 0.7+/-0.07 to 0.5+/-0.04 ms/mm Hg; P<0.001) in the SHRs. Thus, PI3K signaling in the nucleus tractus solitarii of SHR restrains arterial pressure in this animal model of neurogenic hypertension.

Figure 1

Endogenous expression of PI3K catalytic p110β and p110δ subunits in the NTS. A and B, Results of real-time RT PCR for p110β and p110δ, respectively. Data are expressed as the fold difference vs the WKY. *P<0.05 vs WKY.

Figure 2

DNp85α expression in the NTS of SHRs, but not WKY rats, raises arterial blood pressure (ABP). SBP (A), DBP (B), and MBP (C) are significantly elevated 14 days after the injection of LV-EF1α-DNp85α-IRES-eGFP (LV-DNp85α). They remained elevated for the 28-day observation period. No changes were seen in the control SHR, where LV-EF1α-eGFP (LV-eGFP) was injected into NTS or when LV-DNp85α or LV-eGFP was injected into the NTS of WKY rats. In D, NTS injection of LV-DNp85α significantly elevated HR in the SHR vs the LV-eGFP-treated SHR, which, however, showed a significant decrease in HR over time. In the WKY rat, there were no significant differences between the effects produced by either the LV-eGFP or LV-DNp85α on HR, with both groups showing a similar significant decrease in HR over time. In E, sBRG (PI) is significantly reduced after LV-DNp85α NTS injection in the SHR vs the effect of the LV-eGFP injection. There was no effect of either virus injection on sBRG (pulse interval) in the WKY rats. †P<0.05, ††P<0.01, and †††P<0.001 vs “Before.” *P<0.05, **P<0.01, and ***P<0.001 vs LV-eGFP transduction. N=5 per group.

Figure 3

A, Schematic of the area transduced by LV-DNp85α spanning from −14.80 to −13.80 mm from bregma, with the greatest transfection observed at −14.30 to −14.08 mm from bregma. Red box in A indicates the region corresponding with images in B. B, Validation of transgene expression was visualized by eGFP fluorescence (left). Middle panel shows immunostaining for NeuN, and the right panel shows an overlay of eGFP and NeuN; the LV-DNp85α–transduced neurons are indicated by white arrows. The images were taken at approximately −14.30 mm from bregma. CC indicates central canal; Sol, solitary tract; DVN, dorsal vagal motonucleus; AP, area postrema. Scale bar=50 μm.