Decreased expression of neuronal nitric oxide synthase in the nucleus tractus solitarii inhibits sympathetically mediated baroreflex responses in rat

Abstract

Despite numerous studies it remains controversial whether nitric oxide (NO·) synthesized by neuronal NOS (nNOS) plays an excitatory or inhibitory role in transmission of baroreflex signals in the nucleus tractus solitarii (NTS). In the current studies we sought to test the hypothesis that nNOS is involved in excitation of baroreflex pathways in NTS while excluding pharmacological interventions in assessing the influence of nNOS. We therefore developed, validated and utilized a short hairpin RNA (shRNA) to reduce expression of nNOS in the NTS of rats whose baroreflex activity was then studied. We demonstrate downregulation of nNOS through transduction with adeno-associated virus type 2 (AAV2) carrying shRNA for nNOS. When injected bilaterally into NTS AAV2nNOSshRNA significantly reduced reflex tachycardic responses to acute hypotension while not affecting reflex bradycardic responses to acute increases of arterial pressure. Control animals treated with intravenous propranolol to block sympathetically mediated chronotropic responses manifested the same baroreflex responses as animals that had been treated with AAV2nNOSshRNA. Neither AAV2 eGFP nor AAV2nNOScDNA affected baroreflex responses. Blocking cardiac vagal influences with atropine similarly reduced baroreflex-mediated bradycardic responses to increases in arterial pressure both in control animals and in those treated with AAV2nNOSshRNA. We conclude that NO· synthesized by nNOS in the NTS is integral to excitation of baroreflex pathways involved in reflex tachycardia, a largely sympathetically mediated response, but not reflex bradycardia, a largely parasympathetically mediated response. We suggest that, at the basal state, nNOS is maximally engaged. Thus, its upregulation does not augment the baroreflex.

Figure 1. Expression of nNOS was reduced in HEK293 cells by AAVp-nNOSshRNA

A, real time RT-PCR results show that AAVp-nNOSshRNA led to 94% reduction (mean of 3 set of experiments) in induced nNOS mRNA (column AAV2shRNA). A control short hairpin RNA did not affect nNOS expression (data not shown). B, Western blot shows that a decrease in nNOS protein was also observed by AAVp-nNOSshRNA (lane AAV2shRNA). The nNOS protein band (160 kDa) is indicated by the arrow on the left side of the blot.

Figure 2. Confocal immunofluorescence images showing a marked decrease in nNOS- immunoreactivity (IR) in the NTS and nodose ganglion (NG) after AAV2nNOSshRNA injection

AAV2nNOSshRNA (B) injected into the NTS decreased the number of cells and fibres that were positive for nNOS-IR in the NTS and dorsal motor nucleus of vagus (DMV) 2 weeks after the injection when compared with a PBS-injected control rat (A). Similarly, we also saw a decrease in nNOS-IR in the NG (D) as compared to that of a PBS-injected rat (C). Scale bar = 100 μm.

Figure 3. Confocal images of eNOS-IR (A and B), TH-IR (C and D) and PGP9.5-IR (E and F) in the rat NTS show no difference between PBS-injected control (A, C and E) and AAV2nNOSshRNA injected (B, D and F) rats

Arrows in A and B indicate eNOS-IR in the inner lining (endothelial layer) of blood vessels. Scale bar = 50 μm in A and B, 100 μm in C–F.

Figure 4. Real time RT-PCR results showing significant reduction (**P< 0.001) of nNOS mRNA in the rat NTS 2 weeks after AAV2nNOSshRNA (column AAV2shRNA) injection when compared to that of PBS injected controls

There was no difference in eNOS mRNA expression.

Figure 5. Western blot analysis showing decreased nNOS expression in rat NTS 2 weeks after AAV2nNOSshRNA injection

The nNOS protein band (160 kDa, top blot) and the loading control band (GAPDH, 36 kDa, bottom blot) from the same blot analysis are indicated by the arrows on the right side of the blot.

Figure 6. Reflex increases (upper left hand part of regression curves) in heart rate (HR, ordinate) in response to decreases in mean arterial pressure (MAP, abscissa) produced by infusion of nitroprusside are significantly (P < 0.05) attenuated in animals treated with nNOSshRNA (shRNA; r = −0.52) when compared with those seen in animals treated with PBS (r = −1.69), AAV2eGFP (eGFP; r = −1.50), or AAV2nNOScDNA (cDNA; r = −1.63) while reflex decreases (lower right hand part of regression curves) in HR in response to increases in MAP produced by infusion of phenylephrine do not differ between groups

Slopes (ΔHR/ΔMAP) for the tachycardic and bradycardic reflex responses are shown graphically in the bar graph insert at the lower left hand corner with data expressed as means ± SEM. A comparison of slopes of the full baroreflex analysis (including reductions and increases in MAP and reflex changes in HR; not shown) did show that responses in the PBS (r = −1.27), eGFP (r = −1.21), and cDNA nNOS (r = −1.20) groups did not differ from each other but responses in the shRNA group (r = −0.73) differed significantly (P = 0.002; 0.004; and 0.007 respectively) from responses in each of the other groups. Basal MAP and HR did not significantly differ between groups (see Table 3).

Figure 7

Representative physiographic traces of changes in arterial pressure (AP), mean arterial pressure (MAP), and heart rate (HR) in animals that had received bilateral injections of PBS (left panels), AAV2nNOScDNA (middle panels) or AAV2shRNA (right panels) show similar reflex tachycardic responses to depressor effects of intravenous nitroprusside infusion in the two control groups, but diminished reflex tachycardia despite similar depressor responses in an animal treated with shRNA