Inhibition of nischarin expression attenuates rilmenidine-evoked hypotension and phosphorylated extracellular signal-regulated kinase 1/2 production in the rostral ventrolateral medulla of rats

Abstract

Imidazoline (I(1))-evoked hypotension is linked to enhanced phosphorylated extracellular signal-regulated kinase (pERK)1/2 production in the rostral ventrolateral medulla (RVLM). Recent cell culture findings suggest that nischarin is a candidate for the I(1) receptor. In the present study, nischarin antisense oligodeoxynucleotide (ODN) (AS1 or AS2), designed according to nischarin cDNA sequence, was administered intracisternally (i.c., 2 nmol/rat for 2 days) to knockdown central nischarin expression; control rats received the corresponding mismatched ODN (MM1 or MM2) or artificial cerebrospinal fluid (aCSF). We investigated the effects of AS1 or AS2 on nischarin expression in the RVLM, and on the hypotension and RVLM pERK1/2 production elicited by the I(1)-selective agonist rilmenidine (25 mug/rat i.c.). Compared with aCSF, the mismatched ODN (MM1 or MM2) had no significant effect on RVLM nischarin expression or the cardiovascular and cellular (RVLM pERK1/2) responses elicited by rilmenidine. However, either antisense ODN substantially (>80%) reduced nischarin expression in the RVLM (AS1/MM1, 3 +/- 1 versus 32 +/- 2 positive cells; AS2/MM2, 4 +/- 1 versus 31 +/- 2 positive cells) and abrogated rilmenidine (I(1))-evoked hypotension (AS1/MM1, -4.1 +/- 0.9 versus -10.8 +/- 1.9 mm Hg; AS2/MM2, -2.1 +/- 1.1 versus -15.3 +/- 2.5 mm Hg) and ERK1/2 activation in the RVLM (AS1/MM1, 10 +/- 1 versus 15 +/- 2 positive cells; AS2/MM2, 9 +/- 1 versus 18 +/- 2 positive cells). Finally, pERK1/2 generated by central I(1) receptor activation is colocalized with nischarin in the RVLM neurons. This is the first evidence in vivo that nischarin plays a critical role in I(1) receptor-mediated pERK1/2 production in the RVLM and the subsequent hypotension.

Fig. 1

Effect of intracisternal administration of one of the nischarin anti-sense ODNs (AS1 or AS2) or the mismatched ODNs (MM1 or MM2), compared with equal volume of aCSF, on rilmenidine (25 μg/rat i.c.)-evoked hypotension and bradycardia. Values are mean ± S.E., and the number in parentheses indicates the number of rats in each group. * or #, p < 0.05, compared with the corresponding mismatched ODN and aCSF-treated group, respectively.

Fig. 2

Effect of intracisternal administration of one of the nischarin antisense ODNs (AS1 or AS2) or the mismatched ODNs (MM1 or MM2), compared with equal volume of aCSF on the expression of nischarin in the RVLM. The photomicrograph shows the immunohistochemical images of nischarin-immunoreactive neurons in the RVLM. The low-power image demonstrates the location of the RVLM (marked by square) from which the immunoreactive neurons were counted. High-power images display the effects of the different treatments. Scale bar, 100 μm. Bar graphs show the number of nischarin-immunoreactive neurons in the RVLM. Values are expressed as mean ± S.E., and the number in parentheses indicates the number of rats in each group. *, p < 0.05, compared with the aCSF control group. # or @, p < 0.05, compared with the corresponding mismatched ODN group.

Fig. 3

Effect of intracisternal administration of one of the nischarin antisense ODNs (AS1 or AS2) or the mismatched ODNs (MM1 or MM2), compared with equal volume of aCSF, on the phosphorylation of MAPK (pERK1/2) in the RVLM elicited by rilmenidine (25 μg/rat i.c.). The photomicrograph shows the immunohistochemical images of pERK1/2-immunoreactive neurons. The low-power image demonstrates the location of the RVLM (marked by square) from which the pERK1/2-immuno-reactive neurons were counted. Scale bar, 200 μm. High-power images display the effects of the different treatments. Scale bar, 100 μm. Bar graphs show the number of pERK1/2-immunoreactive neurons in the RVLM. Values are expressed as mean ± S.E. *, p < 0.05 compared with the aCSF control group. # or @, p < 0.05 compared with the corresponding mismatched ODN group.

Fig. 4

Colocalization of nischarin protein and pERK1/2 detected by double-labeling immunofluorescence technique. pERK1/2 was detected by phospho-p42/44 MAPK monoclonal antibody with anti-mouse IgG secondary antibody conjugated with Texas Red fluorescent label (red). Nischarin in protein (imidazoline receptor, IR) was identified by nischarin polyclonal antibody with anti-rabbit IgG secondary antibody conjugated with fluorescein isothiocyanate fluorescent label (green). The signal of colocalization (yellow) is visualized by merging the two images.