Eph/ephrin interactions modulate vascular sympathetic innervation

Abstract

Ephs and ephrins are membrane-bound proteins that interact to modulate axon growth and neuronal function. We tested the hypothesis that eph/ephrin interactions affected the growth and function of vascular sympathetic innervation. Using RT-PCR analyses, we detected both classes of ephs (A and B) and both classes of ephrins (A and B) in sympathetic ganglia from neonatal and adult rats. Both classes of ephs (A and B) and both classes of ephrins (A and B) bound to the cell bodies and neurites of dissociated postganglionic sympathetic neurons. Messenger RNAs encoding for both classes of ephs (A and B) and both classes of ephrins (A and B) were also detected in sympathetically innervated arteries from neonatal and adult rats. These data suggest that ephrins/ephs on nerve fibers of postganglionic sympathetic neurons could interact with ephs/ephrins on cells in innervated arteries. We found that ephA4 reduced reinnervation of denervated femoral arteries. Reinnervation in the presence of ephA4-Fc (38.9±6.6%) was significantly less than that in the presence of IgG-Fc (62±10%; n=5; p<0.05; one-tailed unpaired t-test). These data indicate that eph/ephrin interactions modulated the growth of vascular sympathetic innervation. We also found that ephA4 increased basal release of norepinephrine from nerve terminals of isolated tail arteries. These data indicate that eph/ephrin interactions affect the growth and function of vascular sympathetic innervation.

Figure 1. Eph and ephrin expression in postganglionic sympathetic neurons

A) Representative RT-PCR analyses of eph and ephrin mRNA expression in neonatal (n) and adult (a) superior cervical ganglia. B) Representative (n = 2) eph and ephrin binding to dissociated postganglionic sympathetic neurons derived from superior ganglia of neonatal rats.

Figure 2. Eph and ephrin expression in femoral arteries

Representative RT-PCR analyses of eph and ephrin mRNA expression in neonatal (n) and adult (a) femoral arteries and in VSM derived from explants of adult femoral arteries.

Figure 3. Eph/ephrin interactions promote reinnervation of denervated femoral arteries

Reinnervation of femoral arteries 14 days after denervation in the absence (control arteries; open bars) and presence of IgG-Fc (gray bar) or ephA4-Fc (black bar). * indicates that reinnervation in the presence of ephA4-Fc (n = 6) was significantly less than control (p < 0.05; two-tailed unpaired t-test). + indicates that reinnervation in the presence of ephA4-Fc was significantly less than that in the presence of IgG-Fc (n = 5) (p < 0.05; one-tailed unpaired t-test).

Figure 4. Eph/ephrin interactions decrease basal release of norepinephrine

A) Basal release of norephinephrine (NE) from freshly isolated tail arteries incubated for two hours with 10 µg/ml control IgG-Fc (open bars) or ephA4-Fc (closed bars). * indicates that release in the presence of ephA4-Fc was significantly greater than that in the presence of IgG-Fc (n=4; P < 0.05; one-tailed unpaired t-test assuming unequal variances). B) Stimulated release of NE from freshly isolated tail arteries incubated for 2 hours with 10 µg/ml control IgG-Fc (open bars) or ephA4-Fc (closed bars). C) NE uptake by freshly isolated tail arteries incubated for 2 hours with 10 µg/ml control IgG-C (open bars) or ephA4-Fc (closed bars).