Heightened Vascular Reactivity Following Sympathetic Denervation Correlates with Increased Expression of C-Kit and Connexin43
Short-term denervation produces supersensitivity to α-adrenergic agonists that resembles the heightened vascular reactivity observed in hypertensive subjects. Gap junctions, transmembrane channels responsible for cell-to-cell communication, may play a role in maintaining increased vascular reactivity. We examined the effects of denervation with intra-peritoneal reserpine 3 mg/kg/day (N=5) or topical 5% phenol-glycerol (N=7) on agonist-induced oscillatory activity. Wistar-Kyoto (WKY) rat tail arteries were exposed to step-wise addition of norepinephrine (NE) 10-9M-10-5M. The concentration for half-maximal response for NE-induced contraction was lower in reserpine- and phenol-treated vessels than controls (log EC50 [mol]/L control -6.89 vs reserpine -7.16, p<0.05); control -6.86 vs phenol -7.32, p<0.0001). Incubation in K+-free solution produced an 8-fold increase in isometric tension in control vs denervated vessels (138.4±23.2 vs 17.2±4.5 % of contraction to phenylephrine 10-6M, p<0.001). These findings are consistent with adrenergic denervation. Agonist-induced oscillatory activity was observed in 5/5 reserpine- and 4/7 phenol-treated vessels vs 0/12 controls (Fischer’s exact test p<0.05). The highly selective gap junction inhibitor, Gap27 (10 mM), nearly abolished NE-induced vascular smooth muscle oscillations. Reverse transcriptase polymerase chain reaction was carried out for connexin43 (Cx43) and c-Kit, a marker for non-neural interstitial cells that serve as pacemaker cells, using specific oligonucleotide primers. Following denervation, mRNA levels of Cx43 remained unchanged while levels of c-Kit increased markedly (0.16±0.03 vs 0.62±0.13 a.u., p<0.05). Western blot analysis revealed near doubling of Cx43 protein after denervation (80.0±13.7 vs 41.4±5.7 a.u., p<0.05). In conclusion, sympathetic denervation produced increased agonist-induced oscillatory contractile activity in vascular smooth muscle. The heightened vascular reactivity may result from upregulation of pacemaker cells and increased Cx43 expression.