Na+-dependent inactivation of vascular Na+/Ca2+ exchanger responsible for reduced peripheral blood flow in neuropathic pain model

Abstract

Reduced skin blood flow has been reported in neuropathic pain patients as well as various peripheral neuropathic pain model animals. We have previously shown that vasodilators, which improves reduced skin blood flow, correlatively alleviate neuropathic pain in chronic constriction injury (CCI) mice, a model of neuropathic pain from peripheral nerve injury. Here, we sought to elucidate the mechanism underlying the reduced skin blood flow in CCI rats. The skin blood flow of the ipsilateral plantar arteries was significantly reduced compared to that of the contralateral ones 4 weeks after loose ligation of the sciatic nerve. The contraction induced by noradrenaline, serotonin, and U46619, a thromboxane receptor agonist, in the isolated ipsilateral plantar arteries was significantly enhanced compared to that in the contralateral ones. KB-R7943, a Na⁺/Ca²⁺ exchanger (NCX) inhibitor, shifted the concentration-response curves of noradrenaline to the left in the contralateral arteries but had no effect on the ipsilateral side. There was no significant difference in concentration-response curves of noradrenaline between the ipsilateral and contralateral arteries in the presence of KB-R7943. Amiloride, a non-specific inhibitor of Na⁺ channels and transporters, comparably shifted concentration-response curves of noradrenaline to the left in both the contralateral and ipsilateral arteries. One hundred nM of noradrenaline induced intracellular Ca²⁺ elevation in the ipsilateral arteries, which was significantly larger than that induced by 300-nM noradrenaline in the contralateral arteries. These results suggest that reduced peripheral blood flow after nerve injury is due to Na⁺-dependent inactivation of NCX in the ipsilateral plantar arteries.

Keywords: Arteries; Chronic constriction injury; Neuropathic pain; Sodium-calcium exchanger; Vasoconstriction.

Fig. 1

A–C, Concentration-response curves for contraction induced by noradrenaline, U-46619, and 5-HT in the contralateral (open circle) and ipsilateral (closed circle) plantar arteries isolated from CCI rats. Noradrenaline, U46619 and 5-HT were applied in a cumulative manner. D, Typical trace of the contraction induced by cumulative application of noradrenaline in the contralateral and ipsilateral arteries. Data are expressed as mean ± S.E.M. **P < 0.01 vs. the contralateral side. (n = 5–6)

Fig. 2

A, Ca²⁺-induced contraction in the presence of A-23187, a calcium ionophore (10 μM), nifedipine (1 μM), and 2-APB (100 μM) in the contralateral (open circle) and ipsilateral (closed) plantar arteries isolated from CCI rats. A-23187, nifedipine, and 2-APB were applied 15 min before the application of Ca²⁺. B–C, In the contralateral and ipsilateral plantar arteries pre-contracted with 300-nM (B) and 30-nM (C) noradrenaline, respectively, KB-R7943 (closed symbol), a Na⁺/Ca²⁺ exchanger (NCX) inhibitor, and vehicle (open symbol) were applied in a cumulative manner. Enhancement of contraction indued by KB-R 7943 was normalized by noradrenaline-induced pre-contraction. D, Concentration-response curves for noradrenaline-induced contraction in the presence of KB-R 7943 (3 μM) in the contralateral (open circle) and ipsilateral (closed circle) plantar arteries. KB-R7943 was applied 15 min before the application of noradrenaline. Data are expressed as mean ± S.E.M. (n=4–6). E, Immunohistochemical images using antibodies for NCX1 (green) and α-smooth muscle actin (red) and Hoechst (blue) in the contralateral (upper) and ipsilateral (lower) plantar arteries. The rightest images are merged images.

Fig. 3

A–B, Concentration-response curves for noradrenaline in normal buffer (open symbols) and low-Na⁺ buffer (closed symbols) in the contralateral and ipsilateral plantar arteries. C-D, Concentration-response curves for noradrenaline-induced contraction in the absence (open symbols) and presence of ouabain (100 μM; closed symbols) in the contralateral and ipsilateral plantar arteries. E. Concentration-response curves of noradrenaline in the presence of amiloride (300 μM) in the contralateral (open symbols) and ipsilateral (closed symbols) plantar arteries. The application of low-Na⁺ buffer, amiloride, or ouabain was performed 15 min before the application of noradrenaline. Data are expressed as mean ± S.E.M. (n = 4–5).

Fig. 4

A, Representative Fluo-8 images of the contralateral (upper) and ipsilateral (lower) plantar arteries before and after the application of noradrenaline (100 or 300 nM). The rightest images indicate the portion of region of interest. B, Peak amplitude of Ca²⁺ elevation induced by 100 or 300-nM noradrenaline in the contralateral (open column) and ipsilateral (closed column) plantar arteries. C, Peak amplitude of Ca²⁺ elevation induced by 100-nM noradrenaline in the presence of KB-R 7943 (3 μM) in the contralateral (open column) and ipsilateral (closed column) isolated plantar arteries. KB-R7943 was applied 10 min before the application of noradrenaline. Data are expressed as mean ± S.E.M. (n = 4–7). *P < 0.05, **P < 0.01 vs. 100-nM noradrenaline in the ipsilateral arteries.