Reverse-mode Na+/Ca2+ exchange is an important mediator of venous contraction

Abstract

The Na(+)/Ca(2+) exchanger (NCX) is a bi-directional regulator of cytosolic Ca(2+), causing Ca(2+) efflux in forward-mode and Ca(2+) influx in reverse-mode. We hypothesized that reverse-mode NCX is a means of Ca(2+) entry in rat aorta (RA) and vena cava (RVC). NCX protein in RA and RVC was confirmed by immunoprecipitation. To assess NCX function, isometric contraction and intracellular Ca(2+) was measured in RA and RVC rings in response to low extracellular Na(+), endothelin-1 (ET-1), and KCl, in the presence or absence of the NCX antagonist KB-R7943. In RVC, low extracellular Na(+) caused vasoconstriction and an increase in intracellular Ca(2+) that was attenuated by 10μM KB-R7943. KB-R7943 (10 μM) attenuated maximal contraction to ET-1 in RVC (53 ± 9% of control), but not RA (91±1% of control). KB-R7943 (10 μM) reduced the maximal contraction to KCl in RA (48 ± 5%) and nearly abolished it in RVC (9 ± 2%), suggesting that voltage-dependent Ca(2+) influx may be inhibited by KB-R7943 as well. However, the L-type Ca(2+) channel inhibitor nifedipine (1 μM) did not alter ET-1-induced contraction. Our findings suggest that reverse-mode NCX is an important mechanism of Ca(2+) influx in RVC but not RA, especially during ET-1-induced contraction. Also, the effects of KB-R7943 on ET-1-induced contraction of RA and RVC are predominantly mediated by reverse-mode NCX inhibition and not due to off-target inhibition of Ca(2+) channels.

Figure 1

(a) Representative Western blot analysis of immunoprecipitation of NCX1 from 200 μg of whole-tissue protein homogenate from aorta (RA1-3) and vena cava (RVC1-3), isolated from Sprague-Dawley rats. NCX1 protein was immunoprecipitated using protein A/G beads (Santa Cruz Biotechnology, CA USA) and NCX1 antibody (Ab) (Swant, Switzerland). Blots were probed with antibody against NCX1. (b) Densitometry of NCX western blot analysis shows no significant difference in NCX expression between rat aorta (black) and vena cava (white). N.S. = p>0.05; N=6.

Figure 2

(a–c) Representative tracings of rat aorta contraction, in response to rapid exposure to low-Na⁺ (~15 mM) physiological salt solution. Shown are responses from tissues incubated with vehicle (DMSO) (a), 1 μM KB-R7943 (b) and 10 μM KB-R7943 (c). Arrows indicate the baseline and maximal contraction used to measure the responses. (d,e) Summary graphs of low-Na⁺-induced contraction in aorta, in the presence or absence of KB-R7943 (1 μM and 10 μM). All bars represent mean ± SEM for the number of animals indicated. Black bars represent vehicle-exposed tissues (differences between vehicle contractions were not significant). White bars represent tissues exposed to 1 μM KB-R7943 (d) or 10 μM KB-R7943 (e). Results are shown as percentages of initial phenylephrine contraction (PE). N=3–5; * = p<0.05 versus vehicle.

Figure 3

(a–c) Representative tracings of rat vena cava contraction, in response to rapid exposure to low-Na⁺ (~15 mM) physiological salt solution. Shown are responses from tissues incubated with vehicle (a), 1 μM KB-R7943 (b) and 10 μM KB-R7943 (c). Arrows indicate the baseline and maximal contraction used to measure the responses. (d,e) Summary graphs of low-Na⁺-induced contraction in vena cava, in the presence or absence of KB-R7943 (1 μM and 10 μM). All bars represent mean ± SEM for the number of animals indicated. Black bars represent vehicle-exposed tissues (differences between vehicle contractions were not significant). White bars represent tissues exposed to 1 μM KB-R7943 (d) or 10 μM KB-R7943 (e). Results are shown as percentages of initial norepinephrine contraction (NE). N=4–5; * = p<0.05 versus vehicle.

Figure 4

(a,b) Representative measurements of Fura2-AM fluorescence ratio (dashed line, left axis) and contraction (solid line, right axis) in vena cava exposed to low Na⁺ PSS in the presence of vehicle (a) or 10 μM KB-R7943 (b). (c,d) Summary bar graphs indicating the maximum change in fluorescence ratio (c) and contraction (d) from these same experiments. Black bars represent vehicle-exposed tissues. White bars represent tissues exposed to 10 μM KB-R7943. N=5–6; * = p<0.05 versus vehicle.

Figure 5

Measurement of endothelin-1-induced responses in aorta (a) and vena cava (b), exposed to vehicle or KB-R7943 (10 μM). Vehicle or antagonists were incubated with tissue for 1h prior to ET-1 exposure. Points represent mean ± SEM for the number of animals indicated in parentheses. * = p<0.05 versus vehicle.

Figure 6

Measurement of endothelin-1-induced responses in aorta (a) and vena cava (b), exposed to vehicle or KB-R7943 (1 μM). Vehicle or antagonists were incubated with tissue for 1h prior to ET-1 exposure. Points represent mean ± SEM for the number of animals indicated in parentheses. * = p<0.05 versus vehicle.

Figure 7

The effects of KB-R7943 (10 μM) on endothelin-1-induced contraction in aorta (a) and vena cava (b) exposed to L-NNA (100 μM) and indomethacin (5 μM). Vehicle or antagonists were incubated with tissue for 1h prior to ET-1 exposure. Points represent mean ± SEM for the number of animals indicated in parentheses. * = p<0.05 versus vehicle.

Figure 8

Measurement of KCl-induced contraction in aorta (a) and vena cava (b), exposed to vehicle or KB-R7943 (10 μM). Vehicle or antagonists were incubated with tissue for 1h prior to agonist exposure. Points represent mean ± SEM for the number of animals indicated in parentheses. * = p<0.05 versus vehicle.

Figure 9

Measurement of CaCl₂ concentration response curves, in the presence of ET-1 (100 nM), in aorta (a) and vena cava (b). Tissues were first incubated for 30 minutes in Ca²⁺-free buffer with 1 mM EGTA, then in Ca²⁺-free buffer (no EGTA) for 10 minutes before addition of 100nM ET-1. After plateau of any contraction to ET-1, tissues were incubated with vehicle (solid shapes) or 10 μM KB-R7943 (open shapes). Points represent mean ± SEM for the number of animals indicated in parentheses. Boxes represent the CaCl₂ concentration that is equivalent to that of physiological salt solution used in all other experiments. * = p<0.05 versus vehicle.

Figure 10

Top: Measurement of KCl-induced contraction in aorta (a) and vena cava (b), exposed to vehicle or nifedipine (1 μM). Bottom: Measurement of ET-1-induced contraction in aorta (c) and vena cava (d), exposed to vehicle or nifedipine (1 μM). In (ad), vehicle or antagonists were incubated with tissue for 1h prior to agonist exposure. Points represent mean ± SEM for the number of animals indicated in parentheses. * = p<0.05 versus vehicle.