Thapsigargin-induced endothelium-dependent triphasic regulation of vascular tone in the porcine renal artery

Abstract

1. To elucidate the role of thapsigargin-induced Ca2+ entry in endothelial cells in the regulation of vascular tone, changes in Ca2+ and force of smooth muscle were simultaneously monitored in fura-2-loaded strips of porcine renal artery. 2. During phenylephrine-induced sustained contraction, thapsigargin caused an endothelium-dependent triphasic response; an initial relaxation, a subsequent transient contraction, and a sustained relaxation. The initial relaxation and the contraction were associated with a decrease and an increase in [Ca2+]i, respectively. There was no apparent [Ca2+]i decrease during the sustained relaxation. Thapsigargin-induced responses were observed at 10-8 M and higher concentrations, with the maximum response observed at 10-6 M. 3. The transient contraction was inhibited by a cyclo-oxygenase inhibitor (10-5 M indomethacin), a thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor antagonist (10-5 M ONO-3708), and a TXA2 synthase inhibitor (10-5 M OKY-046). 4. During the phenylephrine-induced contraction in the presence of indomethacin, thapsigargin caused an initial, but not a sustained relaxation, in the presence of Nomega-nitro-L-arginine methylester (L-NAME). During the contraction induced by phenylephrine plus 40 mM K+-depolarization in the presence of indomethacin, thapsigargin induced both a transient and a sustained relaxation. However, these relaxations were completely abolished in the presence of L-NAME. 5. Thapsigargin caused a large Ca2+ elevation in cultured endothelial cells of the renal artery. The concentration-response relation was thus similar to that for force development in the arterial strips. 6. In conclusion, thapsigargin-induced Ca2+ entry in endothelial cells led to triphasic changes in the tone of the porcine renal artery. The endothelium-dependent contraction was mediated mainly by TXA2. Nitric oxide and hyperpolarizing factor are both involved in the initial relaxation. However, a sustained relaxation was observed which mainly depended on nitric oxide.

Figure 1

Thapsigargin (TG)-induced endothelium-dependent triphasic changes in [Ca²⁺]_i and force during phenylephrine (Phe)-induced sustained contraction in porcine renal artery. (a) and (b) The representative recordings of changes in [Ca²⁺]_i and force induced by 10⁻⁶ M TG in the strips without (a) and with (b) endothelium. After the reference response to 10⁻⁶ M Phe was recorded and the strip was stimulated once with 118 mM K⁺ depolarization, the precontraction was initiated with 10⁻⁶ M Phe. TG (10⁻⁶ M) was applied at 15 min during the sustained contraction induced by Phe. (c) Summary of the time courses for the endothelium-dependent changes in [Ca²⁺]_i and force induced by 10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M, 10⁻⁵ M TG, and the control. The levels of [Ca²⁺]_i and force at rest and during Phe-induced sustained contraction were designated as 0 and 100%, respectively. All data are the mean±s.e.mean. (n=6).

Figure 2

Concentration-dependency of triphasic changes in [Ca²⁺]_i and force induced by thapsigargin (TG) during phenylephrine (phe)-induced sustained contraction in the porcine renal artery. The levels of [Ca²⁺]_i and force at (a) maximum initial relaxation, at (b) the peak of transient contraction and at (c) 30 min after application of TG during the sustained relaxation are shown. The levels of [Ca²⁺]_i and force at rest and during Phe-induced sustained contraction were designated as 0 and 100%, respectively. All data are the mean±s.e.mean (n=6). ^*Significantly different from the levels of [Ca²⁺]_i and force seen during the sustained phase of control contraction induced by 10⁻⁶  M Phe (P<0.05).

Figure 3

Effects of indomethacin (Ind: a cyclo-oxygenase inhibitor), ONO-3708 (a thromboxane A₂/prostaglandin H₂ receptor antagonist), and OKY-046 (a thromboxane A₂ synthase inhibitor) on thapsigargin (TG)-induced endothelium-dependent contraction. Time courses of TG-induced changes in [Ca²⁺]_i (left panels) and force (right panels) in the presence of (a) 10⁻⁵ M Ind, (b) 10⁻⁵ M ONO-3708, and (c) 10⁻⁵  M OKY-046 in porcine renal artery. After recording the reference response to 10⁻⁶ M phenylephrine (Phe), each inhibitor was applied just prior to the initiation of contraction by Phe. TG was applied at 15 min after the initiation of Phe-precontraction. The levels of [Ca²⁺]_i and force at rest and during Phe-induced sustained contraction were designated as 0 and 100%, respectively. All data are the mean±s.e.mean (n=5–6).

Figure 4

Involvement of nitric oxide and endothelium-derived hyperpolarizing factor in thapsigargin-induced relaxation in the porcine renal artery. Representative traces of changes in [Ca²⁺]_i and force induced by thapsigargin (TG) during the contraction induced by phenylephrine (phe) (a) or phenylephrine plus 40 mM K⁺ (b) in the presence of 10⁻⁵ M indomethacin (Ind) and 3×10⁻⁵ M N^ω-nitro-L-arginine methylester (L-NAME), and during the contraction induced by phenylephrine plus 40 mM K⁺ in the presence of 10⁻⁵ M Ind (c). (d) Summary on the level of forces just before, and 5 min and 30 min after the application of thapsigargin in protocol a, b, and c. The level of force at rest and just prior to the application of thapsigargin during the sustained contraction under each protocol were designated as 0 and 100%. All data are the mean±s.e.mean (n=4).

Figure 5

Effect of N^ω-nitro-L-arginine methylester on the endothelium-dependent relaxation induced by thapsigargin. After recording the reference response to 10⁻⁶ M phenylephrine (Phe), N^ω-nitro-L-arginine methylester (L-NAME) were added just prior to the initiation of precontraction by Phe. Phe-induced changes in [Ca²⁺]_i (left panel) and force (right panel) in the presence of L-NAME were shown. Thapsigargin (TG) was applied at 15 min after the initiation of the precontraction. The levels of [Ca²⁺]_i and force at rest and during Phe-induced sustained contraction were designated as 0 and 100%, respectively. All data are the mean±s.e.mean (n=6).

Figure 6

Ca²⁺ transients induced by thapsigargin (TG) in endothelial cells of renal artery in primary culture. (a) The representative recording of changes in [Ca²⁺]_i induced by 10⁻⁶ M TG in endothelial cells of renal artery in primary culture. The responses to 10⁻⁵ M ATP were recorded as control reference (100%) and TG was applied 15 min later. (b) The time course of changes in [Ca²⁺]_i in endothelial cells in primary culture induced by 10⁻⁸ M, 10⁻⁷ M, 10⁻⁶ M, and 10⁻⁵ M TG. [Ca²⁺]_i levels at rest and at peak response to 10⁻⁵ M ATP were designated as 0 and 100%, respectively. All data are the mean±s.e.mean (n=3).