In the presence of L-NAME SERCA blockade induces endothelium-dependent contraction of mouse aorta through activation of smooth muscle prostaglandin H2/thromboxane A2 receptors

Abstract

1. The mechanism of transient contractions induced by the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) blocker cyclopiazonic acid (CPA) in the presence of L-NAME was investigated in mouse aorta. 2. The contractions elicited by 10 micro M CPA required an intact endothelium, were dependent upon external Ca(2+) and were prevented by 10 micro M indomethacin, the inhibitor of prostaglandin synthesis, or 1 micro M SQ29548, the specific prostaglandin H2/thromboxane A2 (PGH2/TXA2) receptor blocker. 3. A blocker of receptor/store operated Ca(2+) channels and voltage gated calcium channels (VGCC), SK&F 96365 (10 micro M), completely abolished the contractions, while a specific blocker of VGCC nifedipine (1 micro M) inhibited them by one third. 4. Dichlorobenzamyl hydrochloride, a blocker of Na(+)/Ca(2+) exchange effectively prevented return of tension to baseline value. 5. At higher concentrations (30-100 micro M) CPA induced indomethacin-resistant tonic contractions of mouse aorta. The CPA dose response curve for tonic contractions is shifted to the right compared to the transient contractions suggesting that smooth muscle is less sensitive to CPA than endothelium. 6. PGH2/TXA2 receptors in mouse aorta are highly sensitive to the thromboxane analogue U46619 (EC(50) : 1.93 nM). This compound stimulates contractions even in the absence of external Ca(2+), which are abolished by the Rho-kinase inhibitor HA-1077. 7. The results suggest that 10 micro M CPA induced capacitive Ca(2+) entry in endothelial cells stimulating the release of PGH2/TXA2, which subsequently caused smooth muscle contraction dependent on Ca(2+) influx and myofilament sensitization by Rho-kinase. Higher concentrations of CPA (30-100 micro M) directly induced contraction of mouse aortic smooth muscle.

Figure 1

The effect of indomethacin (10 μM) on contraction, induced by 10 μM CPA, in C57BL/6J mouse aortae. (a) Individual traces of force development in Ca²⁺-containing Krebs' buffer. (b) 10 μM CPA-induced contraction initiated by the readmission of Ca²⁺ after preincubation in Ca²⁺-free solution with CPA. (c) Average amplitudes (n=6) of CPA-induced transient contractions and maximal tensions in the presence of indomethacin in the Ca²⁺-contaning buffer and in the Ca²⁺ replenishment protocol. C, CPA-induced transient contractions; Cr, CPA-induced transient contractions after the readmission of Ca²⁺; I, CPA-induced maximal tensions in the presence of indomethacin; Ir, CPA-induced maximal tensions in the presence of indomethacin after the readmission of Ca²⁺. ^*Significantly different from the amplitude in the Ca²⁺-containing buffer with P<0.05.

Figure 2

(a), 10 μM CPA-induced contraction in native and denuded aortae of C57BL/6J mice (representative of n=6). (b) Effect of SQ29548 (1 μM) on the 10 μM CPA-induced contraction of C57BL/6J mouse aorta (representative of n=6).

Figure 3

CPA concentration response curves for the peak amplitudes of transient contractions and the indomethacin-resistant tonic contractions of mouse aortae (CD1mice, 5–8 animals per point). The points were fitted with 3-parameter logistic curves.

Figure 4

10 nM U46619-induced contractions of CD1 mouse aorta. Curve 1, Ca²⁺-containing buffer; Curve 2, Ca²⁺-free buffer; Curve 3, after preincubation with 75 M 2APB in Ca²⁺-free buffer (representative of n=5).

Figure 5

Effect of SKF (10 μM) and HA-1077 (50 μM) on 10 nM U46619-induced contractions of CD1 mouse aorta (representative of n=4).

Figure 6

Effect of 75 μM 2APB on the force developed by CD1 mouse aorta in the presence of 30 μM CPA (representative of n=6).

Figure 7

Effect of ryanodine (100 μM) on 10 μM CPA-induced contraction of C57BL/6J mouse aortae (representative of n=3).

Figure 8

Effect of DCB (10 μM) on 10 μM CPA-induced contraction of C57BL/6J mouse aortae in the presence of indomethacin (10 μM) (representative of n=6).

Figure 9

Endothelial agonist-induced contraction of C57BL/6J mouse aorta in comparison with 10 μM CPA-induced contraction in the presence and the absence of 10 μM indomethacin. Acetylcholine (Ach) 1 μM (n=4); bradykinin (BK) 100 nM (n=5); adenosine 5′-triphosphate (ATP) 300 μM (n=6).

Figure 10

Paracrine effect of arachidonic acid (AA) metabolites in mouse aorta. Sequence of events: (1) CPA binds to SERCA in endothelial cells (ECs) and holds it open depleting the endoplasmic reticulum (ER); (2) depletion of ER opens SOC and induces Ca²⁺ entry into ECs; (3) [Ca²⁺]_i activates phospholipase A2 (PLA2) leading to AA production; (4) AA is transformed by cyclo-oxygenase (COX) into prostaglandin H2, possibly followed by its transformation into thromboxane A2; (5) PGH2/TXA2 activate the PGH2/TXA2 receptors (TXR) of smooth muscle cells thereby initiating signal transduction cascades; (6) TXR activation induces Ca²⁺ entry into the smooth muscle cells through ROC and VGCC with minor contribution of IP3 pathway; (7) elevated [Ca²⁺]_i induces contraction of SMCs; (8) TXR activation induces myofilament sensitization to Ca²⁺.