thapsigargin and manoalide

We examined whether Ca(2+) mobilizers induce endothelium-dependent contraction and relaxation (EDC and EDR) in isolated rabbit intrapulmonary arteries. Ionomycin (10(-7) M) and A-23187 (10(-7) M), both Ca(2+) ionophores, and thapsigargin (10(-6) M), an endoplasmic reticulum Ca(2+)-ATPase inhibitor, caused a contraction in the non-contracted preparations, and a transient relaxation followed by a transient contraction and sustained relaxation in the precontracted preparations. Endothelium-removal abolished the contraction and transient relaxation (EDC and EDR) but not sustained relaxation (endothelium-independent relaxation, EIR). In the noncontracted preparations, ionomycin-induced EDC was significantly attenuated by quinacrine (10(-5) M), manoalide (10(-6) M), both phospholipase A(2) inhibitors, indomethacin (10(-5) M) and aspirin (10(-4) M), both COX inhibitors, and ozagrel (10(-5) M), a TXA(2) synthetase inhibitor. In the precontracted arteries, EDR was markedly reduced by L-NAME (10(-4) M), a NOS inhibitor, and methylene blue (10(-6) M), a guanylate cyclase inhibitor, and was enhanced by indomethacin, aspirin and ozagrel, probably due to inhibition of EDC. ZM230487, a 5-lipoxygenase inhibitor, had no effect on EDR. EIR was not affected by L-NAME, indomethacin or ZM230487. Arachidonic acid (10(-6) M) evoked EDC sensitive to indomethacin and ozagrel. L-Arginine (10(-3) M) caused EDR sensitive to L-NAME in the ionomycin-stimulated preparations. In conclusion, Ca(2+) mobilizers cause EDC and EDR via production of TXA(2) and NO, respectively.

Topics: Animals; Aspirin; Calcimycin; Calcium; Endothelium, Vascular; Enzyme Inhibitors; Indomethacin; Ionomycin; Ionophores; Male; Methacrylates; Methylene Blue; Muscle Relaxation; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Pulmonary Artery; Quinacrine; Rabbits; Terpenes; Thapsigargin

1. Human umbilical vein endothelial cells cultured on a transparent silicone chamber were subjected to a short stretch pulse (ca. 1 s, 5-25% stretch) of their substrate and following increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured by fluorescence intensity ratiometry using fura-2. 2. In response to mechanical stretch, the cells in HEPES buffered saline exhibited a Ca(2+) transient in a dose dependent way. The response was completely dependent on external Ca(2+) and inhibited by gadolinium (Gd(3+)), suggesting that it was mediated by the activation of a stretch activated cation channel (SACatC). 3. Interestingly, the stretch induced Ca(2+) transient was significantly augmented in the presence of basic polypeptide, protamine. This augmented Ca(2+) response was inhibited neither by Gd(3+) nor by the deprivation of external Ca(2+), indicating that the SACatC is not responsible for this phenomenon. 4. In contrast, this augmentation was inhibited by depletion of intracellular Ca(2+) stores with thapsigargin or by the pretreatment with phospholipase inhibitors such as U73122 and manoalide. 5. These results suggest the presence of a metabotropic mechanoreceptor distinct from the SACatC in vascular endothelium. This augmented [Ca(2+)](i) increase may contribute to the vasodilating response induced by protamine during heparin neutralization in cardiac surgery.

Topics: Calcium; Cell Membrane Permeability; Cells, Cultured; Endothelium, Vascular; Estrenes; Gadolinium; Humans; Manganese; Protamines; Pyrrolidinones; Stress, Mechanical; Terpenes; Thapsigargin; Time Factors