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1 To investigate the mechanism underlying the trypsin-induced endothelium-dependent relaxation, cytosolic Ca 2+ concentration ([Ca 2+ ] i ) and tension development of smooth muscle were simultaneously monitored in the porcine coronary artery, and [Ca 2+ ] i of in situ endothelial cells were monitored in the porcine aortic valvular strips, using fura-2¯uorometry. 2 During the contraction induced by 30 nM U46619, a thromboxane A 2 analogue, 100 nM trypsin induced a rapid transient signi®cant decrease in both [Ca 2+ ] i (from 67.9+5.1 to 15.7+4.4%) and tension (from 97.5+9.2 to 16.8+3.5%) of smooth muscle only in the presence of endothelium (100% level was assigned to the level obtained with the 118 mM K + -induced contraction). [Ca 2+ ] i and the tension thus returned to the levels prior to the application of trypsin by 5 and 10 min, respectively. 3 The initial phase of this relaxation was partly inhibited by 100 mM N o -nitro-L-arginine (L-NOARG), and was completely inhibited by L-NOARG plus 40 mM K + or L-NOARG plus 100 nM charybdotoxin and 100 nM apamin, while the late phase of the relaxation was inhibited by L-NOARG alone. 4 Trypsin induced a transient [Ca 2+ ] i elevation in the endothelial cells mainly due to the Ca 2+ release from the intracellular stores, at the concentrations (1 ± 100 nM) similar to those required to induce relaxation. 5 In conclusion, trypsin induced an elevation in [Ca 2+ ] i mainly due to Ca 2+ release in endothelial cells, and thereby caused endothelium-dependent relaxation. The early phase of relaxation was due to nitric oxide and hyperpolarizing factors, while the late phase was mainly due to nitric oxide in the porcine coronary artery.

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1 The mechanism of trypsin-induced contraction in the rat myometrium was investigated using front-surface¯uorimetry on fura-PE3-loaded strips. The expression of protease-activated receptors (PARs) in the rat myometrium was determined by reverse transcription polymerase chain reaction (RT ± PCR). 2 In non-pregnant rats, 10 mM trypsin developed a force of up to 30.5+5.1% of that obtained during the 40 mM K + -depolarization-induced contraction. In pregnant rats, the maximal level of the cytosolic Ca 2+ concentration and tension obtained with 3 mM trypsin was 143.2+6.0% and 63.2+7.9%, respectively. The depletion of the extracellular Ca 2+ abolished the trypsin-induced contraction.3 Trypsin-induced contraction was abolished by the pre-treatment of a serine protease inhibitor. PAR1-activating peptide (PAR1-AP) caused a potent contraction of the myometrium, while neither PAR2-AP nor PAR4-AP induced any contraction. 4 RT ± PCR analysis detected the expression of PAR1 mRNA. However, neither PAR2 nor PAR4 mRNA was detected in the rat myometrium. 5 Once the strips were stimulated with thrombin, the subsequent application of thrombin failed to induce any contraction, while trypsin induced a contraction similar to that observed without the prestimulation with thrombin. Once the strip was stimulated with trypsin, neither trypsin nor thrombin induced any contraction. The response to PAR1-AP remained after the pre-stimulation with thrombin and trypsin. 6 In conclusion, PAR1 was the only known receptor for trypsin expressed in the rat myometrium, but it was suggested to be cleaved and inactivated by trypsin. Trypsin was thus suggested to contract the rat myometrium via a novel type of PAR, which might be upregulated during pregnancy.

The high incidence of VAE highlights the need for practitioners to be aware of this potentially serious event. Use of PDU can aid in the detection of VAE during ERCP and should be considered especially during high-risk therapeutic procedures. Detection may allow appropriate interventions before serious adverse events such as cardiovascular collapse occur.

1 Using fura-2¯uorometry of [Ca 2+ ] i in response to thrombin, trypsin and protease-activated receptor activating peptides (PAR-APs), we determined whether trypsin cleaves protease-activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein. 2 Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca 2+ ] i elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothelial cells. However, the preceding stimulation with trypsin abolished the subsequent response to thrombin, but not to bradykinin or substance P. 3 The response to PAR1-AP (SFLLRNP) was signi®cantly (P50.05) reduced by the preceding stimulation with thrombin and PAR1-AP in the valvular endothelial cells, while, importantly, it remained unaected by the preceding stimulation with either trypsin or PAR2-AP (SLIGRL). The response to PAR2-AP was reduced by the preceding stimulation with trypsin and PAP2-AP. PAR1-AP attenuated the subsequent responses not only to thrombin and PAR1-AP but also to trypsin and PAR2-AP, while PAR2-AP speci®cally attenuated the subsequent responses to trypsin and PAR2-AP. 4 In human umbilical vein endothelial cells, a higher anity PAR1-AP (haPAR1-AP) (Ala-pFArg-Cha-HArg-Tyr-NH 2 ) speci®cally attenuated the responses to thrombin but not trypsin. On the other hand, the response to haPAR1-AP was signi®cantly (P50.05) attenuated by the preceding stimulation with thrombin but not trypsin. 5 In conclusion, trypsin cleaved PAR1 but did not activate it in the endothelial cells. Moreover, the trypsin-cleaved PAR1 was no longer responsive to thrombin.

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