Vascular and endothelial actions of inhibitors of substance P amidation.

Formation of mature active neuropeptides such as substance P (SP) from their glycine extended precursors entails alpha-amidation of peptide precursors by the sequential enzymatic action of peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL). We reported that these two enzymes that can produce mature active neuropeptides are present in cultured bovine aortic endothelial cells (BAECs). We hypothesize that alpha-amidation of peptides occurs in endothelial cells and that these peptides are critically involved in the overall regulation of cardiovascular function. In this study, this hypothesis was tested using specific amidation inhibitors to determine their effects on the actions of SP and its glycine-extended precursor (SP-Gly). We have found that SP and SP-Gly are equipotent in stimulating nitric oxide (NO) release by BAECs. At 10(-5) M, the specific inhibitors of PAM (4-phenyl-3-butenoic acid; PBA) and PGL (5-acetamido-2,4-diketo-6-phenyl-hexanoic acid and its methyl ester) reduced NO basal release by 40, 34, and 45%, respectively. They also reduced the production of NO induced by SP-Gly by 63, 68, and 69%, respectively, but had no effect on NO production in response to either SP or acetylcholine. SP and SP-Gly also were equipotent in relaxing rat aortic segments. The vasorelaxation to SP-Gly was endothelium dependent and inhibited by the NOS antagonist L-nitroarginine methyl ester (L-NAME), but it was not affected by inhibition of prostaglandin synthesis. Inhibitors of both PAM and PGL significantly reduced the vasorelaxing actions of SP-Gly, whereas responses to SP were not affected. A cumulative infusion of PBA into the femoral artery of rabbits, at final concentrations of 2.4, 24, and 240 microM for 20 min each, increased the vascular resistance (VR), indicating the tonic production of vasodilating amidated peptide(s). This effect was maximum at 60 min after infusion (20.5 +/- 4.7 vs. 8.2 +/- 0.7 mm Hg/ml/min; p < 0.05). These results suggest that endothelial cells can produce mature SP from its SP-Gly precursor and that a product of peptide alpha-amidation tonically stimulates endothelial cell NO release to control vascular tone.

Non-epithelial endothelin-A receptors activate adenylate cyclase in rat trachea: biochemical mechanisms and physiological implications.

In the present study, we investigated the mechanisms underlying the differential effects of endothelins (ETs) in the rat trachea. Sarafotoxin-S6c (SRTX-c) and ET-3 were more potent spasmogens to rat tracheal strips than ET-1. The EC50 values were 12, 14.1 and 89.1 nM, respectively. Tension responses to ET-1 and ET-3, but not to SRTX-c, were enhanced by either indomethacin or the ET(A) blocker, BQ-610 (1 microM). In epithelium-intact tracheal rings, both ET-1 and ET-3 activated adenylate cyclase (AC) in a concentration-dependent manner. The activation by ET-1 of AC was significantly higher than that of ET-3. Thus, EC50 values for ET-1 and ET-3 were 71 and 200 nM, and maximal cAMP increments were 196% and 62% above baseline, respectively. SRTX-c, up to 1 microM, did not alter basal cAMP level. Mechanical removal of the epithelium neither had an effect on AC activation by ET-1 or ET-3, nor did it alter the inability of SRTX-c to modulate AC activity. Conversely, pre-incubation of tracheal strips with indomethacin (1 microM) virtually ablated the increments in cAMP by the ETs. Likewise, BQ-610 attenuated AC activation, concentration-dependently (IC50=28.2 nM). Taken together, the present study suggests that ET(A) receptors, from non-epithelial source, are functionally-linked to AC activation via a prostanoid-dependent pathway. This ET(A)-initiated cascade acts to negatively regulate muscle contraction. Such a cross-talk between ET signals most likely accounts for variation of tension responses to ET homologs.

Endothelins-induce cyclicAMP formation in the guinea-pig trachea through an ETA receptor- and cyclooxygenase-dependent mechanism.

1. The non-selective endothelin agonist, endothelin-1 (ET-1), and the selective ETB receptor agonist, sarafotoxin-S6c (SRTX-c), contracted guinea-pig isolated trachea in a concentration-dependent manner. The EC50 value for ET-1 (11 +/- 2.1 nM) was significantly higher than that of SRTX-c (3.2 +/- 0.21 nM) and the maximal developed tension due to SRTX-c was 42.8 +/- 2.3% higher than that produced by ET-1 (P < 0.05). 2. Pretreatment with the ETA antagonist, BQ-610, appreciably enhanced the developed tension due to ET-1 but not SRTX-c. Likewise, the cyclo-oxygenase inhibitor, indomethacin, markedly potentiated the contractile responses to ET-1, but not to SRTX-c. Combining BQ-610 with indomethacin was not more effective than either of them in augmenting ET-1-evoked tension. 3. ET-1 significantly increased cyclic AMP formation in the trachea in concentration- and time-dependent manners. A t1/2 value of 4.3 min, an EC50 value of 20 +/- 3 nM and a maximal cyclic AMP increment of 124% above the basal level, were obtained for ET-1. Similarly but less effectively, ET-3 (0.1 microM) increased cyclic AMP level (35 +/- 3.7% compared to 94 +/- 7.8% for the same concentration of ET-1). By contrast, SRTX-c did not alter the cyclicAMP level when applied in concentrations up to 1 microM. 4. Pre-incubation of the trachea with BQ-610 (1 microM) or indomethacin (1 microM) prevented cyclicAMP formation by either ET-1 or ET-3. 5. The results of the present study indicate a negative regulatory role mediated by the ETA receptor on the ETB-triggered mechanical response. This effect is likely to be mediated by activation of adenylate cyclase through a cyclo-oxygenase-dependent mechanism.