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Abdulmaged M Traish - One of the best experts on this subject based on the ideXlab platform.
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development of human and rabbit vaginal smooth muscle cell cultures effects of vasoactive agents on intracellular levels of Cyclic nucleotides
Molecular Cell Biology Research Communications, 1999Co-Authors: Abdulmaged M Traish, Robert B Moreland, Yuehua Huang, Noel N Kim, Jennifer Berman, Irwin GoldsteinAbstract:Abstract In this study, we subcultured and characterized human and rabbit vaginal smooth muscle cells and investigated the synthesis of second messenger Cyclic nucleotides in response to vasodilators and determined the activity and kinetics of Phosphodiesterase (PDE) type 5 (EC 3.1.4.35 3′,5′-Cyclic GMP Phosphodiesterase). Cultured vaginal cells exhibited growth characteristics typical of smooth muscle cells and immunostained with antibodies against alpha smooth muscle actin. The cells retained functional prostaglandin E and β-adrenergic receptors as demonstrated by increased intracellular cAMP synthesis in response to PGE 1 , or isoproterenol. The response to these vasoactive substances was augmented with forskolin, suggesting stabilization of G-protein-activated adenylyl cyclases. Treatment with the nitric oxide donor, sodium nitroprusside, in the presence of sildenafil, a PDE type 5 inhibitor, enhanced intracellular cGMP synthesis and accumulation. Incubation of rabbit vaginal tissue with sildenafil, sodium nitroprusside, and PGE 1 or forskolin produced a marked increase in intracellular cGMP. These observations were similar to findings with cultured cells and suggest that subcultured cells retain functional characteristics exhibited in intact tissue. The cells retained Phosphodiesterase type 5 expression as shown by specific cGMP hydrolytic activity. Sildenafil and zaprinast inhibited cGMP hydrolysis competitively and bound with high affinity ( K i = 7 and 250 nM, respectively). These observations suggest that cultured human and rabbit vaginal smooth muscle cells retained their metabolic functional integrity and this experimental system should prove useful in investigating the pathway of nitric oxide and PDE type 5 inhibitors in modulating vaginal smooth muscle tone.
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sildenafil inhibits Phosphodiesterase type 5 in human clitoral corpus cavernosum smooth muscle
Biochemical and Biophysical Research Communications, 1998Co-Authors: Kwangsung Park, Robert B Moreland, Irwin Goldstein, Anthony Atala, Abdulmaged M TraishAbstract:Abstract Phosphodiesterases play an important physiological role by regulating the intracellular levels of Cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of Phosphodiesterase (PDE) type 5 (EC 3.1.4.35, 3′,5′-Cyclic GMP Phosphodiesterase) by a novel, high-affinity, selective PDE type 5 inhibitor, sildenafil, in intact cells and in soluble extracts of human clitoral corpus cavernosum smooth muscle cells. Sildenafil inhibited cGMP hydrolysis in the crude extract (Ki= 7.2 ± 2.7) and in partially purified preparations (Ki= 9 nM) in a competitive manner, as determined by Dixon plots. Sildenafil was a more effective PDE type 5 inhibitor than zaprinast (Ki= 400.0 ± 76.4 nM, crude extracts; 250 nM, partially purified). Stimulation of intracellular cGMP synthesis by the nitric oxide donor sodium nitroprusside resulted in a 3.3- and 2.9-fold increase in cGMP concentration in the presence of sildenafil or zaprinast, respectively, compared to sodium nitroprusside treatment alone in intact cells at physiological temperatures. These observations suggest that human clitoral corpus cavernosum smooth muscle tone may be regulated by the synthesis and release of nitric oxide and that this pathway is dependent on PDE type 5 activity.
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sildenafil a novel inhibitor of Phosphodiesterase type 5 in human corpus cavernosum smooth muscle cells
Life Sciences, 1998Co-Authors: Robert B Moreland, Irwin Goldstein, Abdulmaged M TraishAbstract:Abstract In human corpus cavernosum, release of nitric oxide from the non-adrenergic, non-cholinergic nerves and/or the endothelium activates guanylyl cyclase and increases intracellular cGMP levels. The increase in intracellular cGMP modulates intracellular calcium and in turn regulates smooth muscle contractility and erectile function. Phosphodiesterases play an important physiological role by regulating the intracellular levels of Cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of Phosphodiesterase (PDE) type 5 (E.C. 3.1.4.35 3′, 5′-Cyclic GMP Phosphodiesterase) by a novel, high affinity, selective PDE type 5 inhibitor, sildenafil, in soluble extracts of human corpus cavernosum smooth muscle cells. Sildenafil inhibited PDE type 5 cGMP-hydrolytic activity, in the crude extract (K i = 4–6 nM) and in partially purified preparations (K i = 2 nM) in a competitive manner, as determined by Dixon plots. Sildenafil (K i = 2–4 nM) was a more effective PDE type 5 inhibitor than zaprinast (K i = 250 nM). Stimulation of intracellular cGMP synthesis by the nitric oxide donor, sodium nitroprusside, resulted in less than a 5% increase in cGMP levels in the absence of sildenafil and a 35% increase in cGMP levels in the presence of sildenafil, in intact cells at physiological temperatures. These results are in accord with the clinical observations that sildenafil, taken orally, promotes penile erection through increased intracellular cGMP in response to sexual stimulation, potentiating smooth muscle relaxation.
Fiona Burns - One of the best experts on this subject based on the ideXlab platform.
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the effect of Cyclic amp and Cyclic GMP Phosphodiesterase inhibitors on the superoxide burst of guinea pig peritoneal macrophages
British Journal of Pharmacology, 1993Co-Authors: Nicholas C Turner, Fiona Burns, Lorna J Wood, Thomas Gueremy, John E SounessAbstract:1. The Cyclic nucleotide Phosphodiesterase (PDE) activity of guinea-pig peritoneal macrophages was partially characterized and the effects of selective and non-selective inhibitors of adenosine 3':5'-Cyclic monophosphate (Cyclic AMP PDE) and guanosine 3':5'-Cyclic monophosphate (Cyclic GMP PDE) Phosphodiesterases on superoxide generation were investigated using peritoneal macrophages from horse-serum pretreated guinea-pigs. 2. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and the PDE I/V selective inhibitor, zaprinast, inhibited spontaneous superoxide generation with IC50s of 30.7 +/- 11.3 microM and 145 +/- 17 microM respectively (n = 6 and 5). The concentration-response curves for the PDE IV selective inhibitors rolipram and Ro20-1724 were biphasic; mean maximum inhibitions were 56.9 +/- 5.9% and 66.8 +/- 10.5% respectively at 300 microM, but in 2 out of 6 (rolipram) and 2 out of 5 (Ro20-1724) experiments inhibition was < 50%. The PDE III inhibitor SK&F 94120 was without effect. Spontaneous superoxide generation was reduced 57 +/- 10% by 1 microM prostaglandin E2 (PGE2) and 62.6 +/- 3.76% by 1 microM salbutamol. 3. The increase in superoxide generation elicited by FMLP (10(-9)-10(-5)M) was unaffected by any of the PDE inhibitors studied. Inhibition of FMLP-stimulated superoxide generation by PGE2 was enhanced in the presence of 10 microM IBMX. 4. Macrophages were found to contain a predominantly membrane bound Cyclic AMP PDE (90% of total activity) which was unaffected by Cyclic GMP or calcium/calmodulin. The Cyclic AMP PDE activity in the cytosolic fraction was enhanced in the presence of calcium/calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)
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interaction of the catalytic subunit of protein kinase a with the lung type v Cyclic GMP Phosphodiesterase modulation of non catalytic binding sites
Biochemical and Biophysical Research Communications, 1992Co-Authors: Fiona Burns, Nigel J PyneAbstract:We have previously demonstrated that the catalytic sub-unit of protein kinase A can catalyse a potent activation of partially purified Type V Cyclic GMP-specific Phosphodiesterase activity (Burns et al., 1992, Biochem. J. 283, 487-491). We now demonstrate that this Phosphodiesterase most likely has a sub-unit mass of 90kDa, based upon 32P-Cyclic GMP photo-affinity labelling, that activation of the Phosphodiesterase does not require the prior binding of Cyclic GMP to the Phosphodiesterase, and that alkaline phosphatase can reverse the protein kinase A-dependent activation of Phosphodiesterase activity. Zaprinast is a mixed inhibitor of non-activated Cyclic GMP Phosphodiesterase activity. However, inhibition of the protein kinase A-activated Phosphodiesterase is competitive. These results suggest that protein kinase A can modulate the inhibitory effects of zaprinast via perturbations of a non-catalytic binding site.
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the catalytic subunit of protein kinase a triggers activation of the type v Cyclic GMP specific Phosphodiesterase from guinea pig lung
Biochemical Journal, 1992Co-Authors: Fiona Burns, I W Rodger, Nigel J PyneAbstract:The type V Cyclic GMP Phosphodiesterase was partially purified from the high-speed supernatant of guinea-pig lung. The isoenzyme displayed linear kinetics for Cyclic GMP hydrolysis, with Km = 2.2 +/- 0.2 microM and Vmax. = 1.2 +/- 0.08 nmol/min per mg. The selective type V Phosphodiesterase inhibitor Zaprinast inhibited Cyclic GMP hydrolysis with IC50 (concn. giving 50% inhibition) = 0.45 +/- 0.08 microM. Isobutylmethylxanthine promoted a 3-fold increase in the binding of Cyclic GMP to the isoenzyme. The addition of the catalytic subunit of protein kinase A to an activation cocktail containing the partially purified type V Phosphodiesterase resulted in a marked increase in Vmax. for Cyclic GMP hydrolysis (approximately 10-fold at 40 units of protein kinase A). We have suggested that protein kinase A triggers phosphorylation of the Phosphodiesterase, which results in activation of Phosphodiesterase activity. In addition, the sensitivity to inhibition by Zaprinast is severely decreased (the IC50 for inhibition is 7.5 +/- 1.1 microM), suggesting that the potency of Phosphodiesterase inhibitors is effected by phosphorylation of the enzyme.
Robert B Moreland - One of the best experts on this subject based on the ideXlab platform.
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development of human and rabbit vaginal smooth muscle cell cultures effects of vasoactive agents on intracellular levels of Cyclic nucleotides
Molecular Cell Biology Research Communications, 1999Co-Authors: Abdulmaged M Traish, Robert B Moreland, Yuehua Huang, Noel N Kim, Jennifer Berman, Irwin GoldsteinAbstract:Abstract In this study, we subcultured and characterized human and rabbit vaginal smooth muscle cells and investigated the synthesis of second messenger Cyclic nucleotides in response to vasodilators and determined the activity and kinetics of Phosphodiesterase (PDE) type 5 (EC 3.1.4.35 3′,5′-Cyclic GMP Phosphodiesterase). Cultured vaginal cells exhibited growth characteristics typical of smooth muscle cells and immunostained with antibodies against alpha smooth muscle actin. The cells retained functional prostaglandin E and β-adrenergic receptors as demonstrated by increased intracellular cAMP synthesis in response to PGE 1 , or isoproterenol. The response to these vasoactive substances was augmented with forskolin, suggesting stabilization of G-protein-activated adenylyl cyclases. Treatment with the nitric oxide donor, sodium nitroprusside, in the presence of sildenafil, a PDE type 5 inhibitor, enhanced intracellular cGMP synthesis and accumulation. Incubation of rabbit vaginal tissue with sildenafil, sodium nitroprusside, and PGE 1 or forskolin produced a marked increase in intracellular cGMP. These observations were similar to findings with cultured cells and suggest that subcultured cells retain functional characteristics exhibited in intact tissue. The cells retained Phosphodiesterase type 5 expression as shown by specific cGMP hydrolytic activity. Sildenafil and zaprinast inhibited cGMP hydrolysis competitively and bound with high affinity ( K i = 7 and 250 nM, respectively). These observations suggest that cultured human and rabbit vaginal smooth muscle cells retained their metabolic functional integrity and this experimental system should prove useful in investigating the pathway of nitric oxide and PDE type 5 inhibitors in modulating vaginal smooth muscle tone.
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sildenafil inhibits Phosphodiesterase type 5 in human clitoral corpus cavernosum smooth muscle
Biochemical and Biophysical Research Communications, 1998Co-Authors: Kwangsung Park, Robert B Moreland, Irwin Goldstein, Anthony Atala, Abdulmaged M TraishAbstract:Abstract Phosphodiesterases play an important physiological role by regulating the intracellular levels of Cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of Phosphodiesterase (PDE) type 5 (EC 3.1.4.35, 3′,5′-Cyclic GMP Phosphodiesterase) by a novel, high-affinity, selective PDE type 5 inhibitor, sildenafil, in intact cells and in soluble extracts of human clitoral corpus cavernosum smooth muscle cells. Sildenafil inhibited cGMP hydrolysis in the crude extract (Ki= 7.2 ± 2.7) and in partially purified preparations (Ki= 9 nM) in a competitive manner, as determined by Dixon plots. Sildenafil was a more effective PDE type 5 inhibitor than zaprinast (Ki= 400.0 ± 76.4 nM, crude extracts; 250 nM, partially purified). Stimulation of intracellular cGMP synthesis by the nitric oxide donor sodium nitroprusside resulted in a 3.3- and 2.9-fold increase in cGMP concentration in the presence of sildenafil or zaprinast, respectively, compared to sodium nitroprusside treatment alone in intact cells at physiological temperatures. These observations suggest that human clitoral corpus cavernosum smooth muscle tone may be regulated by the synthesis and release of nitric oxide and that this pathway is dependent on PDE type 5 activity.
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sildenafil a novel inhibitor of Phosphodiesterase type 5 in human corpus cavernosum smooth muscle cells
Life Sciences, 1998Co-Authors: Robert B Moreland, Irwin Goldstein, Abdulmaged M TraishAbstract:Abstract In human corpus cavernosum, release of nitric oxide from the non-adrenergic, non-cholinergic nerves and/or the endothelium activates guanylyl cyclase and increases intracellular cGMP levels. The increase in intracellular cGMP modulates intracellular calcium and in turn regulates smooth muscle contractility and erectile function. Phosphodiesterases play an important physiological role by regulating the intracellular levels of Cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of Phosphodiesterase (PDE) type 5 (E.C. 3.1.4.35 3′, 5′-Cyclic GMP Phosphodiesterase) by a novel, high affinity, selective PDE type 5 inhibitor, sildenafil, in soluble extracts of human corpus cavernosum smooth muscle cells. Sildenafil inhibited PDE type 5 cGMP-hydrolytic activity, in the crude extract (K i = 4–6 nM) and in partially purified preparations (K i = 2 nM) in a competitive manner, as determined by Dixon plots. Sildenafil (K i = 2–4 nM) was a more effective PDE type 5 inhibitor than zaprinast (K i = 250 nM). Stimulation of intracellular cGMP synthesis by the nitric oxide donor, sodium nitroprusside, resulted in less than a 5% increase in cGMP levels in the absence of sildenafil and a 35% increase in cGMP levels in the presence of sildenafil, in intact cells at physiological temperatures. These results are in accord with the clinical observations that sildenafil, taken orally, promotes penile erection through increased intracellular cGMP in response to sexual stimulation, potentiating smooth muscle relaxation.
Nigel J Pyne - One of the best experts on this subject based on the ideXlab platform.
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interaction of the catalytic subunit of protein kinase a with the lung type v Cyclic GMP Phosphodiesterase modulation of non catalytic binding sites
Biochemical and Biophysical Research Communications, 1992Co-Authors: Fiona Burns, Nigel J PyneAbstract:We have previously demonstrated that the catalytic sub-unit of protein kinase A can catalyse a potent activation of partially purified Type V Cyclic GMP-specific Phosphodiesterase activity (Burns et al., 1992, Biochem. J. 283, 487-491). We now demonstrate that this Phosphodiesterase most likely has a sub-unit mass of 90kDa, based upon 32P-Cyclic GMP photo-affinity labelling, that activation of the Phosphodiesterase does not require the prior binding of Cyclic GMP to the Phosphodiesterase, and that alkaline phosphatase can reverse the protein kinase A-dependent activation of Phosphodiesterase activity. Zaprinast is a mixed inhibitor of non-activated Cyclic GMP Phosphodiesterase activity. However, inhibition of the protein kinase A-activated Phosphodiesterase is competitive. These results suggest that protein kinase A can modulate the inhibitory effects of zaprinast via perturbations of a non-catalytic binding site.
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the catalytic subunit of protein kinase a triggers activation of the type v Cyclic GMP specific Phosphodiesterase from guinea pig lung
Biochemical Journal, 1992Co-Authors: Fiona Burns, I W Rodger, Nigel J PyneAbstract:The type V Cyclic GMP Phosphodiesterase was partially purified from the high-speed supernatant of guinea-pig lung. The isoenzyme displayed linear kinetics for Cyclic GMP hydrolysis, with Km = 2.2 +/- 0.2 microM and Vmax. = 1.2 +/- 0.08 nmol/min per mg. The selective type V Phosphodiesterase inhibitor Zaprinast inhibited Cyclic GMP hydrolysis with IC50 (concn. giving 50% inhibition) = 0.45 +/- 0.08 microM. Isobutylmethylxanthine promoted a 3-fold increase in the binding of Cyclic GMP to the isoenzyme. The addition of the catalytic subunit of protein kinase A to an activation cocktail containing the partially purified type V Phosphodiesterase resulted in a marked increase in Vmax. for Cyclic GMP hydrolysis (approximately 10-fold at 40 units of protein kinase A). We have suggested that protein kinase A triggers phosphorylation of the Phosphodiesterase, which results in activation of Phosphodiesterase activity. In addition, the sensitivity to inhibition by Zaprinast is severely decreased (the IC50 for inhibition is 7.5 +/- 1.1 microM), suggesting that the potency of Phosphodiesterase inhibitors is effected by phosphorylation of the enzyme.
Irwin Goldstein - One of the best experts on this subject based on the ideXlab platform.
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development of human and rabbit vaginal smooth muscle cell cultures effects of vasoactive agents on intracellular levels of Cyclic nucleotides
Molecular Cell Biology Research Communications, 1999Co-Authors: Abdulmaged M Traish, Robert B Moreland, Yuehua Huang, Noel N Kim, Jennifer Berman, Irwin GoldsteinAbstract:Abstract In this study, we subcultured and characterized human and rabbit vaginal smooth muscle cells and investigated the synthesis of second messenger Cyclic nucleotides in response to vasodilators and determined the activity and kinetics of Phosphodiesterase (PDE) type 5 (EC 3.1.4.35 3′,5′-Cyclic GMP Phosphodiesterase). Cultured vaginal cells exhibited growth characteristics typical of smooth muscle cells and immunostained with antibodies against alpha smooth muscle actin. The cells retained functional prostaglandin E and β-adrenergic receptors as demonstrated by increased intracellular cAMP synthesis in response to PGE 1 , or isoproterenol. The response to these vasoactive substances was augmented with forskolin, suggesting stabilization of G-protein-activated adenylyl cyclases. Treatment with the nitric oxide donor, sodium nitroprusside, in the presence of sildenafil, a PDE type 5 inhibitor, enhanced intracellular cGMP synthesis and accumulation. Incubation of rabbit vaginal tissue with sildenafil, sodium nitroprusside, and PGE 1 or forskolin produced a marked increase in intracellular cGMP. These observations were similar to findings with cultured cells and suggest that subcultured cells retain functional characteristics exhibited in intact tissue. The cells retained Phosphodiesterase type 5 expression as shown by specific cGMP hydrolytic activity. Sildenafil and zaprinast inhibited cGMP hydrolysis competitively and bound with high affinity ( K i = 7 and 250 nM, respectively). These observations suggest that cultured human and rabbit vaginal smooth muscle cells retained their metabolic functional integrity and this experimental system should prove useful in investigating the pathway of nitric oxide and PDE type 5 inhibitors in modulating vaginal smooth muscle tone.
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sildenafil inhibits Phosphodiesterase type 5 in human clitoral corpus cavernosum smooth muscle
Biochemical and Biophysical Research Communications, 1998Co-Authors: Kwangsung Park, Robert B Moreland, Irwin Goldstein, Anthony Atala, Abdulmaged M TraishAbstract:Abstract Phosphodiesterases play an important physiological role by regulating the intracellular levels of Cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of Phosphodiesterase (PDE) type 5 (EC 3.1.4.35, 3′,5′-Cyclic GMP Phosphodiesterase) by a novel, high-affinity, selective PDE type 5 inhibitor, sildenafil, in intact cells and in soluble extracts of human clitoral corpus cavernosum smooth muscle cells. Sildenafil inhibited cGMP hydrolysis in the crude extract (Ki= 7.2 ± 2.7) and in partially purified preparations (Ki= 9 nM) in a competitive manner, as determined by Dixon plots. Sildenafil was a more effective PDE type 5 inhibitor than zaprinast (Ki= 400.0 ± 76.4 nM, crude extracts; 250 nM, partially purified). Stimulation of intracellular cGMP synthesis by the nitric oxide donor sodium nitroprusside resulted in a 3.3- and 2.9-fold increase in cGMP concentration in the presence of sildenafil or zaprinast, respectively, compared to sodium nitroprusside treatment alone in intact cells at physiological temperatures. These observations suggest that human clitoral corpus cavernosum smooth muscle tone may be regulated by the synthesis and release of nitric oxide and that this pathway is dependent on PDE type 5 activity.
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sildenafil a novel inhibitor of Phosphodiesterase type 5 in human corpus cavernosum smooth muscle cells
Life Sciences, 1998Co-Authors: Robert B Moreland, Irwin Goldstein, Abdulmaged M TraishAbstract:Abstract In human corpus cavernosum, release of nitric oxide from the non-adrenergic, non-cholinergic nerves and/or the endothelium activates guanylyl cyclase and increases intracellular cGMP levels. The increase in intracellular cGMP modulates intracellular calcium and in turn regulates smooth muscle contractility and erectile function. Phosphodiesterases play an important physiological role by regulating the intracellular levels of Cyclic nucleotides. In this study, we investigated the kinetic parameters of inhibition of Phosphodiesterase (PDE) type 5 (E.C. 3.1.4.35 3′, 5′-Cyclic GMP Phosphodiesterase) by a novel, high affinity, selective PDE type 5 inhibitor, sildenafil, in soluble extracts of human corpus cavernosum smooth muscle cells. Sildenafil inhibited PDE type 5 cGMP-hydrolytic activity, in the crude extract (K i = 4–6 nM) and in partially purified preparations (K i = 2 nM) in a competitive manner, as determined by Dixon plots. Sildenafil (K i = 2–4 nM) was a more effective PDE type 5 inhibitor than zaprinast (K i = 250 nM). Stimulation of intracellular cGMP synthesis by the nitric oxide donor, sodium nitroprusside, resulted in less than a 5% increase in cGMP levels in the absence of sildenafil and a 35% increase in cGMP levels in the presence of sildenafil, in intact cells at physiological temperatures. These results are in accord with the clinical observations that sildenafil, taken orally, promotes penile erection through increased intracellular cGMP in response to sexual stimulation, potentiating smooth muscle relaxation.
