Cellular mechanisms of cyclic nucleotide-induced vasorelaxation

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Cellular mechanisms of cyclic nucleotide-induced vasorelaxation Colleen M. Brophy, MD, E.Glenn Whitney, MD, Shannon Lamb, BS, Arthur Beall, PhD Journal of Vascular Surgery Volume 25, Issue 2, Pages 390-397 (February 1997) DOI: 10.1016/S0741-5214(97)70361-6 Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 1 Two pathways have been implicated in modulating vascular smooth muscle relaxation. Adenylate cyclase (AC) activation by agents such as prostacyclin (PC), isoproterenol (ISO), and FSK, leads to increases in cAMP. cAMP activates cAMP-dependent kinase (PKA). In addition, NO and NO donors, such as nitroglycerine (NTG) and SNP, activate guanylate cyclase (GC), leading to increases in cGMP. cGMP activates PKG. Protein kinases such as PKA and PKG modulate cellular physiologic mechanisms through the phosphorylation of specific phosphoproteins (SP). A number of substrate proteins have been suggested to mediate vasorelaxation including the IP3 receptor, the plasma membrane Ca2+-pump, phospholamban, and two unidentified 20 kDa proteins. The subsequent events that lead to dissociation of actin and myosin and relaxation have not been clearly determined. Journal of Vascular Surgery 1997 25, 390-397DOI: (10.1016/S0741-5214(97)70361-6) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Representative tracing of BCASM relaxation responses. BCASM were equilibrated in muscle bath and maximal contractile response to KCl (110 mmol/L) was determined. Some strips were then subjected to Ca2+-free conditions and did not contract to a subsequent KCl (110 mmol/L) treatment. PDBu (300 nmol/L) treatment led to a slowly developing sustained contraction that nearly completely relaxed in the presence or absence of Ca2+ with the addition of SNP (10 μmol/L) or FSK (10 μmol/L). Journal of Vascular Surgery 1997 25, 390-397DOI: (10.1016/S0741-5214(97)70361-6) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Representative tracing of BCASM relaxation responses. BCASM were equilibrated in muscle bath and maximal contractile response to KCl (110 mmol/L) was determined. Some strips were then subjected to Ca2+-free conditions and did not contract to a subsequent KCl (110 mmol/L) treatment. PDBu (300 nmol/L) treatment led to a slowly developing sustained contraction that nearly completely relaxed in the presence or absence of Ca2+ with the addition of SNP (10 μmol/L) or FSK (10 μmol/L). Journal of Vascular Surgery 1997 25, 390-397DOI: (10.1016/S0741-5214(97)70361-6) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Representative tracing of BCASM relaxation responses. BCASM were equilibrated in muscle bath and maximal contractile response to KCl (110 mmol/L) was determined. Some strips were then subjected to Ca2+-free conditions and did not contract to a subsequent KCl (110 mmol/L) treatment. PDBu (300 nmol/L) treatment led to a slowly developing sustained contraction that nearly completely relaxed in the presence or absence of Ca2+ with the addition of SNP (10 μmol/L) or FSK (10 μmol/L). Journal of Vascular Surgery 1997 25, 390-397DOI: (10.1016/S0741-5214(97)70361-6) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 2 Representative tracing of BCASM relaxation responses. BCASM were equilibrated in muscle bath and maximal contractile response to KCl (110 mmol/L) was determined. Some strips were then subjected to Ca2+-free conditions and did not contract to a subsequent KCl (110 mmol/L) treatment. PDBu (300 nmol/L) treatment led to a slowly developing sustained contraction that nearly completely relaxed in the presence or absence of Ca2+ with the addition of SNP (10 μmol/L) or FSK (10 μmol/L). Journal of Vascular Surgery 1997 25, 390-397DOI: (10.1016/S0741-5214(97)70361-6) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 3 There were no significant differences in the extent of relaxation in strips under Ca2+-containing (+) or Ca2+-free (−) conditions treated with FSK (10 μmol/L) or SNP (10 μmol/L) in the presence or absence of IBMX (1 mmol/L) (n = 4 to 6 separate animals, 2 to 3 strips per animal). Journal of Vascular Surgery 1997 25, 390-397DOI: (10.1016/S0741-5214(97)70361-6) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 4 BCASM strips precontracted with the agonist serotonin (5-HT, 10 μmol/L) or by depolarization with KCL (110 mmol/L) nearly completely relax with the addition of FSK (10 μmol/L) or SNP (10 μmol/L). However, strips pre-contracted with the phosphatase inhibitor calyculin (CALYC, 100 nmol/L) are refractory to relaxation with the addition of FSK (10 μmol/L) or SNP (10 μmol/L) (n = 4 to 6 separate animals, 2 to 3 strips per animal). Journal of Vascular Surgery 1997 25, 390-397DOI: (10.1016/S0741-5214(97)70361-6) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 5 Intracellular Ca2+ concentrations do not change in BCASM strips loaded with the photoprobe aequorin when stimulated with phorbol ester (DPB, 1 μmol/L) or FSK (50 μmol/L). Journal of Vascular Surgery 1997 25, 390-397DOI: (10.1016/S0741-5214(97)70361-6) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 6 BCASM were treated with FSK (10 μmol/L) and IBMX (1 mmol/L) for 15 minutes at 37° C. Control tissues remained in buffer at 37° C. PKA activity ratios were determined as previously described.13 Basal PKA activity was (0.46 ± 0.04). Stimulation with FSK/IBMX resulted in PKA activation (activity ratio of 1) in Ca2+-free (−Ca2+) or Ca2+-containing (+Ca2+) conditions, and in the presence of calyculin (CALYC) (n = 4 separate experiments). Journal of Vascular Surgery 1997 25, 390-397DOI: (10.1016/S0741-5214(97)70361-6) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

Fig. 7 Strips of BCASM were preincubated with 32P-orthophosphate and then no agonist (Control) (A); PDBu (100 nmol/L, 1 hour) (B); PDBu (100 nmol/L, 1 hour) followed by FSK (10 μmol/L, 15 minutes) (C); or PDBu (100 nmol/L, 1 hour) followed by SNP (10 μmol/L, for 15 minutes) (D) Ca2+-containing (+Ca) or Ca2+-free conditions (−Ca). Phosphoproteins were resolved by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FSK and SNP resulted in an increase in phosphorylation of two 20 kDa phosphoproteins with a pI of approximately 6 (arrows). The 20 kDa myosin light chain (m) is labeled for reference. This blot is representative of three separate experiments. Journal of Vascular Surgery 1997 25, 390-397DOI: (10.1016/S0741-5214(97)70361-6) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions