8-((4-chlorophenyl)thio)cyclic-3--5--gmp and Hypertrophy--Right-Ventricular

The signal transduction mechanisms defining the role of cyclic nucleotides in the regulation of pulmonary vascular tone is currently an area of great interest. Normally, signaling mechanisms that elevate cAMP and guanosine-3',5'-cyclic monophosphate (cGMP) maintain the pulmonary vasculature in a relaxed state. Modulation of the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BK(Ca) channel has been implicated in the development of pulmonary hypertension. Accordingly, studies were done to determine the effect of cAMP-elevating agents on BK(Ca) channel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), a recognized animal model of pulmonary hypertension. Forskolin (10 micro M), a stimulator of adenylate cyclase and an activator of cAMP-dependent protein kinase (PKA), and 8-4-chlorophenylthio (CPT)-cAMP (100 micro M), a membrane-permeable derivative of cAMP, opened BK(Ca) channels in single FHR PASMC. Treatment of FHR PASMC with 300 nM KT5823, a selective inhibitor of cGMP-dependent protein kinase (PKG) activity inhibited the effect of both forskolin and CPT-cAMP. In contrast, blocking PKA activation with 300 nM KT5720 had no effect on forskolin or CPT-cAMP-stimulated BK(Ca) channel activity. These results indicate that cAMP-dependent vasodilators activate BK(Ca) channels in PASMC of FHR via PKG-dependent and PKA-independent signaling pathways, which suggests cross-activation between cyclic nucleotide-dependent protein kinases in pulmonary arterial smooth muscle and therefore, a unique signaling pathway for cAMP-induced pulmonary vasodilation.

Topics: Alkaloids; Animals; Carbazoles; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Hypertrophy, Right Ventricular; Indoles; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Large-Conductance Calcium-Activated Potassium Channels; Male; Membrane Potentials; Muscle, Smooth, Vascular; Patch-Clamp Techniques; Platelet Aggregation Inhibitors; Potassium Channels, Calcium-Activated; Pulmonary Artery; Rats; Rats, Inbred Strains; Thionucleotides; Vasodilator Agents

Recent studies from our laboratory indicate that pulmonary vasodilatory responses to exogenous nitric oxide (NO) are attenuated following chronic hypoxia (CH) and that this NO-dependent vasodilation is mediated by cGMP. Similarly, we have demonstrated that CH attenuates vasodilatory responses to the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). We hypothesized that attenuated pulmonary vasodilation to 8-BrcGMP following CH is mediated by decreased protein kinase G-1 (PKG-1) expression/activity. Therefore, we examined vasodilatory responses to 8-BrcGMP (1 microM) in isolated, saline-perfused lungs from control and CH (4 wk at barometric pressure of 380 mmHg) rats in the presence of the competitive PKG inhibitor Rp-beta-phenyl-1, N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate (30 microM) or the highly specific PKG inhibitor KT-5823 (10 microM). PKG-1 expression and activity were determined in whole lung homogenates from each group, and vascular PKG-1 levels were assessed by quantitative immunohistochemistry. PKG inhibition with either Rp-8-Br-PET-cGMPS or KT-5823 diminished vasodilatory responses to 8-BrcGMP in lungs from both control and CH rats, thus indicating a role for PKG in mediating reactivity to 8-BrcGMP in each group. However, in contrast to our hypothesis, PKG-1 levels were approximately twofold greater in lungs from CH rats vs. controls, and furthermore, this upregulation was localized to the vasculature. This correlates with an increase in PKG activity following CH. We conclude that PKG-1 is involved in 8-BrcGMP-mediated vasodilation; however, attenuated pulmonary vasodilation following CH is not associated with decreased expression/activity of PKG-1.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Alkaloids; Animals; Carbazoles; Chronic Disease; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Immunohistochemistry; Indoles; Lung; Nitric Oxide; Platelet Aggregation Inhibitors; Polycythemia; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Thionucleotides; Up-Regulation; Vascular Resistance; Vasodilation