Role of Cyclic Nucleotide-Dependent Actin Cytoskeletal Dynamics: [Ca2+]i and Force Suppression in Forskolin-Pretreated Porcine Coronary Arteries

Abstract

Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca2+]i) and phosphorylation of myosin light chains (MLC). However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA) prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca2+]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.

The AMP-Related Kinase (AMPK) Induces Ca 2+ -Independent Dilation of Resistance Arteries by Interfering With Actin Filament Formation

“…Of note, of 6 studies focusing especially on VSM, 5 reported increased cofilin phosphorylation in response to contractile stimulation. 39,[44][45][46][47] If one can conclude that vasodilation should lead to opposite changes, the majority of these studies are consistent with cofilin dephosphorylation being a relevant factor in controlling cytoskeletal changes, which ultimately lead to vasodilation. However, one vascular study also reported decreased phosphorylation with contraction.…”

“…39,[44][45][46][47] By contrast, in airway smooth muscle ADF (actin depolymerizing factor)/cofilin dephosphorylation was reported to occur during constriction, that is, a condition that goes along with actin polymerization. 48 We cannot explain the conflicting results at present, but differences between VSM and other tissues or the mode of preactivation of the muscle may play a role here.…”

Heat Shock–Related Protein 20 Peptide Decreases Human Airway Constriction Downstream of β2-Adrenergic Receptor

“…Previous investigations by our laboratory demonstrated that P20 peptide caused relaxation of vascular smooth muscle by inducing actin depolymerization (24,30,33), and we hypothesized that P20 peptide would also cause actin depolymerization in ASM. Therefore, we measured the actin concentration in intact PASM tissue, as PASM samples have less heterogeneity compared with HASM samples.…”

“…Phosphorylation of HSP20 in response to bronchodilators was examined by isoelectric focusing, which separates the phospho and nonphospho forms of HSP20, and phospho-HSP20 level was measured by Western blotting, as described previously (30).…”

“…The ratio of filamentous (F)-actin versus globular (G)-actin in intact porcine ASM (PASM) tissue was measured using the G-actin/F-actin In Vivo Assay kit (Cytoskeleton, Denver, CO), per the manufacturer's protocol, as described previously (30).…”