Kathleen G. Morgan

Vascular smooth muscle: The first recorded Ca2+ transients

The bioluminescent calcium indicator aequor in was successfully loaded into vascular smooth muscle cells ofAmphiuma tridactylum by either microinjection or a new method which makes the cells reversibly hyperpermeable. Both gave similar results; however, the latter method produced larger signals. Vasoconstrictors produced a sustained contraction and a light (calcium) response consisting of two component: a large transient followed by a smaller, sustained response. Electrical stimulation produced a light transient that was much briefer than the contraction. These results suggest that tension can be maintained in smooth muscle in the presence of lower calcium levels than those present during force development.

Smooth muscle signalling pathways in health and disease

•  Introduction •  Mechanisms that regulate LC20 phosphorylation ‐  Regulation of myosin phosphatase ‐  CaMKII •  Mechanisms that regulate the access of myosin to actin ‐  Caldesmon ‐  bCaP function in regulation of PKC and ERK signalling •  Mechanisms that regulate cytoskeletal remodelling •  Conclusions

Alterations in cytoplasmic calcium sensitivity during porcine coronary artery contractions as detected by aequorin.

1. Intracellular‐Ca2+‐force relationships were investigated in porcine epicardial coronary arteries by the simultaneous measurement of aequorin luminescence and isometric force. 2. In response to K+ depolarization and histamine, force and aequorin light rose monophasically. In response to carbachol and serotonin, tonic contractions were accompanied by biphasic aequorin signals consisting of an initial spike followed by a low plateau. Contractions produced by prostaglandin F2 alpha (PGF2 alpha) or the endoperoxide analogue U‐46619 were accompanied by little or no detectable rise in light. 3. Comparison of steady‐state force to steady‐state light levels indicated that agonists gave greater force for a given intracellular Ca2+ concentration ([Ca2+]i) compared to that seen during K+ contractures. 4. In Ca2+‐free bathing media, carbachol produced a transient contraction accompanied by a transient intracellular Ca2+ spike indicating release of Ca2+ from intracellular storage sites. 5. In Ca2+‐free bathing media PGF2 alpha produced a tonic contraction with no detectable change in light. 6. These results suggest that changes in the sensitivity of the contractile apparatus to Ca2+ or other activator systems may be as important a mechanism of contraction as are changes in [Ca2+]i.

A role for MAP kinase in differentiated smooth muscle contraction evoked by alpha-adrenoceptor stimulation.

The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in smooth muscle contraction by monitoring MAP kinase activation, caldesmon phosphorylation, and contractile force during agonist stimulation. Isometric tension in response to KCl and phenylephrine (PE) was measured from strips of ferret aorta. MAP kinase activation was monitored by Western blot using a phosphospecific p44/p42 MAP kinase antibody. Caldesmon phosphorylation was assessed using specific phosphocaldesmon antibodies. We report here that treatment of smooth muscle strips with PD-098059, a specific inhibitor of MAP kinase kinase, did not detectably modify the KCl-evoked contraction but significantly inhibited the contraction to PE in the absence of extracellular Ca2+. In this experimental condition, where the contraction occurs in the absence of increases in 20-kDa myosin light chain phosphorylation, PD-098059 also inhibited significantly MAP kinase and caldesmon phosphorylation. Collectively, these results demonstrate a direct cause-and-effect relationship between MAP kinase activation and Ca2+-independent smooth muscle contraction and support the concept of caldesmon phosphorylation as the missing link between both events.

Calponin and Mitogen-activated Protein Kinase Signaling in Differentiated Vascular Smooth Muscle

Contraction of smooth muscle cells is generally assumed to require Ca2+/calmodulin-dependent phosphorylation of the 20-kDa myosin light chains. However, we report here that in the absence of extracellular calcium, phenylephrine induces a contraction of freshly isolated ferret aorta cells in the absence of increases in intracellular ionized calcium or light chain phosphorylation levels but in the presence of activation of mitogen-activated protein kinase. A protein at 36 kDa co-immunoprecipitated with the mitogen-activated protein kinase and was identified as the actin-binding protein, calponin, by immunoblot. An overlay assay further confirmed an interaction between the kinase and calponin, even though the kinase did not phosphorylate calponin in vitro. Calponin also co-immunoprecipitated from smooth muscle cells with protein kinase C-ε. High resolution digital confocal studies indicated that calponin redistributes to the cell membrane during phenylephrine stimulation at a time when mitogen-activated protein kinase and protein kinase C-ε are targeted to the plasmalemma. These results suggest a role for calponin as a signaling molecule, possibly an adapter protein, linking the targeting of mitogen-activated protein kinase and protein kinase C-ε to the surface membrane.

Calcium and cardiovascular function: Intracellular calcium levels during contraction and relaxation ofmammalian cardiac and vascular smooth muscle as detected with aequorin

Calcium ion (Ca++) plays a central role in excitation-contraction coupling of both cardiac and vascular smooth muscles and have important functional interactions with other cations, including sodium, potassium, and magnesium. Ca++ transients associated with contraction-relaxation cycles of the heart and vasculature can now be recorded directly by use of aequorin, a bioluminescent protein that emits light when it combines with Ca++. After microinjection or chemical loading of aequorin into the sarcoplasm, light output provides an index of intracellular [Ca++]. In cardiac muscle, intracellular Ca++ increases more quickly than tension and decreases toward basal levels by the time peak tension is reached. The calcium transients of working myocardium in both human subjects and other mammalian species appear to be dominated by the release and uptake of Ca++ from intracellular stores under all conditions studied. Drugs and disease states produce marked changes in the amplitude and time course of the Ca++ transient and the corresponding contractile response. In vascular smooth muscle, there are stimulus-specific patterns in intracellular Ca++ associated with tonic contractions. Although Ca++ is related to tension development, the relationship appears to be more complex than that in cardiac muscle. As a result, tension development cannot be used as an index of free Ca++ levels in vascular smooth muscle. Selection of the most effective therapy to reverse a tonic contraction in states of spasm or hypertension may depend on the specific stimulus that caused the increased tone.

Agonist-specific myosin phosphorylation and intracellular calcium during isometric contractions of arterial smooth muscle

The relationship between phosphorylation of the 20-kDa myosin light chain, intracellular calcium levels ([Ca2+]i), and isometric force was studied during prolonged activation of arterial smooth muscle. Aequorin, preloaded into ferret aortic strips, was used as a [Ca2+]i indicator. Two dimensional polyacrylamide gel electrophoresis was used to determine the phosphorylation levels of the 20-kDa myosin light chain (LC20). During the 30-min depolarization of arterial smooth muscle by K+ (21 mM), both LC20 phosphorylation and [Ca2+]i increased significantly at all time points examined as did the steady state stress. A transient rise in LC20 phosphorylation and [Ca2+]i occurred within 30 s, followed by suprabasal levels through the 10-min period during a sustained alpha1-mediated activation by 10− M phenylephrine whereas a higher force was developed at a shorter time compared to K+. An active phorbol ester 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA, 10− M) induced a slow contraction of similar magnitude to that induced by K+ without significantly changing either [Ca2+]i or LC20 phosphorylation over a 90-min period. These results demonstrate that the amount of LC20 phosphorylation correlates with the [Ca2+]i in all three types of activation. The initial levels of [Ca2+]i and LC20 phosphorylation correlate with the onset of force development but not the magnitude of steady state stress, suggesting a role for [Ca2+]i and LC20 phosphorylation in regulating the cross bridge cycling rate during tension development. The lack of a detectable increase in [Ca2+]i and LC20 phosphorylation during DPBA activation suggests that sites other than LC20, phosphorylated by protein kinase C, may be involved in regulating smooth muscle contraction.

Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent

Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

Differential Association and Localization of Myosin Phosphatase Subunits During Agonist-Induced Signal Transduction in Smooth Muscle

It has been known for some time that agonist-induced contractions of vascular smooth muscle are often associated with a sensitization of the contractile apparatus to intracellular Ca2+. One mechanism that has been suggested to explain Ca2+ sensitization is inhibition of myosin phosphatase activity. In the present study, we tested the hypothesis that differential localization of the phosphatase might be associated with its inhibition. Quantitative confocal microscopy of freshly dissociated, fully contractile smooth muscle cells was used in parallel with measurements of myosin light chain and myosin phosphatase phosphorylation. The results indicate that, in the smooth muscle cells, the catalytic and targeting subunits of the phosphatase are dissociated from each other in an agonist-specific manner and that the dissociation is accompanied by a slower rate of myosin phosphorylation. Targeting of myosin phosphatase to the cell membrane precedes the dissociation of subunits and is associated with phosphorylation of the targeting subunit at a Rho-associated kinase (ROK) phosphorylation site. The phosphorylation and membrane translocation of the targeting subunit are inhibited by a ROK inhibitor. This dissociation of subunits may provide a mechanism for the decreased phosphatase activity of phosphorylated myosin phosphatase.

Requirement for protein kinase C theta for cell cycle progression and formation of actin stress fibers and filopodia in vascular endothelial cells.

Activation of the protein kinase C (PKC) family with phorbol esters induces endothelial proliferation and angiogenesis, but which of the events that constitute angiogenesis are affected by individual members of the PKC family is unknown. In rat capillary endothelial (RCE) cells, serum stimulation increased expression of a single PKC isoenzyme, PKCθ, and its translocation to the periphery. Conditional overexpression of a dominant-negative mutant of PKCθ markedly inhibited RCE proliferation, as well as closure of a “wound” by RCE migration and formation of capillary rings and tubules in vitro. PKCθ inhibition delayed the endothelial cell cycle at the G2/M phase and prevented formation of actin stress fibers and filopodia but not lamellipodia. The defect in cell morphology and wound closure in PKCθ-kn cells was reversed by overexpressing kinase-active PKCθ, indicating that these RCE functions depend upon PKCθ substrates. Thus, PKCθ is required for multiple processes essential for angiogenesis and wound repair, including endothelial mitosis, maintenance of a normal actin cytoskeleton, and formation of an enclosed tube.