Melatonin Attenuates Vascular Smooth Muscle Contraction Through the γ-Secretase/Notch Intracellular Domain/Myocardin Pathway.

ABSTRACT: Inositol 1, 4, 5-trisphosphate (IP3) signaling-mediated calcium release drives the contraction of vascular smooth muscles and hence regulates blood vessel volume and blood pressure. Melatonin supplementation has been suggested to be beneficial for hypertension. To determine whether the blood pressure-lowering effect of melatonin was accounted for by IP3 signaling, we evaluated the vasoconstriction response and IP3 signaling in isolated mouse thoracic aortic rings during melatonin incubation. C57BL/6 mice were given intraperitoneal injections daily with melatonin, and the systolic blood pressure and contractility of aortic rings from melatonin-treated mice were decreased, and the contraction suppression effect of melatonin was attributed to the impaired expression of contractile proteins in vascular smooth muscle cells rather than IP3 signaling. Our results further showed that melatonin increased the expression of γ-secretase, which could cleave and release the notch intracellular domain, and the notch intracellular domain prevented the transcription of contractile genes by interfering with the interaction between serum response factor and myocardin, the master regulator of contractile protein. In this article, we report a novel mechanism by which melatonin regulates smooth muscle contractility that does not depend on IP3 signaling.

Atorvastatin regulates vascular smooth muscle cell phenotypic transformation by epigenetically modulating contractile proteins and mediating Akt/FOXO4 axis.

Atherosclerosis (AS) is a prevalent cardiovascular disease with severe morbidity and high mortality. Phenotypic regulation of vascular smooth muscle cells (VSMCs) from the contractile and quiescent phenotype to the synthetic type is a critical step for the vascular remodeling of AS. Atorvastatin, as a 3­hydroxy­3­methyl­glutaryl coenzyme A reductase inhibitor, presents an anti­inflammatory effect to improve vascular endothelial functions. The aim of the present study was to examine the effect of atorvastatin on VSMCs phenotypic transformation and the underlying mechanism. The rat primary VSMCs were isolated and identified. The protein expression of contractile proteins, such as α­SMA, SM­MHC, and SM22α, was reduced by angiotensin II (AngII) and enhanced by atorvastatin, in which atorvastatin could reverse the effect of AngII in the VSMCs. The treatment of HDAC inhibitor trichostatin A was able to enhance AngII­inhibited expression of α­SMA and SM­MHC. Atorvastatin regulated AngII­associated VSMCs phenotypic transformation by epigenetically regulating contractile proteins. Moreover, atorvastatin modulated platelet­derived growth factor­BB (PDGF­BB)­induced VSMC phenotypic transformation by modulating the Akt/forkhead Box O4 (FOXO4) axis. Immunofluorescence analysis revealed that PDGF­BB enhanced the accumulation of FOXO4 in the VSMCs, while the treatment of atorvastatin was able to attenuate this effect and the co­treatment of Akt inhibitor LY294002 could further inhibit the phenotype. The treatment of PDGF­BB enhanced the interaction of SRF with FOXO4 and myocardin in the VSMCs, in which the co­treatment of atorvastatin and LY294002 could reverse the effect of PDGF­BB in the system. Thus, atorvastatin regulates VSMCs phenotypic transformation by epigenetically modulating contractile proteins and mediating the Akt/FOXO4 axis. Findings of the present study provide new insights into the mechanism by which atorvastatin modulates VSMCs, providing valuable evidence for the application of atorvastatin in the treatment of AS.

ANLN-induced EZH2 upregulation promotes pancreatic cancer progression by mediating miR-218-5p/LASP1 signaling axis.

BACKGROUND: Pancreatic cancer is a highly lethal malignancy with poor prognosis. Anillin (ANLN), an actin binding protein, is upregulated and plays an important role in many malignant tumors. However, the precise role of ANLN in pancreatic cancer remains unclear. METHODS: The expression of ANLN and its association with pancreatic cancer patient survival were analyzed using an online database and confirmed by immunohistochemistry. The ANLN protein expression in pancreatic cancer cell lines was detected by Western blot. Cell proliferation, colony formation and transwell assays in vitro and in vivo tumor growth were used to determine the role of ANLN in pancreatic cancer. Gene expression microarray analysis and a series of in vitro assays were used to elucidate the mechanisms of ANLN regulating pancreatic cancer progression. RESULTS: We found that the ANLN expression was significantly upregulated in pancreatic cancer tissues and cell lines. The high expression of ANLN was associated with tumor size, tumor differentiation, TNM stage, lymph node metastasis, distant metastasis and poor prognosis in pancreatic cancer. ANLN downregulation significantly inhibited cell proliferation, colony formation, migration, invasion and tumorigenicity in nude mice. Meanwhile, we found that ANLN knockdown inhibited several cell-cell adhesion related genes, including the gene encoding LIM and SH3 protein 1 (LASP1). LASP1 upregulation partially reversed the tumor-suppressive effect of ANLN downregulation on pancreatic cancer cell progression. Moreover, we found that ANLN downregulation induced the expression of miR-218-5p which inhibited LASP1 expression through binding to its 3'UTR. We also found that ANLN-induced enhancer of zeste homolog 2 (EZH2) upregulation was involved in regulating miR-218-5p/LASP1 signaling axis. EZH2 upregulation or miR-218-5p downregulation partially reversed the tumor-suppressive effect of ANLN downregulation on pancreatic cancer cell progression. CONCLUSION: ANLN contributed to pancreatic cancer progression by regulating EZH2/miR-218-5p/LASP1 signaling axis. These findings suggest that ANLN may be a candidate therapeutic target in pancreatic cancer.

Anillin Promotes Cell Contractility by Cyclic Resetting of RhoA Residence Kinetics.

RhoA stimulates cell contractility by recruiting downstream effectors to the cortical plasma membrane. We now show that direct binding by anillin is required for effective signaling: this antagonizes the otherwise labile membrane association of GTP-RhoA to promote effector recruitment. However, since its binding to RhoA blocks access by other effectors, we demonstrate that anillin must also concentrate membrane phosphoinositide-4,5-P2 (PIP2) to promote signaling. We propose and test a sequential pathway where GTP-RhoA first binds to anillin and then is retained at the membrane by PIP2 after it disengages from anillin. Importantly, re-binding of membrane GTP-RhoA to anillin, regulated by the cortical density of anillin, creates cycles through this pathway. These cycles repeatedly reset the dissociation kinetics of GTP-RhoA, substantially increasing its dwell time to recruit effectors. Thus, anillin regulates RhoA signaling by a paradigm of kinetic scaffolding that may apply to other signals whose efficacy depends on their cortical dwell times.

Microfibril-associate glycoprotein-2 (MAGP-2) promotes angiogenic cell sprouting by blocking notch signaling in endothelial cells.

Angiogenesis is highly sensitive to the composition of the vascular microenvironment, however, our understanding of the structural and matricellular components of the vascular microenvironment that regulate angiogenesis and the molecular mechanisms by which these molecules function remains incomplete. Our previous results described a novel pro-angiogenic activity for Microfibril-Associated Glycoprotein-2 (MAGP-2), but did not address the molecular mechanism(s) by which this is accomplished. We now demonstrate that MAGP-2 promotes angiogenic cell sprouting by antagonizing Notch signaling pathways in endothelial cells. MAGP-2 decreased basal and Jagged1 induced expression from the Notch sensitive Hes-1 promoter in ECs, and blocked Jagged1 stimulated Notch1 receptor processing in transiently transfected 293T cells. Interestingly, inhibition of Notch signaling by MAGP-2 seems to be restricted to ECs since MAGP-2 increased Hes-1 promoter activity and Notch1 receptor processing in heterologous cell types. Importantly, constitutive activation of the Notch signaling pathway blocked the ability of MAGP-2 to promote angiogenic cell sprouting, as well as morphological changes associated with angiogenesis. Collectively, these observations indicate that MAGP-2 promotes angiogenic cell spouting in vitro by antagonizing Notch signaling pathways in ECs.

A 90 kDa fragment of filamin A promotes Casodex-induced growth inhibition in Casodex-resistant androgen receptor positive C4-2 prostate cancer cells.

Prostate tumors are initially dependent on androgens for growth, but the majority of patients treated with anti-androgen therapy progress to androgen-independence characterized by resistance to such treatment. This study investigates a novel role for filamin A (FlnA), a 280 kDa cytoskeletal protein (consisting of an actin-binding domain (ABD) followed by 24 sequential repeats), in androgen-independent (AI) growth. Full-length FlnA is cleaved to 170 kDa (ABD+FlnA1-15) and 110 kDa fragments (FlnA16-24); the latter is further cleaved to a 90 kDa fragment (repeats 16-23) capable of nuclear translocation and androgen receptor (AR) binding. Here, we demonstrate that in androgen-dependent LNCaP prostate cancer cells, the cleaved 90 kDa fragment is localized to the nucleus, whereas in its AI subline C4-2, FlnA failed to cleave and remained cytoplasmic. Transfection of FlnA16-24 cDNA in C4-2 cells restored expression and nuclear localization of 90 kDa FlnA. Unlike LNCaP, C4-2 cells proliferate in androgen-reduced medium and in the presence of the AR-antagonist Casodex. They also exhibit increased Akt phosphorylation compared to LNCaP, which may contribute to their AI phenotype. Nuclear expression of 90 kDa FlnA in C4-2 cells decreased Akt phosphorylation, prevented proliferation in androgen-reduced medium and restored Casodex sensitivity. This effect was inhibited by constitutive activation of Akt indicating that FlnA restored Casodex sensitivity in C4-2 cells by decreasing Akt phosphorylation. In addition, FlnA-specific siRNA which depleted FlnA levels, but not control siRNA, induced resistance to Casodex in LNCaP cells. Our results demonstrate that expression of nuclear FlnA is necessary for androgen dependence in these cells.

Effects of profilin and thymosin beta4 on the critical concentration of actin demonstrated in vitro and in cell extracts with a novel direct assay.

The free actin concentration at steady state, Ac, is a variable that determines how actin regulatory proteins influence the extent of actin polymerization. We describe a novel method employing fluorescence anisotropy to directly measure Ac in any sample after the addition of a trace amount of labeled thymosin beta4 or thymosin beta4 peptide. Using this assay, we confirm earlier theoretical work on the helical polymerization of actin and confirm the effects of actin filament-stabilizing drugs and capping proteins on Ac, thereby validating the assay. We also confirm a controversial prior observation that profilin lowers the critical concentration of Mg2+-actin. A general mechanism is proposed to explain this effect, and the first quantitative dose-response curve for the effect of profilin on Ac facilitates its evaluation. This mechanism also predicts the effect of profilin on critical concentration in the presence of the limited amount of capping protein, which is the condition often found in cells, and the effect of profilin on critical concentration in cell extracts is demonstrated for the first time. Additionally, nonlinear effects of thymosin beta4 on the steady state amount of F-actin are explained by the observed changes in Ac. This assay has potential in vivo applications that complement those demonstrated in vitro.

Dominant negative mutants of filamin A block cell surface expression of the D2 dopamine receptor.

Protein interaction screens have revealed an interaction between the D2 dopamine receptor and the actin cross-linking protein filamin A. However, the physiological significance of this interaction has not been explained. To better understand the role of filamin A in D2 receptor-mediated signaling, we examined the effect of disrupting filamin A/D2 receptor interaction. Overexpression of a truncated form of filamin A (repeat units 18-19 containing the D2, but not the actin, binding domain) caused a marked reduction in both the number and half-life of cell surface D2 receptors. These results suggest that disruption of the linkage between D2 receptors and the actin cytoskeleton destabilizes plasma membrane-associated D2 receptors. Several missense mutations within repeat unit 19 of filamin A were identified that abrogate filamin A/D2 receptor interaction. Introduction of mutant and wild-type filamin A into filamin A-deficient M2 cells demonstrated that wild-type filamin A, but not the filamin A-binding mutants, was able to promote cell-surface expression of D2 receptors. Together, these studies provide evidence that filamin A/D2 receptor interaction is required for the proper targeting or stabilization of D2 dopamine receptors at the plasma membrane.

Comparison of filamin A-induced cross-linking and Arp2/3 complex-mediated branching on the mechanics of actin filaments.

We compared the effects of human filamin A (FLNa) and the activated human Arp2/3 complex on mechanical properties of actin filaments. As little as 1 FLNa to 800 polymerizing actin monomers induces a sharp concentration-dependent increase in the apparent viscosity of 24 microm actin, a parameter classically defined as a gel point. The activated Arp2/3 complex, at concentrations up to 1:25 actins had no detectable actin gelation activity, even in the presence of phalloidin, to stabilize actin filaments against debranching. Increasing the activated Arp2/3 complex to actin ratio raises the FLNa concentration required to induce actin gelation, an effect ascribable to Arp2/3-mediated actin nucleation resulting in actin filament length diminution. Time lapse video microscopy of microparticles attached to actin filaments or photoactivation of fluorescence revealed actin filament immobilization by FLNa in contrast to diffusion of Arp2/3-branched actin filaments. The experimental results support theories predicting that polymer branching absent cross-linking does not lead to polymer gelation and are consistent with the observation that cells deficient in actin filament cross-linking activity have unstable surfaces. They suggest complementary roles for actin branching and cross-linking in cellular actin mechanics in vivo.

Androgen receptor nuclear translocation is facilitated by the f-actin cross-linking protein filamin.

The human androgen receptor (hAR) is a ligand-dependent transcription factor responsible for the development of the male phenotype. The mechanism whereby nuclear translocation of the hAR is induced by its natural ligand 5alpha-dihydrotestosterone is a phenomenon not fully understood. The two-hybrid interaction trap assay has been used to isolate proteins that interact with the hAR in an attempt to identify molecules involved in hAR transactivation and movement. We have identified the actin-binding protein filamin, a 280-kDa component of the cytoskeleton, as an hAR interacting protein. This interaction is ligand independent but is enhanced in its presence. The functional significance of this interaction was analyzed using a cell line deficient in filamin via transient expression of a green fluorescent protein-hAR chimera. In filamin-deficient cells this revealed that hAR remained cytoplasmic even after prolonged exposure to synthetic ligand. Nuclear shuttling was restored when this cell line regained wild-type expression of filamin. These data suggest a novel role for filamin, implicating it as an important molecule in AR movement from the cytoplasm to the nucleus.

The bi-directional regulation of filamin on the ATPase activity of smooth muscle myosin.

OBJECTIVE: The aim of this study is to investigate the functional relationship between filamin, a known actin binding protein, and myosin and the effects of filamin on the interaction between myosin and actin. METHODS: Ultra-centrifugation method was used to investigate the binding of filamin to both phosphorylated and unphosphorylated myosins. Mg-ATPase activities of both phosphorylated and unphosphorylated myosins in the presence and absence of actin were measured to observe the effects resulted from filamin-actin and filamin-myosin interactions. RESULTS: It was found that filamin is also a myosin binding protein. Filamin inhibited the actin activated Mg-ATPase activity of phosphorylated myosin and stimulated Mg-ATPase of phosphorylated myosin in the absence of actin; in addition, filamin stimulated Mg-ATPase activity of unphosphorylated myosin in both the presence or absence of actin. CONCLUSION: The result suggest that the effects of filamin on the myosin Mg-ATPase activities are bi-directional, i.e., stimulatory via binding to myosin and inhibitory via binding to actin.

Actin filament organization is required for proper cAMP-dependent activation of CFTR.

Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl(-) currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl(-) conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl(-) currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(-)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl(-) channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl(-) channel activity in excised patches of ABP(-)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.

A novel anti-inflammatory peptide inhibits endothelial cell cytoskeletal rearrangement, nitric oxide synthase translocation, and paracellular permeability increases.

The endothelial cell (EC) membrane-cytoskeletal interface in part maintains plasma membrane integrity and promotes cell-cell apposition. Nonmuscle filamin (ABP-280), an actin crosslinking protein, promotes orthogonal branching of F-actin and is the major protein that links the peripheral actin network to the plasma membrane through its C-terminal glycoprotein binding site. In response to bradykinin, filamin translocates from the cell periphery to the cytosol within 1 min. A synthetic peptide, corresponding to filamin's C-terminal calcium/calmodulin-dependent protein kinase II phosphorylation site (CaM peptide), prevents calcium-activated filamin translocation in permeabilized bovine pulmonary artery EC. The myristoylated permeable form of this peptide inhibits bradykinin-induced filamin translocation and F-actin rearrangement in cultured intact ECs. In addition, bradykinin-induced paracellular gap formation is significantly attenuated by CaM peptide, which suggests that the presence of a filamin-based peripheral F-actin network is essential for maintaining EC barrier function. Moreover, CaM peptide reduces wound-induced EC migration rate by 40%, which indicates that F-actin rearrangement is required for efficient cell motility. The CaM peptide affects other bradykinin-induced inflammatory responses. EC nitric oxide synthase (eNOS) translocates from the cell membrane to the nuclear fraction within 1-2 min of bradykinin treatment. Pretreatment with CaM peptide inhibits eNOS translocation. However, the peptide has no effect on bradykinin-induced von Willebrand Factor release. In summary, the CaM peptide exhibits several anti-inflammatory properties that include maintaining EC junctional stability and inhibiting eNOS translocation.

Contrasting effects of betaxolol and propranolol on Ca(2+)-activated contractions in skinned fibers from canine coronary arteries and ventricular muscles.

The effects of some beta-adrenoceptor antagonists (atenolol, betaxolol, bunitrolol, labetalol, pindolol, and propranolol) on Ca2+ (pCa 5.8)-activated tension development in chemically skinned fibers from canine coronary artery and right ventricular trabeculae were studied. In skinned coronary arteries, Ca(2+)-activated tension development was decreased by betaxolol and propranolol at concentration of more than 10(-5) and 10(-4) M, respectively. The pCa-tension relationships were shifted to the right and down by betaxolol. In contrast, in skinned cardiac muscle Ca(2+)-activated tension development was increased by betaxolol and propranolol at the same range of concentrations as in coronary arteries, with no change in maximum tension. The pCa-tension relation was shifted to the left by betaxolol. Other beta-adrenoceptor antagonists (atenolol, bunitrolol, labetalol, and pindolol) had no effect on Ca(2+)-induced contraction in either muscle type. These results indicate that among beta-adrenoceptor antagonists, only betaxolol and propranolol can directly modulate the Ca2+ sensitivity of myofilaments and have opposite effects on the contractile system in canine cardiac and vascular smooth muscle.

cAMP-independent regulation of CFTR by the actin cytoskeleton.

Protein kinase A (PKA)-activation of epithelial Na+ channels requires actin filaments. Mouse mammary adenocarcinoma cells expressing the human cystic fibrosis transmembrane conductance regulator (CFTR) or mock transfectants were used to determine whether CFTR is also modulated by the actin cytoskeleton. The actin filament disrupter cytochalasin D (CD; approximately 5 micrograms/ml) readily activated whole cell currents in CFTR but not in mock-transfected (MOCK) cells. Addition of actin to the cytosolic side of quiescent excised inside-out patches of CFTR but not MOCK cells also activated CFTR. The actin-activated Cl- channels (symmetrical Cl-) had a linear conductance of 9.3 pS and were inhibited by diphenylamine-2-carboxylate and monoclonal antibodies raised against CFTR. Channel activity was also blocked by addition of the actin-binding proteins deoxyribonuclease I and filamin. Incubation of CFTR cells with CD (approximately 15 micrograms/ml) for > 6 h prevented CFTR activation by the addition of either 8-bromoadenosine 3',5'-cyclic monophosphate plus forskolin under whole cell conditions or PKA under excised inside-out conditions. However, CFTR activation was restored by subsequent addition of actin. The data indicate that CFTR is regulated by actin filaments whose effect may, in turn, be associated with the PKA-dependent pathway.

Binding of divalent cation and nucleotide to G-actin in the presence of profilin.

The effect of profilin, a G-actin binding protein, on the mechanism of exchange of the tightly bound metal ion and nucleotide on G-actin, has been investigated. 1) In low ionic strength buffer, profilin increases the rates of Ca2+ and Mg2+ dissociation from G-actin 250- and 50-fold, respectively. On the profilin-actin complex as well as on G-actin alone, nucleotide exchange is dependent on the concentration of divalent metal ion and is kinetically limited, at low concentration of metal ion, by the dissociation of the metal ion. 2) Under physiological ionic conditions, nucleotide exchange on G-actin is 1 order of magnitude faster than at low ionic strength. The rate of MgATP dissociation is increased by profilin from 0.05 s-1 to 2 s-1, the rate of MgADP dissociation is increased from 0.2 s-1 to 24 s-1. The dependences of the exchange rates on profilin concentration are consistent with a high affinity (5 x 10(6) to 10(7) M-1) of profilin for ATP-G-actin, and a 20-fold lower affinity for ADP-G-actin. Profilin binding to actin lowers the affinity of metal-nucleotide by about 1 order of magnitude. These results restrain the possible roles of profilin in actin assembly in vivo.

Filamin and gelsolin influence Ca(2+)-sensitivity of smooth muscle thin filaments.

Sheep aorta thin filaments were prepared by ultracentrifugation of an ATP-containing extract in the presence of different concentrations of ethanediol. Thin filaments prepared without ethanediol contained small quantities of tropomyosin (0.027 Tm:actin) and caldesmon (0.017 CD:actin) and activated the MgATPase of skeletal myosin independently of Ca2+. Ultracentrifugation in the presence of 10-20% ethanediol resulted in preparation of thin filaments with increased content of tropomyosin (0.17 Tm:actin) and caldesmon (0.04 CD:actin). These thin filaments possessed high Ca(2+)-sensitivity in activation of skeletal muscle myosin ATPase. Besides actin, tropomyosin and caldesmon, thin filaments contained gelsolin and filamin. Gelsolin content (0.007 gelsolin:actin) was independent of the presence of ethanediol. The filamin content decreased from 0.015 to 0.007 mol:mol actin when the ethanediol concentration was increased from 0 to 20%, and was negatively correlated with the Ca2+ sensitivity of thin filaments. In a reconstituted system, pure filamin or gelsolin affected caldesmon regulation of actomyosin ATPase. Gelsolin (0.01:actin) reduced the inhibition of actomyosin ATPase caused by caldesmon and increased the potency of Ca(2+)-calmodulin in reversing this inhibition. Filamin (0.007:actin) also decreased the inhibitory action of caldesmon on actin-activated myosin ATPase and also potentiated the reversal of this inhibition by calmodulin. We conclude that minor components of smooth muscle thin filaments (gelsolin and filamin) significantly modify caldesmon mediated regulation of actomyosin ATPase. We suggest a tropomyosin-mediated mechanism by which filamin or gelsolin could exert similar effects.

Effects of pH and inorganic phosphate on force production in alpha-toxin-permeabilized isolated rat uterine smooth muscle.

1. Strips of longitudinal smooth muscle isolated from rat uterus were permeabilized using crude alpha-toxin from the bacterium Staphylococcus aureus. This treatment rendered the surface membrane permeable to small molecular weight substances. Simultaneous measurements of tension and calcium concentration ([Ca2+]) (using indo-1 fluorescence) were used to investigate the effects of pH and inorganic phosphate concentration ([Pi]) on Ca(2+)-activated force generated by the contractile proteins. 2. Raising the [Pi] from 1 to 11 mM at a pH of 7.2 depressed both maximal and submaximal Ca(2+)-activated force. This effect of Pi was concentration dependent having the majority of its effect by 6 mM. 3. Further experiments at a submaximal [Ca2+] showed that Ca(2+)-activated force was enhanced by raising [Pi] from 6 to 11 mM suggesting that Pi increased the Ca2+ sensitivity of tension production. Based on these results, calculations indicate that the apparent affinity constant of Ca2+ for the contractile proteins increased from 4 x 10(6) M-1 to 6 x 10(6) M-1 on raising [Pi] from 1 to 11 mM. 4. Lowering pH from 7.2 to 6.7 at a [Pi] of 1 mM potentiated Ca(2+)-activated force with a small depression in the apparent Ca2+ sensitivity of tension production. This effect of pH on maximum (100 microM Ca2+) and submaximum (0.3 microM Ca2+) Ca(2+)-activated force was observed over a range of acidic pHs (7.0-6.7). 5. Increasing pH from 7.2 to 7.7 at a [Pi] of 1 mM depressed Ca(2+)-activated force with no effect on Ca2+ sensitivity of tension production. 6. Spontaneous contractions in intact rat myometrium are abolished under hypoxic conditions. Under these same conditions intracellular [Pi] rises and pH falls. The results of this study suggest that taken individually neither the effect of a rise in [Pi] nor a fall in pH on Ca(2+)-activated force generated by the contractile proteins can account for the effect of hypoxia on spontaneous contractions.

Modulation of the interaction between G-actin and thymosin beta 4 by the ATP/ADP ratio: possible implication in the regulation of actin dynamics.

The interaction of G-actin with thymosin beta 4 (T beta 4), the major G-actin-sequestering protein in motile and proliferating cells, has been analyzed in vitro. T beta 4 is found to have a 50-fold higher affinity for MgATP-actin than for MgADP-actin. These results imply that in resting platelets and neutrophils, actin is sequestered by T beta 4 as MgATP-G-actin. Kinetic experiments and theoretical calculations demonstrate that this ATP/ADP dependence of T beta 4 affinity for G-actin can generate a mechanism of desequestration of G-actin by ADP, in the presence of physiological concentrations of T beta 4 (approximately 0.1 mM). The desequestration of G-actin by ADP is kinetically enhanced by profilin, which accelerates the dissociation of ATP from G-actin. Whether a local drop in the ATP/ADP ratio can allow local, transient desequestration and polymerization of actin either close to the plasma membrane, following platelet or neutrophil stimulation, or behind the Listeria bacterium in the host cell, while the surrounding cytoplasm contains sequestered ATP-G-actin, is an open issue raised by the present work.