Bridging structures spanning the junctioning gap at the triad of skeletal muscle.

The membrane systems of skeletal muscle were examined after tannic acid fixation. A new structure consisting of bridges spanning the junctional gap is described, and a model is proposed in which the cytoplasmic but not the luminal membrane leaflets of the transverse tubule and of the junctional sarcoplasmic reticulum (SR) are continuous. The globular particles (presumably the Ca-binding proteins) within the terminal cisternae were arranged in longitudinal rows and appeared adherent to the junctional membrane. The junctional gap was present in negatively stained, frozen thin sections of fixed muscles. Negatively staining material occured within the junctional gap. The cytoplasmic leaflets of the longitudinal, intermediate, and terminal cisterna regions of the SR exhibited a thick coat of densely staining material compatible with the presence of the Ca-ATPase. Similar bridges were also observed at the surface membrane-SR close coupling sites of vascular smooth muscle.

Dense bodies and actin polarity in vertebrate smooth muscle.

The arrangement of cytoplasmic dense bodies in vertebrate smooth muscle and their relationship to the thin filaments was studied in cells from rabbit vas deferens and portal vein which were made hyperpermeable (skinned) with saponin and incubated with myosin subfragment 1 (S-1). The dense bodies were obliquely oriented, elongated structures sometimes appearing as chains up to 1.5 microns in length; they were often continuous across the cell for 200 to 300 nm and were interconnected by an oblique network of 10-nm filaments. The arrowheads, formed by S-1 decoration of actins, which inserted into both the sides and ends of dense bodies, always pointed away from the dense body, similar to the polarity of the thin filaments at the Z-bands of skeletal muscle. These results show that the cytoplasmic dense bodies function as anchoring sites for the thin filaments and indicate that the thin filaments, thick filaments, and dense bodies constitute a contractile unit.

Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm.

Electron probe analysis of dry cryosections was used to determine the composition of the cytoplasm and organelles of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle. All analytical values given are in mmol/kg wt +/- SEM. Cytoplasmic concentrations in normal, resting muscles were: K, 611 +/- 1.7; Na, 167 +/- 2.7; Cl, 278 +/- 1.0; Mg, 36 +/- 1.1; Ca, 1.9 +/- 0.5; and P, 247 +/- 1.1. Hence, the sum of intracellular Na + K exceeded cytoplasmic Cl by 500 mmol/kg dry wt, while the calculated total, nondiffusible solute was approximately 50 mmol/kg. Cytoplasmic K and Cl were increased in smooth muscles incubated in solutions containing an excess (80 mM) of KCl. Nuclear and cytoplasmic Na and Ca concentrations were not significantly different. The mitochondrial Ca content in normal fibers was low, 0.8 +/- 0.5, and there was no evidence of mitochondrial Ca sequestration in muscles frozen after a K contracture lasint 30 min. Transmitochondrial gradients of K, Na, and Cl were small (0.9--1.2). In damaged fibers, massive mitochondrial Ca accumulation of up to 2 mol/kg dry wt in granule form and associated with P could be demonstrated. Our findings suggest (a) that the nonDonnan distribution of Cl in smooth muscle is not caused by sequestration in organelles, and that considerations of osmotic equilibrium and electroneutrality suggest the existence of unidentified nondiffusible anions in smooth muscle, (b) that nuclei do not contain concentrations of Na or Ca in excess of cytoplasmic levels, (c) that mitochondria in PAMV smooth muscle do not play a major role in regulating cytoplasmic Ca during physiological levels of contraction but can be massively Ca loaded in damaged cells, and (d) that the in situ transmitochondrial gradients of K, Na, and Cl do not show these ions to be distributed according to a large electromotive Donnan force.

Elemental distribution in striated muscle and the effects of hypertonicity. Electron probe analysis of cryo sections.

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-mum diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.

Sarcoplasmic reticulum and excitation-contraction coupling in mammalian smooth muscles.

The sarcoplasmic reticulum (SR) was studied in the smooth muscles of rabbit main pulmonary artery, mesenteric vein, aorta, mesenteric artery, taenia coli, guinea pig mesenteric artery, and human uterus, and correlated with contractions of the smooth muscles in Ca-free media. SR volumes were determined in main pulmonary artery (5.1%), aorta (5%), portal-anterior mesenteric vein (2.2%), taenia coli (2%), and mesenteric artery (1.8%): because of tangentially sectioned membranes these estimates are subject to a correction factor of up to +50% of the values measured. Smooth muscles that contained a relatively large volume of SR maintained significant contractile responses to drugs in the virtual absence of extracellular calcium at room temperatures, while smooth muscles that had less SR did not. The unequal maximal contractions of main pulmonary artery elicited by different drugs were also observed in Ca-free, high potassium-depolarizing solution, indicating that they were secondary to some mechanism independent of changes in membrane potential or calcium influx. Longitudinal tubules of SR run between and are fenestrated about groups of surface vesicles separated from each other by intervening dense bodies. Extracellular markers (ferritin and lanthanum) entered the surface vesicles, but not the SR. The peripheral SR formed couplings with the surface membrane: the two membranes were separated by gaps of approximately 10 nm traversed by electron-opaque connections suggestive of a periodicity of approximately 20-25 nm. These couplings are considered to be the probable sites of electromechanical coupling in twitch smooth muscles. Close contacts between the SR and the surface vesicles may have a similar function, or represent sites of calcium extrusion. The presence of both thick and thin myofilaments and of rough SR in smooth muscles supports the dual, contractile and morphogenetic, function of smooth muscle.

Hypertrophy-induced increase of intermediate filaments in vascular smooth muscle.

The distribution of filaments was studied in hypertrophied rabbit vascular smooth muscle. Hypertrophy was induced by partial ligation of the portal-anterior mesenteric vein. 14 d after ligation, there was an approximately threefold increase in the number of intermediate filaments per cross-sectional area, as compared to control values. The actin:intermediate:myosin filament ratio was 15:1.1:1 in control and 15:3.5:0.5 in hypertrophied portal-anterior mesentric vein vascular smooth muscle. Comparison of the filament ratios with the increase in volume density of the hypertrophied cells suggests that the number of myosin filaments per cell profile remained approximately the same as in controls, whereas the number of actin filaments increased in proportion to the increase in cell volume.

Localization of calcium in presynaptic nerve terminals. An ultrastructural and electron microprobe analysis.

Ultrastructural techniques and electron probe microanalysis were used to determine whether or not the smooth endoplasmic reticulum (SER) within presynaptic nerve terminals is a Ca-sequestering site. The three-dimensional structure of the SER was determined from serial sections of synaptosomes. The SER consists of flattened cisterns that may branch and are frequently juxtaposed to mitochondria. To investigate intraterminal Ca sequestration, synaptosomes were treated with saponin to disrupt the plasmalemmal permeability barrier. When these synaptosomes were incubated in solutions containing Ca, ATP, and oxalate, electrondense Ca oxalate deposits were found in intraterminal mitochondria, SER cisterns, and large vesicular profiles. Saponin-treated synaptosomes that were incubated in the presence of mitochondrial poisons contained electron-dense deposits within SER cisterns and large vesicular profiles, but very rarely in mitochondria. Similar deposits were observed within saponin-treated synaptosomes that were not post-fixed with OSO4, and within saponin-treated synaptosomes that were prepared for analysis by freeze-substitution. Electron-probe microanalyses of these deposits confirmed the presence of large concentrations of Ca. When oxalate was omitted from the incubation solutions, no electron-dense deposits were present in saponin-treated synaptosomes. In other control experiments, either the Ca ionophore A23187 or the Ca chelator EGTA was added to the incubation media; electron-dense deposits were very rarely observed within the intraterminal organelles of these saponin-treated synaptosomes. The data indicate that presynaptic nerve terminal SER is indeed a Ca-sequestering organelle.

Golgi organelle response to the antibiotic X537A.

The effects of the ionophoric antibiotic X537A on cell structure were studied with phase-contrast, fluorescence, and electron microscopy. X537A induced selective vacuolation of the Golgi apparatus of vascular and intestinal smooth muscle, epithelium, plasma cells, and cultured chick heart and guinea pig vascular smooth muscle cells. The swelling of the Golgi apparatus induced by X537A was reversible in the systems examined for reversibility: vascular smooth muscle and cultured chick heart. Myelin figures were common in the Golgi apparatus vacuolated by X537A. Fluorescence microscopy of cultured cells incubated with X537A showed the characteristic blue X537A fluorescence associated with lipid globules in the cultured cells. Incubation of cultured chick heart cells with X537A reduced the beating rate and, after 24-72 h, abolished the sarcomere pattern. The swelling of the Golgi membranes produced by X537A in cultured vascular smooth muscle was associated with inhibition of D-[6-3H]glucosamine and [35S]sulfate incorporation into glycosaminoglycans.

Sarcoplasmic reticulum and the temperature-dependent contraction of smooth muscle in calcium-free solutions.

The contractile response of turtle oviduct smooth muscle to acetylcholine after 30 min of incubation of muscles in Ca-free, 4 mM ethylene (bis) oxyethylenenitrilotetraacetic acid (EGTA) solutions at room temperature was greater than the contractile response after 30 min of incubation in the Ca-free medium at 37 degrees C. Incubation in Ca-free solution at 37 degrees C before stimulation with acetylcholine in Ca-free solutions at room temperature also reduced the contractile response, suggesting that activator calcium was lost from the fibers at a faster rate at higher temperatures. Electron micrographs of turtle oviduct smooth muscle revealed a sarcoplasmic reticulum (SR) occupying approximately 4% of the nucleus- and mitochondria-free cell volume. Incubation of oviduct smooth muscle with ferritin confirmed that the predominantly longitudinally oriented structures described as the SR did not communicate with the extracellular space. The SR formed fenestrations about the surface vesicles, and formed close contacts (couplings) with the surface membrane and surface vesicles in oviduct and vena caval smooth muscle; it is suggested that these are sites of electromechanical coupling. Calculation of the calcium requirements for smooth muscle contraction suggest that the amount of SR observed in the oviduct smooth muscle could supply the activator calcium for the contractions observed in Ca-free solutions. Incubation of oviduct smooth muscle in hypertonic solutions increased the electron opacity of the fibers. A new feature of some of the surface vesicles observed in oviduct, vena caval, and aortic smooth muscle was the presence of approximately 10 nm striations running approximately parallel to the openings of the vesicles to the extracellular space. Thick, thin, and intermediate filaments were observed in turtle oviduct smooth muscle, although the number of thick filaments seen in the present study appeared less than that previously found in mammalian smooth muscles.

Primary role of sarcoplasmic reticulum in phasic contractile activation of cardiac myocytes with shunted myolemma.

Homogeneous populations of single myocytes showing good preservation of ultrastructure were obtained by enzymatic digestion of rabbit and rat hearts, and maintained in a relaxed state in the presence of free Ca2+ concentrations less than 10(-7) M. Ultrastructural details such as a cytoskeleton of 100-A filaments connected to the sarcolemma at the Z lines were demonstrated especially well in these preparations. In spite of seemingly normal structure, electron probe analysis of cryosections reveals similar concentrations of electrolytes in the medium and in the cytoplasm, indicating the presence of electrochemical shunting across the external membrane. The dissociated myocytes display Ca uptake and phasic contractions that are apparently dependent on mitochondrial respiration, but are not affected by mitochondrial uncouplers when ATP and phosphocreatine are added. The uptake is augmented by oxalate and, based on identification of calcium oxalate crystals by electron microscopy and electron probe analysis, is localized to the sarcoplasmic reticulum (SR). An advantageous feature of the dissociated myocytes is that they are suitable for experiments using large numbers of cells in suspension. Thereby, velocities of calcium transport were measured directly by isotopic tracer and filtration methods. It was then found that the lowest CA2+ concentrations (5 x 10(-7) M for the rabbit and 1 x 10(-7) M for the rat) sustaining Ca transport also induce phasic contractile activity in all myocytes, even though the external membrane is electrochemically shunted. A stepwise rise in the Ca2+ concentration of up to one order of magnitude, increases transport velocities in parallel with the rates of phasic contractions. Both these parameters are affected by Mg2+, temperature, cyclic-AMP, and methylxanthines, even though the Ca2+ concentration is maintained constant in the medium. Therefore, Ca transport by SR is a requirement and a rate limiting factor for the occurrence of phasic contractile activation in dissociated cardiac cells retaining an electrochemically shunted external membrane. It is suggested that transient Ca release required for phasic contractile activation is due to equilibrium oscillations across the SR membrane. The sequential pattern of sarcomere activation is consistent with a self propagating mechanism of calcium release. SR in dissociated skeletal muscle cells sustains a greater Ca transport activity than in dissociated heart cells. However, the heart cells display a much higher phasic contractile activity, indicating that cardiac SR has a greater tendency to release accumulated calcium. If free Ca2+ in the medium is raised above 10(-6) M, both cardiac and skeletal myocytes undergo contractures and degenerative phenomena, accompanied by Ca, Mg, and phosphate accumulation in cardiac mitochondria.

Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron-probe study.

Approximately 60-70% of the total fiber calcium was localized in the terminal cisternae (TC) in resting frog muscle as determined by electron-probe analysis of ultrathin cryosections. During a 1.2 s tetanus, 59% (69 mmol/kg dry TC) of the calcium content of the TC was released, enough to raise total cytoplasmic calcium concentration by approximately 1 mM. This is equivalent to the concentration of binding sites on the calcium-binding proteins (troponin and parvalbumin) in frog muscle. Calcium release was associated with a significant uptake of magnesium and potassium into the TC, but the amount of calcium released exceeded the total measured cation accumulation by 62 mEq/kg dry weight. It is suggested that most of the charge deficit is apparent, and charge compensation is achieved by movement of protons into the sarcoplasmic reticulum (SR) and/or by the movement of organic co- or counterions not measured by energy dispersive electron-probe analysis. There was no significant change in the sodium or chlorine content of the TC during tetanus. The unchanged distribution of a permeant anion, chloride, argues against the existence of a large and sustained transSR potential during tetanus, if the chloride permeability of the in situ SR is as high as suggested by measurements on fractionated SR. The calcium content of the longitudinal SR (LSR) during tetanus did not show the LSR to be a major site of calcium storage and delayed return to the TC. The potassium concentration in the LSR was not significantly different from the adjacent cytoplasmic concentration. Analysis of small areas of I-band and large areas, including several sarcomeres, suggested that chloride is anisotropically distributed, with some of it probably bound to myosin. In contrast, the distribution of potassium in the fiber cytoplasm followed the water distribution. The mitochondrial concentration of calcium was low and did not change significantly during a tetanus. The TC of both tetanized and resting freeze-substituted muscles contained electron-lucent circular areas. The appearance of the TC showed no evidence of major volume changes during tetanus, in agreement with the estimates of unchanged (approximately 72%) water content of the TC obtained with electron-probe analysis.

Electron microscopy and electron probe analysis of mitochondrial cation accumulation in smooth muscle.

The contractile responses to barium and the ultrastructure and ionic composition of mitochondria were studied in vascular smooth muscle. In normal rabbit portal anterior mesenteric vein (PAMV) and main pulmonary artery (MPA) smooth muscle mitochondria were frequently associated with the surface vesicles. The average distance between the outer mitochondrial and inner surface vesicle membrane was 4-5 nm. Ba contractures of MPA were tonic and of PAMV were phasic. Incubation of MPA and PAMV with Ba resulted in the accumulation of mitochondrial granules, followed in the MPA by massive mitochondrial swelling. Oligomycin and anoxia inhibited the appearance of mitochondrial electron-opaque granules and prevented the Ba-induced mitochondrial swelling in the MPA. Electron probe analysis of mitochondria in PAMV incubated with Ba and containing granules showed characteristic Ba signals over the mitochondria. Electron probe X-ray microanalysis also showed a highly significant (P < 0.001) correlation of P with mitochondrial Ba, in an estimated elemental ratio of approximately 3 Ba/4 P. Mitochondrial granules were still prominent after block staining of the osmium-fixed, Ba-loaded PAMV, but electron probe microanalysis showed no Ba, but only U, emissions. Tissues incubated with strontium had electron-opaque mitochondrial granules and deposits in the sarcoplasmic reticulum. X-ray microanalysis of mitochondria containing granules showed the presence of characteristic Sr and Ca emissions. The presence of Sr was similarly verified in the sarcoplasmic reticulum. These findings indicate the energy dependent uptake of divalent cations, in association with phosphate, by mitochondria in vascular smooth muscle in situ and the possibility that mitochondria may contribute to the regulation of intracellular divalent cation levels in smooth muscle.

Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.

Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a beta1-integrin (probably alphaVbeta1) and alphaVbeta5. When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

Cyclic adenosine monophosphate: potassium-dependent action on vascular smooth muscle membrane potential.

Dibutyryl cyclic adenosine monophosphate and theophylline hyperpolarize smooth muscle of rabbit main pulmonary artery in low concentrations of potassium (1 millimole per liter) but do not have a significant effect on the membrane potential in the presence of high concentrations of potassium (10 millimoles per liter). The dependence of the hyperpolarizing effect on a low external concentration of potassium is similar to that observed with isoproterenol. Prior treatment with theophylline potentiated the hyperpolarizing action of isoproterenol. These findings are compatible with the assumption that potassium-dependent, beta-adrenergic hyperpolarization is mediated by cyclic adenosine monophosphate.

Strontium accumulation by sarcoplasmic reticulum and mitochondria in vascular smooth muscle.

Electron-opaque deposits of strontium were observed in the sarcoplasmic reticulum and in mitochondria of spontaneously contracting vascular smooth muscles that had been incubated in a strontium-containing solution prior to fixation. The deposits were present in those elements of the sarcoplasmic reticulum that are in close contact with the surface membrane and also in more centrally located portions. In vascular smooth muscle that does not contract spontaneously, similar deposits of strontium were only seen if the muscle was depolarized during or glycerinated before exposure to the strontium-containing solution. Strontium was also deposited in the sarcoplasmic reticulum of the endothelium. It is suggested that translocation of calcium from the sarcoplasmic reticulum that is in close contact with the surface membrane, and now shown to accumulate divalent cations, is responsible for the action potential-triggered contractions of rabbit and guinea pig mesenteric veins. Strontium may also be a suitable marker for identifying sites that accumulate calcium in other types of cells in which translocation of calcium plays a major regulatory function.

Cyclic adenosine and guaosine monophosphates and gucagon: effect on liver membrane potentials.

Cyclic adenosine monophosphate, cyclic guanosine monophosphate, glucagon, and isoproterenol each hyperpolarized perfused rat liver cells. The hyperpolarization followed a time course similar to the stimulated increase in potassium efflux and was preceded by the increase in calcium efflux. The hyperpolarization induced by cyclic adenosine monophosphate was blocked by tetracaine. The similarity of the action of the cyclic nucleotides to that of glucagon supports the hypothesis that cyclic adenosine monophosphate is the secondary messenger mediating the action of glucagon.

LPP expression during in vitro smooth muscle differentiation and stent-induced vascular injury.

Lipoma preferred partner (LPP) has been identified as a protein highly expressed in smooth muscle (SM) tissues. The aim of the present study was to determine mechanisms that regulate LPP expression in an in vitro model of SM cell (SMC) differentiation and in stent-induced pig coronary vessel injury. All trans-retinoic acid treatment of A404 cells induced a strong increase in LPP, as well as SM alpha-actin, SM myosin heavy chain, and smoothelin mRNA levels, in a Rho kinase (ROK)-dependent manner. Adenovirus mediated overexpression of myocardin in A404 cells significantly increased LPP mRNA expression. Interestingly, inactivation of RhoA with C3-exoenzyme or treatment with ROK inhibitors strongly inhibited myocardin mRNA expression in retinoic acid-treated A404 cells or human iliac vein SMCs. LPP silencing with short interfering RNA significantly decreased SMC migration. LPP expression was also markedly decreased in focal adhesion kinase (FAK)-null cells known to have impaired migration but rescued with inducible expression of FAK. LPP expression in FAK-null fibroblasts enhanced cell spreading. In stented pig coronary vessels, LPP was expressed in the neointima of cells lacking smoothelin and showed expression patterns identical to those of SM alpha-actin. In conclusion, LPP appears to be a myocardin-, RhoA/ROK-dependent SMC differentiation marker that plays a role in regulating SMC migration.

Down-regulation of G-protein-mediated Ca2+ sensitization in smooth muscle.

Prolonged treatment with guanosine 5'-[gamma-thio]triphosphate (GTP gamma S; 5-16 h, 50 microM) of smooth muscle permeabilized with Staphylococcus aureus alpha-toxin down-regulated (abolished) the acute Ca2+ sensitization of force by GTP gamma S, AIF-4, phenylephrine, and endothelin, but not the response to phorbol dibutyrate or a phosphatase inhibitor, tautomycin. Down-regulation also abolished the GTP gamma S-induced increase in myosin light chain phosphorylation at constant [Ca2+] and was associated with extensive translocation of p21rhoA to the particulate fraction, prevented its immunoprecipitation, and inhibited its ADP ribosylation without affecting the immunodetectable content of G-proteins (p21rhoA, p21ras, G alpha q/11, G alpha i3, and G beta) or protein kinase C (types alpha, beta 1, beta 2, delta, epsilon, eta, theta, and zeta). We conclude that the loss of GTP gamma S- and agonist-induced Ca2+ sensitization through prolonged treatment with GTP gamma S is not due to a decrease in the total content of either trimeric (G alpha q/11, G alpha i3, and G beta) or monomeric (p21rhoA and p21ras) G-protein or protein kinase C but may be related to a structural change of p21rhoA and/or to down-regulation of its (yet to be identified) effector.

Inhibition of RhoA translocation and calcium sensitization by in vivo ADP-ribosylation with the chimeric toxin DC3B.

Pretreatment of intact rabbit portal vein smooth muscle with the chimeric toxin DC3B (10(-6) M, 48 h; ; ) ADP-ribosylated endogenous RhoA, including cytosolic RhoA complexed with rhoGDI, and inhibited the tonic phase of phenylephrine-induced contraction and the Ca2+-sensitization of force by phenylephrine, endothelin and guanosine triphosphate (GTP)gammaS, but did not inhibit Ca2+-sensitization by phorbol dibutyrate. DC3B also inhibited GTPgammaS-induced translocation of cytosolic RhoA () to the membrane fraction. In DC3B-treated muscles the small fraction of membrane-associated RhoA could be immunoprecipitated, even after exposure to GTPgammaS, which prevents immunoprecipitation of non-ADP-ribosylated RhoA. Dissociation of cytosolic RhoA-rhoGDI complexes with SDS restored the immunoprecipitability and ADP ribosylatability of RhoA, indicating that both the ADP-ribosylation site (Asn 41) and RhoA insert loop (Wei et al., 1997) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that RhoA plays a significant role in G-protein-, but not protein kinase C-mediated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo the Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.

Pharmacomechanical coupling: the role of calcium, G-proteins, kinases and phosphatases.

The concept of pharmacomechanical coupling, introduced 30 years ago to account for physiological mechanisms that can regulate contraction of smooth muscle independently of the membrane potential, has since been transformed from a definition into what we now recognize as a complex of well-defined, molecular mechanisms. The release of Ca2+ from the SR by a chemical messenger, InsP3, is well known to be initiated not by depolarization, but by agonist-receptor interaction. Furthermore, this G-protein-coupled phosphatidylinositol cascade, one of many processes covered by the umbrella of pharmacomechanical coupling, is part of complex and general signal transduction mechanisms also operating in many non-muscle cells of diverse organisms. It is also clear that, although the major contractile regulatory mechanism of smooth muscle, phosphorylation/dephosphorylation of MLC20, is [Ca2+]-dependent, the activity of both the kinase and the phosphatase can also be modulated independently of [Ca2+]i. Sensitization to Ca2+ is attributed to inhibition of SMPP-1M, a process most likely dominated by activation of the monomeric GTP-binding protein RhoA that, in turn, activates Rho-kinase that phosphorylates the regulatory subunit of SMPP-1M and inhibits its myosin phosphatase activity. It is likely that the tonic phase of contraction activated by a variety of excitatory agonists is, at least in part, mediated by this Ca(2+)-sensitizing mechanism. Desensitization to Ca2+ can occur either through inhibitory phosphorylation of MLCK by other kinases or autophosphorylation and by activation of SMPP-1M by cyclic nucleotide-activated kinases, probably involving phosphorylation of a phosphatase activator. Based on our current understanding of the complexity of the many cross-talking signal transduction mechanisms that operate in cells, it is likely that, in the future, our current concepts will be refined, additional mechanisms of pharmacomechanical coupling will be recognized, and those contributing to the pathologenesis diseases, such as hypertension and asthma, will be identified.