Periodic organization of the contractile apparatus in smooth muscle revealed by the motion of dense bodies in single cells.

To study the organization of the contractile apparatus in smooth muscle and its behavior during shortening, the movement of dense bodies in contracting saponin skinned, isolated cells was analyzed from digital images collected at fixed time intervals. These cells were optically lucent so that punctate structures, identified immunocytochemically as dense bodies, were visible in them with the phase contrast microscope. Methods were adapted and developed to track the bodies and to study their relative motion. Analysis of their tracks or trajectories indicated that the bodies did not move passively as cells shortened and that nearby bodies often had similar patterns of motion. Analysis of the relative motion of the bodies indicated that some bodies were structurally linked to one another or constrained so that the distance between them remained relatively constant during contraction. Such bodies tended to fall into laterally oriented, semirigid groups found at approximately 6-microns intervals along the cell axis. Other dense bodies moved rapidly toward one another axially during contraction. Such bodies were often members of separate semirigid groups. This suggests that the semirigid groups of dense bodies in smooth muscle cells may provide a framework for the attachment of the contractile structures to the cytoskeleton and the cell surface and indicates that smooth muscle may be more well-ordered than previously thought. The methods described here for the analysis of the motion of intracellular structures should be directly applicable to the study of motion in other cell types.

The sarcoplasmic reticulum calcium pump is functionally altered in dystrophic muscle.

In Duchenne muscular dystrophy, muscle cells, which lack the protein dystrophin, have been reported to have elevated resting intracellular calcium levels. It has also been noted that, compared to normal muscle, intracellular [Ca2+] in dystrophic muscle returns more slowly to its resting level following contractile stimulation. Consistent with this, it has been suggested that dystrophin is directly involved in the regulation of Ca2+ influx. A secondary alteration in the sarcoplasmic reticulum Ca2+ pump, however, could also contribute to, or be responsible for, the abnormal Ca2+ handling seen. To determine whether the Ca2+ pump is functionally altered in dystrophic muscle, we examined Ca2+ uptake by vesicles derived from skeletal muscle sarcoplasmic reticulum of normal and dystrophic (mdx) mice. The Hill coefficient and the Ca2+ sensitivity of the Ca2+- ATPase were the same in both cases. The maximum velocity of Ca2+ uptake, however, normalized to the ATPase content of the vesicles, was less for mdx muscle.

Direct measurement of Ca2+ uptake and release by the sarcoplasmic reticulum of saponin permeabilized isolated smooth muscle cells.

To make direct measurements of Ca2+ uptake and release by the sarcoplasmic reticulum (SR) of isolated smooth muscle cells, a fluorometric method for monitoring Ca2+ uptake by striated muscle SR vesicles (Kargacin, M.E., C.R. Scheid, and T.W. Honeyman. 1988. American Journal of Physiology. 245:C694-C698) was modified. With the method, it was possible to make continuous measurements of SR function in saponin-skinned smooth muscle cells in suspension. Calcium uptake by the SR was inhibited by thapsigargin and sequestered Ca2+ could be released by Br-A23187 and thapsigargin. From the rate of Ca2+ uptake by the skinned cells and the density of cells in suspension, it was possible to calculate the Ca2+ uptake rate for the SR of a single cell. Our results indicate that the SR Ca2+ pump in smooth muscle cells can remove Ca2+ at a rate that is 45-75% of the rate at which Ca2+ is removed from the cytoplasm of intact cells during transient Ca2+ signals. From estimates of SR volume reported by others and our measurements of the amount of Ca2+ taken up by the skinned cells, we conclude that the SR of a single cell can store greater than 10 times the amount of Ca2+ needed to elicit a single transient contractile response.

Physiological and structural properties of saponin-skinned single smooth muscle cells.

The study of the fundamental events underlying the generation and regulation of force in smooth muscle would be greatly facilitated if the permeability of the cell membrane were increased so that the intracellular environment of the contractile apparatus could be manipulated experimentally. To initiate such an analysis, we developed a saponin permeabilization procedure that was used to "skin" isolated smooth muscle cells from the stomach of the toad, Bufo marinus. Suspensions of single cells isolated enzymatically were resuspended in high-K+ rigor solution (0 ATP, 5 mM EGTA) and exposed for 5 min to 25 micrograms/ml saponin. Virtually all the cells in a suspension were made permeable by this procedure and shortened to less than one-third their initial length when ATP and Ca++ were added; they re-extended when free Ca++ was removed. Analysis of the protein content of the skinned cells revealed that, although their total protein was reduced by approximately 30%, they retained most of their myosin and actin. Skinning was accompanied by a rearrangement of actin and myosin filaments within the cells such that a fine fibrillar structure became visible under the light microscope and a tight clustering of acting filaments around myosin filaments was revealed by the electron microscope. Face-on views of saponin-treated cell membranes revealed the presence of 70-80-A-wide pits or holes. The shortening rate of skinned cells was sensitive to [Ca++] between pCa 7 and pCa 5 and was half-maximal at approximately pCa 6.2. Shortening was also dependent on [ATP] but could be increased at low [ATP] by pretreatment with adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), which suggests that myosin phosphorylation was more sensitive to low substrate concentrations than was cross-bridge cycling. To determine whether a significant limitation to free diffusion existed in the skinned cells, a computer model of the cell and the unstirred layer surrounding it was developed. Simulations revealed that the membrane, even in skinned cells, could, for short time intervals, significantly inhibit the movement of substances into and out of cells.

Effects of anti-oestrogens and beta-estradiol on calcium uptake by cardiac sarcoplasmic reticulum.

1. Tamoxifen and a group of structurally similar non-steroidal, triphenolic compounds inhibit the oestrogen receptor. In addition to this action, these anti-oestrogens are known to inhibit some types of plasma membrane ion channels and other proteins through mechanisms that do not appear to involve their interactions with the estrogen receptor but could be the result of their effect on membrane lipid structure or fluidity. 2. We studied the effects of beta-estradiol and three anti-oestrogens (tamoxifen, 4-hydroxytamoxifen and clomiphene) on Ca(2+) uptake into sarcoplasmic reticulum (SR) vesicles isolated from canine cardiac ventricular tissue. 3. The antiestrogens all inhibit SR Ca(2+) uptake in a concentration-dependent manner (order of potency: tamoxifen > 4-hydroxytamoxifen > or = clomiphene). Although these compounds rapidly inhibit net Ca(2+) uptake they do not have a similar rapid effect on the ATPase activity of the SR Ca pump. beta-estradiol has no effect on Ca(2+) uptake nor does it alter the inhibitory action of tamoxifen on the SR. 4. The differences in the effects of beta-estradiol and the anti-oestrogens on cardiac SR Ca(2+) uptake do not correlate with differences in the ways in which these compounds have been reported to interact with membrane lipids. Our results are consistent, however, with direct effects on a membrane protein (possibly an SR Cl(-) or K(+) channel).

Calcium signaling in restricted diffusion spaces.

One- and two-dimensional models of Ca2+ diffusion and regulation were developed and used to study the magnitudes and the spatial and temporal characteristics of the Ca2+ transients that are likely to develop in smooth muscle cells in restricted diffusion spaces between the plasma membrane and intracellular organelles. Simulations with the models showed that high [Ca2+] (on the order of several microM) can develop in such spaces and persist for 100-200 ms. These Ca2+ transients could: 1) facilitate the coupling of Ca2+ influx to intracellular Ca2+ release; 2) provide a mechanism for the regulation of stored Ca2+ that does not affect the contractile state of smooth muscle; 3) locally activate specific signal transduction pathways, before, or without activating other Ca2+ dependent pathways in the central cytoplasm of the cell. The latter possibility suggests that independent enzymatic processes in cells could be differentially regulated by the same intracellular second messenger.

Methods for determining cardiac sarcoplasmic reticulum Ca2+ pump kinetics from fura 2 measurements.

We explored the use of four methods for analyzing real and simulated fura 2 measurements of Ca2+ uptake by membrane vesicles derived from the sarcoplasmic reticulum (SR) of cardiac muscle. Uptake velocity was calculated 1) directly from the raw data, 2) after segmenting the raw data and averaging the data points in each segment, 3) after smoothing of the raw data by moving-window averaging, and 4) by Savitsky-Golay convolution. Methods 2, 3, and 4 could be used to determine maximum uptake velocity, the Hill coefficient, and the Ca2+ concentration at half-maximal pump velocity from Ca2+ concentration vs. time and velocity curves that were too noisy to analyze directly. Data analysis using these methods should have general applicability to biological experiments, especially those in which large numbers of measurements are made. The fluorometric method we describe for measuring Ca2+ uptake by cardiac SR vesicles opens up the possibility of studying SR function from very small starting tissue samples.

Light-evoked contraction of the photosensitive iris of the frog.

Smooth muscle cells of the frog iris sphincter contain rhodopsin and contract in response to light. The mechanism of light-evoked contraction was studied with particular attention paid to the role of calcium. The contractile proteins of the sphincter smooth muscle cell can be activated by an increase in intracellular calcium. Light-evoked contraction, however, is not accompanied by a measurable change in membrane potential and occurs in the absence of extracellular Na+ and/or Ca2+, as well as in the presence of isotonic KCI. Maximum light-evoked tension is reduced by exposure to Ca2+-free solutions containing EGTA and high K+ and is restored by incubation in solutions containing Ca2+. The restored response, which persists after return to Ca2+-free solution, depends on the concentration of Ca2+ in the incubating solution and on the duration of the incubation. The results support the conclusion that light produces contraction of the iris sphincter by causing the release of Ca2+ from an intracellular storage site.

Predicted changes in concentrations of free and bound ATP and ADP during intracellular Ca2+ signaling.

High Ca2+ concentrations can develop near Ca2+ sources during intracellular signaling and might lead to localized regulation of Ca2+-dependent processes. By shifting the amount of Ca2+ and other cations associated with ATP, local high Ca2+ concentrations might also alter the substrate available for membrane-associated and cytoplasmic enzymes. To study this, simultaneous equations were solved over a range of Ca2+ and Mg2+ concentrations to determine the general effects of Ca2+ on the concentrations of free and Ca2+- and Mg2+-bound forms of ATP. To obtain a more specific picture of the changes that might occur in smooth muscle cells, mathematical models of Ca2+ diffusion and regulation were used to predict the magnitude and time course of near-membrane Ca2+ transients and their effects on the free and bound forms of ATP near the membrane. The results of this work indicate that changes in free Ca2+ concentration over the range of 50 nM-100 microM would result in significant changes in free ATP concentration, MgATP concentration, and the CaATP-to-MgATP concentration ratio.

Ruthenium red reduces the Ca2+ sensitivity of Ca2+ uptake into cardiac sarcoplasmic reticulum.

Ruthenium red inhibits mitochondrial Ca2+ uptake and is widely used as an inhibitor of ryanodine-sensitive Ca2+ channels that function to release Ca2+ from the sarcoplasmic reticulum (SR) of muscle cells. It also has effects on other Ca2+ channels and ion transporters. To study the effects of ruthenium red on Ca2+ transport into the SR of cardiac muscle cells, fluorescence measurements of Ca2+ uptake into cardiac SR vesicles were made. Ruthenium red significantly decreased the Ca2+ sensitivity of SR uptake in a dose-dependent manner at concentrations ranging from 5 microM to 20 microM. There were no significant effects of ruthenium red on the maximum velocity or the Hill coefficient of SR Ca2+ uptake.

Chloride channel blockers inhibit Ca2+ uptake by the smooth muscle sarcoplasmic reticulum.

Despite the fact that Ca2+ transport into the sarcoplasmic reticulum (SR) of muscle cells is electrogenic, a potential difference is not maintained across the SR membrane. To achieve electroneutrality, compensatory charge movement must occur during Ca2+ uptake. To examine the role of Cl- in this charge movement in smooth muscle cells, Ca2+ transport into the SR of saponin-permeabilized smooth muscle cells was measured in the presence of various Cl- channel blockers or when I-, Br-, or SO42- was substituted for Cl-. Calcium uptake was inhibited in a dose-dependent manner by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and by indanyloxyacetic acid 94 (R(+)-IAA-94), but not by niflumic acid or 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Smooth muscle SR Ca2+ uptake was also partially inhibited by the substitution of SO42- for Cl-, but not when Cl- was replaced by I- or Br-. Neither NPPB nor R(+)-IAA-94 inhibited Ca2+ uptake into cardiac muscle SR vesicles at concentrations that maximally inhibited uptake in smooth muscle cells. These results indicate that Cl- movement is important for charge compensation in smooth muscle cells and that the Cl- channel or channels involved are different in smooth and cardiac muscle cells.

Peptide modulators of myosin light chain kinase affect smooth muscle cell contraction.

To examine the importance of myosin light chain kinase (MLCK) in the initiation of contraction in smooth muscle, we used a constitutively active form of MLCK (IMLCK) and two specific peptide inhibitors of MLCK to study the activation of skinned single smooth muscle cells. Although unregulated by Ca-calmodulin, IMLCK, in vitro, was found to have biochemical properties like those of MLCK. Upon photolysis of caged ATP, IMLCK caused Ca-free shortening of skinned cells similar in time course and extent to that induced by Ca2+. Two peptide probes, RS-20 and SM-1, patterned after the Ca-calmodulin binding site and a pseudosubstrate inhibitory site, respectively, of the native MLCK molecule, were shown to specifically inhibit MLCK in in vitro experiments. Both peptides dose dependently inhibited Ca-induced shortening of skinned single cells. These results indicate that MLCK plays an essential role in the activation process in the smooth muscle cell in that activation of this enzyme is both necessary and sufficient for the initiation of contraction.

Characterization and confocal imaging of calponin in gastrointestinal smooth muscle.

Calponin isolated from chicken gizzard smooth muscle binds in vitro to actin in a Ca(2+)-independent manner and thereby inhibits the actin-activated Mg(2+)-adenosinetriphosphatase of smooth muscle myosin. This inhibition is relieved when calponin is phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II, suggesting that calponin is involved in thin filament-associated regulation of smooth muscle contraction. To further examine this possibility, calponin was isolated from toad stomach smooth muscle, characterized biochemically, and localized in intact isolated cells. Toad stomach calponin had the same basic biochemical properties as calponin from other sources. Confocal immunofluorescence microscopy revealed that calponin in intact smooth muscle cells was localized to long filamentous structures that were colabeled by antibodies to actin or tropomyosin. Preservation of the basic biochemical properties of calponin from species to species suggests that these properties are relevant for its in vivo function. Its colocalization with actin and tropomyosin indicates that calponin is associated with the thin filament in intact smooth muscle cells.

Intracellular mechanisms involved in the regulation of vascular smooth muscle tone.

Vascular smooth muscle contraction is thought to occur by a mechanism similar to that described for striated muscles, i.e., via a cross-bridge cycling--sliding filament mechanism. This symposium focused on Ca2+ signalling and the role of intracellular free Ca2+ concentration, [Ca2+]i, in regulating vascular tone: how contractile stimuli leading to an increase in [Ca2+]i trigger vasoconstriction and how relaxant signals reduce [Ca2+]i causing vasodilation. M.P. Walsh opened the symposium with an overview emphasizing the central role of myosin phosphorylation-dephosphorylation in the regulation of vascular tone and identifying recent developments concerning regulation of [Ca2+]i, Ca2+ sensitization and desensitization of the contractile response, Ca(2+)-independent protein kinase C induced contraction, and direct regulation of cross-bridge cycling by the thin filament associated proteins caldesmon and calponin. The remainder of the symposium focused on three specific areas related to the regulation of vascular tone: Ca2+ signalling in relation to smooth muscle structure, structure-function relations of myosin, and the role of cyclic GMP (cGMP) dependent protein kinase. G.J. Kargacin described how smooth muscle cells are structured and how second messenger signals such as Ca2+ might be modified or influenced by this structure. J. Kendrick-Jones then discussed the results of mutagenesis studies aimed at understanding how the myosin light chains, particularly the phosphorylatable (Ca(2+)-calmodulin dependent) regulatory light chains, control myosin. The vasorelaxant effects of signalling molecules such as beta-adrenergic agents and nitrovasodilators are mediated by cyclic nucleotide dependent protein kinases, leading principally to a reduction in [Ca2+]i. T.M. Lincoln described the roles of cyclic nucleotide dependent protein kinases, in particular cyclic GMP dependent protein kinase, in vasodilation.

Calponin and smooth muscle regulation.

Calponin has been implicated in the regulation of smooth muscle contraction as a result of its ability to inhibit the actin-activated Mg ATPase of smooth muscle myosin. This inhibitory effect is abolished by phosphorylation of calponin by Ca2+/calmodulin-dependent protein kinase II or protein kinase C, and restored following dephosphorylation by a type 2A protein phosphatase. Confocal immunofluorescent images of isolated smooth muscle cells colabeled with antibodies to calponin and actin or to calponin and tropomyosin indicate that calponin is present on thin filaments throughout the cell cytoplasm. Both calponin phosphorylation and myosin light chain phosphorylation increased in intact smooth muscle tissue strips when they contracted in response to carbachol or the phosphatase inhibitor okadaic acid. These results support the hypothesis that calponin phosphorylation-dephosphorylation plays a role in regulating smooth muscle contraction.

Iodide and bromide inhibit Ca(2+) uptake by cardiac sarcoplasmic reticulum.

Recent studies indicate that the Ca(2+) permeability of the sarcoplasmic reticulum (SR) can be affected by its anionic environment. Additionally, anions could directly modulate the SR Ca(2+) pump or the movement of compensatory charge across the SR membrane during Ca(2+) uptake or release. To examine the effect of anion substitution on cardiac SR Ca(2+) uptake, fluorometric Ca(2+) measurements and spectrophotometric ATPase assays were used. Ca(2+) uptake into SR vesicles was inhibited in a concentration-dependent manner when Br(-) or I(-) replaced extravesicular Cl(-) (when Br(-) completely replaced Cl(-), uptake velocity was approximately 70% of control; when I(-) completely replaced Cl(-), uptake velocity was approximately 39% of control). Replacement of Cl(-) with SO(2)(-4) had no effect on SR uptake. Although both I(-) and Br(-) inhibited net Ca(2+) uptake, neither anion directly inhibited the SR Ca(2+) pump nor did they increase the permeability of the SR membrane to Ca(2+). Our results support the hypothesis that an anionic current that occurs during SR Ca(2+) uptake is reduced by the substitution of Br(-) or I(-) for Cl(-).

Comparison of the caldesmon content of cardiac and smooth muscle.

The high abundance of caldesmon in smooth muscle and its ability to inhibit actomyosin ATPase activity have led to the hypothesis that caldesmon modulates contractile activity. It has also been proposed, however, that caldesmon acts as a structural protein in muscle and non-muscle cells. We have determined the caldesmon content of mammalian cardiac muscle and have found that caldesmon is 200-fold less abundant in cardiac muscle than it is in gizzard smooth muscle. This finding argues against a role for caldesmon in the modulation of cardiac contractility.

Identification and localization of caldesmon in cardiac muscle.

Caldesmon has been detected in smooth muscle and in a number of non-muscle cells. It binds both actin and myosin and may act as a regulator of contraction or a structural element in smooth muscle. The presence of caldesmon in striated muscle has not been well established. To address this issue, polyclonal antibodies and a panel of monoclonal antibodies were raised against chicken gizzard smooth muscle caldesmon and used to demonstrate that caldesmon is present in adult cardiac muscle of a variety of mammalian species. Western-blot analysis revealed the presence of caldesmon in ventricular myocytes isolated from rat heart. The epitopes for the individual monoclonal antibodies were mapped to the caldesmon primary structure using chymotryptic and 2-nitro-5-thiocyanatobenzoic acid fragments. Bovine and rat cardiac caldesmons were recognized only by a subset of these monoclonal antibodies, indicating primary sequence differences from the chicken smooth muscle protein. Immunofluorescence labelling of isolated myocytes from rat, rabbit and guinea pig cardiac muscle revealed a striated pattern of fluorescence labelling. Dual labelling of caldesmon and myosin or caldesmon and alpha-actinin demonstrated that caldesmon was present at the centre of the I-band rather than in the A-band, as might have been expected from the myosin binding properties of the smooth muscle protein. These results suggest a structural role for caldesmon in cardiac muscle cells.

Tamoxifen inhibits Ca2+ uptake by the cardiac sarcoplasmic reticulum.

Ca2+ transients in isolated cardiac ventricular myocytes and the amount of Ca2+ that could be released from the sarcoplasmic reticulum (SR) in these cells by caffeine were reduced in the presence of tamoxifen. To examine the effects of tamoxifen on the cardiac muscle SR directly, isolated SR vesicles and fluorimetry methods were used to measure the uptake of Ca2+ by the SR and the ATPase activity of the SR Ca2+ pump. SR Ca2+ uptake was inhibited by tamoxifen at concentrations greater than 2.4 microM. Half-maximal inhibition was seen at approximately 5 microM. Inhibition of uptake was not due to the development of a substantial tamoxifen-dependent leak of Ca2+ from the SR or to a direct inhibitory effect of tamoxifen on the ATPase activity of the SR Ca2+ pump. In addition to its effect on SR Ca2+ uptake, tamoxifen also reduced the rate at which stored Ca2+ could be released from the SR by the Ca2+ ionophore 4-bromo A23187. Our results are consistent with the hypothesis that tamoxifen inhibits an ion current that accompanies Ca2+ movement across the SR membrane. This possibility is also consistent with the known inhibitory action of tamoxifen on some types of Cl- and K+ channels.

Calponin phosphorylation in vitro and in intact muscle.

Calponin, a thin-filament-associated protein implicated in the regulation of smooth-muscle contraction, is phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II [Winder and Walsh (1990) J. Biol. Chem. 265, 10148-10155] and dephosphorylated by a type 2A protein phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203]. Unphosphorylated calponin binds to actin and inhibits the actin-activated myosin MgATPase; these properties are lost on phosphorylation. Although both serine and threonine residues in calponin are phosphorylated, the major site of phosphorylation by either kinase is Ser-175. Calponin also undergoes phosphorylation when bound to actin in synthetic thin filaments, in a reconstituted actomyosin system, in washed myofibrils and in tissue extracts; this results in dissociation of calponin from actin. Tryptic phosphopeptide mapping indicates that the same sites are phosphorylated in the bound as in the isolated protein. Toad stomach calponin exists in at least three isoforms which differ in charge but exhibit the same molecular mass on SDS/PAGE. In a toad stomach extract, all three isoforms are phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II as shown by two-dimensional gel electrophoresis (non-equilibrium pH-gradient gel electrophoresis and SDS/PAGE). Calponin phosphorylation also occurs in intact toad stomach smooth-muscle strips metabolically labelled with 32Pi and stimulated to contract with carbachol. These results support the hypothesis that calponin may be regulated in vivo by phosphorylation-dephosphorylation.