The formation of intravesicular calcium phosphate deposits in microsomes of smooth muscle

Summary The calcium uptake in the microsomial fraction isolated from the smooth muscle of the antrum of the pig stomach is stimulated by phosphate. The microsomial vesicles which are loaded with calcium phosphate can be purified by differential centrifugation. A purification of 36 times in terms of calcium content was reached. Electron microscopy of the freshly prepared material revealed calcium phosphate deposits in the form of needles of crystalline calcium phosphate. This structure differs from that of the deposits which appear in the fragmented sarcoplasmic reticulum of skeletal muscle. Their morphology is that of non-crystalline calcium phosphate. However, on standing these deposits convert slowly into crystalline calcium phosphate. This difference reflects different kinetics of crystallization of the precipitates in the two preparations. After negative staining of the calcium phosphate loaded microsomes of skeletal and of smooth muscle, only few deposits are preserved because a release of calcium occurs as a consequence of the action of the stain and also of the dilution and warming up of the suspension. Smooth muscle microsomes partially purified by loading with calcium phosphate were studied by freeze etching and rotary replication. Membrane fragments displaying subunit intramembrane particles similar to those observed in sarcoplasmic reticulum of skeletal muscle could be identified. However, in the smooth muscle microsomes the intramembrane particles were much less densely packed. Part of these particles could correspond to calcium transport sites.     

Detrusor smooth muscle cells of the guinea-pig are functionally coupled via gap junctions in situ and in cell culture

Intercellular communication between smooth muscle cells is crucial for contractile behaviour in normal and pathologically altered urinary bladder. Since the study of coupling is difficult in situ, we established cell cultures of bladder smooth muscle cells to analyse coupling mechanisms. Microinjection of Lucifer yellow demonstrated syncytia composed of only a few to several dozen cells. Electron-microscopic examination of freeze-fracture specimens and ultrathin sections revealed that the dye-coupling was based on typical gap junction formation between the cultured smooth muscle cells. Furthermore, we were able to demonstrate gap junctions within the tissue fragments from which the primary cultures were grown. By Western blotting, we found connexin-43-positive protein bands both in native tissue probes from the guinea-pig urinary bladder and in smooth muscle cell cultures. Extracellular electrical stimulation of single cells evoked calcium transients, as visualized by fura-2 ratiofluorimetry. Calcium waves propagated throughout the syncytia with a declining amplitude, showing that the calcium signal was not regenerative. Therefore, the calcium signal was probably transmitted by a diffusible factor. These findings correlated well with the dye-coupling that we found between detrusor smooth muscle cells in situ. The use of smooth muscle cell cultures therefore seems to be a feasible approach for studying coupling behaviour in vitro.     

Simultaneous measures of contraction and intracellular calcium in single, cultured smooth muscle cells

Summary Simple methods are presented for quantitating contraction and intracellular calcium simultaneously in single, cultured smooth muscle cells. These methods are the first to demonstrate that reliable velocities of cell shortening can be measured in cultured smooth muscle cells and that cells in vitro exhibit shortening velocities comparable to those measured in the fastest phasic muscles in situ. Temporal relationships between changes in intracellular calcium and shortening within single cells were determined with a resolution of 100 ms and were consistent with measures in more “classical” preparations. Intracellular calcium rose quickly and transiently 10-fold above the basal level of 80–90 nM in response to the muscarinic agonist, carbachol. Shortening of the cells occurred 200 ms after intracellular calcium began to rise. The sensitivity and reliability of these methods allowed the effects of different stimuli to be easily resolved. The present report demonstrates that genuine contractility need not be ignored in cultured smooth muscle cells and that the temporal relations between shortening and intracellular calcium mobilization can be quantitatively assessed in controlled in vitro environments.     

Modified cardiovascular L-type channels in mice lacking the voltage-dependent Ca2+ channel beta3 subunit

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.     

Calcium homeostasis and nerve growth factor secretion from vascular and bladder smooth muscle cells

Bladder and vascular smooth muscle cells cultured from four rat strains (WKY, SHR, WKHA, WKHT) differing in rates of nerve growth factor (NGF) production were used to determine whether a relationship exists between intracellular calcium and NGF secretion. Basal cytosolic calcium was related to basal NGF secretion rates in bladder and vascular smooth muscle cells from all four strains with the exception of WKHT bladder muscle cells. Thrombin is a calcium-mobilizing agent and increases NGF production from vascular but not bladder smooth muscle cells. Strain differences were found in the magnitude of the calcium peak induced by thrombin in vascular smooth muscle cells, but these differences did not correlate with NGF secretion. Thrombin caused a calcium response in bladder smooth muscle cells without influencing NGF production. Quenching the calcium transient with a calcium chelator had no effect on thrombin-inducted NGF secretion rates in vascular smooth muscle cells. Thus, basal intracellular calcium may establish a set point for NGF secretion from smooth muscle. In addition, transient elevations in cytosolic calcium were unrelated to the induction of NGF output.     

Modulation of electrical activity in gastrointestinal smooth muscle by peptides.

A rise in intracellular calcium is the predominant signal that leads to the activation of the contractile machinery in gastrointestinal smooth muscle. The primary sources of activating calcium are illustrated in Fig. 2. Voltage- and peptide-mediated release of intracellular calcium contribute to activation of some gastrointestinal smooth muscles. However, the primary source of activating calcium appears to be an influx of calcium across the plasma membrane. The degree of modulation of electrical activity by peptides varies depending upon the region of the gastrointestinal tract studied. Second messenger systems are undoubtly involved in the transduction pathway for receptor-mediated changes in ion channel activity in gastrointestinal smooth muscle. However, in comparison to other excitable cell types, little is known about the coupling mechanisms whereby peptide-receptor binding alters ion channel activity in gastrointestinal smooth muscle. This represents one of the challenging areas to be studied in the field of gastrointestinal smooth muscle. One disease in which a better appreciation of the regulation of ion channel activity could lead to therapeutic benefit is irritable bowel syndrome. A coupling of smooth muscle electrical activity to hypermotility in irritable bowel syndrome has been reported. CCK increases the level of spike activity which triggers hypermotility [40]. It would follow that inhibition of calcium influx should reduce spiking and, therefore, hypermotility. In fact, the calcium channel blockers nifedipine and nicardipine have been shown to decrease colonic motility in irritable bowel syndrome patients [62-64]. As our understanding of gastrointestinal smooth muscle ion channels expands, development of a gastrointestinal selective calcium channel blocker may be possible. This class of agents would be effective in the treatment of irritable bowel syndrome and potentially other peptide-related spastic smooth muscle disorders.     

International Commission for Protection Against Environmental Mutagens and Carcinogens. ICPEMC Working Paper 7/1/1. Mutational events in the etiology of arteriosclerotic plaques

The arteriosclerotic plaque is the lesion most often associated with cardiovascular disease, which is the leading cause of death in North America and Western Europe. Plaques are composed of cells (mostly smooth muscle cells but also macrophages and some lymphocytes) and formed elements (cellular debris, collagen, elastin, glycosaminoglycans, lipid droplets, cholesterol crystals and sometimes calcium deposits). Proliferation of smooth muscle cells is essential to plaque formation and development. Most theories of plaque development have viewed this proliferation as a secondary event following an initiating stimulus (e.g., endothelial injury). According to this view, the proliferating smooth muscle cells are otherwise identical to the large number of non-proliferating smooth muscle cells in the artery wall. The 'monoclonal' hypothesis of plaque formation presents a fundamentally different view; namely, that the cell proliferation critical to plaque development follows the stable transformation of smooth muscle cells and that the plaques can therefore be viewed as benign smooth muscle cell tumors of the artery wall. Environmental agents, including viruses and chemicals that have been previously associated with cell transformation and tumorigenesis may therefore also contribute directly to plaque development. Data are provided from in vivo and in vitro studies in support of this proposition. Evidence is also presented that in standardized assays human and animal plaque DNAs elicit responses similar to those elicited by tumor DNAs. Thus, both plaque formation and tumorigenesis may share common mechanisms.     

Histopathological and ultrastructural alterations in the aorta in experimental Solanum malacoxylon poisoning

A histopathological and electron microscopic study of the aortic wall of rabbits intoxicated with Solanum malacoxylon was performed. Histological examination showed local loss of the normal waviness of the elastic fibers and calcium deposits. Electron microscopic study of the corresponding areas showed a modified aspect of the smooth muscle cells with loss of some of their differential characteristics, especially their intracytoplasmic fibrils and densifications. The neighbouring elastic fibers showed an electron-dense peripheral band and sometimes a crystal deposit. X-ray microanalysis revealed the presence of a large amount of calcium in these crystals. Collagen fibers played no apparent role in this calcification. These findings support the idea that a local cellular alteration is necessary prior to elastic calcification. Two hypothetical mechanisms are proposed.     

Ca2+ uptake, Ca2+-ATPase activity, phosphoprotein formation and phosphate turnover in a microsomal fraction of smooth muscle

Vesicles capable of phosphate-stimulated calcium uptake were isolated from the microsomal fraction of the smooth muscle of the pig stomach according to a previously described procedure which consists in increasing the density of the vesicles by loading them with calcium phosphate and isolating them by centrifugation [Raeymaekers, L., Agostini, B., and Hasselbach, W. (1981) Histochemistry, 70, 139--150]. These vesicles, which contain calcium phosphate deposits, are able to accumulate an additional amount of calcium. This calcium uptake is accompanied by calcium-stimulated ATPase activity and by the formation of an acid-stable phosphoprotein. The acid-denatured phosphoprotein is dephosphorylated by hydroxylamine, which indicates that an acylphosphate is formed. This phosphoprotein probably represents a phosphorylated transport intermediate similar to that seen with the Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle. As with the Ca2+-ATPase of sarcoplasmic reticulum vesicles, this vesicular fraction catalyses an exchange between inorganic phosphate and the gamma-phosphate of ATP (ATP-Pi exchange) which is dependent on the presence of intravesicular calcium, and an exchange of phosphate between ATP and ADP (ATP-ADP exchange). The results further indicate that the turnover rate of the calcium pump, calculated from the ratio of calcium-stimulated ATPase activity to the steady-state level of phosphoprotein, is similar to that of Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle.     

CD38/cyclic ADP-ribose signaling: role in the regulation of calcium homeostasis in airway smooth muscle

The contractility of airway smooth muscle cells is dependent on dynamic changes in the concentration of intracellular calcium. Signaling molecules such as inositol 1,4,5-trisphosphate and cyclic ADP-ribose play pivotal roles in the control of intracellular calcium concentration. Alterations in the processes involved in the regulation of intracellular calcium concentration contribute to the pathogenesis of airway diseases such as asthma. Recent studies have identified cyclic ADP-ribose as a calcium-mobilizing second messenger in airway smooth muscle cells, and modulation of the pathway involved in its metabolism results in altered calcium homeostasis and may contribute to airway hyperresponsiveness. In this review, we describe the basic mechanisms underlying the dynamics of calcium regulation and the role of CD38/cADPR, a novel pathway, in the context of airway smooth muscle function and its contribution to airway diseases such as asthma.     

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

The role of the sodium-calcium exchanger for calcium extrusion in coronary arteries

Calcium ionophores, such as the A23187, cause endothelium-dependent relaxation of arterial strips with intact endothelium, whereas the effect of the ionophore should result from the combination of a relaxation caused by the endothelium-dependent factors and of a contraction of the smooth muscles. In addition, the application of a calcium ionophore to a strip of pig coronary arteries without endothelium does not change cytosolic free calcium concentration and force developed by the smooth muscle cells. To explain these paradoxes, the hypothesis that active calcium extrusion would match the entry of extracellular calcium caused by the ionophore was tested. We see that the sodium-calcium exchanger extrudes calcium that enters the smooth muscle cells in the absence of the ionophore. This exchanger is efficient enough to expel the increased influx of calcium created by the additional calcium carriers formed by the ionophore. This explains the inefficiency of calcium ionophores to increase cytosolic free calcium of smooth muscle cells and consequently, the fact that the ionophore does not cause a contraction of a strip without endothelium. This makes evident that a calcium ionophore fully relaxes, in an endothelium-dependent manner. an intact strip of porcine coronary artery.     

Influence of calcium on myosin thick filament formation in intact airway smooth muscle

Myosin thick filaments have been shown tobe structurally labile in intact smooth muscles. Although the mechanismof thick filament assembly/disassembly for purified myosins in solution has been well described, regulation of thick filament formation inintact muscle is still poorly understood. The present study investigates the effect of resting calcium level on thick filament maintenance in intact airway smooth muscle and on thick filament formation during activation. Cross-sectional density of the thick filaments measured electron microscopically showed that the density increased substantially (144%) when the muscle was activated. Theabundance of filamentous myosins in relaxed muscle was calcium sensitive; in the absence of calcium (with EGTA), the filament densitydeceased by 35%. Length oscillation imposed on the muscle underzero-calcium conditions produced no further reduction in the density.Isometric force and filament density recovered fully after reincubationof the muscle in normal physiological saline. The results suggest thatin airway smooth muscle, filamentous myosins exist in equilibrium withmonomeric myosins; muscle activation favors filament formation, and theresting calcium level is crucial for preservation of the filaments inthe relaxed state.

     

Endothelin-1 increases intracellular calcium mobilization but not calcium uptake in rabbit vascular smooth muscle cells

Conflicting evidence has been reported regarding the role of endothelin-1, a potent vasconstrictor peptide, in stimulating extracellular calcium influx in rabbit vascular smooth muscle. The objective of this study was to elucidate the effects of endothelin-1 on transmembrane 45Ca2+ influx and intracellular calcium mobilization in cultured rabbit aortic smooth muscle cells. In calcium containing buffer, endothelin-1 induced a concentration-dependent 45Ca2+ efflux response over the range of 10 pM to 100 nM with an EC50 of approximately 60 pM. Maximum endothelin-stimulated 45Ca2+ efflux was not affected by the absence of extracellular calcium or the presence of 1 microM verapamil. Endothelin-1 did not induce transplasmalemmal 45Ca2+ uptake at times up to 30 min. These findings suggest that an alteration in intracellular calcium handling, rather than extracellular calcium influx, is responsible for the endothelin-stimulated increase in intracellular calcium concentration in rabbit aortic smooth muscle cells.     

Ryanodine receptors in muscarinic receptor-mediated bronchoconstriction

Ryanodine receptors (RyRs), intracellular calcium release channels essential for skeletal and cardiac muscle contraction, are also expressed in various types of smooth muscle cells. In particular, recent studies have suggested that in airway smooth muscle cells (ASMCs) provoked by spasmogens, stored calcium release by the cardiac isoform of RyR (RyR2) contributes to the calcium response that leads to airway constriction (bronchoconstriction). Here we report that mouse ASMCs also express the skeletal muscle and brain isoforms of RyRs (RyR1 and RyR3, respectively). In these cells, RyR1 is localized to the periphery near the cell membrane, whereas RyR3 is more centrally localized. Moreover, RyR1 and/or RyR3 in mouse airway smooth muscle also appear to mediate bronchoconstriction caused by the muscarinic receptor agonist carbachol. Inhibiting all RyR isoforms with > or = 200 microM ryanodine attenuated the graded carbachol-induced contractile responses of mouse bronchial rings and calcium responses of ASMCs throughout the range of carbachol used (50 nM to > or = 3 microM). In contrast, inhibiting only RyR1 and RyR3 with 25 microM dantrolene attenuated these responses caused by high (>500 nM) but not by low concentrations of carbachol. These data suggest that, as the stimulation of muscarinic receptor in the airway smooth muscle increases, RyR1 and/or RyR3 also mediate the calcium response and thus bronchoconstriction. Our findings provide new insights into the complex calcium signaling in ASMCs and suggest that RyRs are potential therapeutic targets in bronchospastic disorders such as asthma.     

Cyclic stretch decreases TRPC4 protein and capacitative calcium entry in rat vascular smooth muscle cells

We investigated whether cyclic stretch affects TRPC4 or TRPC6 expression and calcium mobilization in cultured vascular smooth muscle cells. In aortic and mesenteric smooth muscle cells isolated from male Sprague-Dawley rats, TRPC4 expression was decreased after 5 h stretch and remained suppressed through 24 h stretch. After removal of the stretch stimulus, TRPC4 expression recovered within 2 h. Stretch did not affect TRPC6 expression. Stretch also decreased capacitative calcium entry, while agonist-induced calcium influx was increased. Similar results were obtained in primary aortic smooth muscle cells. TRPC4 mRNA levels were not decreased in response to mechanical strain. TRPC4 downregulation was also achieved by increasing extracellular calcium and was attenuated by gadolinium and MG132, suggesting that TRPC4 protein is regulated by intracellular calcium concentration and/or the ubiquitin-proteasome pathway. These data suggest that stretch-induced downregulation of TRPC4 protein expression and capacitative calcium entry may be a protective mechanism to offset stretch-induced increases in intracellular calcium.     

Intracellular calcium gradients in cultured human uterine smooth muscle: a functionally important subplasmalemmal space

The plasma membrane contains the key elements for the control of coupling excitation to contraction in smooth muscle. The superficial calcium buffer barrier, initially proposed by van Breemen for vascular smooth muscle, may participate in the regulation of calcium entry in other smooth muscle types. To investigate the relationship between the sarcoplasmic reticulum (SR) and the plasma membrane in myometrial smooth muscle cells, we performed experiments using videofluorescence imaging and cell-attached electrophysiology. The cell-attached patch was used as a reporter for the free calcium in the subplasmalemmal space by monitoring openings of the Maxi-K channel. Calcium green-1 was used to simultaneously monitor changes of the deep cytosolic calcium concentrations. The cell with the patch attached was stimulated via an intercellular calcium wave from an adjacent cell. In this fashion, release of SR calcium was accomplished with minimal disturbance of the plasma membrane and the subplasmalemmal space of the cell studied. With physiological bathing solution, six of seven calcium waves activated Maxi-K channels. Surprisingly, the Maxi-K channels began opening 6.3 +/- 4.7s (range 2.6-15.0s) after the wave passed the pipette location. When plasma membrane calcium fluxes were inhibited with 100 microM lanthanum, no Maxi-K channel openings were observed in six of seven experiments. These results are best explained by a subplasmalemmal space in which the calcium concentration is largely controlled by store-operated channels. These results suggest the superficial buffer barrier as merely one aspect of subplasmalemmal regulation of calcium dynamics, and emphasize the importance of store-operated calcium channels during dynamic calcium changes.     

Electron cytochemistry of oxalate-stimulated calcium uptake in microsomes from the smooth muscle of the pig stomach

Summary A microsomal fraction was isolated from the smooth muscle of the antrum of the pig stomach by differential centrifugation. Electron microscopy of the negatively stained material showed that this fraction is heterogeneous in composition. The microsomes accumulated calcium in the presence of ATP, magnesium and oxalate. The amount of calcium taken up per mg protein was in the same range as observed for other smooth muscle microsomal preparations. Although this amount is much smaller than that in the microsomal fraction of skeletal muscle, calcium oxalate crystals were formed in some vesicles, as occurs in the skeletal muscle fragmented sarcoplasmic reticulum. Through the presence of the calcium oxalate crystals, many of these vesicles acquired sufficient mass and density to allow them to be isolated by centrifugation. A purification of about 40 fold in terms of calcium content was reached.     

Ontogeny of the ryanodine receptor in rabbit urinary bladder smooth muscle

Bladder smooth muscle contraction is mediated by both direct calcium entry through the cell membrane, and by calcium induced calcium release (CICR) from the sarcoplasmic reticulum (SR) storage sites. Ryanodine is a neutral plant alkaloid which binds to an ion channel located on the SR membrane. Its effects in cardiac skeletal muscle are well characterized where it inibits the efflux of intracellular calcium stores, and thus it serves as a negative inotrope. It has also been shown that in the develpping rabbit myocardium, there is a gradual increase in the expression of this ion channel. Little has been written about the expression and function of the ryanodine sensitive ion channel in smooth muscle. Recently we have shown that neonatal rabbit bladder smooth muscle is not very sensitive to ryanodine, while that from mature rabbits is extremely sensitive. This leads us to quantify the expression of the ryanodine sensitive ion channel. In this paper we demonstrate that the Kd values do not change to any significant degree with normal rabbit bladder development. However the Bmax values for 3 day, 2, 4, 6, and 8 week rabbit bladder smooth muscle are 7, 10, 15, 29, and 44 fmol specifically bound ryanodine/mg protein. The differences between the neonatal groups and the mature groups are significant (P<0.5). This increase in ryanodine sensitive ion channel expression with normal growth would suggest that with normal maturation, the bladder smooth muscle cell acquires an increased pool of sequestrered intracellular calcium. This would follow a similar pattern of development that has already been described in rabbit myocardium.