A new selective ultrahistochemical method for the demonstration of calcium using N,N-naphthaloylhydroxylamine

Summary A new, simple, rapid, and highly sensitive and selective method for the ultrahistochemical detection of calcium is described. The reagent N,N-Naphthaloylhydroxylamine (1,8-C₁₀H₆CON(ONa)CO) sodium salt was employed in this study for the demonstration of calcium at the subcellular level in relaxed and contracted muscles (smooth muscle of the stomach, thoracic aorta, and myocardial muscle cells) of the rat (in vitro as well as in vivo) and in the human vascular smooth muscle of the aorta with atherosclerotic calcification.Direct evidence of the presence of calcium in the electron-dense reaction products (calcium N,N-Naphthaloylhydroxylamine) is given by X-ray microanalysis of 1,500–2,000 Å thick sections.The significance of distributional differences in the localization of calcium in subcellular structures of relaxed and contracted muscles in discussed in relation to the role of calcium in the control of the muscle activity during the contraction-relaxation cycle.     

Ultrastructure of a molluscan smooth muscle during phasic contractions and in the relaxed state

Tension and X-ray diffraction patterns are not always correlated in the smooth anterior retractor muscle (ABRM) of Mytilus edulis. The muscle produces equatorial intensity profiles of X-ray diffraction patterns corresponding to either a relaxed or a contracted structure. During phasic contractions, comprising a contracted as well a a relaxed phase, the diffracted intensity on the equator at 0.003 A^?1 changes within the first 10s after onset of stimulation. The tension reaches a maximum after about the same time. The time dependence of this intensity change during phasic contraction has been measured. It shows that the tension decays within 10s, but the relaxed structure needs 30–40 s to reestablish. There is no difference between the observed intensities from the tonic and phasic contracted states. Inactivated muscles with minimum tension, normally termed relaxed, can have either a “contracted” or a relaxed structure.     

Dephosphorylation of myosin by the catalytic subunit of a type-2 phosphatase produces relaxation of chemically skinned uterine smooth muscle

It is now well-established that phosphorylation of the 20,000-dalton light chain of smooth muscle myosin (LC20) is a prerequisite for muscle contraction. However, the relationship between myosin dephosphorylation and muscle relaxation remains controversial. In the present study, we utilized a highly purified catalytic subunit of a type-2, skeletal muscle phosphoprotein phosphatase (protein phosphatase 2A) and a glycerinated smooth muscle preparation to determine if myosin dephosphorylation, in the presence of saturating calcium and calmodulin, would cause relaxation of contracted uterine smooth muscle. Addition of the phosphatase catalytic subunit (0.28 microM) to the muscle bath produced complete relaxation of the muscle. The phosphatase-induced relaxation could be reversed by adding to the muscle bath either purified, thiophosphorylated, chicken gizzard 20,000-dalton myosin light chains or purified, chicken gizzard myosin light chain kinase. Incubation of skinned muscles with adenosine 5'-O-(thiotriphosphate) prior to the addition of phosphatase resulted in the incorporation of 0.93 mol of PO4/mol of LC20 and prevented phosphatase-induced relaxation. Under all of the above conditions, changes in steady-state isometric force were associated with parallel changes in myosin light chain phosphorylation over a range of phosphorylation extending from 0.01 to 0.97 mol of PO4/mol of LC20. We found no evidence that dephosphorylation of contracted uterine smooth muscles, in the presence of calcium and calmodulin, could produce a latch-state where isometric force was maintained in the absence of myosin light chain phosphorylation. These results show that phosphorylation or dephosphorylation of the 20,000-dalton myosin light chain is adequate for the regulation of contraction or relaxation, respectively, in glycerinated uterine smooth muscle.     

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.

     

Effects of extracellular calcium on canine tracheal smooth muscle

Strips of canine tracheal smooth muscle were studied in vitro to determine the effects of changes in the extracellular calcium (Cao) concentration on tonic contractions induced by acetylcholine and 5-hydroxytryptamine. Strips were contracted with graded concentrations of the above agents in 2.4 mM Ca, after which CaCl2 was administered to achieve final concentrations of 5.0, 10.0, and 20.0 mM. Increases in Cao to 5 mM or above caused significant relaxation of muscles contracted with 5-hydroxytryptamine but did not significantly relax muscles contracted with acetylcholine. Increases in Cao also caused significant relaxation of muscles contracted with low concentrations of K+ (20 or 30 mM). However, in 60 or 120 mM K+, increases in Cao resulted predominantly in muscle contraction. Inhibition of the Na+-K+-ATPase by ouabain (10(-5) M) or K+ depletion reversed the effects of Cao from relaxation to contraction in tissues contracted with 5-hydroxytryptamine. Increases in Cao also caused contraction rather than relaxation in the presence of verapamil (10(-6) M). We conclude that calcium has both excitatory and inhibitory effects on the contractile responses of canine tracheal smooth muscle. The inhibitory effects of Ca2+ appear to be linked to the activity of the membrane Na+-K+-ATPase.     

ULTRASTRUCTURAL STUDIES ON THE CONTRACTILE MECHANISM OF SMOOTH MUSCLE

Fresh taenia coli and chicken gizzard smooth muscle were studied in the contracted and relaxed states. Thick and thin filaments were observed in certain (but not all) cells fixed in contraction. Relaxed smooth muscle contained only thin filaments. Several other morphological differences were observed between contracted and relaxed smooth muscle. The nuclear chromatin is clumped in contraction and evenly dispersed in the relaxed state. The sarcolemma is more highly vesiculated in contraction than in relaxation. In contraction, the sarcoplasm also appears more electron opaque. Over-all morphological differences between cells fixed in isometric and in unloaded contraction were also noticeable. The results suggest a sliding filament mechanism of smooth muscle contraction; however, in smooth muscle, unlike striated muscle, the thick filaments appear to be in a highly labile condition in the contractile process. The relation between contraction and a possible change in pH is also discussed.     

The effects of prostacyclin (PGI2) on tissues which detect prostaglandins (PG'S).

The effects of prostacyclin (PGI2) and its stable metabolite 6-oxo-PGF1alpha on various bioassay tissues are compared with those of PGE2 and PGF2alpha, using the cascade superfusion method. On vascular smooth muscle, PGI2 caused relaxation of all tissues tested except the rabbit aorta. PGE2 relaxed rabbit coeliac and mesenteric artery but contracted bovine coronary artery and had no effect on rabbit aorta. 6-oxo-PGF1alpha was ineffective at the concentrations tested. On gastro-intestinal smooth muscle, PGI2 contracted strips of rat and hamster stomach and the chick rectum. It was less potent than PGE2 or PGF2alpha. None of these substances contracted the cat terminal ileum. 6-oxo-PGF1alpha was inactive on these tissues at the doses tested. PGI2 was less active than PGE2 or PGF2alpha in contracting guinea-pig trachea and rat uterus; 6-oxo-PGF1alpha was active only on the rat uterus. Thus, PGI2 can be distinguished from the other stable prostaglandins using the cascade method of superfusion.     

Decreased sensitivity of spontaneously hypertensive rat aortic smooth muscle to vasorelaxation by atriopeptin III

The effects of a synthetic form of Atrial Natriuretic Factor (ANF) on spontaneously hypertensive rat aortic smooth muscle were investigated using either an alpha-adrenoceptive agonist (phenylephrine) or an agent which partially depolarized the plasma membrane (20mM KCl) as a contractile agent. The relaxant response was studied under conditions resembling normal physiological calcium ion levels (1.5mM) as well as over a range of calcium ion concentrations (0.1-2.5mM). The results demonstrate a hyporesponsiveness of hypertensive aorta to vasorelaxation induced by synthetic ANF, which is more apparent when the tissue is contracted with KCl. The results also suggest that ANF, which has been shown previously to inhibit intracellular and receptor operated calcium channel mobilization only, may additionally work through a mechanism which is related to the voltage induced calcium flux across the membrane, which also is inhibited less in hypertensive smooth muscle.     

Analysis of some effects of aortic smooth muscle on<Emphasis Type="Italic">in vivo</Emphasis> aortic pressure curves

When aortic pressure curves were predicted previously on the basis of a newly developed model of visco-elastic properties of the aorta, it was necessary to use published viscoelastic constants. These were usually obtained from longitudinal strips of blood vessels long removed from the animal, and therefore probably containing deteriorated smooth muscle. The predicted curves had the same form as actual tracings, substantiating the analysis somewhat, but the pressure levels were low. These low levels, if due to inadequate visco-elastic constants, could be attributed to the use of longitudinal rather than circumferential segments as well as to the use of segments with deteriorated muscle. The present analysis uses data collected by the author testing circumferential viscoelastic properties of fourteen different aortic regions in a way suggested by the author's model of an aortic wall. Moreover, the constants were measured on segments containing muscle relaxed by EDTA solutions and on similar segments containing muscle contracted by neosynephrine. These visco-elastic constants were used in the author's nonlinear differential equation of motion of the aortic wallin vivo to predictin vivo pressure curves. The predicted curves were low in any given aortic region if relaxed constants were used, but at normal levels with contracted constants. In fact, pressure curves predicted using constants obtained from aortic segments containing contracted muscle resembled actual tracings in form and pressure levels. Even the observed variations in the form of the systolic pressure curve down the aorta were predicted by this analysis.     

Effects of prostaglandin E1, isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings

The role of cyclic AMP in the control of vascular smooth muscle tone was studied by monitoring the effects of prostaglandin E1 (PGE1), isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings. PGE1, isoproterenol and forskolin all increased cyclic AMP levels in rabbit aortic rings. Isoproterenol and forskolin relaxed phenylephrine-contracted aortic rings, but PGE1 contracted the rings in the presence or absence of phenylephrine. Isoproterenol relaxed these PGE1-contracted aortic rings without further change in total cyclic AMP levels, which were already elevated by the PGE1 alone. Pretreatment with forskolin potentiated the effects of PGE1 on cyclic AMP levels. PGE1 caused contractions in muscles partially relaxed by forskolin, even though very large increases in cyclic AMP levels (30 fold) were produced by PGE1 in the presence of forskolin. Isoproterenol was able to relax these forskolin-treated, PGE1-contracted muscles with no further increase in cyclic AMP levels. Thus, there does not appear to be a good correlation between total tissue levels of cyclic AMP and tension in these experiments. Our results suggest that, if cyclic AMP is responsible for relaxation of smooth muscle, some form of functional compartmentalization of cyclic AMP must exist in this tissue.     

Actin filament organization and crossbridge decoration in a contracting molluscan smooth muscle

Equatorial intensity distributions of x-ray diffraction patterns from relaxed and contracted states of the anterior byssus retractor muscle, ABRM, are compared with distributions of non-physiological reference states and with calculations based on various packing models of the actin filaments. Relaxed and contracted muscles provide distributions that agree with models, in which actin filaments are packed hexagonally in discrete areas containing 12 to 16 filaments. The crystalline fractions of actin filaments in the relaxed and contracted states are 0.91 and 0.57 respectively. Contracting muscles, however, show deviations from the calculated distributions at small angles of diffraction. This is interpreted as being due to the fact that actin filaments, outside crystalline areas, are decorated by crossbridges as about every 6th actin monomer.     

Nonmuscle Myosin motor of smooth muscle

Nonmuscle myosin can generate force and shortening in smooth muscle, as revealed by studies of the urinary bladder from mice lacking smooth muscle myosin heavy chain (SM-MHC) but expressing the nonmuscle myosin heavy chains A and B (NM-MHC A and B; Morano, I., G.X. Chai, L.G. Baltas, V. Lamounier-Zepter, G. Lutsch, M. Kott, H. Haase, and M. Bader. 2000. Nat. Cell Biol. 2:371-375). Intracellular calcium was measured in urinary bladders from SM-MHC-deficient and SM-MHC-expressing mice in relaxed and contracted states. Similar intracellular [Ca2+] transients were observed in the two types of preparations, although the contraction of SM-MHC-deficient bladders was slow and lacked an initial peak in force. The difference in contraction kinetics thus do not reflect differences in calcium handling. Thick filaments were identified with electron microscopy in smooth muscle cells of SM-MHC-deficient bladders, showing that NM-MHC can form filaments in smooth muscle cells. Maximal shortening velocity of maximally activated, skinned smooth muscle preparations from SM-MHC-deficient mice was significantly lower and more sensitive to increased MgADP compared with velocity of SM-MHC-expressing preparations. Active force was significantly lower and less inhibited by increased inorganic phosphate. In conclusion, large differences in nucleotide and phosphate binding exist between smooth and nonmuscle myosins. High ADP binding and low phosphate dependence of nonmuscle myosin would influence both velocity of actin translocation and force generation to promote slow motility and economical force maintenance of the cell.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.     

Distribution of contractile proteins in single isolated smooth muscle cells from the ascidian body-wall muscle

1. Relaxed cells isolated from ascidian body-wall muscle were morphologically very similar to relaxed common smooth muscle cells. 2. The contracted cells, however, possessed striations which were resolved into a repeating pattern of light and dark bands using phase contrast microscope. 3. The relaxed ascidian cells treated with Triton X-100 were contracted and showed the striations by adding Ca2+. 4. By an indirect immunofluorescence method, it was clearly seen that antiactin spread uniformly in the relaxed cells, while this antibody was concentrated on the dark bands of striations in the contracted cells.     

Serotonergic modulation of murine fundic tone

Fundic tone is maintained through a balance of excitatory and inhibitory input to fundic smooth muscle. The aim of this study was to determine the role of serotonin (5-HT) and 5-HT receptors in modulating murine fundic tone. Muscle strips were prepared from the murine fundus. Intracellular recordings were made from circular smooth muscle cells, and the effects of 5-HT on tone and excitatory and inhibitory junction potentials evoked by electrical field stimulation (EFS) were determined. 5-HT induced a concentration-dependent contraction and smooth muscle depolarization that was tetrodotoxin resistant. The 5-HT(1B/D) receptor antagonists GR-127935 and BRL-155172 significantly inhibited 5-HT-induced contractions. The 5-HT(1B/D) agonist sumatriptan contracted murine fundic muscle. The 5-HT(1A) receptor agonist buspirone relaxed fundic smooth muscle, and the relaxation was inhibited by WAY-100135 but not by N(omega)-nitro-l-arginine or tetrodotoxin. 5-HT enhanced both the excitatory and inhibitory responses to EFS. The 5-HT(3) receptor antagonist MDL-72222 partly inhibited both the excitatory and inhibitory response elicited by EFS, whereas the 5-HT(4) receptor antagonist GR-113808 partly inhibited the EFS-evoked inhibitory response. The 5-HT reuptake inhibitor fluoxetine contracted smooth muscle strips, a contraction that was partially inhibited by GR-127935 and abolished by tetrodotoxin. In conclusion, the data suggest that 5-HT modulates murine fundic contractile activity through several different receptor subtypes. Sustained release of 5-HT maintains fundic tone through postjunctional 5-HT(1B/D) receptors. 5-HT(3) receptors modulate excitatory neural input to murine fundic smooth muscle, and both 5-HT(3) and 5-HT(4) receptors modulate inhibitory neural input to murine fundic smooth muscle.     

Aminoacyl-tRNA synthetases: inter-site interaction as a possible proofreading mechanism

Smooth muscle cell energetics of taenia caeci during relaxation, activity and maximal contraction were investigated using ³¹P-NMR. In relaxed muscle obtained in calcium-free medium, [ATP], [phosphocreatine] and [sugar phosphate] were 4.4 mM, 7.7 mM and 2.8 mM, respectively. There was only a small difference in the energetics of spontaneously active and maximally contracted muscles, but under both conditions substantial changes occurred as compared with relaxed muscles. The internal pH in relaxed muscle was found to be 7.05, which acidified to 6.5 during contraction. The level of sugar phosphates was found to be not a limiting factor in energetics.     

Presence of specific bradykinin receptors in arterial and venous smooth muscle

The relationship between bradykinin action and its concentration was examined on isolated rings of the rabbit aorta, femoral artery, jugular vein and on isolated strips of the rat portal vein. The sensitivity of femoral artery and portal vein smooth muscles to bradykinin was disclosed. Venous smooth muscles were more sensitive to bradykinin as compared with arterial smooth muscles. Dissociation constants for the rabbit aorta, femoral artery, jugular vein and for the rat portal vein were 3.98 X 10(-6), 6.3 X 10(-6), 1.26 X 10(-7), and 7.6 X 10(-9)M, respectively. Effects of endogenous bradykinin in vivo might result from its primary action on the venous smooth muscle, action on the arterial smooth muscle and veno-arterial interactions.     

Muscular system of a peculiar parasitic cnidarian Polypodium hydriforme: a phalloidin fluorescence study

Musculature of the free-living stages of Polypodium hydriforme has been studied using phalloidin fluorescence method and confocal microscopy. P. hydriforme is a unique cnidarian possessing only smooth muscle cells situated within the mesoglea, not epithelial muscle cells, like the rest of cnidarians. Phalloidin fluorescence on whole mount preparations demonstrates an extensively developed subepidermal muscle system mostly consisting of long parallel fibers running along the tentacles. For the first time along with contracted muscle fibers we could clearly demonstrate relaxed fibers looking as long spirals. System of thin parallel circular F-actin positive fibers has been discovered outside of longitudinal muscles. The body of the animal and the mouth cone contain weakly developed parallel muscles. No special attachment of the muscle fibers to the tips of the tentacles or to the rim of the mouth has been observed. The results are discussed in connection with the "triploblastic" organization of P. hydriforme and its phylogenetic position.     

Dissociation of cGMP accumulation and relaxation in myometrial smooth muscle: effects of S-nitroso-N-acetylpenicillamine and 3-morpholinosyndonimine

In guinea pig, primate and man, nitric oxide (NO)-induced regulation of myometrial smooth muscle contraction is distinct from other smooth muscles because cyclic guanosine 3',5'-cyclic monophosphate (cGMP) accumulation is neither necessary nor sufficient to relax the tissue. To further our understanding of the mechanism of action of NO in myometrium, we employed the NO donors, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosyndonimine (SIN-1) proposed to relax airway smooth muscle by disparate mechanisms involving elevation in intracellular calcium ([Ca(2+)](i)) or cGMP accumulation, respectively. Treatment of guinea pig myometrial smooth muscle with either NO donor at concentrations thought to produce maximal relaxation of smooth muscles resulted in significant elevations in cGMP that were accompanied by phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein (VASP), shown here for the first time to be present and phosphorylated in myometrium. Stimulation of myometrial strips with oxytocin (OT, 1 microM) produced an immediate increase in contractile force that persisted in the continued presence of the agonist. Addition of SNAP (100 microM) in the presence of OT relaxed the tissue completely as might be expected of an NO donor. SIN-1 failed to relax the myometrium at any concentration tested up to 300 microM. In Fura-2 loaded myometrial cells prepared from guinea pig, addition of SNAP (100 microM) in the absence of other agonists caused a significant, reproducible elevation of intracellular calcium while SIN-1 employed under the same conditions did not. Our data further support the notion that NO action in myometrium is distinct from that in other smooth muscles and underscores the possibility that discrete regional changes in [Ca(2+)](i), rather than cGMP, signal NO-induced relaxation of the muscle.     

Effects of TRIM on tension, intracellular calcium and nitrergic transmission in the rat anococcygeus muscle.

The effects of the putatively selective inhibitor of neuronal nitric oxide synthase (nNOS) 1-(2-trifluoromethylphenyl) imidazole (TRIM) were investigated on contractility, intracellular calcium and nitrergic relaxations in the rat anococcygeus muscle. TRIM (100-1000 microM) reduced the tension of rat anococcygeus muscles when contracted with guanethidine (10 microM) and clonidine (0.1 microM). Relaxations to TRIM persisted in the presence of the non-selective NOS inhibitor L-NAME (100 microM) and the inhibitor of soluble guanylate cyclase ODQ (1 microM). TRIM also reduced tension when muscles were contracted with phenylephrine (3 microM), noradrenaline (3 microM) or high K physiological salt solution (high KPSS; 60mM). Influx of calcium ([Ca(2+)](i)) in response to high KPSS was significantly reduced in the presence of TRIM (1mM). TRIM also inhibited the influx of (45)Ca(2+) induced by KPSS, but had no effect on the influx induced by phenylephrine (10 microM). TRIM (300 microM) had a modest, but significant, inhibitory effect on nitrergic relaxations that were evoked by electrical field stimulation (1-10 Hz, 15 V, 10s trains) in muscles contracted with guanethidine and clonidine. In contrast, L-NAME (1-100 microM) inhibited these nitrergic responses with an IC(50) of 9.31+/-0.87 microM (n=4). The results suggest that the smooth muscle relaxant effect of TRIM in the rat anococcygeus muscle may affect the entry of Ca(2+) possibly through voltage-operated calcium channels. Furthermore, the relatively modest effect of TRIM on nitrergic responses indicates that it is not a particularly reliable inhibitor of nNOS.