Fibronectin dependence of the contraction of collagen lattices by human skin fibroblasts

The role of fibronectin in the contraction of collagen lattices by human skin fibroblasts has been investigated. Incubation of lattice cultures in Dulbecco's modified Eagle's medium supplemented with increasing concentrations of non-dialysed or dialysed fetal calf serum demonstrated that the rate of contraction was dependent on non-dialysable serum components. The suppression of contraction observed when fibronectin was eliminated from serum, either by affinity chromatography on gelatin-agarose columns or by precipitation with anti-fibronectin antibodies, showed that fibronectin is critical for the contraction. When collagen lattices were incubated in a serum-free culture medium totally devoid of fibronectin, no contraction occurred. When fibronectin was added to this medium, their contraction was correlated with the concentration of fibronectin added. The contraction was inhibited by cycloheximide, tunicamycin, and monensin. These results demonstrate that the contraction of collagen lattices by human skin fibroblasts is dependent on fibronectin, and that other protein factors synthesized by the cells or contained in serum are also necessary.     

Galanin induces contraction of isolated cells from circular muscle layer of pig ileum.

The effects of galanin and its interaction with cholecystokinin and acetylcholine on smooth muscle cells were studied in vitro on isolated cells obtained from pig ileum circular muscle layer. Galanin induced a concentration-dependent cell contraction with a maximal contraction (24.5% decrease in cell length from control) obtained at 1 nM. The concentration of galanin inducing a half-maximal contraction was 3 pM. Tetrodotoxin (10 microM) failed to inhibit cell contraction induced by galanin (1 nM), pentagastrin (10 nM) and acetylcholine (1 microM). Atropine abolished the contraction induced by acetylcholine (1 microM), but had no effect on galanin- and pentagastrin-induced contraction. L 364,718 inhibited the contraction induced by CCK8 but not the galanin-induced contraction. At the uneffective concentration of 10 fM, galanin had a synergistic effect with an uneffective concentration of CCK8 (1 pM). These results suggest that (i) galanin contracts smooth muscle cells from pig ileum by acting on a specific receptor; (ii) galanin and either CCK or acetylcholine may act in a synergistic way to induce cell contraction.     

Differential regulation of smooth muscle contraction in rabbit internal anal sphincter by substance P and bombesin.

Substance P and bombesin induce contraction of isolated IAS smooth muscle cells by different intracellular mechanisms. The cells contracted in a dose dependent manner to both peptides. The kinetics of contraction were different. Substance P induced contraction peaked at 30 seconds and declined in a time dependent manner while bombesin induced contraction peaked at 30 seconds and was maintained for up to 8 minutes. The absence of extracellular calcium in the medium (0 calcium and 2 mM EGTA) had no affect on substance P induced contraction while it blocked bombesin induced contraction. Substance P induced contraction was blocked by the calmodulin antagonist W7 (10(-9)M) and was not affected by the PKC antagonist H7 (10(-6)M). Bombesin induced contraction was blocked by the PKC antagonist H7 and was not affected by the calmodulin antagonist W7. Our data indicate that substance P induces a transient contraction utilizing intracellular calcium and a calmodulin dependent pathway, while bombesin induces a sustained contraction utilizing calcium from extracellular sources and a calmodulin independent pathway.     

The interaction of human papillary and reticular fibroblasts and human keratinocytes in the contraction of three-dimensional floating collagen lattices

Fibroblasts derived from the papillary and reticular dermis of human skin and human keratinocytes show differences in their abilities to contract floating three-dimensional gels constructed from type I collagen. Reticular fibroblasts produce greater gel contraction than papillary fibroblasts. When equal numbers of papillary and reticular fibroblasts are mixed in the gels, papillary fibroblasts consistently inhibit gel contraction by reticular fibroblasts indicating interaction between these cell types in the contraction process. Surprisingly, keratinocytes alone produce greater gel contraction than that produced by either fibroblast type. Cooperativity in the gel contraction process is observed when fibroblasts are incorporated into the collagen matrix and keratinocytes are seeded onto the gel surface. Keratinocytes and dermal fibroblasts adhere to the collagen fibril to induce gel contraction by different mechanisms. Fibroblast contraction of collagen gels does not require fibronectin but is a serum-dependent reaction. In contrast, keratinocyte contraction of collagen gels occurs in a serum-free environment. Polyclonal, affinity-purified antibodies to human plasma fibronectin at high concentrations do not inhibit gel contraction by keratinocytes, making unlikely the possibility that fibronectin synthesized by the keratinocyte is a significant factor in the gel contraction process. We are currently examining the possibilities either that keratinocytes are synthesizing other adhesion proteins or that receptors on the cell surface can interact directly with the collagen fiber.     

Relationship of diaphragmatic contractility to diaphragmatic blood flow in newborn lambs

We determined the relationship of diaphragmatic contraction rate to diaphragmatic blood flow (Qdi), metabolism, and contractility in nine open-chested mechanically ventilated newborn lambs. The diaphragm was paced for 15 min at slow (20/min) and fast (100/min) contraction rates each followed by a 30-min rest period. There was a mild reduction in transdiaphragmatic pressure (Pdi) during the slow contraction period accompanied by a shift to the right of the curve relating stimulation frequency (10-100 Hz) to Pdi. Pdi returned to control at the start of the fast contraction period, but then fell by 30% within 2 min with continued fast contraction rates. The frequency-Pdi curve was significantly shifted to the right. Qdi, O2 transport, and O2 consumption increased during slow contraction and to an even greater extent during fast contraction. Fractional O2 extraction reached an apparent maximum during slow contraction. Lactate efflux from the right phrenic vein during slow contraction remained unchanged from control. During fast contraction lactate efflux rose proportionately more than did O2 consumption. We conclude that the energy demands at fast rates of diaphragmatic contraction in newborn lambs cannot be met by aerobic metabolism alone despite increasing O2 transport to the diaphragm.     

Effects of bile acids on the muscle functions of guinea pig gallbladder

Hydrophobic bile acids impair gallbladder emptying in vivo and inhibit gallbladder muscle contraction in response to CCK-8 in vitro. This study was aimed at determining the mechanisms of muscle cell dysfunction caused by bile acids in guinea pig gallbladders. Muscle cells were obtained by enzymatic digestion. Taurochenodeoxycholic acid (TCDC), a hydrophobic bile acid, caused a contraction of up to 15% and blocked CCK-induced contraction. Indomethacin abolished the TCDC-induced contraction. Hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had no effect on muscle contraction but prevented the TCDC-induced contraction and its inhibition on CCK-induced contraction. Pretreatment with NADPH oxidase inhibitor PH2I, xanthine oxidase inhibitor allopurinol, and free-radical scavenger catalase also prevented TCDC-induced contraction and its inhibition of the CCK-induced contraction. TCDC caused H2O2 production, lipid peroxidation, and increased PGE2 synthesis and activities of catalase and SOD. These changes were significantly inhibited by pretreatment of PH2I or allopurinol. Inhibitors of cytosolic phospholipase A2 (cPLA2), protein kinase C (PKC), and mitogen-activating protein kinase (MAPK) also blocked the TCDC-induced contraction. It is concluded that hydrophobic bile acids cause muscle cell dysfunction by stimulating the formation of H2O2 via activation of NADPH and xanthine oxidase. H2O2 causes lipid peroxidation and activates cPLA2 to increase PGE2 production, which, in turn, stimulates the synthesis of free-radical scavengers through the PKC-MAPK pathway.     

High-speed video cinematographic demonstration of stalk and zooid contraction of Vorticella convallaria.

Stalk contraction and zooid contraction of living Vorticella convallaria were studied by high-speed video cinematography. Contraction was monitored at a speed of 9000 frames per second to study the contractile process in detail. Complete stalk contraction required approximately 9 ms. The maximal contraction velocity, 8.8 cm/s, was observed 2 ms after the start of contraction. We found that a twist appeared in the zooid during contraction. As this twist unwound, the zooid began to rotate like a right-handed screw. The subsequent stalk contraction steps, the behavior of which was similar to that of a damped harmonic oscillator, were analyzed by means of the equation of motion. From the beginning of stalk contraction, the Hookean force constant increased, and reached an upper limit of 2.23 x 10(-4) N/m 2-3 ms after the start of contraction. Thus, within 2 ms, the contraction signal spread to the entire stalk, allowing the stalk to generate the full force of contraction. The tension of an extended stalk was estimated to be 5.58 x 10(-8) N from the Hookean force constant of a stalk. This value coincides with that of the isometric tension of a glycerol-treated V. convallaria, confirming that the contractile system of V. convallaria is well preserved despite glycerol treatment.     

Contribution of Na+ and membrane depolarization to contraction induced by adrenaline in the guinea pig vas deferens

The contribution of Na+ and membrane depolarization to biphasic contractions induced by adrenaline were investigated in the smooth muscle of guinea pig vas deferens. Adrenaline (5 X 10(-6) M) produced an initial small contraction (first contraction) followed by a large tonic contraction (second contraction) with subsequent rhythmic activity. The entire response to adrenaline was largely inhibited by phentolamine (5 X 10(-6) M). By adding an appropriate concentration of Mn2+ (2 X 10(-4) M) or nifedipine (3 X 10(-7) M), a Ca2+ blocker, the second contraction was strongly reduced, accompanied by abolishment of the rhythmic contraction, whereas the first contraction was virtually unaffected. However, the first contraction was markedly suppressed by a higher concentration of Mn2+. All contractions produced by adrenaline were greatly reduced in Ca2+-free solution containing 0.5 mM EGTA. By lowering external Na+ concentration, the first contraction was markedly increased without greatly affecting the second contraction. By exposure to Na+-free isotonic high K+ solution, which elicited a greater depolarization of the membrane, the first contraction produced by adrenaline was also greatly potentiated, while the second and rhythmic contractions were eliminated. These results suggest that the adrenaline-evoked first contraction may be due to an influx of membrane bound Ca2+ which is independent of membrane depolarization, while the second (rhythmic) contraction is due to an influx of extracellular Ca2+ which is dependent upon depolarization.     

Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin.

We have identified the low MW 27 kD heat shock protein as a major phosphoprotein constituent of smooth muscle and have investigated its potential role in agonist induced smooth muscle contraction. The neuropeptides bombesin and substance P, which are present in neurons of the anorectal region, induce contraction of isolated smooth muscle cells from this region by activating different intracellular pathways. Substance P-induced contraction is 1,4,5-inositol trisphosphate (IP3)/calmodulin dependent, while contraction induced by bombesin is mediated by a protein kinase C (PKC)-dependent pathway. The sustained contraction induced by bombesin or exogenous PKC was blocked by preincubation of cells with monoclonal antibodies to hsp27, while the transient contraction induced by substance P or IP3 was unaffected by the antibodies. Preincubation with isotype matched control antibodies had no inhibitory effect on contraction induced in response to the agents used. These data support a novel role for hsp27 in the non calmodulin mediated sustained contraction induced by bombesin or PKC.     

Absence of effect of somatostatin on the guinea pig gallbladder

The effect of somatostatin (GH-RIH) on cholecystokinin octapeptide (OP-CCK) or acetylcholine (ACh) induced contraction of the guinea pig gallbladder was evaluated in vitro. GH-RIH failed to inhibit the muscle contraction induced by OP-CCK or ACh. To correlate with the in vitro results, the effect of GH-RIH on OP-CCK induced contraction of the gallbladder was evaluated in the guinea pig in vivo. GH-RIH did not affect the OP-CCK induced contraction of the gallbladder. Our results suggest that GH-RIH does not have direct inhibitory effect on the contraction of the guinea pig gallbladder induced by OP-CCK or ACh.     

Evidence for the role of p38 MAP kinase in hypoxia-induced pulmonary vasoconstriction

Mitogen-activated protein (MAP) kinases regulate smooth muscle cell contraction. Hypoxia contracts pulmonary arteries by mechanisms that are incompletely understood. We hypothesized that hypoxic contraction of pulmonary arteries involves activation of the MAP kinases. To test this hypothesis, we studied the effects of SB-202190, a p38 MAP kinase inhibitor, PD-98059 and UO-126, two structurally different MEKK inhibitors, and anisomycin, a stimulator of p38 MAP kinase on acute hypoxia-induced contraction in rat conduit pulmonary artery rings precontracted with phenylephrine or KCl. Hypoxia induced a transient contraction, followed by a relaxation, and then a slowly developing sustained contraction. Hypoxia also significantly increased phosphorylation of p38 MAP kinase. SB-202190 did not affect the transient phase but abrogated the sustained phase of hypoxic contraction, whereas anisomycin enhanced both phases of contraction. SB-202190 also attenuated and anisomycin enhanced the phenylephrine-induced contraction. In contrast, PD-98059 and UO-126 had minimal effects on either hypoxic or phenylephrine-induced contraction. None of the treatments modified KCl-induced contraction. We conclude that p38, but not the ERK1/ERK2 MAP kinase pathway, mediates the sustained phase of hypoxic contraction in isolated rat pulmonary arteries.     

Uncoupling of respiration-linked contraction in corn mitochondria

Respiration-linked contraction of corn mitochondria is not noticeably reduced by low, uncoupling concentrations of dinitrophenol. However, if a contraction/respiration ratio is calculated, the contraction proves to be uncoupled. Previous statements that contraction cannot be uncoupled from respiration are in error.

The uncoupling of contraction is consistent with the concept that dinitrophenol attacks a primary non-phosphorylated high energy intermediate (I~X). It is proposed that this intermediate is linked to some contractile mechanism such that the degree of contraction reflects the level of intermediate.

     

Swelling and contraction of corn mitochondria

A survey has been made of the properties of corn mitochondria in swelling and contraction. The mitochondria swell spontaneously in KCl but not in sucrose. Aged mitochondria will swell rapidly in sucrose if treated with citrate or EDTA. Swelling does not impair oxidative phosphorylation if bovine serum albumin is present.

Contraction can be maintained or initiated with ATP + Mg or an oxidizable substrate, contraction being more rapid with the substrate. Magnesium is not required for substrate powered contraction. Contraction powered by ATP is accompanied by the release of phosphate. Oligomycin inhibits both ATP-powered contraction and the release of phosphate. However, it does not affect substrate-powered contraction. Substrate powered contraction is inhibited by electron-transport inhibitors. The uncoupler, carbonyl cyanide m-chlorophenyl hydrazone, accelerates swelling and inhibits both ATP-and substrate-powered contraction. However, the concentrations required are well in excess of those required to produce uncoupling and to accelerate adenosine triphosphatase; the concentrations required inhibit respiration in a phosphorylating medium.

Phosphate is a very effective inhibitor of succinate-powered contraction. Neither oligomycin nor Mg affects the phosphate inhibition. Phosphate is less inhibitory with the ATP-powered contraction.

The results are discussed in terms of a hypothesis that contraction is associated with a nonphosphorylated high energy intermediate of oxidative phosphorylation.

     

Histamine-induced depolarization: ionic mechanisms and role in sustained contraction of rabbit cerebral arteries

The role of membrane depolarization in the histamine-induced contraction of the rabbit middle cerebral artery was examined by simultaneous measurements of membrane potential and isometric force. Histamine (1-100 microM) induced a concentration-dependent sustained contraction associated with sustained depolarization. Action potentials were observed during depolarization caused by histamine but not by high-K(+) solution. K(+)-induced contraction was much smaller than sustained contraction associated with the same depolarization caused by histamine. Nifedipine attenuates histamine-induced sustained contraction by 80%, with no effect on depolarization. Inhibition of nonselective cation channels with Co(2+) (100-200 microM) reversed the histamine-induced depolarization and relaxed the arteries but induced only a minor change in K(+)-induced contraction. In the presence of Co(2+) and in low-Na(+) solution, histamine-evoked depolarization and contraction were transient. We conclude that nonselective cation channels contribute to histamine-induced sustained depolarization, which stimulates Ca(2+) influx through voltage-dependent Ca(2+) channels participating in contraction. The histamine-induced depolarization, although an important and necessary mechanism, cannot fully account for sustained contraction, which may be due in part to augmentation of currents through voltage-dependent Ca(2+) channels and Ca(2+) sensitization of the contractile process.     

Effects of morphine and methionine-enkephalin on the smooth muscle tonus and the contraction induced by transmural stimulation in the carp (Cyprinus carpio) intestinal bulb

The effects of morphine and methionine-enkephalin (met-enkephalin) on the smooth muscle tonus and the contraction induced by transmural stimulation were investigated in the isolated intestinal bulb of carp in vitro. Morphine (30 nM-3 microM) and met-enkephalin (3 nM-5 microM) caused dose-dependent non-sustained contraction. Naloxone (10 nM) inhibited the contraction induced by morphine or met-enkephalin in a competitive manner. Tetrodotoxin (400 nM) or atropine (500 nM) did not inhibit the contraction induced by morphine or met-enkephalin. Cooling of the bath fluid from 20 to 10 degrees C decreased nicotine- and transmural stimulation-induced contraction. But met-enkephalin-induced contraction was not affected. Transmural stimulation-induced contraction (3 Hz) was not affected by pretreatment with morphine, met-enkephalin or naloxone. The results demonstrated that morphine or met-enkephalin caused contraction of the smooth muscle directly through the activation of opiate receptors on the smooth muscle cells and neither morphine nor met-enkephalin regulated the cholinergic neurotransmission presynaptically.     

Ca2+-independent contraction of uterine smooth muscle

Rat uterine smooth muscle shows sustained contraction to oxytocin in Ca2+-free medium with EGTA, that is called "Ca-free contraction"(1). Participation of the rise in cytosolic free Ca2+ in this Ca-free contraction was tested. In Ca-free contraction, the cytosolic free Ca2+ level was not changed at all as measured with fura-2. Further, the chelation of cytosolic free Ca2+ with quin-2 did not at all affect Ca-free contraction. These results strongly suggest that Ca-free contraction is not triggered by Ca2+.     

Local longitudinal muscle shortening of the human esophagus from high-frequency ultrasonography

We analyzed local longitudinal shortening by combining concurrent ultrasonography and manometry with basic principles of mechanics. We applied the law of mass conservation to quantify local axial shortening of the esophageal wall from ultrasonically measured cross-sectional area concurrently with measured intraluminal pressure, from which correlations between local contraction of longitudinal and circular muscle are inferred. Two clear phases of local longitudinal shortening were observed during bolus transport. During luminal filling by bolus fluid, the muscle layer distends and the muscle thickness decreases in the absence of circular or longitudinal muscle contraction. This is followed by local contraction, first in longitudinal muscle, then in circular muscle. Maximal longitudinal shortening occurs nearly coincidently with peak intraluminal pressure. Longitudinal muscle contraction begins before and ends after circular muscle contraction. Larger longitudinal shortening is correlated with higher pressure amplitude, suggesting that circumferential contractile forces are enhanced by longitudinal muscle shortening. We conclude that a peristaltic wave of longitudinal muscle contraction envelops the wave of circular muscle contraction as it passes through the middle esophagus, with peak longitudinal contraction aligned with peak circular muscular contraction. Our results suggest that the coordination of the two waves may be a physiological response to the mechanical influence of longitudinal shortening, which increases contractile force while reducing average muscle fiber tension by increasing circular muscle fiber density locally near the bolus tail.     

ATP-induced cell contraction in dermal fibroblasts: effects of cAMP and myosin light-chain kinase

When 1 mM ATP is added to human dermal fibroblasts (DF) in monolayer culture permeabilized by glycerol, they undergo a rapid reduction in length and their intracellular actin filaments aggregate. This process is referred to as cell contraction. Treating glycerol-permeabilized DF with alkaline phosphatase before adding 1 mM ATP should cause dephosphorylation. Dephosphorylated preparations do not undergo cell contraction initiated by ATP. When myosin light-chain kinase (MLCK) isolated from turkey gizzard is added with cofactors to cells dephosphorylated by alkaline phosphatase treatment, contraction is restored. DF incubated for 24 h with db cAMP or cholera toxin show elevated intracellular concentrations of cAMP and little cell contraction. Contraction is reestablished when MLCK with cofactors is incubated with these preparations before ATP is added. Fibroblasts from Epidermolysis Bullosa dystrophica recessive patients produce excess cAMP. Those cells show minimal contraction, however; treating them with MLCK and cofactors renews contraction brought about by ATP. When DF are incubated with trifluoperazine to block calmodulin-dependent enzyme reactions, cell contraction is inhibited. Adding cytochalasin B disrupts microfilaments and also inhibits contraction. This work supports the idea that myosin ATPase is critical to cell contraction. Myosin ATPase is dependent on the phosphorylation of the regulatory peptide, myosin light chain. Elevating intracellular concentrations of cAMP or treatment of permeabilized cell preparations with alkaline phosphatase may inhibit myosin ATPase activity. The restoration of phosphorylation by adding MLCK with cofactors served to reestablish cell contraction.     

Micromechanics of fibroblast contraction of a collagen-GAG matrix

The contractile force developed by fibroblasts has been studied by measuring the macroscopic contraction of porous collagen-GAG matrices over time. We have identified the microscopic deformations developed by individual fibroblasts which lead to the observed macroscopic matrix contraction. Observation of live cells attached to the matrix revealed that matrix deformation occurred as a result of cell elongation. The time dependence of the increase in average fibroblast aspect ratio over time corresponded with macroscopic matrix contraction, further linking cell elongation and matrix contraction. The time dependence of average fibroblast aspect ratio and macroscopic matrix contraction was found to be the result of the stochastic nature of cell elongation initiation and of the time required for cells to reach a final morphology (2-4 h). The proposed micromechanics associated with observed buckling or bending of individual struts of the matrix by cells may, in part, explain the observation of a force plateau during macroscopic contraction. These findings indicate that the macroscopic matrix contraction measured immediately following cell attachment is related to the extracellular force necessary to support cell elongation, and that macroscopic time dependence is not directly related to microscopic deformation events.     

Free fatty acids and dialyzed serum alterations of fibroblast populated collagen lattice contraction.

Fibroblast populated collagen lattices (FPCL) have facilitated the in vitro study of wound contraction and scar contracture. Mixing fibroblasts, serum containing culture medium and soluble collagen, together and then incubating the mixture at 37 degrees C produces a FPCL. The fibroblasts elongate and spread within the collagen matrix, and by forces associated with cell locomotion they reorganize the collagen fibers. The reorganization of the collagen produces a reduction in size of the FPCL, called lattice contraction. It was also found that dialyzed fetal bovine serum did not support lattice contraction. Supplementing dialyzed serum with fatty acids accelerated lattice contraction. The fatty acid composition of the fibroblast plasma membrane influences that membrane fluidity. These studies demonstrated that lattice contraction was enhanced by the additions of saturated fatty acids in the order of laurate (C-12), palmitic (C-16), and stearate (C-18). With unsaturated fatty acids additions, the order of enhanced lattice contraction was arachidonate (4 C = C), linoleate (2 C = C) and oleate (1 C = C). The addition of dialyzed serum with or without fatty acids neither altered ATP-induced cell contraction activity nor cell proliferation. It was concluded that free fatty acid additions do not modulate FPCL contraction by enhancing microfilaments contraction or increasing cell numbers. The mechanism of action was proposed to be by altering cell membrane fluidity. This finding further supports the theory that the mechanism for lattice contraction is cell locomotion, rather than cell contraction.