Ca binding of intestinal smooth muscle myosin B.

By means of both centrifugation and filtration techniques, the Ca binding activity of intestinal myosin B was studied. The binding capacity of myosin B was Ca dependent and was approximately linear when the concentration of Ca in the medium ranged from 10(-4) to 10(-7) M. The Ca sensitivity of ATPase activity in the same range of Ca concentration exhibited a sigmoid curve. The Scatchard plot of Ca binding showed that intestinal myosin B had at least two types of binding sites. One of these was defined as a high affinity site with an apparent affinity constant of 2.5 x 10(6) M-1. The other was supposed to be a low affinity site of Ca binding. Mild trypsin treatment reduced the Ca binding capacity of intestinal myosin B by 1.45-2.44 nmol/mg protein. These values are approximately the concentration of the high affinity Ca binding sites in the intestinal myosin B. A major concern regarding the effect of trypsin is that the reduction of Ca binding surely accompanied the elimination of Ca sensitivity of myosin B ATPase activity. From these results, it seems likely that the high affinity sites of Ca binding identified in this study are based on the troponin-like component included in intestinal myosin B.     

Effect of procaine on potassium permeability of canine tracheal smooth muscle

Procaine depolarized the cell membrane, and initiated oscillations and spike-like potentials in canine tracheal smooth muscle, at concentrations between 1 and 5 mM, while higher concentrations of this drug suppressed the spontaneous activities. Inhibitory effects of procaine on⁸⁶Rb-efflux were not evident in normal solution. In high K solution, procaine decreased the rate of⁸⁶Rb-efflux, in a dose dependent manner, and a Scatchard plot suggested two sites of action for procaine. The site with a higher affinity (KD=0.26 mM) may directly regulate the K permeability and interact with procaine in a one to one manner. In high concentrations (10 mM), procaine interacted at another site and the tracheal smooth muscle which contracted in high K solution relaxed almost completely. Thus, the decrease in⁸⁶Rb-efflux by high concentrations of procaine may partly result from a decrease in intracellular Ca concentration. It is proposed that procaine has the dual effect by inhibiting the K conductance: (1) depolarization of cell membrane, (2) increase in membrane excitability. These actions would explain the spontaneous electrical activity induced by procaine in canine tracheal smooth muscle.     

Stimulation of albumin endocytosis by cationized ferritin in cultured aortic smooth muscle cells.

Anionic microdomains within the aortic smooth muscle cell (SMC) surface glycocalyx represent a potential barrier to the endocytosis of anionic plasma proteins. Cultured SMCs exposed briefly to cationized ferritin (CF) exhibit ultrastructural aggregations of surface anionic sites resulting in intervening areas essentially devoid of anionic charge. Preincubation of cultured aortic medial SMCs with 0.2 mg/ml CF for 1 minute at 37 C resulted in a 4-fold increase in binding and a 13-fold increase in internalization of 125I-human serum albumin (125I-HSA) relative to cells pretreated with native ferritin. When both the CF preincubation and the endocytosis were performed at 4 C, the influence of CF was abolished. Studies at 4 C indicated that CF pretreatment of SMC at 37 C induced high affinity (Kd = 1.5 nM) saturable 125I-HSA binding, in addition to low-affinity nonsaturable binding. These results were further confirmed by binding competition studies using increasing concentrations of unlabeled HSA. In contrast, low-density lipoprotein, a large anionic molecule, failed to compete with 125I-HSA for binding sites on CF-pretreated SMCs at either 4 or 37 C. Pulse-chase studies at 37 C indicated that 20-30% of internalized 125I-HSA was degraded, and 40-50% exocytosed within 24 hours in CF-treated cells. CF pretreatment of the SMCs did not significantly enhance the uptake of 14C-sucrose as a measure of fluid-phase endocytosis at 30 and 60 minutes. The results of these studies emphasize the potentially important regulatory roles of cell-surface anionic charge distribution and cationic molecules in cellular endocytosis.     

Stimulation of vascular smooth muscle cell migration by macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is a well known proinflammatory factor that influences the migration and proliferation of various cell types, predominantly monocytes and macrophages. Recent evidence suggests an important role for MIF in the progression of atherosclerosis and restenosis. For this reason, we studied the effect of MIF on platelet-derived growth factor-BB (PDGF-BB)-induced migration and PDGF receptor protein expression in vascular smooth muscle cells (VSMCs). Furthermore, the possibility of MIF influencing the migration of VSMCs was investigated. Our results show that short-term incubation of MIF is able to enhance PDGF-BB-induced migration. Long-term incubation decreases PDGF-BB-induced migration, but preserves a short-term stimulatory effect. These effects are not regulated at the level of PDGF receptor protein expression. MIF also acts as a chemoattractant for VSMCs, with a maximum response at 15 ng/ml. In contrast, the proliferation of VSMCs was unaffected by MIF. We conclude that MIF has a biphasic effect on VSMC migration. It remains unclear whether this effect is direct or involves the secretion of unidentified promigratory factors. Exogenous MIF does not stimulate VSMC proliferation; however, a role for MIF in proliferation cannot be fully ruled out. In view of the known key contributions of macrophage-derived MIF and VSMCs, the observed effects may well play a role in the progression of atherosclerosis and restenosis.     

Electrogenic sodium pumping in rabbit small intestinal smooth muscle.

Oscillations in the activity of an electrogenic Na pump has been suggested as the ionic mechanism underlying the intestinal control potential (slow wave). We investigated the electrogenicity of this pump in rabbit jejunal smooth muscle. Potassium admission to Na-rich tissues caused a large increase in membrane potential which after 10--20 min decreased toward values comparable with those of normal tissues. This hyperpolarization far exceeded EK and could be prevented by cooling or by ouabain. No hyperpolarization occurred upon K admission to Li-rich tissues in the absence of Na. Thus, the pump in this tissue can operate electrogenically. Goldman's equation was modified so as to account for the pump's contribution to the membrane potential. Using this equation, the calculated contribution of the pump, under normal "steady-state" conditions, is unlikely to exceed a few millivolts. It is concluded that although the pump in this tissue can be electrogenic, its contribution may be smaller than that required if the intestinal control potential resulted from rhythmic turning off and on of the electrogenic Na pump.     

Electrical activity of small intestinal smooth muscle and its temperature dependence.

Some important features of the intracellularly recorded electrical control activity of rabbit jejunal smooth muscle and its temperature dependence are reported in this study. This activity consisted of repetitive 18-mV depolarizations (control potentials (CP) or slow waves), which at 37degreesC lasted 2 s and had a frequency of 18/min and arose from a membrane potential of --55 mV. In some cells periods between CP's exhibited "diastolic" progressive depolarizations (intercontrol-potential depolarization), which may be the trigger of the CP in driving cells. While CP was usually monophasic, some cells persistently exhibited a notch early in the plateau phase. We suggest that CP consists of two components, an "initial depolarization" and a "secondary depolarization," which are usually fused together to give a monophasic potential. Cooling reduced CP frequency and prolonged its duration and caused more cells to show notching. While amplitude and rate of CP initial depolarization had low Q10's, duration and rates of onset and offset of the secondary depolarization had higher Q10's. Thus, the process responsible for secondary depolarization is more sensitive to temperature thant that underlying initial depolarization of the CP.     

Collagen production by human smooth muscle cells isolated during intestinal organogenesis

Summary The extracellular matrix influences organogenesis by modulating cell behavior. In humans, collagen is the major matrix constituent of the adult intestinal wall and is synthesized by smooth muscle cells. The objective of the current study was to examine collagen production by fetal human intestinal smooth muscle cells isolated during intestinal morphogenesis. Techniques were developed for the isolation and culture of human fetal intestinal smooth muscle cells. The cultured cells were confirmed as muscle by immunohistochemical stains for cytoskeletal filaments and documentation of contractile behavior. In culture, these cells stained for mesenchymal and muscle cytoskeletal proteins: vimentin, actin, and desmin, and did not stain for neural or epithelial markers. The muscle cells contracted in response to acetylcholine, in contrast to human fetal dermal fibroblasts which did not contract appreciably. Collagen production was assayed by the uptake of [³H]-proline into collagenase-digestible protein. Collagen production was greatest at 11 weeks gestation, the youngest age studied. By 20 weeks gestation, collagen production had decreased to adult levels. However, when compared to another matrix-producing fetal mesenchymal cell, the dermal fibroblast, intestinal smooth muscle cells produced twice as much collagen. Collagen types were determined by polyacrylamide slab gel electrophoresis. Smooth muscle cells predominantly produced types I and III collagen chains. Therefore, collagen production is a significant function of human fetal intestinal smooth muscle cells, and probably plays a major role in the development of intestinal structure. The in vitro model presented here provides a means of studying the regulation of this collagen production throughout intestinal organogenesis.     

Block of quantal end-plate currents of mouse muscle by physostigmine and procaine.

of quantal end-plate currents of mouse muscle by physostigmine and procaine. Quantal endplate currents (qEPCs) were recorded from hemidiaphragms of mice by means of a macro-patch-clamp electrode. Excitation was blocked with tetrodotoxin, and quantal release was elicited by depolarizing pulses through the electrode. Physostigmine (Phys) or procaine (Proc) was applied to the recording site by perfusion of the electrode tip. Low concentrations of Phys increased the amplitude and prolonged the decay time constants of qEPCs from approximately 3 to approximately 10 ms, due to block of acetylcholine-esterase. With 20 microM to 2 mM Phys or Proc, the decay of qEPCs became biphasic, an initial short time constant taus decreasing to <1 ms with 1 mM Phys and to approximately 0.3 ms with 1 mM Proc. The long second time constant of the decay, taul, reached values of 相似文献   

A study of pace-maker activity in intestinal smooth muscle

1. Electrical activity of longitudinal muscle from cat intestine was recorded in the double sucrose gap.2. Approximately 20% of the preparations demonstrated slow, spontaneous fluctuations of membrane voltage, slow waves. This activity, although quite uniform in a given preparation, showed considerable inter-preparation variation with respect to amplitude, frequency and wave form.3. Application of steady hyperpolarizing current decreased slow-wave frequency and increased slow-wave amplitude while depolarizing currents increased frequency and decreased amplitude.4. Some preparations with no spontaneous slow-wave activity developed slow waves when the membrane was hyperpolarized into a given range which, depending on the preparation, varied in size from 10 to 40 mV. Step or ramp depolarization of the membrane from hyperpolarized levels triggered slow waves in some preparations.5. When the membrane potential of a slow-wave generating preparation was clamped at the resting potential, spontaneous inward-directed current transients were observed.6. No changes in membrane conductance were observed during the course of a slow wave.7. The slow-wave pattern was simulated for individual preparations by applying the membrane current measured under voltage clamp to the passive membrane resistance and capacitance measured independently under current clamp.8. In addition to the defined slow-wave activity, voltage-dependent oscillations in membrane potential were sometimes observed.9. Application of 10(-5)M ouabain irreversibly blocked slow waves and produced a membrane depolarization equal to or slightly greater than the slow wave crest. Repolarization of the membrane to the resting potential, or hyperpolarization, failed to restore slow-wave activity.10. Removal of external potassium produced a reversible sequence of events almost identical to those following ouabain application.11. Replacement of 50% of the external sodium chloride with sucrose produced no changes in slow-wave activity with respect to rates of rise or fall, maximum amplitude or frequency. Sucrose replacement of all external sodium chloride eliminated slow waves after 5 min; however, activity could be restored by a slight hyperpolarization. Longer exposures to the modified bath abolished activity.12. Following a conditioning exposure to potassium-free Krebs solution, readmission of potassium at normal concentration produced a mean hyperpolarization of 20.5 mV and in spontaneous preparations an arrest of activity.13. Pump current in sodium-loaded, non-spontaneously active preparations was measured by voltage clamp and was observed to be voltage-dependent.14. The results of this study indicate that an electrogenic pump is present in longitudinal muscle of cat duodenum, and that oscillations in the level of pump current produce slow waves.     

Stimulation of45Ca efflux from smooth muscle cells by metabolic inhibition and high K depolarization

Summary The characteristics of the extracellular and cellular calcium exchange in taenia coli have been studied by efflux experiments under different experimental conditions. The exchange of extracellularly bound calcium is accelerated by the presence of calcium in the external solution. If a Ca-free solution is used as washing solution, the slowly exchanging extracellular calcium also contributes appreciably to the later phase of the Ca efflux and obscures the changes of the cellular calcium exchange. There is no evidence for a Ca–Ca exchange diffusion.Most of the⁴⁵Ca bound at extracellular binding sites can be released by a 10 min exposure to 2 mM EGTA or to 10 mM La³⁺. This La concentration moreover largely inhibits the release of⁴⁵Ca from the cellular compartment by metabolic depletion. A release of cellular⁴⁵Ca can be induced by metabolic depletion or by K depolarization. Both procedures probably act at the same sequestering sites. However, while DNP+IAAa cts in the absence of external Ca, it is observed that K depolarization can only cause a Ca release if external Ca can enter the cells.This work was supported by research grant 20.487 from the F.W.G.O. (Belgium).Established investigator of the American Heart Association