Caffeine-induced electrical responses of intact endothelial cells

The mechanism of caffeine-induced endothelial-dependent relaxation of vascular smooth muscle cells has been studied by recording caffeine application-induced electrical responses from intact guinea pig aortic endothelial cells. Depending on the values of the membrane potential, caffeine evoked either hyperpolarizing responses (V _m<−45 mV, 88.9% of the cells tested), or depolarizing reactions (V _m>−45 mV). The mean amplitude of caffeine-induced hyperpolarization of endothelial cells was 11.2±5.5 mV, which is comparable with the amplitude of ATP-induced hyperpolarization. The amplitude of caffeine-induced depolarization was 8.9±3.4 mV, on average. It was shown that caffeine-induced hyperpolarization of endothelial cells is a result of calcium release from the intracellular stores with subsequent activation of calcium-dependent potassium channels. Intracellular calcium stores involved in caffeine-induced responses are different from those involved in ATP responses. It is concluded that calcium mobilization from the intracellular stores of endothelial cells and, possibly, activation of calcium entry contributes to the caffeine-induced endothelial-dependent relaxation of vascular smooth muscle cells.     

Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells

The ciliary muscle which is involved in accommodation and regulation of aqueous humour outflow resistance resembles smooth muscle in other parts of the body. In the present investigation we used an established primary cell line (H7CM) to study the effects of endothelin, a novel vasoconstrictor peptide, on membrane voltage (V) and intracellular calcium in cultured human ciliary muscle cells. Membrane voltage was measured in confluent monolayers of H7CM cells using conventional microelectrodes. Intracellular calcium concentration [( Ca]i) was measured in single H7CM cells using the fluorescent calcium indicator fura-2. Under resting conditions V averaged -66.9 +/- 0.7 mV (mean +/- SEM, n = 125). Endothelin (10(-10)-10(-6)M) induced a dose-dependent reversible membrane voltage depolarization and a dose-dependent rise in [Ca]i. The initial calcium peak was followed by a recovery phase during which oscillations of [Ca]i occurred. The initial calcium peak was not dependent on the presence of extracellular calcium and was not abolished in the presence of the calcium antagonist verapamil (10(-4)M). Thus it is probably mediated by a release of calcium from intracellular reservoirs. We conclude that cultured human ciliary muscle cells express a functional endothelin receptor.     

Smooth Muscle Strips for Intestinal Tissue Engineering

Functionally contracting smooth muscle is an essential part of the engineered intestine that has not been replicated in vitro. The purpose of this study is to produce contracting smooth muscle in culture by maintaining the native smooth muscle organization. We employed intact smooth muscle strips and compared them to dissociated smooth muscle cells in culture for 14 days. Cells isolated by enzymatic digestion quickly lost maturity markers for smooth muscle cells and contained few enteric neural and glial cells. Cultured smooth muscle strips exhibited periodic contraction and maintained neural and glial markers. Smooth muscle strips cultured for 14 days also exhibited regular fluctuation of intracellular calcium, whereas cultured smooth muscle cells did not. After implantation in omentum for 14 days on polycaprolactone scaffolds, smooth muscle strip constructs expressed high levels of smooth muscle maturity markers as well as enteric neural and glial cells. Intact smooth muscle strips may be a useful component for engineered intestinal smooth muscle.     

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.     

Force–Velocity Curves of Motor Proteins Cooperating In Vivo

Motor proteins convert chemical energy into work, thereby generating persistent motion of cellular and subcellular objects. The velocities of motor proteins as a function of opposing loads have been previously determined in vitro for single motors. These single molecule “force–velocity curves” have been useful for elucidating motor kinetics and for estimating motor performance under physiological loads due to, for example, the cytoplasmic drag force on transported organelles. Here we report force–velocity curves for single and multiple motors measured in vivo. Using motion enhanced differential interference contrast (MEDIC) movies of living NT2 (neuron-committed teratocarcinoma) cells at 37°C, three parameters were measured—velocity (v), radius (a), and effective cytoplasmic viscosity (η′)—as they applied to moving vesicles. These parameters were combined in Stokes’ equation, F = 6πaη′v, to determine the force, F, required to transport a single intracellular particle at velocity, v. In addition, the number of active motors was inferred from the multimodal pattern seen in a normalized velocity histogram. Using this inference, the resulting in vivo force–velocity curve for a single motor agrees with previously reported in vitro single motor force–velocity curves. Interestingly, however, the curves for two and three motors lie significantly higher in both measured velocity and computed force, which suggests that motors can work cooperatively to attain higher transport forces and velocities. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Influence of low-intensity magnetic fields on the development of satellite muscle cells of a newborn rat in primary culture

The influence of Earth magnetic field shielded down to 0.3 μT and static magnetic field (60–160 μT) on the proliferation and differentiation of satellite muscle cells in primary culture has been investigated. A stimulatory effect of static magnetic fields on the rate of the formation of massive multinucleate myotubes and an increase in the intracellular calcium concentration ([Ca²⁺] _i ) have been detected for magnetic fields of the microtesla range. On the other hand, it was shown that the reduction of earth magnetic fields to 0.3 μT leads to inhibition of proliferation and differentiation of skeletal muscle cells in primary culture. Since the formation of contractile myotubes during in vitro experiments is similar to the regeneration of skeletal muscle fibers under muscle damage in vivo, it may be concluded that weak magnetic fields have a strong effect on intracellular processes by influencing all phases of muscle fiber formation. It is necessary to take this fact into consideration when forecasting probable complications of skeletal muscle regeneration during long-term exposure of man to low-intensity magnetic fields and also for the potential use of low static magnetic fields as a tool to recover the affected myogenesis.     

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.     

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.     

Biochemical markers of contraction in human myometrial smooth muscle cells in culture

Summary Phosphorylation of a light chain subunit of myosin by Ca²⁺ and calmodulin-dependent myosin light chain kinase is believed to be essential for smooth muscle contraction. The biochemical properties of the myosin phosphorylation system in human myometrial smooth muscle cells in monolayer culture were compared with those of human myometrial tissue and nonmuscle cells in culture. Native myosin was isolated from other cellular proteins of crude homogenates by polyacrylamide gel electrophoresis (in the presence of pyrophosphate) and quantified by densitometry. The myosin content of myometrial smooth muscle cells in culture and that of myometrial tissue were similar and four- to five-fold greater than that of human endometrial stromal cells or skin fibroblasts in culture. The specific activities of myosin light chain kinase in homogenates of myometrial smooth muscle cells that were maintained in culture and in myometrial tissue were similar (2.05±0.18 and 1.60±0.37 nmol phosphate incorporated per min per mg protein, respectively). On the other hand, enzyme activity in skin fibroblasts was only 5% of that in myometrial smooth muscle cells. Myosin light chain kinase activity in myometrial smooth muscle cells was dependent upon Ca²⁺ and was inhibited reversibly by the calmodulin antagonist, calmidazolium. The intracellular Ca²⁺ concentration measured by quin2 fluorescence was 0.12 μM in resting cells and increased in a concentration-dependent manner with KC1 to a maximal value of 0.47 μM. These results indicate that biochemical processes important for smooth muscle contraction are retained in human myometrial smooth muscle cells in culture. This research was supported by grants HL26043, HD11149, and GM07062 from the National Institutes of Health, Bethesda, MD.     

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.     

Calcium Movements in CGRP-treated Cultured Skeletal Muscle Cells: Is There a Role for CGRP in Tension Headaches?

It was previously determined that the site of action of calcitonin gene-related peptide (CGRP) in cardiomyocytes was predominantly at the sarcolemmal calcium release channel, and studies have shown that CGRP has major effects on intracellular cardiomyocyte calcium concentrations. We postulated that CGRP would have similar effects on striated skeletal muscle and determined the effects of CGRP on calcium levels in cultured chick myotubes by fluorescence imaging. Myoblasts were cultured until they were continuous myotubes. Deconvolution fluorescence imaging was employed to visualize subcellular organelles and construct 3D renditions. Myotubes were treated with a high (1 μM) and a low (1 nM) concentration of CGRP for 1 h or 24 h time periods, and real-time fluorescence spectrophotometry with a calcium specific fluoroprobe permitted the acquisition of images and calcium transients. Experiments also used CGRP 8–37 to ensure specificity of action of the full-length neuropeptide. CGRP localizations by image stacking were made using fluorescence deconvolution microscopy and distributions on the myotubes were shown. Myotube contractions and intracellular calcium levels were dose dependent, a high CGRP concentration producing calcium overload. CGRP 8–37 had no effect on contractions or calcium levels. Reconstructed images revealed the neuropeptide to be localized to juxta-nuclear areas, supporting the likelihood of site specific actions. CGRP has dramatic effects on intracellular calcium in striated muscle, high concentrations producing sustained contractions and calcium overload. The results give support to a mechanistic role for CGRP in muscle tension headaches, and underscore the importance in the development of CGRP analogues or receptor antagonists for treatment.     

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.     

Effects of endothelial growth media on proepicardial cell gene expression and morphogenesis in 3D collagen matrices

Proepicardial cells (PE) contribute to embryonic coronary vessel and epicardial development. Cells from the PE region can differentiate into coronary vascular smooth muscle cells and fibroblasts in vitro, but the endothelial specification capability of these cells is controversial. We sought to examine the effects of endothelial cell growth media on gene expression and the morphogenic properties of proepicardial cells in three-dimensional (3D) matrices. A primary culture of avian PE cells was subjected to molecular characterization with selected endothelial specific markers. Morphogenic properties of PE cells were assessed by in vitro assays of coronary vasculogenesis and invasion, which utilized highly defined, serum free, three-dimensional matrix conditions. PE cells maintained mixed cell population properties in the culture based on morphogenic features, immunohistochemistry, and the gene expression data. When suspended in a 3D vasculogenesis in vitro assay, PE cells formed intracellular vacuoles and assembled into multicellular tubes. Further, ultrastructural analysis revealed the presence of pinocytic vacuoles, intercellular junctions, and endothelial specific Weibel Palade bodies. In the invasion assay, PE cells spontaneously invaded control matrices. This invasion was markedly enhanced by lysophosphatidic acid (94 ± 9.6 vs. 285.6 ± 54.9, p < 0.05) and was completely blocked with synthetic broad-spectrum metalloproteinase inhibitor GM6001. Isolated PE cells grown in endothelial cell media represent mixed-cell population, characterized by both smooth muscle and endothelial gene expression. When placed in 3D in vitro assays, PE cells manifest morphogenic properties, including multicellular tube assembly and invasion.     

Charge-coupled device imaging of rapid calcium transients in cultured arterial smooth muscle cells

Transient changes in the concentration of intracellular free calcium are associated with the transduction of primary signals and the subsequent employment of Ca2+ as a second messenger in a multitude of cell types. These transients, typically monitored with the calcium-sensitive fluorescent dye Fura-2, are known to occur with a time course in the order of seconds. In order to accurately monitor such rapid changes in intracellular free calcium concentration in both single cells and simultaneously in several cells in a single field, we have developed a digital fluorescence imaging system based on a charge-coupled device (CCD) camera. We report here on the detailed kinetics of calcium increases in cultured arterial swine smooth muscle cells in response to the agonist ATP.     

Does endothelin mobilize calcium from intracellular store sites in rat aortic vascular smooth muscle cells in primary culture?

In the presence of endothelin, there was a rapid increase in the 45Ca++ efflux from primary cultured rat vascular smooth muscle cells, both in physiological salt solution and in calcium free medium containing 2 mM EGTA. The 45Ca++ influx was not affected. The endothelin-induced, transient increase in cytosolic calcium concentration is probably mainly due to release of calcium from the intracellular store in vascular smooth muscle cells.     

Calcium channels of amphibian stomach and mammalian aorta smooth muscle cells.

Whole-cell and single-channel calcium currents were studied using single smooth muscle cells enzymatically-isolated from stomach of Amphiuma tridactylum and from guinea-pig aorta. These cells have a high specific resistance and can sustain calcium action potentials after suppression of potassium currents. Dialyzed Amphiuma smooth muscle cells had calcium currents which were stable for several hours whereas the calcium currents of aortic cells ran down quickly. Single channel calcium currents in cell-attached patches behaved similarly for the two cell types. Calcium channel conductance in 110 mM barium was 12 pS and the mean open time was 1.4 ms at a nominal membrane potential of +10 mV. Exposure of both cell types to BAY K8644 resulted in a dramatic prolongation of the calcium channel open times and a shift in the probability of opening to more negative potentials. Low-threshold calcium channels were not identified in the extensively studied amphibian cells. High-threshold calcium channels therefore appear to be the primary pathway for the calcium influx that produces contraction in these smooth muscle cells.     

Effect of different zinc sources and levels on inhibition of the apoptosis induced by glucocorticoid of thymocytes in vitro

This experiment was conducted to investigate the effects of zinc sulfate and zinc methionine (Zn-Met) and their levels on apoptosis induced by glucocorticoid of thymocytes and the possible mechanism. Dexamethasone was used to make the apoptosis model of thymocytes; zinc sulfate and zinc methionine were supplemented to the medium at levels of 0,50, 100, 500, and 1000 μM. The activity of cells,Cu,Zn superoxide dismutase (Cu,Zn-SOD), DNA ladder pattern, intracellular calcium concentration, and the percentage of apoptosis nuclei were determined. Both ZnSO₄ and Zn-Met could modulate apoptosis; they inhibited apoptosis and decreased DNA fragmentation. The regulation was concentration dependent. At levels of 50 and 100 μM, the effect of Zn-Met on inhibiting apoptosis was less efficient than that of ZnSO₄ (p<0.05), but the activity of the cells cultured with Zn-Met was higher than those cultured with ZnSO₄; they showed no difference in modulating apoptosis when added at levels of 500 and 1000 μM to the medium (p>0.05). Intracellular calcium concentrations of cells cultured with Zn-Met were higher than those cultured with ZnSO₄ at the same levels. Zinc supplementation decreased the concentration of intracellular calcium significantly (p<0.05) and increased the activity of Cu,Zn-SOD in the extract of the cells (p<0.05). Both zinc sulfate and Zn-Met could modulate apoptosis of thymocytes induced by glucocorticoid; the mechanism might involve the exchange of intracellular calcium, the redox of cells, and the two forms of zinc might go different ways in the regulations.     

A method for isolating adult and neonatal airway smooth muscle cells and measuring shortening velocity

Methods are described for isolating smooth muscle cells from thetracheae of adult and neonatal sheep and measuring the single-cell shortening velocity. Isolated cells were elongated,Ca²⁺ tolerant, and contractedrapidly and substantially when exposed to cholinergic agonists, KCl,serotonin, or caffeine. Adult cells were longer and widerthan preterm cells. Mean cell length in 1.6 mMCaCl₂ was 194 ± 57 (SD) µm(n = 66) for adult cells and 93 ± 32 µm (n = 20) for preterm cells(P < 0.05). Mean cell width at thewidest point of the adult cells was 8.2 ± 1.8 µm(n = 66) and 5.2 ± 1.5 µm(n = 20) for preterm cells(P < 0.05). Cells were loaded into aperfusion dish maintained at 35°C and exposed to agonists, andcontractions were videotaped. Cell lengths were measured from 30 videoframes and plotted as a function of time. Nonlinear fitting of celllength to an exponential model gave shortening velocities faster thanmost of those reported for airway smooth muscle tissues. For a sampleof 10 adult and 10 preterm cells stimulated with 100 µM carbachol,mean (± SD) shortening velocity of the preterm cells was notdifferent from that of the adult cells (0.64 ± 0.30 vs. 0.54 ± 0.27 s¹, respectively), butpreterm cells shortened more than adult cells (68 ± 12 vs. 55 ± 11% of starting length, respectively;P < 0.05). The preparative andanalytic methods described here are widely applicable to other smoothmuscles and will allow contraction to be studied quantitatively at thesingle-cell level.

     

Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression.

cGMP is a second messenger that produces its effects by interacting with intracellular receptor proteins. In smooth muscle cells, one of the major receptors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG). PKG has been shown to catalyze the phosphorylation of a number of physiologically relevant proteins whose function it is to regulate the contractile activity of the smooth muscle cell. These include proteins that regulate free intracellular calcium levels, the cytoskeleton, and the phosphorylation state of the regulatory light chain of smooth muscle myosin. Other studies have shown that vascular smooth muscle cells (VSMCs) that are cultured in vitro may cease to express PKG and will, coincidentally, acquire a noncontractile, synthetic phenotype. The restoration of PKG expression to the synthetic phenotype VSMC results in the cells acquiring a more contractile phenotype. These more recent studies suggest that PKG controls VSMC gene expression that, in turn, regulates phenotypic modulation of the cells. Therefore, the regulation of PKG gene expression appears to be linked to phenotypic modulation of VSMC. Because several vascular disorders are related to the accumulation of synthetic, fibroproliferative VSMC in the vessel wall, it is likely that changes in the activity of the nitric oxide/cGMP/PKG pathway is involved the development of these diseases.