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PubMed2000

Implications of mechanical stretch on wound repair of gastric smooth muscle cells in vitro.

Gastric smooth muscle cells continually receive repetitive physical stretching by food storage, peristalsis and fasting contraction; therefore mechanical stretch can not be disregarded in gastric events. The aim of this study was to clarify the effects of mechanical stretch on wound repair using a rabbit gastric smooth muscle cell sheet. Mechanical stretch was imposed on adherent cells on a flexible membrane in order to increase elongation by an average of 5% and 10%, respectively, at 5 cycles per minute after artificial wounding. Adherent cells not subjected to mechanical stretch served as controls. The restoration process was monitored by measuring wound size for 48 h. Proliferation was assessed by BrdU staining and the influence on the cytoskeletal system was assessed by actin staining. The speed of restoration was highest in controls and lowest in the 10% stretch groups. Proliferation was almost equal to that of controls in the stretch groups. Under the condition of mechanical stretch, stress fibers appeared weakened and the direction of fibers was not consistent but random. In conclusion, mechanical stretch inhibited the migration of gastric smooth muscle cells, leading to cytoskeletal dysfunction. It is suggested that physical stretch, such as mechanical stretch, might be an important factor in the process of gastric wound repair.

Animals↗
PubMed2005

Remodelling of airway smooth muscle in asthma: what sort do you have?

BACKGROUND: Shortening of smooth muscle around airways is the basis of symptoms and abnormal lung function in asthma. The airway wall is increased in thickness in small and large airways asthma, in relation to the clinical severity of asthma and the airway smooth muscle layer is the main contributor to this thickening. The relative contributions of more airway smooth muscle cells, bigger cells or more extracellular matrix to the increased thickness of the smooth muscle layer in asthma is not clear and has been examined in only a small number of cases, Studies of the natural history of asthma suggest that the clinical severity of asthma is relatively constant over time, deficits in lung function compared with nonasthmatic subjects occur early in the course of the disease and the decline in lung function with age is increased in asthma. HYPOTHESIS: The observations from studies of the quantitative pathology and the natural history of asthma might be combined in the hypothesis that the severity of asthma is determined early (in its natural history) and is related mainly to increased volume density of airways smooth muscle cells (hyperplasia) and that later deposition of extracellular matrix from larger, hypertrophic smooth muscle cells results in fixed and increasing deficits in lung function. SPECULATION: The relative contribution of more smooth muscle cells, bigger cells and extracellular matrix will be determined by unbiased stereological measurements in many cases of asthma of varying severity. The outcomes of such studies will be methods of monitoring and of treatment that will be tailored to the sort of smooth muscle modelling that is present in individual cases.

Asthma↗
PubMed1990

Phosphorylation of myosin as a regulatory mechanism in smooth muscle.

Contractile activity of the smooth muscle cell is regulated by the concentration of intracellular Ca2+. The Ca2+ transients are sensed by the target protein, calmodulin, and via activation of myosin light chain kinase (by Ca2(+)-calmodulin) transmitted to the contractile apparatus. Phosphorylation of myosin increases its actin-activated ATPase activity and in smooth muscle fibers is thought to initiate contraction. The effects of phosphorylation on the conformation of myosin are not established, but at least two areas of the molecule are influenced by phosphorylation of the two light chains. These are at the actin-binding site and at the head-neck junction. The latter site is important in regulating ATPase activity and a working hypothesis is that phosphorylation increases flexibility at this site and facilitates cross-bridge cycling. The phosphorylation theory has extensive experimental support, and is accepted as a major regulatory component in smooth muscle. However, the simplest interpretation of this scheme cannot adequately account for the varied physiological responses. Either there are aspects of the phosphorylation theory that are not considered, or an additional regulatory mechanism is involved. Both possibilities are discussed.

Animals↗
PubMed1977

Studies on isolated smooth muscle cells: The contractile apparatus.

Smooth muscle cells may be isolated from the taenia coli muscle of the guinea pig which, when made permeable by treatment with Triton X-100 (0-05%) show a sensitivity to Ca for contraction with MgATP. The rate of contraction, about 10 micron s-1, corresponds closely to the maximum velocity of shortening of the intact muscle. Electron microscopy of such partially demembranated muscle cells shows that myosin filaments of about 16-nm diameter are present in both the rigor and the relaxes states. In addition, the actin and myosin filaments are commonly seen to be associated in groups corresponding approximately in size to the fibrils recognizable in cells in rigor in the light microscope. The dense bodies and the 10-nm filaments are found located between the actin-myosin filament groups. The thick myosin filaments may be isolated by fragmentation of the cells under relaxing conditions. These native filaments range up to about 8 micron in length and show the same structural organization as filaments aseembled from purified smooth muscle myosin: there is no central bare zone and bare edges, about 0-2 micrin long, occur at the filament ends. The lack of bipolarity of the native smooth muscle muosin filaments and the absence, in the contractile apparatus, of actin-associated structures equivalent to Z-lines suggests that the amount of shearing that can occur between the actin and myosin filaments is considerably greater than in skeletal muscle.

Animals↗
PubMed2005

'Sarcomeres' of smooth muscle: functional characteristics and ultrastructural evidence.

Smooth muscle cells line the walls of hollow organs and control the organ dimension and mechanical function by generating force and changing length. Although significant progress has been made in our understanding of the molecular mechanism of actomyosin interaction that produces sliding of actin (thin) and myosin (thick) filaments in smooth muscle, the sarcomeric structure akin to that in striated muscle, which allows the sliding of contractile filaments to be translated into cell shortening has yet to be elucidated. Here we show evidence from porcine airway smooth muscle that supports a model of malleable sarcomeric structure composed of contractile units assembled in series and in parallel. The geometric organization of the basic building blocks (contractile units) within the assembly and the dimension of individual contractile units can be altered when the muscle cells adapt to different lengths. These structural alterations can account for the different length-force relationships of the muscle obtained at different adapted cell lengths. The structural malleability necessary for length adaptation precludes formation of a permanent filament lattice and explains the lack of aligned filament arrays in registers, which also explains why smooth muscle is 'smooth'.

Actins↗
PubMed1992

Bradykinin B2 receptor-mediated phosphoinositide hydrolysis in bovine cultured tracheal smooth muscle cells.

1. Bovine tracheal smooth muscle cells were established in culture to study agonist-induced phosphoinositide (PI) hydrolysis in this tissue. 2. Bradykinin (0.1 nM-10 microM) evoked a concentration-dependent increase (log EC50 (M) = -9.4 +/- 0.2; n = 8) in the accumulation of total [3H]-inositol phosphates in cultured tracheal smooth muscle cells whereas the selective B1 receptor agonist des-Arg9-bradykinin (10 microM) was significantly less effective (16% of bradykinin maximal response; relative potency = 0.2 with respect to bradykinin = 100). 3. The bradykinin-induced increase in PI hydrolysis was unaffected by the B1 receptor antagonist des-Arg9[Leu8]-bradykinin (1 nM-1 microM) but showed marked attenuation in the presence of the B2 receptor antagonists D-Arg,[Hyp3,D-Phe7]-bradykinin (10 nM-10 microM) or D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin (10 nM-10 microM). The estimated KB values obtained for these two compounds, assuming competitive antagonism, were 40 +/- 14 nM and 8.6 +/- 2.8 nM for D-Arg,[Hyp3,D-Phe7]-bradykinin and D-Arg[Hyp3,Thi5,8,D-Phe7]-bradykinin respectively. 4. We conclude that bradykinin B2 receptors are expressed in cultured bovine tracheal smooth muscle cells and are coupled to PI hydrolysis mechanisms.

Animals↗
PubMed1996

Myosin filament structure in vertebrate smooth muscle.

The in vivo structure of the myosin filaments in vertebrate smooth muscle is unknown. Evidence from purified smooth muscle myosin and from some studies of intact smooth muscle suggests that they may have a nonhelical, side-polar arrangement of crossbridges. However, the bipolar, helical structure characteristic of myosin filaments in striated muscle has not been disproved for smooth muscle. We have used EM to investigate this question in a functionally diverse group of smooth muscles (from the vascular, gastrointestinal, reproductive, and visual systems) from mammalian, amphibian, and avian species. Intact muscle under physiological conditions, rapidly frozen and then freeze substituted, shows many myosin filaments with a square backbone in transverse profile. Transverse sections of fixed, chemically skinned muscles also show square backbones and, in addition, reveal projections (crossbridges) on only two opposite sides of the square. Filaments gently isolated from skinned smooth muscles and observed by negative staining show crossbridges with a 14.5-nm repeat projecting in opposite directions on opposite sides of the filament. Such filaments subjected to low ionic strength conditions show bare filament ends and an antiparallel arrangement of myosin tails along the length of the filament. All of these observations are consistent with a side-polar structure and argue against a bipolar, helical crossbridge arrangement. We conclude that myosin filaments in all smooth muscles, regardless of function, are likely to be side-polar. Such a structure could be an important factor in the ability of smooth muscles to contract by large amounts.

Animals↗
PubMed2005

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

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

ADP-ribosyl Cyclase↗
PubMed2004

Effects of volatile anesthetics on store-operated Ca(2+) influx in airway smooth muscle.

BACKGROUND: In airway smooth muscle (ASM), volatile anesthetics deplete sarcoplasmic reticulum (SR) Ca(2+) stores by increasing Ca(2+) "leak." Accordingly, SR replenishment becomes dependent on Ca(2+) influx. Depletion of SR Ca(2+) stores triggers Ca(2+) influx via specific plasma membrane channels, store-operated Ca(2+) channels (SOCC). We hypothesized that anesthetics inhibit SOCC triggered by increased SR Ca(2+) "leak," preventing SR replenishment and enhancing ASM relaxation. METHODS: In porcine ASM cells, SR Ca was depleted by cyclopiazonic acid or caffeine in 0 extracellular Ca(2+), nifedipine and KCl (preventing Ca(2+) influx through L-type and SOCC channels). Extracellular Ca(2+) was rapidly introduced to selectively activate SOCC. After SOCC activation, SR was replenished and the protocol repeated in the presence of 1 or 2 minimum alveolar concentration halothane, isoflurane, or sevoflurane. In other cells, characteristics of SOCC and interactions between acetylcholine (Ach) and volatile anesthetics were examined. RESULTS: Cyclopiazonic acid produced slow SR leak, whereas the caffeine response was transient in ASM cells. Reintroduction of extracellular Ca(2+) rapidly increased [Ca(2+)]i. This influx was insensitive to nifedipine, SKF-96365, and KBR-7943, inhibited by Ni and blockade of inositol 1,4,5-triphosphate-induced SR Ca(2+) release, and enhanced by ACh. Preexposure to 1 or 2 minimum alveolar concentration halothane completely inhibited Ca(2+) influx when extracellular Ca(2+) was reintroduced, whereas isoflurane and sevoflurane produced less inhibition. Only halothane and isoflurane inhibited ACh-induced augmentation of Ca(2+) influx. CONCLUSION: Volatile anesthetics inhibit a Ni/La-sensitive store-operated Ca(2+) influx mechanism in porcine ASM cells, which likely helps maintain anesthetic-induced bronchodilation.

Acetylcholine↗
PubMed2005

pH effects on high conductance Ca2+-activated K+ channels (BK(Ca)) in human internal mammary artery smooth muscle cells.

INTRODUCTION: In vascular smooth muscle cells, different types of K+ channels participate in the regulation of membrane potential and consequently in the contractile behavior of the vessel. There is little information about the properties and role of K+ channels in human internal mammary artery (HIMA), the vessel of choice for coronary revascularization. METHODS: Patch-clamp technique on isolated HIMA smooth muscle cells was used. RESULTS: This work presents for the first time single-channel properties of the high conductance Ca2+-activated K+ channel (BK(Ca)) of HIMA. It presents a single-channel conductance of 228+/-4 pS (n=44, 8 cells), is sensitive to 100 nM iberiotoxin, and its open probability is Ca2+- and voltage-dependent. Inside-out results show that BK(Ca) channels in HIMA are directly activated by increasing the pH of intracellular media (NPo=0.096+/-0.032 at pH 7.4 and NPo=0.459+/-0.111 at pH 7.6, n=12 cells, p<0.05) and inhibited by lowering this pH (NPo=0.175+/-0.067 at pH 7.4 and NPo=0.051+/-0.019 at pH 6.8, n=13 cells, p<0.05). CONCLUSIONS: The evidences presented about single-channel properties and intracellular pH sensitivity of BK(Ca) from HIMA smooth muscle cells provide useful information to elucidate physiological or pathological mechanisms in this vessel, as well as for future studies where drugs could have BK(Ca) channels as targets for pharmacological therapies.

Analysis of Variance↗
PubMed1998

Involvement of small GTPases in the regulation of smooth muscle contraction.

Neurohumoral stimulation of smooth muscle leads to an increased responsiveness of the myofilaments to Ca2+. This review provides a summary of the data that suggest that the signalling from the membrane-bound serpentine receptors to the contractile apparatus leading to the increase in Ca(2+)-sensitivity requires the activation of the Ras-related low molecular mass GTPase Rho. In smooth muscle permeabilized with alpha-toxin or beta-escin, the increase in force elicited by different agonists at fixed [Ca2+] (Ca(2+)-sensitization) can be inhibited by bacterial toxins (EDIN, and exoenzyme C3) which ADP-ribosylate and inactivate Rho proteins. Moreover, the agonist-induced increase in Ca(2+)-sensitivity can be mimicked by constitutively active recombinant Rho proteins. The physiological relevance of this mechanism is suggested by the fact that toxins that are internalized into intact cells (toxin B from C. difficile and a chimeric toxin (DC3B) consisting of C3 and the (non-catalytic) B fragment of diphteria toxin (inhibit the tonic phase of an agonist-induced contraction. Toxin B inhibits contraction without affecting the intracellular Ca(2+)-transient determined with fura-2. However, it inhibits phosphorylation of the regulatory light chains of myosin (MLC). Rho has been suggested to activate a Rho-associated kinase which in turn phosphorylates the myosin binding subunit of the myosin light chain phosphatase. This would lead to an increase in phosphorylation of MLC and hence of force at constant Ca2+. The Ca(2+)-sensitizing effect of agonists is also inhibited by tyrosine kinase inhibitors. This suggests the possibility that in smooth muscle, like in non-muscle cells, there is a cross-talk between Rho and tyrosine kinases.

Actin Cytoskeleton↗
PubMed2005

Functional and molecular analysis of L-type calcium channels in human esophagus and lower esophageal sphincter smooth muscle.

Excitation of human esophageal smooth muscle involves the release of Ca(2+) from intracellular stores and influx. The lower esophageal sphincter (LES) shows the distinctive property of tonic contraction; however, the mechanisms by which this is maintained are incompletely understood. We examined Ca(2+) channels in human esophageal muscle and investigated their contribution to LES tone. Functional effects were examined with tension recordings, currents were recorded with patch-clamp electrophysiology, channel expression was explored by RT-PCR, and intracellular Ca(2+) concentration was monitored by fura-2 fluorescence. LES muscle strips developed tone that was abolished by the removal of extracellular Ca(2+) and reduced by the application of the L-type Ca(2+) channel blocker nifedipine (to 13 +/- 6% of control) but was unaffected by the inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). Carbachol increased tension above basal tone, and this effect was attenuated by treatment with CPA and nifedipine. Voltage-dependent inward currents were studied using patch-clamp techniques and dissociated cells. Similar inward currents were observed in esophageal body (EB) and LES smooth muscle cells. The inward currents in both tissues were blocked by nifedipine, enhanced by Bay K8644, and transiently suppressed by acetylcholine. The molecular form of the Ca(2+) channel was explored using RT-PCR, and similar splice variant combinations of the pore-forming alpha(1C)-subunit were identified in EB and LES. This is the first characterization of Ca(2+) channels in human esophageal smooth muscle, and we establish that L-type Ca(2+) channels play a critical role in maintaining LES tone.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Initial biochemical and functional characterization of cyclic nucleotide phosphodiesterase isozymes in canine colonic smooth muscle.

Cyclic nucleotides mediate relaxation of gastrointestinal smooth muscle. The intracellular concentration of these second messengers is determined by a balance between their synthesis and metabolism. Because cyclic nucleotide phosphodiesterase enzymes (PDE) are the sole enzymes responsible for their degradation, it is essential to determine the role of the various PDE isozymes in regulating cyclic nucleotide content of gastrointestinal smooth muscle. To examine the role of different PDE isozymes in colonic smooth muscle motility, soluble PDE activity was measured in fractions obtained from homogenates of canine colon using DEAE sepharose chromatography. PDE activity was determined using [3H]cyclic AMP (cAMP) (1 microM) or [3H]cyclic GMP (cGMP) (1 microM) as a substrate. Results indicated that colonic smooth muscle contains at least two forms of PDE with a high affinity for cGMP. One form was stimulated by calmodulin (type I) and the other was inhibited by low concentrations of zaprinast (type V). In addition, colonic smooth muscle contains at least two isozymes that prefer cAMP as a substrate. One form was inhibited by SB 94120 and cGMP (type III) and the other by rolipram (type IV). An additional peak of PDE activity was identified. The hydrolysis of cAMP by this peak was greatly enhanced by the presence of cGMP, suggesting that this activity belonged to type II or cGMP-stimulated PDE. The functional role of these isozymes was evaluated by determining the ability of selective PDE inhibitors to antagonize a carbachol (0.3 microM)-induced contraction of isolated circular colonic muscle strips in the presence of forskolin (0.2 microM). Concentration-dependent decreases in contractile activity were observed with the following potency order: rolipram > Ro 20-1724 > isobutyl methylxanthine > SB 94120 > zaprinast. These results demonstrate that colonic smooth muscle contains several PDE isozymes and that selective inhibition of PDE isozymes can increase cyclic nucleotide content and antagonize contractile responses. Functionally, PDE IV appears to be very important in reducing contractile activity, suggesting that selective PDE IV inhibitors might be useful in the treatment of gut hypermotility disorders.

1-Methyl-3-isobutylxanthine↗
PubMed1986

A dynamic model of smooth muscle contraction.

A dynamic model of smooth muscle contraction is presented and is compared with the mechanical properties of vascular smooth muscle in the rat portal vein. The model is based on the sliding filament theory and the assumption that force is produced by cross-bridges extending from the myosin to the actin filaments. Thus, the fundamental aspects of the model are also potentially applicable to skeletal muscle. The main concept of the model is that the transfer of energy via the cross-bridges can be described as a 'friction clutch' mechanism. It is shown that a mathematical formulation of this concept gives rise to a model that agrees well with experimental observations on smooth muscle mechanics under isotonic as well as isometric conditions. It is noted that the model, without any ad hoc assumptions, displays a nonhyperbolic force-velocity relationship in its high-force portion and that it is able to maintain isometric force in conditions of reduced maximum contraction velocity. Both these findings are consistent with new experimental observations on smooth muscle mechanics cannot be accounted for by the classical Hill model.

Animals↗
PubMed2004

Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation.

BACKGROUND: Airway smooth muscle proliferation plays an important role in airway remodeling in asthma. But little is known about the intracellular signal pathway in the airway smooth muscle cell proliferation in asthma. The objective of this paper is to investigate the contribution of protein kinase C (PKC) and its alpha isoform to passively sensitized human airway smooth muscle cells (HASMCs) proliferation. METHODS: HASMCs in culture were passively sensitized with 10% serum from asthmatic patients, with non-asthmatic human serum treated HASMCs used as the control. The proliferation of HASMCs was examined by cell cycle analysis, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining. The effect of PKC agonist phorbol 12-myristate 13-acetate (PMA) and PKC inhibitor Ro-31-8220 on the proliferation of HASMCs exposed to human asthmatic serum and non-asthmatic control serum was also examined by the same methods. The protein and mRNA expression of PKC-alpha in passively sensitized HASMCs were detected by immunofluorescence staining and reverse transcription-polymerase chain reaction. RESULTS: The percentage of S phase, absorbance (value A) and the positive percentage of PCNA protein expression in HASMCs passively sensitized with asthmatic serum were (16.30 +/- 2.68)%, 0.430 +/- 0.060 and (63.4 +/- 7.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [(10.01 +/- 1.38)%, 0.328 +/- 0.034 and (37.2 +/- 4.8)%, respectively] (P < 0.05). After HASMCs were passively sensitized with asthmatic serum, they were treated with PMA, the percentage of S phase, value A and the positive percentage of PCNA protein expression were (20.33 +/- 3.39)%, 0.542 +/- 0.065 and (76.0 +/- 8.7)% respectively, which were significantly increased compared with asthmatic serum sensitized HASMCs without PMA(P < 0.05). After HASMCs passively sensitized with asthmatic serum were treated with Ro-31-8220, the percentage of S phase, value A and the positive percentage of PCNA protein expression were (11.21 +/- 1.56)%, 0.331 +/- 0.047 and (38.8 +/- 6.0)% respectively, which were significantly decreased compared with asthmatic serum sensitized HASMCs without Ro-31-8220 (P < 0.05). The relative ratio of value A of PKC-alpha mRNA and the positive percentage of PKC-alpha protein expression in passively sensitized HASMCs were 1.23 +/- 0.10 and (61.1 +/- 9.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [1.05 +/- 0.09 and (34.9 +/- 6.7)%, respectively] (P < 0.05). CONCLUSIONS: The proliferation of HASMCs passively sensitized with human asthmatic serum is increased. PKC and its alpha isoform may contribute to this proliferation.

Asthma↗
PubMed2004

Nucleotide dependent intrinsic fluorescence changes of W29 and W36 in smooth muscle myosin.

The intrinsic fluorescence of smooth muscle myosin is sensitive to both nucleotide binding and hydrolysis. We have examined this relationship by making MDE mutants containing a single tryptophan residue at each of the seven positions found in the wild-type molecule. Previously, we have demonstrated that a conserved tryptophan residue (W512) is a major contributor to nucleotide-dependent changes of intrinsic fluorescence in smooth muscle myosin. In this study, an MDE containing all the endogenous tryptophans except W512 (W512 KO-MDE) decreases in intrinsic fluorescence upon nucleotide binding, demonstrating that the intrinsic fluorescence enhancement of smooth muscle myosin is not solely due to W512. Candidates for the observed quench of intrinsic fluorescence in W512 KO-MDE include W29 and W36. Whereas the intrinsic fluorescence of W36-MDE is only slightly sensitive to nucleotide binding, that of W29-MDE is paradoxically both quenched and blue-shifted upon nucleotide binding. Steady-state and time-resolved experiments suggest that fluorescence intensity changes of W29 involve both excited-state and ground-state quenching mechanisms. These results have important implications for the role of the N-terminal domain (residues 1-76) in smooth muscle myosin in the molecular mechanism of muscle contraction.

Actins↗
PubMed1976

Studies on the composition of phospholipids in rat small intestinal smooth muscle.

The phospholipid composition of rat small intestinal smooth muscle was investigated in comparison with those of the mucosa and liver. Phospholipid content per g of the wet smooth muscle was almost identical with that of the mucosa and was about 1/4 of that in the liver. The phospholipid/protein ratio of the smooth muscle was about 1/2 of the value in the liver. Sphingomyelin content was significantly high and amounted to 18% of total phospholipids. This value was about twice that in the mucosa and 4 times higher than that in the liver. On the other hand, the percent distribution of phosphatidylcholine was lowest in the smooth muscle. Distribution patterns of phosphatidylserine and phosphatidylinositol in the smooth muscle as well as in the mucosa were different from those in the liver. The occurrence of vinyl-ether and ether phospholipids was clearly demonstrated in the smooth muscle as well as in the mucosa. A major part of the ether lipids was detected in the phosphatidylethanolamine fraction, in which they amounted to about 50%; 40% as alkenyl-acyl type and 12% as alkyl-acyl type. A high content of ether lipids was also observed in the phosphatidylethanolamine fraction from mucosa, but the distribution was reversed, that is, 14% alkenyl-acyl type and 28% alkyl-acyl type. Fatty aldehydes, fatty alcohols, and fatty acids were also determined by gas-liquid chromatography. The compositions of fatty aldehydes in the phosphatidylethanolamine fraction from smooth muscle and from mucosa were similar, whereas the compositions of long chain fatty alcohol and fatty acids were clearly different. The compositions of fatty alcohols and fatty acids of the phosphatidylcholine fraction from smooth muscle showed significantly different patterns from those of the phosphatidylethanolamine fraction and from those of the same phospholipid fraction in the mucosa.

Aldehydes↗
PubMed1988

Phorbol ester increases the dihydropyridine-sensitive calcium conductance in a vascular smooth muscle cell line.

In vascular smooth muscle, phorbol esters cause a slowly developing contraction and an associated transmembrane calcium flux, both of which are inhibited by dihydropyridine calcium channel antagonists. In the A7r5 cultured vascular cell line, we used the whole-cell voltage-clamp technique to identify voltage-dependent calcium conductances and investigate the effect of phorbol esters on that conductance having characteristic dihydropyridine sensitivity (slowly inactivating, high-threshold, "L-type"). With barium as the charge carrier, large-amplitude (100-800 pA) inward currents of two types were characterized by their kinetics and voltage dependence. With holding potential--80 mV, a rapidly inactivating, low-threshold current ("T-type") was activated by depolarizations above-40 mV and was maximal at -10 mV. With holding potential -30 mV, this component was inactivated, and a second slowly inactivating, high-threshold current was activated above -10 mV and was maximal at +10 to +20 mV. These currents are similar to the T-type and L-type currents previously described in vascular smooth muscle cells. When added to the bath, the active phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (100 nM) increased the slowly inactivating (L-type) current by 32 +/- 20% (n = 8, +/- SD). Phorbol-12,13-dibutyrate (100 nM) caused a similar effect, but the inactive phorbol, 4-alpha-phorbol (100 nM), did not. We conclude that at least two distinct calcium conductances are expressed in A7r5 vascular smooth muscle cells, and that the dihydropyridine-sensitive calcium conductance is acutely modulated by phorbol esters, presumably acting through stimulation of protein kinase C. Such modulation may play a role in increasing transmembrane calcium influx mediated by agonist-receptor interactions that lead to activation of protein kinase C and may help to sustain or amplify calcium-dependent cell responses.

Animals↗