Phosphorylation Of Smooth Muscle Myosin Research Articles

Ardipusilloside-I stimulates gastrointestinal motility and phosphorylation of smooth muscle myosin by myosin light chain kinase.

Ardipusilloside-I is a natural triterpenoid saponin, which was isolated from Ardisia pusilla A. DC. The aim of the study was to evaluate the stimulation of ardipusilloside-I on gastrointestinal motility in vitro and in vivo. The experiment of smooth muscle contraction directly monitored the contractions of the isolated jejunal segment (IJS) in different contractile states, and the effects of ardipusilloside-I on myosin were measured in the presence of Ca2+-calmodulin using the activities of 20 kDa myosin light chain (MLC20) phosphorylation and myosin Mg2+-ATPase. The effects of ardipusilloside-I on gastro emptying and intestinal transit in constipation-predominant rats were observed, and the MLCK expression in jejuna of constipated rats was determined by western blot. The results showed that, ardipusilloside-I increased the contractility of IJS in a dose-dependent manner and reversed the low contractile state (LCS) of IJS induced by low Ca2+, adrenaline, and atropine respectively. There were synergistic effects on contractivity of IJS between ardipusilloside-I and ACh, high Ca2+, and histamine, respectively. Ardipusilloside-I could stimulate the phosphorylation of MLC20 and Mg2+-ATPase activities of Ca2+- dependent phosphorylated myosin. Ardipusilloside-I also stimulated the gastric emptying and intestinal transit in normal and constipated rats in vivo, respectively, and increased the MLCK expression in the jejuna of constipation-predominant rats. Briefly, the findings demonstrated that ardipusilloside-I could effectively excite gastrointestinal motility in vitro and in vivo.

Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle.

Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot-labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto-myosin and MLCK-myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting "stuck" on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle.

Kinetics of Myosin Light Chain Kinase Activation of Smooth Muscle Myosin in an in Vitro Model System

During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²⁺CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (<0.1-0.2 s⁻¹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.

MLCK Induces Actin Filament Bundling and Moves on Bundled Actin and Stress Fibers of Smooth Muscle Cells

We are interested in the mechanism of phosphorylation of smooth muscle myosin (SMM) by the myosin light chain kinase -calmodulin-Ca2+ complex (MLCK-CaM-Ca2+). This reaction is required for activation of SMM catalytic activity and smooth muscle contraction. We have been studying the interactions between MLCK, actin, SMM and smooth muscle stress fibers at a single molecule level using total internal reflectance fluorescence microscopy (TIRF). We observed that MLCK induces actin filament bundling. The number and length of the bundles depends upon the MLCK concentration and incubation time. MLCK also induced actin-tropomyosin complex (actin-Tm) bundling, but with longer bundle length. The N-terminal 1-75 peptide of MLCK (GST-N1-75 MLCK), which has three DFRxxL actin binding motifs does not cause actin or actin-Tm bundling, suggesting that other actin binding domains on MLCK are required for bundling. These findings may suggest that MLCK plays a role in stabilizing stress fibers in smooth muscle cells.With quantum dots labeled MLCK (QD-MLCK), we have observed that MLCK not only causes actin bundling, but moves along the actin bundles and actin-Tm bundles. In a more physiological system, skinned human airway smooth muscle cells, direct observations of single QD-MLCK molecules show clearly that MLCK co-localizes with and can move along the actin- and myosin-containing stress fibers, at high ionic strength, or at physiological ionic strength with CaM-Ca2+ and ATP. GST-N1-75 MLCK also moves along smooth muscle cell stress fibers. The diffusion coefficient, calculated from mean-squared-displacement (MSD) data from MLCK-QDs' trajectories, indicates that the mechanism by which one MLCK phosphorylates multiple SMMs may involve MLCK movement along thin and/or thick filaments on a time scale measured in seconds.

Imaging of Fluorescent ATP-Stabilized Smooth Muscle Myosin Filaments, Actin Filaments/Bundles and Single Molecules of Myosin Light Chain Kinase (MLCK) under Conditions of SMM Phosphorylation

We are using fluorescence microscopy to characterize the interaction of single active MLCK molecules with smooth muscle myosin (SMM) filaments and acto-SMM bundles in an in vitro¬ model system to determine the mechanism by which MLCK phosphorylates SMM. Since ATP is required for the SMM ATPase and the MLCK kinase activity, this requires unphosphorylated SMM filaments that are stable in the presence of ATP, a condition that normally causes disassembly. We have prepared and characterized modified SMM filaments for this study. They are 1) covalently labeled with NHS-Rhodamine for ease of imaging, 2) lightly cross-linked with EDC on the heavy chain so they are stable to ATP-induced disassembly, 3) similar to native SMM filaments in length, 4) fully regulated by phosphorylation measured by acto-SMM ATPase activity, and the ability to move actin in an in vitro motility assay, and 5) able to dissociate from actin in an [ATP]-dependent manner. Interestingly, these modified SMM filaments are capable of bundling actin filaments in the presence of ATP, calmodulin (CaM), and Ca2+; conditions that are sufficient for MLCK to phosphorylate SMM. SMM filaments preferentially bind to actin bundles rather than single actin filaments, and as ATP is titrated into a preparation of acto-SMM bundles, they dissociate leaving short actin filaments which are predominantly bound to SMM filaments. We have imaged the interaction of single quantum dot-labeled MLCK (QD-MLCK) molecules with these SMM filaments and acto-SMM bundles using total internal reflection fluorescence (TIRF) microscopy to visualize single QD-MLCK. We observed QD-MLCK colocalizing with SMM filaments and acto-SMM bundles and will characterize the QD-MLCK interaction with both proteins in the presence of ATP before and after activation of MLCK by Ca2+-CaM.

MLCK moves on Actin Filament and Stress Fiber of Smooth Muscle Cells

We are interested in the mechanism of phosphorylation of smooth muscle myosin (SMM) by the myosin light chain kinase -calmodulin-Ca2+ complex (MLCK-CaM-Ca2+). This reaction is required for activation of SMM catalytic activity and smooth muscle contraction. In previous studies we characterized tightly-bound SMM-MLCK-CaM complexes in an in vitro model system and demonstrated that SMM-MLCK-CaM complexes co-purified from smooth muscle were functional, i.e. MLCK was able to phosphorylate SMM and the phosphorylated SMM resulted in actin filament motility in an in vitro assay.

Investigation of the Myosin Light Chain Kinase Mechanism of Phosphorylation using Reconstituted Smooth Muscle Myosin Filaments

We are investigating the mechanism by which the Myosin Light Chain Kinase-Calmodulin (MLCK-CaM) complex phosphorylates Smooth Muscle Myosin (SMM). MLCK-CaM phosphorylation of SMM is the primary regulator of smooth muscle contraction, and is required for the catalytic activity of the myosin motor. We have previously shown that MLCK-CaM-SMM forms a tightly-associated complex and that MLCK-CaM co-purifies with SMM. This co-purified MLCK-CaM can phosphorylate SMM which was shown by actin filament motility in an in vitro motility assay. In addition, using total internal reflectance fluorescence (TIRF) microscopy, we were able to characterize dynamic interactions of MLCK with monomeric SMM using Quantum Dots (QD) to label MLCK.The goal of our current study is to determine how a small amount of MLCK is capable of phosphorylating a much larger amount of SMM. The ratio of MLCK to SMM in smooth muscle cells is approximately 1:70, yet the MLCK is able to produce contractions in the cell within seconds. To answer this question, we are investigating the interaction of MLCK-QD with reconstituted SMM filaments. This approach is moving more towards the in vivo conditions, and will help us answer the long term question of how MLCK is phosphorylating SMM within smooth muscle cells.We have to reconstituted SMM filaments which are identical in structure to native SMM filaments, and are capable of being phosphorylated by MLCK. Our reconstituted filaments are fluorescently labeled and are lightly cross-linked, which allows them to be stable in the presence of ATP and/or high ionic strength. We have shown that QD-MLCK co-localizes with our reconstituted SMM filaments. Our data proposes a mechanism of SMM phosphorylation by the physical movement of MLCK along a contractile fiber within the smooth muscle cell.

Update on Corpus Cavernosum Smooth Muscle Contractile Pathways in Erectile Function: A Role for Testosterone?

Normal erectile function (EF) involves a coordinated relaxation of the arteries that supply the penis and the corpus cavernosum smooth muscle (CCSM), resulting in expansion of the sinusoids and increased intracavernous pressure. But the CCSM spends the majority of its time in the contracted state which is mediated by norepinephrine released from nerve endings and other vasoconstrictors like endothelins released from the endothelium. These agents cause smooth muscle myosin (SMM) phosphorylation by elevating intracellular calcium. When calcium returns to basal levels, the calcium sensitivity increases and prevents myosin dephosphorylation, which involves the RhoA/Rho-kinase (ROK) mechanism, thus maintaining force. Although mounting evidences demonstrate that androgens have a major influence on EF that is not just centrally mediated, this notion remains quite controversial. To summarize the current knowledge on CCSM contractile pathways, the role they play in modulating EF, and the influence of androgens. The article reviews the literature and contains some previously unpublished data on CCSM contraction signaling including the role that androgens are known to play in modulating these pathways. Data from peer-reviewed publications and previously unpublished observations. In addition to downregulation of many pro-erectile molecular mechanisms, decreased testosterone (T) levels upregulate CCSM contractility, including hyperresponsiveness to α-adrenergic agonists, increased SMM phosphorylation, alteration of SMM isoform composition, activation of RhoA/ROK signaling and modulation of sphingosine-1-phosphate regulation of CCSM tone. Decreased T levels upregulate CCSM contractile signaling. Meanwhile, it downregulates CCSM relaxation pathways synergizing to produce erectile dysfunction (ED). Although some urologists and researchers are still skeptical of the influence of androgens on penile erection, understanding these molecular control mechanisms as well as the influence that androgens have on these pathways should provide new evidence supporting the roles of androgens in EF and enhance the discovery of novel targets for drug development to treat ED.

Determining Mechanism of Phosphorylation of Smooth Muscle Myosin by Calmodulin-Myosin Light Chain Kinase Using an in vitro Model System

We have shown that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM (up-SMM) from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73 ± 9), the ratio was ∼ 23-37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at ∼1 nM free [Ca2+]. There were two MLCK pools that bound up-SMM with Kd ∼10 μM and 0.2 μM and phosphorylated SMM with a Kd ∼ 20 μM and 0.2 μM. Using motility assays, co-sedimentation assays, and on-coverslip ELISA assays, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain. The bound MLCK can phosphorylate SMM in a Ca2+-dependent manner with a pCa50 ∼ 6 as measured by in vitro motility, similar to in vivo results. After activation of SMM-bound MLCK/CaM with Ca2+ and ATP, both motility (0.5 μm/sec) and phosphorylation (>15%) of SMM reach a maximum after ∼15-30 min, inconsistent with a free diffusion mechanism. Actin movement over the SMM is not required for this phosphorylation process. Experiments are underway to test the idea that SMM heads proximal to the MLCK-SMM become phosphorylated by a tethered diffusion mechanism.

Characterization of Tightly Associated Smooth Muscle Myosin–Myosin Light-Chain Kinase–Calmodulin Complexes

A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by myosin light-chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73±9), the ratio was ∼23–37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at ∼1 nM free [Ca2+]. There were two MLCK pools that bound unphosphorylated SMM with Kd∼10 and 0.2 μM and phosphorylated SMM with Kd∼20 and 0.2 μM. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK–CaM was activated by Ca2+ and phosphorylation of SMM occurred at a pCa50 of 6.1 and at a Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK–CaM–SMM. Using motility assays, co-sedimentation assays, and on-coverslip enzyme-linked immunosorbent assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin-binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.

Mammalian SEPT2 Is Required for Scaffolding Nonmuscle Myosin II and Its Kinases

Mammalian septin SEPT2 belongs to a conserved family of filamentous GTPases that are associated with actin stress fibers in interphase cells and the contractile ring in dividing cells. Although SEPT2 is essential for cytokinesis, its role in this process remains undefined. Here, we report that SEPT2 directly binds nonmuscle myosin II (myosin II), and this association is important for fully activating myosin II in interphase and dividing cells. Inhibition of the SEPT2-myosin II interaction in interphase cells results in loss of stress fibers, while in dividing cells this causes instability of the ingressed cleavage furrow and dissociation of the myosin II from the Rho-activated myosin kinases ROCK and citron kinase. We propose that SEPT2-containing filaments provide a molecular platform for myosin II and its kinases to ensure the full activation of myosin II that is necessary for the final stages of cytokinesis.

The action of PKA on smooth muscle myosin phosphorylation

The aim of the study is to reveal the characterization of PKA acting on myosin. We found: (a) in the absence of Ca 2+/CaM, PKA slightly phosphorylated MLC 20 and stimulated the Mg 2+-ATPase activity of myosin, which was strengthened significantly by arachidonic acid (ACAD); (b) Ca 2+-independent phosphorylation of myosin by PKA was obviously less efficient than both Ca 2+-dependent and independent phosphorylation of myosin by MLCK; (c) micro-amount of calponin could not increase the precipitation of myosin phosphorylated by PKA, but it increased the precipitation of myosin phosphorylated by MLCK, suggesting the presence of conformational differences between the myosins phosphorylated by PKA and by MLCK.

Commentary

commentary

Selected contribution: roles of focal adhesion kinase and paxillin in the mechanosensitive regulation of myosin phosphorylation in smooth muscle.

The increase in intracellular Ca(2+) and myosin light chain (MLC) phosphorylation in response to the contractile activation of tracheal smooth muscle is greater at longer muscle lengths (21). However, MLC phosphorylation can also be stimulated by Ca(2+)-insensitive signaling pathways (19). The cytoskeletal proteins paxillin and focal adhesion kinase (FAK) mediate a Ca(2+)-independent length-sensitive signaling pathway in tracheal smooth muscle (30). We used alpha-toxin-permeabilized tracheal smooth muscle strips to determine whether the length sensitivity of MLC phosphorylation can be regulated by a Ca(2+)-insensitive signaling pathway and whether the length sensitivity of active tension depends on the length sensitivity of myosin activation. Although active tension remained length sensitive, ACh-induced MLC phosphorylation was the same at optimal muscle length (L(o)) and 0.5 L(o) when intracellular Ca(2+) was maintained at pCa 7. MLC phosphorylation was also the same at L(o) and 0.5 L(o) in strips stimulated with 10 microM Ca(2+). In contrast, the Ca(2+)-insensitive tyrosine phosphorylation of FAK and paxillin stimulated by ACh was higher at L(o) than at 0.5 L(o). We conclude that the length-sensitivity of MLC phosphorylation depends on length-dependent changes in intracellular Ca(2+) but that length-dependent changes in MLC phosphorylation are not the primary mechanism for the length sensitivity of active tension.

Invited review: regulation of myosin phosphorylation in smooth muscle.

Phosphorylation of the regulatory light chains of myosin II (rMLC) by the Ca(2+)/calmodulin-dependent myosin light-chain kinase (MLCK) and dephosphorylation by a type 1 phosphatase (MLCP), which is targeted to myosin by a regulatory subunit (MYPT1), are the predominant mechanisms of regulation of smooth muscle tone. The activities of both enzymes are modulated by several protein kinases. MLCK is inhibited by the Ca(2+)/calmodulin-dependent protein kinase II, whereas the activity of MLCP is increased by cGMP and perhaps also cAMP-dependent protein kinases. In either case, this results in a decrease in the Ca(2+) sensitivity of rMLC phosphorylation and force production. The activity of MLCP is inhibited by Rho-associated kinase, one of the effectors of the monomeric GTPase Rho, and protein kinase C, leading to an increase in Ca(2+) sensitivity. Hence, smooth muscle tone appears to be regulated by a network of activating and inactivating intracellular signaling cascades.

Functional Role of the C-Terminal Domain of Smooth Muscle Myosin Light Chain Kinase on the Phosphorylation of Smooth Muscle Myosin

Smooth muscle myosin light chain kinase (MLCK) is known to bind to thin filaments and myosin filaments. Telokin, an independently expressed protein with an identical amino acid sequence to that of the C-terminal domain of MLCK, has been shown to bind to unphosphorylated smooth muscle myosin. Thus, the functional significance of the C-terminal domain and the molecular morphology of MLCK were examined in detail. The C-terminal domain was removed from MLCK by alpha-chymotryptic digestion, and the activity of the digested MLCK was measured using myosin or the isolated 20-kDa light chain (LC20) as a substrate. The results showed that the digestion increased K(m) for myosin 3-fold whereas it did not change the value for LC20. In addition, telokin inhibited the phosphorylation of myosin by MLCK by increasing K(m) but only slightly increased K(m) for LC20. Electron microscopy indicated that MLCK was an elongated molecule but was flexible so as to form folded conformations. MLCK was crosslinked to unphosphorylated heavy meromyosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the absence of Ca(2+)/calmodulin (CaM), and electron microscopic observation of the products revealed that the MLCK molecule bound to the head-tail junction of heavy meromyosin. These results suggest that MLCK binds to the head-tail junction of unphosphorylated myosin through its C-terminal domain, where LC20 can be promptly phosphorylated through its catalytic domain following the Ca(2+)/CaM-dependent activation.

Force relaxes before the fall of cytosolic calcium in the photomechanical response of rat sphincter pupillae.

In the rat sphincter pupillae, as in other smooth muscles, the primary signal transduction cascade for agonist activation is receptor --> G protein --> phospholipase C --> inositol trisphosphate --> intracellular Ca(2+) concentration ([Ca(2+)](i)) --> calmodulin --> myosin light chain kinase --> phosphorylated myosin --> force development. Light stimulation of isolated sphincters pupillae can be very precisely controlled, and precise reproducible photomechanical responses (PMRs) result. This precision makes the PMR ideal for testing models of regulation of smooth muscle myosin phosphorylation. We measured force and [Ca(2+)](i) concurrently in sphincter pupillae following stimulation by light flashes of varying duration and intensity. We sampled at unusually short (0.01-0.02 s) intervals to adequately test a PMR model based on the myosin phosphorylation cascade. We found, surprisingly, contrary to the behavior of intestinal muscle and predictions of the phosphorylation model, that during PMRs force begins to decay while [Ca(2+)](i) is still rising. We conclude that control of contraction in the sphincter pupillae probably involves an inhibitory process as well as activation by [Ca(2+)](i).

S12–4 - Novel signal transduction in smooth muscle myosin phosphorylation

S12–4 - Novel signal transduction in smooth muscle myosin phosphorylation

Skeletal muscle contractions stimulate cGMP formation and attenuate vascular smooth muscle myosin phosphorylation via nitric oxide

Nitric oxide generated by neuronal nitric oxide synthase in contracting skeletal muscle fibers may regulate vascular relaxation via a cGMP-mediated pathway. Neuronal nitric oxide synthase content is greatly reduced in skeletal muscles from mdx mice. cGMP formation increased in contracting extensor digitorum longus muscles in vitro from C57 control, but not mdx mice. The increase in cGMP content was abolished with N G-nitro- l-arginine. Sodium nitroprusside treatment increased cGMP levels in muscles from both C57 and mdx mice. Skeletal muscle contractions also inhibited phenylephrine-induced phosphorylation of smooth muscle myosin regulatory light chain. Arteriolar dilation was attenuated in contracting muscles from mdx but not C57 mice. NO generated in contracting skeletal muscle may contribute to vasodilation in response to exercise.

Signal Transduction in Lung Cells

Part 1 General concepts: molecular diversity and functional correlates in transmembrane signalling by G proteins, Lutz Birnbaumer et al regulation of G protein-linked receptors - analysis from the genome to the cell membrane, Craig C. Malbon and John R. Hadcock G proteins in pulmonary and cardiovascular signal transduction systems, Vsevolod A. Tkachuk et al small GTPases, Burton F. Dickey and Jeffrey B. Rubins phosphoinositides and intracellular calcium, Frank S. Menniti et al protein-tyrosine kinases, Timothy O'Hare et al protein tyrosine phosphatases - a multigene family of diverse structure and function, Deborah E. Cool purinergic signal transduction - extracellular ATP and adenosine as agonists for receptor-mediated signalling, George R. Dubyak. Part 2 Epithelial cells: epithelial cells Na+ and Cl- channels, Sadis Matalon et al protein phosphorylation and dephosphorylation systems in alveolar epithelial cells, David Warburton et al growth factors and pulmonary epithelia, Rita M. Ryan and Jacob N. Finkelstein expression and regulation of chemotactic cytokine genes by nonimmune pulmonary cells, Steven L. Kunkel et al cellular and biochemical mechanisms of airway epithelial mucin release, K. Chul Kim dysregulation of cellular genes by latent viral genes, Lois J. Geist and Gary W. Hunninghake. Part 3 Smooth muscle cells: role of calcium in the regulation of smooth muscle tone, Katheleen G. Morgan the role of myosin phosphorylation in smooth muscle and macrophage signalling, Primal de Lanerolle et al growth factors and signal transduction in smooth muscle systems, Morley D. Hollenberg. Part 4 Endothelial cells: ionic currents in the membranes of cultured endothelial cells, P. Bregestovski et al receptor-dependent regulation of [Ca2+] and phospholipase C in vascular endothelial cells, Pavel Avdonin et al secretion profiles of vasoactive amine-induced leukocyte chemoattractants - evidence for endothelial cell heterogeneity, Dennis J. Beer and Harrison W. Farber high-altitude pulmonary hypertension and signal transduction in the cardiovascular system, A.A. Aldashev. Part 5 Cells of the immune system: alveolar macrophage growth-factor signalling systems, James C. Bonner and Arnold R. Brody the role of the GTPase superfamily in T-lymphocyte signal transduction, Thomas C. Ryan and William W. Cruikshank mechanisms of mast cell signalling, Stephen I. Wasserman et al beta-adrenergic receptors on alveolar macrophages and lymphocytes - studies with cells from asthmatics, Sergey A. Rudchenko et al.