Graeme Nixon

Bio: Graeme Nixon is an academic researcher from University of Aberdeen. The author has contributed to research in topics: Vascular smooth muscle & Religious education. The author has an hindex of 23, co-authored 62 publications receiving 2090 citations. Previous affiliations of Graeme Nixon include University of Virginia & Western Infirmary.

Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle

Abstract: The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S])-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein. The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio]diphosphate (GDP[beta-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase.

Sphingolipids in inflammation: pathological implications and potential therapeutic targets.

Abstract: Sphingolipids are formed via the metabolism of sphingomyelin, a constituent of the plasma membrane, or by de novo synthesis. Enzymatic pathways result in the formation of several different lipid mediators, which are known to have important roles in many cellular processes, including proliferation, apoptosis and migration. Several studies now suggest that these sphingolipid mediators, including ceramide, ceramide 1-phosphate and sphingosine 1-phosphate (S1P), are likely to have an integral role in inflammation. This can involve, for example, activation of pro-inflammatory transcription factors in different cell types and induction of cyclooxygenase-2, leading to production of pro-inflammatory prostaglandins. The mode of action of each sphingolipid is different. Increased ceramide production leads to the formation of ceramide-rich areas of the membrane, which may assemble signalling complexes, whereas S1P acts via high-affinity G-protein-coupled S1P receptors on the plasma membrane. Recent studies have demonstrated that in vitro effects of sphingolipids on inflammation can translate into in vivo models. This review will highlight the areas of research where sphingolipids are involved in inflammation and the mechanisms of action of each mediator. In addition, the therapeutic potential of drugs that alter sphingolipid actions will be examined with reference to disease states, such as asthma and inflammatory bowel disease, which involve important inflammatory components. A significant body of research now indicates that sphingolipids are intimately involved in the inflammatory process and recent studies have demonstrated that these lipids, together with associated enzymes and receptors, can provide effective drug targets for the treatment of pathological inflammation.

Comparison of Sphingosine 1-Phosphate–Induced Intracellular Signaling Pathways in Vascular Smooth Muscles: Differential Role in Vasoconstriction

Abstract: Sphingosine 1-phosphate (S1P), a lipid released from activated platelets, influences physiological processes in the cardiovascular system via activation of the endothelial differentiation gene (EDG/S1P) family of 7 transmembrane G protein-coupled receptors. In cultured vascular smooth muscle (VSM) cells, S1P signaling has been shown to stimulate proliferative responses; however, its role in vasoconstriction has not been examined. In the present study, the effects of S1P and EDG/S1P receptor expression were determined in rat VSM from cerebral artery and aorta. S1P induced constriction of cerebral artery, which was partly dependent on activation of p160(ROCK) (Rho-kinase). S1P also induced activation of RhoA in cerebral artery with a similar time course to contraction. In aorta, S1P did not produce a constriction or RhoA activation. In VSM myocytes from cerebral arteries, stimulation with S1P gives rise to a global increase in [Ca2+]i, initially generated via Ca2+ release from the sarcoplasmic reticulum by an inositol 1,4,5-trisphosphate-dependent pathway. In aorta VSM, a small increase in [Ca2+]i was observed after stimulation at higher concentrations of S1P. S1P induced activation of p42/p44(mapk) in aorta and cerebral artery VSM. Subtype-specific S1P receptor antibodies revealed that the expression of S1P3/EDG-3 and S1P2/EDG-5 receptors is 4-fold higher in cerebral artery compared with aorta. S1P(1)/EDG-1 receptor expression was similar in both types of VSM. Therefore, the ability of S1P to act as a vasoactive mediator is dependent on the activation of associated signaling pathways and may vary in different VSM. This differential signaling may be related to the expression of S1P receptor subtypes.

Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle.

Abstract: Although agonist stimulation leads to an increase in inositol 1,4,5-trisphosphate (InsP3) and decreased calcium in peripherally and centrally located sarcoplasmic reticulum in smooth muscle, the distribution of InsP3 receptors is unknown. InsP3 receptor and the calcium binding protein, calsequestrin were localized by immunolabelling in a tonic and a phasic smooth muscle. InsP3 receptor labelling was predominantly localized at the cell periphery, where most of the sarcoplasmic reticulum is localized in vas deferens (phasic muscle). Elements of central sarcoplasmic reticulum, where present, were also labelled. Distribution of calsequestrin in vas deferens was similar to that of the InsP3 receptor. In aorta (tonic muscle) the InsP3 receptor labelling was proportional to sarcoplasmic reticulum distribution: predominantly central. No labelling of sections or immunoblots was observed with the anti-calsequestrin antibody in aorta. InsP3 and caffeine, but not cyclic ADP-ribose, released intracellular Ca2+ in permeabilized vas deferens and aorta. The ultrastructure of the sarcoplasmic reticulum, investigated in stereo views of semi-thick and thin sections of osmium ferricyanide stained tissue, is shown to have several distinctive features, such as fenestrated sheets (single or in stacks), as well as numerous regions of continuity between central and peripheral sarcoplasmic reticulum, suggesting a single compartment within the smooth muscle cell. Regions of the sarcoplasmic reticulum were closely apposed to and often ensheathed mitochondria. We conclude that InsP3 receptors are present in both the central and the peripheral sarcoplasmic reticulum of tonic and phasic smooth muscle, consistent with electron probe analysis results showing calcium release from both regions.

Tumor Necrosis Factor-α–Induced Activation of RhoA in Airway Smooth Muscle Cells: Role in the Ca2+ Sensitization of Myosin Light Chain20 Phosphorylation

Abstract: Tumor necrosis factor-alpha (TNF), an inflammatory cytokine, has a potentially important role in the pathogenesis of bronchial asthma and may contribute to airway hyper-responsiveness Recent evidence has revealed that TNF can increase the Ca(2+) sensitivity of agonist-stimulated myosin light chain(20) (MLC(20)) phosphorylation and contractility in guinea pig airway smooth muscle (ASM) In the present study, the potential intracellular pathways responsible for this TNF-induced Ca(2+) sensitization were investigated In permeabilized cultured guinea pig ASM cells, recombinant human TNF stimulated an increase in Ca(2+)-activated MLC(20) phosphorylation under Ca(2+) "clamp" conditions This increased MLC(20) phosphorylation was inhibited by preincubation with the Rho-kinase inhibitor Y27632 TNF also increased the proportion of GTP-bound RhoA, as measured using rhotekin Rho-binding domain, in a time course compatible with a role in the TNF-induced Ca(2+) sensitization In cultured human ASM cells, recombinant human TNF also activated RhoA with a similar time course In addition, TNF stimulated phosphorylation of the regulatory subunit of the myosin phosphatase, which was inhibited by Y27632 Although human ASM cells expressed both receptor subtypes, TNF-R1 and TNF-R2, the activation of RhoA was predominantly via stimulation of the TNF-R1, although RhoA did not immunoprecipitate with the TNF-R1 In conclusion, the TNF-induced increase in the Ca(2+) sensitivity of MLC(20) phosphorylation is through stimulation of the TNF-R1 receptor and via a RhoA/Rho-kinase pathway leading to inhibition of the myosin light chain phosphatase This intracellular mechanism may contribute to TNF-induced airway hyper-responsiveness

Myofibroblasts and mechano-regulation of connective tissue remodelling

Abstract: During the past 20 years, it has become generally accepted that the modulation of fibroblastic cells towards the myofibroblastic phenotype, with acquisition of specialized contractile features, is essential for connective-tissue remodelling during normal and pathological wound healing. Yet the myofibroblast still remains one of the most enigmatic of cells, not least owing to its transient appearance in association with connective-tissue injury and to the difficulties in establishing its role in the production of tissue contracture. It is clear that our understanding of the myofibroblast its origins, functions and molecular regulation will have a profound influence on the future effectiveness not only of tissue engineering but also of regenerative medicine generally.

Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension

Abstract: Abnormal smooth-muscle contractility may be a major cause of disease states such as hypertension, and a smooth-muscle relaxant that modulates this process would be useful therapeutically. Smooth-muscle contraction is regulated by the cytosolic Ca2+ concentration and by the Ca2+ sensitivity of myofilaments: the former activates myosin light-chain kinase and the latter is achieved partly by inhibition of myosin phosphatase. The small GTPase Rho and its target, Rho-associated kinase, participate in this latter mechanism in vitro, but their participation has not been demonstrated in intact muscles. Here we show that a pyridine derivative, Y-27632, selectively inhibits smooth-muscle contraction by inhibiting Ca2+ sensitization. We identified the Y-27632 target as a Rho-associated protein kinase, p160ROCK. Y-27632 consistently suppresses Rho-induced, p160ROCK-mediated formation of stress fibres in cultured cells and dramatically corrects hypertension in several hypertensive rat models. Our findings indicate that p160ROCK-mediated Ca2+ sensitization is involved in the pathophysiology of hypertension and suggest that compounds that inhibit this process might be useful therapeutically.

Small GTP-Binding Proteins

Abstract: Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.

Ca2+ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase

Abstract: Somlyo, Andrew P., and Avril V. Somlyo. Ca2+ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase. Physiol Rev 83: 1325-1358, 2003; 10.1152...

Signal transduction and regulation in smooth muscle

Abstract: Smooth muscle cells in the walls of many organs are vital for most bodily functions, and their abnormalities contribute to a range of diseases. Although based on a sliding-filament mechanism similar to that of striated muscles, contraction of smooth muscle is regulated by pharmacomechanical as well as by electromechanical coupling mechanisms. Recent studies have revealed previously unrecognized contractile regulatory processes, such as G-protein-coupled inhibition of myosin light-chain phosphatase, regulation of myosin light-chain kinase by other kinases, and the functional effects of smooth muscle myosin isoforms. Abnormalities of these regulatory mechanisms and isoform variations may contribute to diseases of smooth muscle, and the G-protein-coupled inhibition of protein phosphatase is also likely to be impor-tant in regulating non-muscle cell functions mediated by cytoplasmic myosin II.