A novel inflammatory biomarker, GlycA, associates with disease activity in rheumatoid arthritis and cardio-metabolic risk in BMI-matched controls

BACKGROUND: RA and CVD both have inflammation as part of the underlying biology. Our objective was to explore the relationships of GlycA, a measure of glycosylated acute phase proteins, with inflammation and cardiometabolic risk in RA, and explore whether these relationships were similar to those for persons without RA. METHODS: Plasma GlycA was determined for 50 individuals with mild-moderate RA disease activity and 39 controls matched for age, gender, and body mass index (BMI). Regression analyses were performed to assess relationships between GlycA and important markers of traditional inflammation and cardio-metabolic health: inflammatory cytokines, disease activity, measures of adiposity and insulin resistance. RESULTS: On average, RA activity was low (DAS-28 = 3.0 ± 1.4). Traditional inflammatory markers, ESR, hsCRP, IL-1β, IL-6, IL-18 and TNF-α were greater in RA versus controls (P < 0.05 for all). GlycA concentrations were significantly elevated in RA versus controls (P = 0.036). In RA, greater GlycA associated with disease activity (DAS-28; RDAS-28 = 0.5) and inflammation (RESR = 0.7, RhsCRP = 0.7, RIL-6 = 0.3: P < 0.05 for all); in BMI-matched controls, these inflammatory associations were absent or weaker (hsCRP), but GlycA was related to IL-18 (RhsCRP = 0.3, RIL-18 = 0.4: P < 0.05). In RA, greater GlycA associated with more total abdominal adiposity and less muscle density (Rabdominal-adiposity = 0.3, Rmuscle-density = -0.3, P < 0.05 for both). In BMI-matched controls, GlycA associated with more cardio-metabolic markers: BMI, waist circumference, adiposity measures and insulin resistance (R = 0.3-0.6, P < 0.05 for all). CONCLUSIONS: GlycA provides an integrated measure of inflammation with contributions from traditional inflammatory markers and cardio-metabolic sources, dominated by inflammatory markers in persons with RA and cardio-metabolic factors in those without.

Modification of P13K- and MAPK-dependent chemotaxis in aortic vascular smooth muscle cells by protein kinase CBII

Hyperglycemia increases expression of platelet-derived growth factor (PDGF)-beta receptor and potentiates chemotaxis to PDGF-BB in human aortic vascular smooth muscle cells (VSMCs) via PI3K and ERK/MAPK signaling pathways. The purpose of this study was to determine whether increased activation of protein kinase C (PKC) isoforms had a modulatory effect on the PI3K and ERK/MAPK pathways, control of cell adhesiveness, and movement. All known PKC isoforms were assessed but only PKC alpha and PKC beta II levels were increased in 25 mmol/L glucose. However, only PKC beta II inhibition affected (decreased) PI3K pathway and MAPK pathway activities and inhibited PDGF-beta receptor upregulation in raised glucose, and specific MAPK inhibition was required to completely block the effect of glucose. In raised glucose conditions, activity of the ERK/MAPK pathway, PI3K pathway, and PKC beta II were all sensitive to aldose reductase inhibition. Chemotaxis to PDGF-BB (360 pmol/L), absent in 5 mmol/L glucose, was present in raised glucose and could be blocked by PKC beta II inhibition. Formation of lamellipodia was dependent on PI3K activation and filopodia on MAPK activation; both lamellipodia and filopodia were eliminated when PKC beta II was inhibited. FAK phosphorylation and cell adhesion were reduced by PI3K inhibition, and although MAPK inhibition prevented chemotaxis, it did not affect FAK phosphorylation or cell adhesiveness. In conclusion, chemotaxis to PDGF-BB in 25 mmol/L glucose is PKC beta II-dependent and requires activation of both the PI3K and MAPK pathways. Changes in cell adhesion and migration speed are mediated mainly through the PI3K pathway.

Internal mammary artery smooth muscle cells resist migration and possess high antioxidant capacity

Objective: This study investigated whether differences exist in atherogen-induced migratory behaviors and basal antioxidant enzyme capacity of vascular smooth muscle cells (VSMC) from human coronary (CA) and internal mammary (IMA) arteries. Methods: Migration experiments were performed using the Dunn chemotaxis chamber. The prooxidant [NAD(P)H oxidase] and antioxidant [NOS, superoxide dismutase, catalase and glutathione peroxidase] enzyme activities were determined by specific assays. Results: Chemotaxis experiments revealed that while both sets of VSMC migrated towards platelet-derived growth factor-BB (1-50 ng/ml) and angiotensin II (1-50 nM), neither oxidized-LDL (ox-LDL, 25-100 ng/ml) nor native LDL (100 ng/ml) affected chemotaxis in IMA VSMC. However, high dose ox-LDL produced significant chemotaxis in CAVSMC that was inhibited by pravastatin (100 nM), mevastatin (10 nM), losartan (10 nM), enalapril (1 micro.M), and MnTBAP (a free radical scavenger, 50 micro.M). Microinjection experiments with isoprenoids i.e. geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) showed distinct involvement of small GTPases in atherogeninduced VSMC migration. Significant increases in antioxidant enzyme activities and nitrite production along with marked decreases in NAD(P)H oxidase activity and superoxide levels were determined in IMA versus CA VSMC. Conclusions: Enhanced intrinsic antioxidant capacity may confer on IMAVSMC resistance to migration against atherogenic agents. Drugs that regulate ox-LDL or angiotensin II levels also exert antimigratory effects.

Role of IP(3) in modulation of spontaneous activity in pacemaker cells of rabbit urethra.

Isolated interstitial ("pacemaker") cells from rabbit urethra were examined using the perforated-patch technique. Under voltage clamp at -60 mV, these cells fired large spontaneous transient inward currents (STICs), averaging -860 pA and >1 s in duration, which could account for urethral pacemaker activity. Spontaneous transient outward currents (STOCs) were also observed and fell into two categories, "fast" (1 s in duration). The latter were coupled to STICs, suggesting that they shared the same mechanism, while the former occurred independently at faster rates. All of these currents were abolished by cyclopiazonic acid, caffeine, or ryanodine, suggesting that they were activated by Ca(2+) release. When D-myo-inositol 1,4,5-trisphosphate (IP(3))-sensitive stores were blocked with 2-aminoethoxydiphenyl borate, the STICs and slow STOCs were abolished, but the fast STOCs remained. In contrast, the fast STOCs were more nifedipine sensitive than the STICs or the slow STOCs. These results suggest that while fast STOCs are mediated by a mechanism similar to STOCs in smooth muscle, STICs and slow STOCs are driven by IP(3). These results support the hypothesis that pacemaker activity in the urethra is driven by the IP(3)-sensitive store. PMID: 11287348 [PubMed - indexed for MEDLINE]

Ca(2+)-activated Cl(-) current in sheep lymphatic smooth muscle.

Freshly dispersed sheep mesenteric lymphatic smooth muscle cells were studied at 37 degrees C using the perforated patch-clamp technique with Cs(+)- and K(+)-filled pipettes. Depolarizing steps evoked currents that consisted of L-type Ca(2+) [I(Ca(L))] current and a slowly developing current. The slow current reversed at 1 +/- 1.5 mV with symmetrical Cl(-) concentrations compared with 23.2 +/- 1.2 mV (n = 5) and -34.3 +/- 3.5 mV (n = 4) when external Cl(-) was substituted with either glutamate (86 mM) or I(-) (125 mM). Nifedipine (1 microM) blocked and BAY K 8644 enhanced I(Ca(L)), the slow-developing sustained current, and the tail current. The Cl(-) channel blocker anthracene-9-carboxylic acid (9-AC) reduced only the slowly developing inward and tail currents. Application of caffeine (10 mM) to voltage-clamped cells evoked currents that reversed close to the Cl(-) equilibrium potential and were sensitive to 9-AC. Small spontaneous transient depolarizations and larger action potentials were observed in current clamp, and these were blocked by 9-AC. Evoked action potentials were triphasic and had a prominent plateau phase that was selectively blocked by 9-AC. Similarly, fluid output was reduced by 9-AC in doubly cannulated segments of spontaneously pumping sheep lymphatics, suggesting that the Ca(2+)-activated Cl(-) current plays an important role in the electrical activity underlying spontaneous activity in this tissue. PMID: 11029279 [PubMed - indexed for MEDLINE]

The smooth muscle pharmacology of maximakinin, a receptor-selective, bradykinin-related nonadecapeptide from the venom of the Chinese toad, Bombina maxima.

Structural homologues of vertebrate regulatory peptides found in defensive skin secretions of anuran amphibians often display enhanced bioactivity and receptor binding when compared with endogenous mammalian peptide ligands. Maximakinin, a novel N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the skin venom of a Chinese toad (Bombina maxima), displays such activity enhancement when compared with bradykinin but is additionally highly selective for mammalian arterial smooth muscle bradykinin receptors displaying a 50-fold increase in molar potency in this smooth muscle type. In contrast, a 100-fold decrease in molar potency was observed at bradykinin receptors in intestinal and uterine smooth muscle preparations. Maximakinin has thus evolved as a “smart” defensive weapon in the toad with receptor/tissue selective targeting. Natural selection of amphibian skin venom peptides for antipredator defence, through inter-species delivery by an exogenous secretory mode, produces subtle structural stabilisation modifications that can potentially provide new insights for the design of selectively targeted peptide therapeutics.

Pachymedusa dacnicolor tryptophyllin-1 (PdT-1): structural characterization, pharmacological activity and cloning of precursor cDNA.

Tryptophyllins are a heterogenous group of amphibian skin peptides originally identified in skin extracts of Neotropical leaf frogs, Phyllomedusa sp., by chemical means. Until now, biosynthetic precursor structure and biological activity remain unreported. Here we describe the isolation of a novel, post-translationally modified tryptophyllin, Lys-Pro-Hyp-Ala-Trp-Val-Pro.amide (PdT-1), from the skin secretion of the Mexican leaf frog, Pachymedusa dacnicolor. Using a 3'- and 5'-RACE strategy and an in vitro skin cDNA library, the PdT-1-encoding precursor was cloned and found to consist of an open-reading frame of 62 amino acids with a single copy of PdT-1 located towards the C-terminus. A synthetic replicate of PdT-1 was found to be a potent myoactive agent, relaxing mammalian arterial smooth muscle and contracting small intestinal smooth muscle at nanomolar concentrations. PdT-1 is thus the first amphibian skin tryptophyllin to be pharmacologically characterized and the first whose precursor cDNA has been cloned.

Kit-positive interstitial cells in the rabbit urethra: structural relationships with nerves and smooth muscle.

OBJECTIVE: To identify interstitial cells (ICs) in the wall of the rabbit urethra using antibodies to the Kit receptor, and to examine their location, morphology and relationship with nerves and smooth muscle cells (SMCs), as studies of enzymatically isolated cells from the rabbit urethra have established that there are specialized cells that show spontaneous electrical activity and have morphological properties of ICs. MATERIALS AND METHODS: Urethral tissues from rabbits were fixed, labelled with antibodies and examined with confocal microscopy. Some specimens were embedded in paraffin wax and processed for histology. Histological sections from the most proximal third and mid-third region of rabbit urethra were stained with Masson's Trichrome to show their cellular arrangement. RESULTS: Sections from both regions had outer longitudinal and inner circular layers of SM, and a lamina propria containing connective tissue and blood vessels; the lumen was lined with urothelial cells. The mid-third region had a more developed circular SM layer than the most-proximal samples, and had extensive inner longitudinal SM bundles in the lamina propria. Labelling with anti-Kit revealed immunopositive cells within the wall of the rabbit urethra, in the circular and longitudinal layers of the muscularis. Double-labelling with an antibody to SM myosin showed Kit-positive cells on the boundary of the SM bundles, orientated parallel to the axis of the bundles. Others were in spaces between the bundles and often made contact with each other. Kit-positive cells were either elongated, with several lateral branches, or stellate with branches coming from a central soma. Similar cells could be labelled with vimentin antibodies. Their relationship with intramural nerves was examined by double immunostaining with an anti-neurofilament antibody. There were frequent points of contact between Kit-positive cells and nerves, with similar findings in specimens double-immunostained with anti-neuronal nitric oxide synthase (nNOS). CONCLUSION: Kit-positive ICs were found within the SM layers of the rabbit urethra, in association with nerves, on the edge of SM bundles and in the interbundle spaces. The contact with nNOS-containing neurones might imply participation in the nitrergic inhibitory neurotransmission of the urethra. PMID: 17212607 [PubMed - indexed for MEDLINE]

Characterization of T-type calcium current and its contribution to electrical activity in rabbit urethra.

Rabbit urethral smooth muscle cells were studied at 37 degrees C by using the amphotericin B perforated-patch configuration of the patch-clamp technique, using Cs(+)-rich pipette solutions. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca(2+) currents, were recorded. Fitting steady-state inactivation curves for the L current with a Boltzmann equation yielded a V(1/2) of -41 +/- 3 mV. In contrast, the T current inactivated with a V(1/2) of -76 +/- 2 mV. The L currents were reduced by nifedipine (IC(50) = 225 +/- 84 nM), Ni(2+) (IC(50) = 324 +/- 74 microM), and mibefradil (IC(50) = 2.6 +/- 1.1 microM) but were enhanced when external Ca(2+) was substituted with Ba(2+). The T current was little affected by nifedipine at concentrations

Effector dynamics of rhythmic wrist activity and its implications for (modeling) bimanual coordination

To examine the role of the effector dynamics of the wrist in the production of rhythmic motor activity, we estimated the phase shifts between the EMG and the task-related output for a rhythmic isometric torque production task and an oscillatory movement, and found a substantial difference (45-52degrees) between the two. For both tasks, the relation between EMG and task-related output (torque or displacement) was adequately reproduced with a physiologically motivated musculoskeletal model. The model simulations demonstrated the importance of the contribution of passive structures to the overall dynamics and provided an account for the observed phase shifts in the dynamic task. Additional simulations of the musculoskeletal model with added load suggested that particular changes in the phase relation between EMG and movement may follow largely from the intrinsic muscle dynamics, rather than being the result of adaptations in the neural control of joint stiffness. The implications of these results are discussed in relation to (models of) interlimb coordination in rhythmic tasks. (C) 2004 Elsevier B.V. All rights reserved.

Changes in muscle recruitment patterns during skill acquisition

The control of movement is predicated upon a system of constraints of musculoskeletal and neural origin. The focus of the present study was upon the manner in which such constraints are adapted or superseded during the acquisition of motor skill. Individuals participated in five experimental sessions, ill which they attempted to produce abduction-adduction movements of the index finger in time with an auditory metronome. During each trial, the metronome frequency was increased in eight steps from an individually determined base frequency. Electromyographic (EMC) activity was recorded from first dorsal interosseous (FDI), first volar interosseous (FVI), flexor digitorum superficialis (FDS), and extensor digitorum communis (EDC) muscles. The movements produced on the final day of acquisition more accurately matched the required profile, and exhibited greater spatial and temporal stability, than those generated during initial performance. Tn the early stages of skill acquisition, an alternating pattern of activation in FDI and FVI was maintained, even at the highest frequencies. Tn contrast, as the frequency of movement was increased, activity in FDS and EDC was either tonic or intermittent. As learning proceeded, alterations in recruitment patterns were expressed primarily in the extrinsic muscles (EDC and FDS). These changes took the form of increases in the postural role of these muscles, shifts to phasic patterns of activation, or selective disengagement of these muscles. These findings suggest that there is considerable flexibility in the composition of muscle synergies, which is exploited by individuals during the acquisition of coordination.

Deletion of TRPC4 and TRPC6 in Mice Impairs Smooth Muscle Contraction and Intestinal Motility In Vivo.

BACKGROUND & AIMS: Downstream effects of muscarinic receptor stimulation in intestinal smooth muscle include contraction and intestinal transit. We thought to determine whether classic transient receptor potential (TRPC) channels integrate the intracellular signaling cascades evoked by the stimulated receptors and thereby contribute to the control of the membrane potential, Ca-influx, and cell responses. METHODS: We created trpc4-, trpc6-, and trpc4/trpc6-gene-deficient mice and analyzed them for intestinal smooth muscle function in vitro and in vivo. RESULTS: In intestinal smooth muscle cells TRPC4 forms a 55 pS cation channel and underlies more than 80% of the muscarinic receptor-induced cation current (mI(CAT)). The residual mI(CAT) depends on the expression of TRPC6, indicating that TRPC6 and TRPC4 determine mI(CAT) channel activity independent of other channel subunits. In TRPC4-deficient ileal myocytes the carbachol-induced membrane depolarizations are diminished greatly and the atropine-sensitive contraction elicited by acetylcholine release from excitatory motor neurons is reduced greatly. Additional deletion of TRPC6 aggravates these effects. Intestinal transit is slowed down in mice lacking TRPC4 and TRPC6. CONCLUSIONS: In intestinal smooth muscle cells TRPC4 and TRPC6 channels are gated by muscarinic receptors and are responsible for mI(CAT). They couple muscarinic receptors to depolarization of intestinal smooth muscle cells and voltage-activated Ca(2+)-influx and contraction, and thereby accelerate small intestinal motility in vivo.

Protein kinase B/Akt activity is involved in renal TGF-beta 1-driven epithelial-mesenchymal transition in vitro and in vivo

The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-beta (TGF-beta1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-beta1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-beta1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473 phosphorylation and GSK-3beta phosphorylation. TGF-beta1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and alpha-smooth muscle actin expression, consistent with the fibrotic actions of TGF-beta1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-beta1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3beta, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-beta1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.

Rho kinase and protein kinase C involvement in vascular smooth muscle myofilament calcium sensitization in arteries from diabetic rats.

BACKGROUND AND PURPOSE: Diabetes mellitus (DM) causes multiple dysfunctions including circulatory disorders such as cardiomyopathy, angiopathy, atherosclerosis and arterial hypertension. Rho kinase (ROCK) and protein kinase C (PKC) regulate vascular smooth muscle (VSM) Ca(2+) sensitivity, thus enhancing VSM contraction, and up-regulation of both enzymes in DM is well known. We postulated that in DM, Ca(2+) sensitization occurs in diabetic arteries due to increased ROCK and/or PKC activity. EXPERIMENTAL APPROACH: Rats were rendered hyperglycaemic by i.p. injection of streptozotocin. Age-matched control tissues were used for comparison. Contractile responses to phenylephrine (Phe) and different Ca(2+) concentrations were recorded, respectively, from intact and chemically permeabilized vascular rings from aorta, tail and mesenteric arteries. KEY RESULTS: Diabetic tail and mesenteric arteries demonstrated markedly enhanced sensitivity to Phe while these changes were not observed in aorta. The ROCK inhibitor HA1077, but not the PKC inhibitor chelerythrine, caused significant reduction in sensitivity to agonist in diabetic vessels. Similar changes were observed for myofilament Ca(2+) sensitivity, which was again enhanced in DM in tail and mesenteric arteries, but not in aorta, and could be reduced by both the ROCK and PKC blockers. CONCLUSIONS AND IMPLICATIONS: We conclude that in DM enhanced myofilament Ca(2+) sensitivity is mainly manifested in muscular-type blood vessels and thus likely to contribute to the development of hypertension. Both PKC and, in particular, ROCK are involved in this phenomenon. This highlights their potential usefulness as drug targets in the pharmacological management of DM-associated vascular dysfunction.