Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide. Materials and Methods: Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay. Results: A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical -KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation. Conclusion: IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.

Evidence for the presence of G-proteins, adenylyl cyclase and phospholipase C activities in lymphatic smooth muscle cell membranes

In plasma membranes derived from bovine mesenteric lymphatic smooth muscle cells, guanine nucleotide and forskolin stimulated adenylyl cyclase (AC) activity in a concentration-dependent manner, indicative of the presence of the stimulatory G-protein G(s) linked to AC. There was no significant enzyme inhibition by low concentrations of guanine nucleotide and no effect on basal or guanine nucleotide-stimulated activity following pertussis toxin treatment of cells, suggesting the absence of G(1) linked to inhibition of AC. Furthermore, there was no effect of adrenaline, isoprenaline or clonidine on basal or forskolin-stimulated activities, nor was there any specific binding of the beta-adrenoceptor ligand [I-125]cyanopindolol to membranes, suggesting that cate-cholamine receptors do not modulate AC activity in these membranes. Pertussis toxin-mediated ADP ribosylation of membrane proteins and Western immunoblotting analysis revealed the presence of G-protein subunits G(alpha l2), G(alpha q), G(alpha 11) and G(beta 1). In experiments designed to identify a possible effector enzyme for these G-proteins, membranes were screened with a range of antibodies raised against phospholipase C (PLC) beta, gamma and delta isozymes. Though no evidence was obtained by Western blotting for any of these proteins, PLC activity was concentration-dependently stimulated by Ca2+, but not by AlF4-, GTP[S], or purified G(beta gamma) subunits. Finally, no specific binding to membranes of the alpha(1)-adrenoceptor ligand [H-3]prazosin or the alpha(2)-adrenoceptor ligand [H-3]yohimbine was obtained. In conclusion, this study provides evidence for a G(s)-dependent stimulation of AC, and for the presence of G(2) and G(q11), which do not appear to regulate a PLC activity also identified in lymphatic smooth muscle cell membranes. Furthermore, neither AC nor PLC appear to be associated with catecholamine receptors. Copyright(C) 1996 Elsevier Science Inc.

Comparative study of the spatial relationship between nicotinamide adenine dinucleotide phosphate-diaphorase activity, serotonin immunoreactivity, and GYIRFamide immunoreactivity and the musculature of the adult liver fluke, Fasciola hepatica (Digenea, Fasciolidae)

This is the first detailed description of the nitrergic nervous system in a fluke. In this study, the authors analysed the distribution of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity in neuronal and nonneuronal tissues of the adult fluke Fasciola hepatica and compared this with the distribution of the musculature using tetramethylrhodamine isothiocyanate-phalloidin. To assess the correlation between the number of muscle cells in different parts of the fluke and the NADPH-d-stained cells, the nuclei were stained with Hoechst 333 42, which is specific for chromatin. The spatial relation between the NADPH-d-positive nerves and the 5-hydroxytryptamine (serotonin; 5-HT)-immunoreactive (-IR) and GYIRFamide-IR nervous elements was also examined. The methods complement each other. NADPH-d-positive staining occurs in both in neuronal tissue and nonneuronal tissue. Large, NADPH-d-stained neurones were localised in the nervous system. The oral and ventral suckers are innervated with many large NADPH-d-stained neurones. Ln addition, the NADPH-d staining reaction follows closely the muscle fibres in both the suckers, in the body, and in the ducts of the reproductive organs. The presence of NADPH-d activity along muscle fibres in F. hepatica and in other flatworms supports a possible myoinhibitory role for nitric oxide. Neuronal nitric oxide synthase in flatworms may form a novel drug target, which would facilitate the development of a novel anthelminthic. (C) 2001 Wiley-Liss, Inc.

Importance of the proline residue to the functional activity and metabolic stability of the nematode FMRFamide-related peptide, KPNFIRFamide (PF4)

PF4 has previously been shown to have potent inhibitory effects on myoactivity of somatic muscle strips from the nematode, Ascaris suum. This study examined the bioactivity and metabolic stability of position 2- and position 5-modified analogues of PF4. Although the analogues [Leu(5)] PF4, [Ala(2)]PF4, [Gly(2)]PF4, [Ala(2),Leu(5)]PF4, and [Gly(2),Leu(5)]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishable and had the order of potency: PF4 = [Leu(5)] PF4 >> [Ala(2)]PF4 = [Ala(2),Leu(5)] PF4 >> [Gly(2)] PF4 = [Gly(2),Leu(5)] PF4. Leu(5) for Ile(5) substitutions in PF4 did not alter the activity of this peptide; however, Gly(2)/Ala(2) for Pro(2) substitutions reduced, but did not abolish, peptide activity. Peptide stability studies revealed that [Gly(2)]PF4(2-7) and -(3-7) and [Ala(2)]PF4(2-7), -(3-7), and -(4-7) fragments were generated following exposure to A. suum somatic muscle strips. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases. Observed analogue metabolism appeared to be due to the activity of released aminopeptidases as identical fragments were generated by incubation in medium that had been exposed to somatic muscle strips and from which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of the tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reveal that Pro(2) in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the actions of native nematode aminopeptidases. Copyright (C) 1996 Elsevier Science Inc.

Physiological effects of platyhelminth RFamide peptides on muscle-strip preparations of Fasciola hepatica (Trematoda: Digenea)

The effects of each of the known platyhelminth neuropeptides were determined on muscle-strip preparations from the liver fluke, Fasciola hepatica. The activity of synthetic replicates of the C-terminal nonapeptide of neuropeptide F (NPF9, Moniezia expansa), and the FMRFamide-related peptides (FaRPs), GNFFRFamide, RYIRFamide, GYIRFamide and YIRFamide, were examined. Muscle-strip activity was recorded from 1 mm segments of muscle prepared from 28 to 32-day-old worms, using a photo-optic transducer system. None of the peptides (less than or equal to 10 mu M) altered baseline tension significantly; however, each of the peptides increased the amplitude and frequency of muscle contraction. The threshold for activity of each of the peptides examined was, respectively, 1 nM (RYIRFamide), 0.3 mu M (GYIRFamide and YIRFamide), and 10 mu M (GNFFRFamide and NPF9). All of the effects were reversible and repeatable, following wash-out. Muscle-strip integrity was tested following experimentation, using arecoline (10 mu M) and high-K+ bathing medium (90 mM K+).

Brain activity during ankle proprioceptive stimulation predicts balance performance in young and older adults

Proprioceptive information from the foot/ankle provides important information regarding body sway for balance control, especially in situations where visual information is degraded or absent. Given known increases in catastrophic injury due to falls with older age, understanding the neural basis of proprioceptive processing for balance control is particularly important for older adults. In the present study, we linked neural activity in response to stimulation of key foot proprioceptors (i.e., muscle spindles) with balance ability across the lifespan. Twenty young and 20 older human adults underwent proprioceptive mapping; foot tendon vibration was compared with vibration of a nearby bone in an fMRI environment to determine regions of the brain that were active in response to muscle spindle stimulation. Several body sway metrics were also calculated for the same participants on an eyes-closed balance task. Based on regression analyses, multiple clusters of voxels were identified showing a significant relationship between muscle spindle stimulation-induced neural activity and maximum center of pressure excursion in the anterior-posterior direction. In this case, increased activation was associated with greater balance performance in parietal, frontal, and insular cortical areas, as well as structures within the basal ganglia. These correlated regions were age- and foot-stimulation side-independent and largely localized to right-sided areas of the brain thought to be involved in monitoring stimulus-driven shifts of attention. These findings support the notion that, beyond fundamental peripheral reflex mechanisms, central processing of proprioceptive signals from the foot is critical for balance control.

Contribution of reverse Na+/Ca2+ exchange to spontaneous activity in interstitial cells of cajal in the rabbit urethra

Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit regular Ca2+ oscillations that are associated with spontaneous transient inward currents (STICs) recorded under voltage clamp. Their frequency is known to be very sensitive to external Ca2+ concentration but the mechanism of this has yet to be elucidated. In the present study experiments were performed to assess the role of Na+-Ca2+ exchange (NCX) in this process. Membrane currents were recorded using the patch clamp technique and measurements of intracellular Ca2+ were made using fast confocal microscopy. When reverse mode NCX was enhanced by decreasing the external Na+ concentration [Na+]o from 130 to 13 mM, the frequency of global Ca2+ oscillations and STICs increased. Conversely, inhibition of reverse mode NCX by KB-R7943 and SEA0400 decreased the frequency of Ca2+ oscillations and STICs. Application of caffeine (10 mM) and noradrenaline (10 microM) induced transient Ca2+-activated chloride currents (I(ClCa)) at -60 mV due to release of Ca2+ from ryanodine- and inositol trisphosphate (IP3)-sensitive Ca2+ stores, respectively, but these responses were not blocked by KB-R7943 or SEA0400 suggesting that neither drug blocked Ca2+-activated chloride channels or Ca2+ release from stores. Intact strips of rabbit urethra smooth muscle develop spontaneous myogenic tone. This tone was relaxed by application of SEA0400 in a concentration-dependent fashion. Finally, single cell RT-PCR experiments revealed that isolated ICC from the rabbit urethra only express the type 3 isoform of the Na+-Ca2+ exchanger (NCX3). These results suggest that frequency of spontaneous activity in urethral ICC can be modulated by Ca2+ entry via reverse NCX.

Na+/H+ exchange inhibitor cariporide attenuates skeletal muscle infarction when administered before ischemia or reperfusion

Administration of Na(+)/H(+) exchange isoform-1 (NHE-1) inhibitors before ischemia has been shown to attenuate myocardial infarction in several animal models of ischemia-reperfusion injury. However, controversy still exists as to the efficacy of NHE-1 inhibitors in protection of myocardial infarction when administered at the onset of reperfusion. Furthermore, the efficacy of NHE-1 inhibition in protection of skeletal muscle from infarction (necrosis) has not been studied. This information has potential clinical applications in prevention or salvage of skeletal muscle from ischemia-reperfusion injury in elective and trauma reconstructive surgery. The objective of this research project is to test our hypothesis that the NHE-1 inhibitor cariporide is effective in protection of skeletal muscle from infarction when administered at the onset of sustained ischemia or reperfusion and to study the mechanism of action of cariporide. In our studies, we observed that intravenous administration of cariporide 10 min before ischemia (1 or 3 mg/kg) or reperfusion (3 mg/kg) significantly reduced infarction in pig latissimus dorsi muscle flaps compared with the control, when these muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion (P <0.05; n = 5 pigs/group). Both preischemic and postischemic cariporide treatment (3 mg/kg) induced a significant decrease in muscle myeloperoxidase activity and mitochondrial-free Ca(2+) content and a significant increase in muscle ATP content within 2 h of reperfusion (P <0.05; n = 4 pigs/group). Preischemic and postischemic cariporide treatment (3 mg/kg) also significantly inhibited muscle NHE-1 protein expression within 2 h of reperfusion after 4 h of ischemia, compared with the control (P <0.05; n = 3 pigs/group). These observations support our hypothesis that cariporide attenuates skeletal muscle infarction when administered at the onset of ischemia or reperfusion, and the mechanism involves attenuation of neutrophil accumulation and mitochondrial-free Ca(2+) overload and preservation of ATP synthesis in the early stage of reperfusion.

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning:efficacy and mechanism

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P <0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P <0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P <0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P <0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.

Splicing of HDAC7 modulates the SRF-myocardin complex during stem-cell differentiation towards smooth muscle cells

Histone deacetylases (HDACs) have a central role in the regulation of gene expression. Here we investigated whether HDAC7 has an impact on embryonic stem (ES) cell differentiation into smooth muscle cells (SMCs). ES cells were seeded on collagen-IV-coated flasks and cultured in the absence of leukemia inhibitory factor in differentiation medium to induce SMC differentiation. Western blots and double-immunofluorescence staining demonstrated that HDAC7 has a parallel expression pattern with SMC marker genes. In ex vivo culture of embryonic cells from SM22-LacZ transgenic mice, overexpression of HDAC7 significantly increased beta-galactosidase-positive cell numbers and enzyme activity, indicating its crucial role in SMC differentiation during embryonic development. We found that HDAC7 undergoes alternative splicing during ES cell differentiation. Platelet-derived growth factor enhanced ES cell differentiation into SMCs through upregulation of HDAC7 splicing. Further experiments revealed that HDAC7 splicing induced SMC differentiation through modulation of the SRF-myocardin complex. These findings suggest that HDAC7 splicing is important for SMC differentiation and vessel formation in embryonic development.

Sp1-dependent activation of HDAC7 is required for platelet-derived growth factor-BB-induced smooth muscle cell differentiation from stem cells

We have previously demonstrated that histone deacetylase 7 (HDAC7) expression and splicing play an important role in smooth muscle cell (SMC) differentiation from embryonic stem (ES) cells, but the molecular mechanisms of increased HDAC7 expression during SMC differentiation are currently unknown. In this study, we found that platelet-derived growth factor-BB (PDGF-BB) induced a 3-fold increase in the transcripts of HDAC7 in differentiating ES cells. Importantly, our data also revealed that PDGF-BB regulated HDAC7 expression not through phosphorylation of HDAC7 but through transcriptional activation. By dissecting its promoters with progressive deletion analysis, we identified the sequence between -343 and -292 bp in the 5'-flanking region of the Hdac7 gene promoter as the minimal PDGF-BB-responsive element, which contains one binding site for the transcription factor, specificity protein 1 (Sp1). Mutation of the Sp1 site within this PDGF-BB-responsive element abolished PDGF-BB-induced HDAC7 activity. PDGF-BB treatment enhanced Sp1 binding to the Hdac7 promoter in differentiated SMCs in vivo as demonstrated by the chromatin immunoprecipitation assay. Moreover, we also demonstrated that knockdown of Sp1 abrogated PDGF-BB-induced HDAC7 up-regulation and SMC differentiation gene expression in differentiating ES cells, although enforced expression of Sp1 alone was sufficient to increase the activity of the Hdac7 promoter and expression levels of SMC differentiation genes. Importantly, we further demonstrated that HDAC7 was required for Sp1-induced SMC differentiation of gene expression. Our data suggest that Sp1 plays an important role in the regulation of Hdac7 gene expression in SMC differentiation from ES cells. These findings provide novel molecular insights into the regulation of HDAC7 and enhance our knowledge in SMC differentiation and vessel formation during embryonic development.

Fruit and vegetable consumption and muscle strength and power during adolescence: a cross-sectional analysis of the Northern Ireland Young Hearts Project 1999-2001

Objectives: To examine the association between fruit and vegetable (FV) consumption and muscle strength and power in an adolescent population. Methods: We conducted a cross-sectional analysis among 1019 boys and 998 girls, aged 12 and 15 years, who participated in The Young Hearts Project. FV consumption (excluding potatoes) was assessed by 7-d diet history. Grip strength and jump power was assessed with a dynamometer and Jump-MD meter, respectively. Associations between FV consumption and strength and power were assessed by regression modelling. Results: Boys and girls with the highest FV intakes (>237.71 g/d and >267.57 g/d, respectively, based on the highest tertile) had significantly higher jump power than those with the lowest intakes (<135.09 g/d and <147.43 g/d, respectively), after adjustment for confounding factors. Although girls with the highest FV intakes had higher grip strength than those with the lowest intakes, no significant independent associations were evident between FV intake and grip strength in boys or girls. Similar findings were observed when FV were analysed separately.Conclusions: Higher FV consumption in this group of adolescents was positively associated with muscle power. There was no independent association between higher FV consumption and muscle strength. Intervention studies are required to determine whether muscle strength and power can be improved through increased FV consumption.

Tullis Rennie's Muscle Memory: Listening to the Act of Listening

This paper explores a recent, broadly 'electroacoustic', fixed medium composition by Tullis Rennie, which uses his background in ethnographic fieldwork to explore (in this case through auto-ethnography) modes of listening, and the role of technologies in mediating this listening. Muscle Memory: A conversation about jazz, with Graham South (trumpet) (2014) begins to answer questions about how one work can comment on and analyse or critique another through its own agency as music, bringing composition and ethnography together in fruitful collision, and illuminating the human capacity to manipulate and be manipulated by musical activity. The paper uses the piece to test the extent to which four functions, identified by Simon Frith (1987. Towards an aesthetic of popular music. In R. Leppert & S. McClary (Eds.), Music and society (pp. 133-49). Cambridge: Cambridge University Press) as crucial to the meaningfulness of popular music may, in the context of ubiquitously technologised music, have broader application than he originally intended.

Autonomic modification of intestinal smooth muscle contractility

Intestinal smooth muscle contracts rhythmically in the absence of nerve and hormonal stimulation because of the activity of pacemaker cells between and within the muscle layers. This means that the autonomic nervous system modifies rather than initiates intestinal contractions. The practical described here gives students an opportunity to observe this spontaneous activity and its modification by agents associated with parasympathetic and sympathetic nerve activity. A section of the rabbit small intestine is suspended in an organ bath, and the use of a pressure transducer and data-acquisition software allows the measurement of tension generated by the smooth muscle of intestinal walls. The application of the parasympathetic neurotransmitter ACh at varying concentrations allows students to observe an increase in intestinal smooth muscle tone with increasing concentrations of this muscarinic receptor agonist. Construction of a concentration-effect curve allows students to calculate an EC50 value for ACh and consider some basic concepts surrounding receptor occupancy and activation. Application of the hormone epinephrine to the precontracted intestine allows students to observe the inhibitory effects associated with sympathetic nerve activation. Introduction of the drug atropine to the preparation before a maximal concentration of ACh is applied allows students to observe the inhibitory effect of a competitive antagonist on the physiological response to a receptor agonist. The final experiment involves the observation of the depolarizing effect of K+ on smooth muscle. Students are also invited to consider why the drugs atropine, codeine, loperamide, and botulinum toxin have medicinal uses in the management of gastrointestinal problems.

GLUT4, AMP kinase, but not the insulin receptor, are required for hepatoportal glucose sensor-stimulated muscle glucose utilization.

Recent evidence suggests the existence of a hepatoportal vein glucose sensor, whose activation leads to enhanced glucose use in skeletal muscle, heart, and brown adipose tissue. The mechanism leading to this increase in whole body glucose clearance is not known, but previous data suggest that it is insulin independent. Here, we sought to further determine the portal sensor signaling pathway by selectively evaluating its dependence on muscle GLUT4, insulin receptor, and the evolutionarily conserved sensor of metabolic stress, AMP-activated protein kinase (AMPK). We demonstrate that the increase in muscle glucose use was suppressed in mice lacking the expression of GLUT4 in the organ muscle. In contrast, glucose use was stimulated normally in mice with muscle-specific inactivation of the insulin receptor gene, confirming independence from insulin-signaling pathways. Most importantly, the muscle glucose use in response to activation of the hepatoportal vein glucose sensor was completely dependent on the activity of AMPK, because enhanced hexose disposal was prevented by expression of a dominant negative AMPK in muscle. These data demonstrate that the portal sensor induces glucose use and development of hypoglycemia independently of insulin action, but by a mechanism that requires activation of the AMPK and the presence of GLUT4.