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.

Surveillance study of a number of synthetic and natural growth promoters in bovine muscle samples using liquid chromatography-tandem mass spectrometry

A simple, new method permitting the simultaneous determination and confirmation of trace residues of 24 different growth promoters and metabolites using liquid chromatography-mass spectrometry was developed and validated. The compounds were extracted from bovine tissue using acetonitrile; sodium sulphate was also added at this stage to aid with purification. The resulting mixture was then evaporated to approximately 1 ml and subsequently centrifuged at high speed and an aliquot injected onto the LC-MS/MS system. The calculated CC values ranged between 0.11 and 0.46 mu g kg-1; calculated CC were in the range 0.19-0.79 mu g kg-1. Accuracy, measurement of uncertainty, repeatability and linearity were also determined for each analyte. The analytical method was applied to a number of bovine tissue samples imported into Ireland from third countries. Levels of progesterone were found in a number of samples at concentrations ranging between 0.28 and 30.30 mu g kg-1. Levels of alpha- and beta-testosterone were also found in a number of samples at concentrations ranging between 0.22 and 8.63 mu g kg-1 and between 0.16 and 2.08 mu g kg-1 respectively.

Feedback via Ca²⁺-activated ion channels modulates endothelin 1 signaling in retinal arteriolar smooth muscle

PURPOSE: To investigate the role of feedback by Ca²?-sensitive plasma-membrane ion channels in endothelin 1 (Et1) signaling in vitro and in vivo. Methods. Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using two-dimensional (2-D) confocal laser microscopy. Vasoconstrictor responses to intravitreal injections of Et1 were recorded in the absence and presence of appropriate ion channel blockers using fluorescein angiograms imaged using a confocal scanning laser ophthalmoscope. Results. Et1 (10 nM) increased both basal [Ca²?](i) and the amplitude and frequency of Ca²?-waves in retinal arterioles. The Ca²?-activated Cl?-channel blockers DIDS and 9-anthracene carboxylic acid (9AC) blocked Et1-induced increases in wave frequency, and 9AC also inhibited the increase in amplitude. Iberiotoxin, an inhibitor of large conductance (BK) Ca²?-activated K?-channels, increased wave amplitude in the presence of Et1 but had no effect on frequency. None of these drugs affected basal [Ca²?](i). The voltage-operated Ca²?-channel inhibitor nimodipine inhibited wave frequency and amplitude and also lowered basal [Ca²?](i) in the presence of Et1. Intravitreal injection of Et1 caused retinal arteriolar vasoconstriction. This was inhibited by DIDS but not by iberiotoxin or penitrem A, another BK-channel inhibitor. Conclusions. Et1 evokes increases in the frequency of arteriolar Ca²?-waves in vitro, resulting in vasoconstriction in vivo. These responses, initiated by release of stored Ca²?, also require positive feedback via Ca²?-activated Cl?-channels and L-type Ca²?-channels.

DETECTION OF CLENBUTEROL RESIDUES IN BOVINE LIVER, MUSCLE, RETINA AND URINE USING GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

A gas chromatographic/mass spectrometric method is described for the detection of clenbuterol residues in liver, muscle, urine and retina. Tissue samples are first digested using protease and any clenbuterol present is extracted using a simple liquid/liquid extraction procedure. The dried extracts are then derivatized using methylboronic acid and the derivatives are subjected to gas chromatography/mass spectrometry on a magnetic sector instrument. The detection limit of the assay is 0.05 ng g-1 clenbuterol in liver, muscle or urine using a 10 g sample size, and 4 ng g-1 in retina using a 0.5 g sample size. The assay is made very specific by using selected ion monitoring of three ions at a resolution of 3500 and by ion ratio measurements. The precision and reproducibility of the assay are enhanced by the use of a deuterated internal standard, with a typical coefficient of variation of 3%.

THE ACTION OF SEROTONIN AND THE NEMATODE NEUROPEPTIDE KSAYMRFAMIDE ON THE PHARYNGEAL MUSCLE OF THE PARASITIC NEMATODE, ASCARIS-SUUM

The pharyngeal component of the enteric nervous system of the parasitic nematode, Ascaris suum exhibits immunoreactivity for serotonin (5-hydroxytryptamine or 5-HT) and for FMRFamide-like peptides. This paper describes the application of an in vitro pharmacological approach to investigate the functional role of 5-HT and FMRFamide-like peptides. The pharyngeal pumping behaviour of Ascaris suum was monitored using a modified pressure transducer system which measures pharyngeal pressure changes and therefore pumping. The pharynx did not contract spontaneously; however, 5-HT (10-1000 mu M) stimulated pumping at a frequency of 0 . 5 Hz. FMRFamide had no apparent effect on pharyngeal pumping. The native nematode FMRFamide-related peptide (FaRP), KSAYMRFamide inhibited the pumping elicited by 5-HT. The duration of inhibition was dose-dependent (0 . 1-1000 nM) with a threshold of 0 . 1 nM. In 4 preparations, the inhibition of the pharyngeal muscle was preceded by an initial excitation and increase in the amplitude of pharyngeal pressure changes. The pharynx is involved in various nematode processes, including feeding, regulation of hydrostatic pressure and excretion. The role of 5-HT and KSAYMRFamide in the pharyngeal function of nematodes is discussed.

Gross anatomy of the muscle systems of Fasciola hepatica as visualized by phalloidin-fluorescence and confocal microscopy

Neuropeptides, biogenic amines and acetylcholine are expressed abundantly within the nervous systems of parasitic flatworms, and are particularly evident in the innervation of the musculature. Such associations have implicated the nervous system in locomotion, host attachment and reproductive co-ordination. Information on the muscle systems of parasitic flatworms is generally sparse, in particular those muscles associated with the reproductive system, intestinal tract and attachment apparatus. Also, the use of sectioned material has left description of the 3-dimensional organization of the musculature largely unrecorded. Using fluorescein isothiocyanate (FITC)-labelled phalloidin as a site-specific probe for filamentous actin, applied to whole-mount preparations of adult Fasciola hepatica and examined by confocal scanning laser microscopy, the present work reports on the organization of the major muscle systems in this trematode parasite. A highly regular array of outer circular, intermediate longitudinal and inner diagonal fibres distinguishes the body wall musculature, which is also involved in the development of both ventral and oral suckers. Circular fibres dominate the duct walls of the male and female reproductive systems, whereas the muscles of the intestinal tract have a somewhat diffuse arrangement of fibres. An understanding of the structural complexity of the muscle systems of parasitic flatworms is considered as fundamental to the interpretation of results from physiological experiments.

Structure-activity relationships of FMRFamide-related peptides contracting Schistosoma mansoni muscle

This study reports the potent myoactivity of flatworm FMRFamide-related peptides (FaRPs) on isolated muscle fibers of the human blood fluke, Schistosoma mansoni. The turbellarian peptides YIRFamide (EC50 4 eta M), GYIRFamide (EC50 1 eta M). and RYIRFamide (EC50 7 eta M), all induced muscle contraction more potently than the cestode FaRP GNFFRFamide (EC50 500 eta M). Using a series of synthetic analogs of the flatworm peptides YIRFamide, GYIRFamide and RYIRFamide, the structure-activity relationships of the muscle FaRP receptor were examined. With a few exceptions, each residue in YIRFamide is important in the maintenance of its myoactivity. Alanine scans resulted in peptides that were inactive (Ala(1), Ala(2), Ala(3) and Ala(4) YIRFamide; Ala(4) and Ala(5) RYIRFamide) or had much reduced potencies (Ala(1), Ala(2) and Ala(3) RYIRFamide). Substitution of the N-terminal (Tyr(1)) residue of YIRFamide with the non-aromatic residues Thr or Arg produced analogs with greatly reduced potency. Replacement of the N-terminal Tyr with aromatic amino acids resulted in myoactive peptides (FIRFamide, EC50 100 eta M; WIRFamide, EC50 0.5 eta M). The activity of YIRFamide analogs which possessed a Leu(2), Phe(2) or Met(2) residue (EC50's 10, 1 and 3 eta M, respectively) instead of Ile(2) was not significantly altered, whereas, YVRFamide had a greatly reduced (EC50 200 eta M) activity. Replacement of the Phe(4) with a Tyr(4) (YIRYamide) also greatly lowered potency. Truncated analogs were either inactive (FRFamide, YRFamide, HRFamide, RFamide, Famide) or had very low potency (IRFamide and MRFamide), with the exception of nLRFamide (EC50 20 eta M). YIRF free acid was inactive. In summary, these data show the general structural requirements of this schistosome muscle FaRP receptor to be similar, but not identical, to those of previously characterized molluscan FaRP receptors. (C) 1997 Elsevier Science Inc.

Pharmacological effects of nematode FMRFamide-related peptides (FaRPs) on muscle contractility of the trematode, Fasciola hepatica

The physiological effects of synthetic replicates of the nematode FaRPs, AF1 (KNEFIRFamide), AF2 (KHEYLRFamide), PF1 (SDPNFLRFamide), PF2 (SADPNFLRFamide), AF8/PF3 (KSAYMRFamide) and PF4 (KPNFIRFamide) were examined on muscle preparations of the liver fluke, Fasciola hepatica. Changes in contractility following the addition of the test compound were recorded using a photo-optic transducer system. Unlike the varied effects these peptides have on nematode somatic musculature, all were found to induce excitatory responses in the muscle activity of F. hepatica. While qualitative effects of the nematode peptides were similar in that they induced increases in both the amplitude and frequency of F. hepatica muscle contractions, they varied considerably in the potency of their excitatory effects. The threshold activity for each peptide was as follows: 10 mu M, PF1 and PF2; 3 mu M, AF1 and PF3; 1 mu M, AF2; and 30 nM, PF4. The results demonstrate, for the first time, the cross-phyla activity of nematode neuropeptides on the neuromuscular activity of a trematode.