In vitro analysis of metabolites from the untreated tissue of Torpedo californica electric organ by mid-infrared laser ablation electrospray ionization mass spectrometry

The neuromuscular junction (NMJ), where a motor neuron intercepts and activates a muscle fiber, is a highly versatile and complex subcellular region. Genomic and proteomic approaches using the large (>1 kg) electric organ of Torpedo californica have helped advancing our understanding of this minute (30–50 μm) electric synapse. However, the majority of these studies have focused on mRNA and proteins, therefore neglecting small signaling molecules involved in muscle-nerve ‘dialogue’. We developed a novel technique, mid-infrared laser ablation electrospray ionization (LAESI) mass spectrometry (MS), with the potential of detecting a diversity of small signaling molecules in vitro. LAESI uses the native water in the tissue as the matrix to couple the laser pulse energy into the target for the ablation process and enables its direct analysis essentially without sample preparation. Here, we report the detection of metabolites from the untreated frozen tissue of the Torpedo electric organ with LAESI MS at atmospheric pressure. A total of 24 metabolites were identified by accurate mass measurements, natural isotope patterns, and tandem mass spectrometry. Most of the identified metabolites were related to the cholinergic function of the electric synapse (acetylcholine and choline), fatty acid metabolism and acetyl transfer (carnitine and acetylcarnitine), the mitigation of osmotic stress (betaine and trimethylamine N-oxide), and energy production (creatine and creatinine). The biosynthetic precursors of these metabolites and their expected degradation products were also detected indicating that LAESI MS is well suited for tissue metabolomics with the ultimate goal of imaging and in vivo studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microsatellite markers characterized in the barn owl (Tyto alba) and of high utility in other owls (Strigiformes: AVES)

We have identified 15 polymorphic microsatellite loci for the barn owl (Tyto alba), five from testing published owl loci and 10 from testing non‐owl loci, including loci known to be of high utility in passerines and shorebirds. All 15 loci were sequenced in barn owl, and new primer sets were designed for eight loci. The 15 polymorphic loci displayed two to 26 alleles in 56–58 barn owls. When tested in 10 other owl species (n = 1–6 individuals), between four and nine loci were polymorphic per species. These loci are suitable for studies of population structure and parentage in owls.     

Delta 12-prostaglandin J2, a plasma metabolite of prostaglandin D2, causes eosinophil mobilization from the bone marrow and primes eosinophils for chemotaxis

PGD(2), a major mast cell mediator, is a potent eosinophil chemoattractant and is thought to be involved in eosinophil recruitment to sites of allergic inflammation. In plasma, PGD(2) is rapidly transformed into its major metabolite delta(12)-PGJ(2), the effect of which on eosinophil migration has not yet been characterized. In this study we found that delta(12)-PGJ(2) was a highly effective chemoattractant and inducer of respiratory burst in human eosinophils, with the same efficacy as PGD(2), PGJ(2), or 15-deoxy-delta(12,14)-PGJ(2). Moreover, pretreatment of eosinophils with delta(12)-PGJ(2) markedly enhanced the chemotactic response to eotaxin, and in this respect delta(12)-PGJ(2) was more effective than PGD(2). delta(12)-PGJ(2)-induced facilitation of eosinophil migration toward eotaxin was not altered by specific inhibitors of intracellular signaling pathways relevant to the chemotactic response, phosphatidylinositol 3-kinase (LY-294002), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (U-0126), or p38 mitogen-activated protein kinase (SB-202190). Desensitization studies using calcium flux suggested that delta(12)-PGJ(2) signaled through the same receptor, CRTH2, as PGD(2). Finally, delta(12)-PGJ(2) was able to mobilize mature eosinophils from the bone marrow of the guinea pig isolated perfused hind limb. Given that delta(12)-PGJ(2) is present in the systemic circulation at relevant levels, a role for this PGD(2) metabolite in eosinophil release from the bone marrow and in driving eosinophil recruitment to sites of inflammation appears conceivable.     

Identification of a functionally important conformation-sensitive region of the secretory Na+-K+-2Cl- cotransporter (NKCC1)

The secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a member of a small gene family of electroneutral salt transporters that play essential roles in salt and water homeostasis in many mammalian tissues. We have identified a highly conserved residue (Ala-483) in the sixth membrane-spanning segment of rat NKCC1 that when mutated to cysteine renders the transporter sensitive to inhibition by the sulfhydryl reagents 2-aminoethyl methanethiosulfonate (MTSEA) and 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The mutation of Ala-483 to cysteine (A483C) results in little or no change in the affinities of NKCC1 for substrate ions but produces a 6-fold increase in sensitivity to the inhibitor bumetanide, suggesting a specific modification of the bumetanide binding site. When residues surrounding Ala-483 were mutated to cysteine, only I484C was sensitive to inhibition by MTSEA and MTSET. Surprisingly I484C showed increased transport activity in the presence of low concentrations of mercury (1-10 microm), whereas A483C showed inhibition. The inhibition of A483C by MTSEA was unaffected by the presence or absence of sodium and potassium but required the presence of extracellular chloride. Taken together, our results indicate that Ala-483 lies at or near an important functional site of NKCC1 and that the exposure of this site to the extracellular medium is dependent on the conformation of the transporter. Specifically, our results indicate that the cysteine introduced at residue 483 is only available for interaction with MTSEA when chloride is bound to NKCC1 at the extracellular surface.     

Evaluation of peristalsis in multiple segments of the guinea-pig isolated small intestine: optimisation of tissue use by refined in vitro methodology

Peristalsis is the aboral movement by which the intestine propels its contents. Since pharmacological research requires an experimental model with which drug-induced modifications of peristalsis can be reliably quantified, we set out to develop and validate an in vitro method for studying peristalsis in multiple gut segments. In our arrangement, up to four 10cm segments isolated from the guinea-pig jejunum and ileum can be set up in parallel and their lumens perfused. Peristalsis was elicited by pressure-evoked wall distension, and the peristalsis-induced changes in the intraluminal pressure were evaluated with software that determined the peristaltic pressure threshold, the frequency, maximal acceleration and amplitude of the peristaltic waves, and the residual baseline pressure. Validation experiments showed that the peristalsis parameters at baseline and after modification by morphine (0.01-10microM) did not differ between segments from the jejunum and ileum, or between segments examined in a consecutive manner. In conclusion, our work succeeded in optimising the use of the guinea-pig jejunum and ileum for multiple recordings of peristalsis in vitro, and in refining the recording and evaluation of peristaltic motility. This system promises to be particularly useful in the pharmacological screening and testing of drugs which modify peristalsis.     

Different roles of PKC and MAP kinases in arteriolar constrictions to pressure and agonists

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinases have been implicated in the modulation of agonist-induced contractions of large vessels. However, their role in pressure- and agonist-induced constrictions of skeletal muscle arterioles, which have a major role in regulating peripheral resistance, is not clearly elucidated. Thus constrictions of isolated rat gracilis muscle arterioles (approximately 80 microm in diameter) to increases in intraluminal pressure and to norepinephrine (NE) or angiotensin II (ANG II) were assessed in the absence or presence of chelerythrine, PD-98058, and SB-203580 (inhibitors of PKC, p42/44 and p38 MAP kinase pathways, respectively). Arteriolar constriction to NE and ANG II were significantly reduced by chelerythrine (by approximately 90%) and unaffected by SB-203580, whereas PD-98058 decreased only ANG II-induced constrictions (by approximately 60%). Pressure-induced increases in wall tension (from 0.1 to 0.7 N/m) resulted in significant arteriolar constrictions (50% maximum) that were abolished by chelerythrine without altering smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) (fura 2 microfluorimetry). PD-98058 and SB-203580 significantly decreased the magnitude of myogenic tone (by 20% and 60%, respectively) and reduced the sensitivity of the myogenic mechanism to wall tension, causing a significant rightward shift in the wall tension-myogenic tone relationship without affecting smooth muscle [Ca(2+)i]. MAP kinases were demonstrated with Western blotting. Thus in skeletal muscle arterioles 1) PKC is involved in both myogenic and agonist-induced constrictions, 2) PD-98058-sensitive p42/44 MAP kinases modulate both wall tension-dependent and ANG II-induced constrictions, whereas 3) a SB-203580-sensitive p38 MAP kinase pathway seems to be specifically involved in the mechanotransduction of wall tension.     

Signal Transduction in Smooth Muscle: Selected Contribution: NO released to flow reduces myogenic tone of skeletal muscle arterioles by decreasing smooth muscle Ca2+ sensitivity

To clarify the contribution of intracellularCa²⁺ concentration([Ca²⁺]_i)-dependent and -independentsignaling mechanisms in arteriolar smooth muscle (aSM) to modulation ofarteriolar myogenic tone by nitric oxide (NO), released in response toincreases in intraluminal flow from the endothelium, changes in aSM[Ca²⁺]_i and diameter of isolated rat gracilismuscle arterioles (pretreated with indomethacin) were studied byfluorescent videomicroscopy. At an intraluminal pressure of 80 mmHg, [Ca²⁺]_i significantly increased andmyogenic tone developed in response to elevations of extracellularCa²⁺ concentration. The Ca²⁺ channelinhibitor nimodipine substantially decreased[Ca²⁺]_i and completely inhibited myogenictone. Dilations to intraluminal flow (that were inhibited byN-nitro-L-arginine methyl ester)or dilations to the NO donorS-nitroso-N-acetyl-DL-penicillamine (that were inhibited by the guanylate cyclase inhibitor1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) were notaccompanied by substantial decreases in aSM[Ca²⁺]_i. 8-Bromoguanosine cGMP and thecGMP-specific phosphodiesterase inhibitor zaprinast significantlydilated arterioles yet elicited only minimal decreases in[Ca²⁺]_i. Thus flow-induced endothelialrelease of NO elicits relaxation of arteriolar smooth muscle by acGMP-dependent decrease of the Ca²⁺ sensitivity of thecontractile apparatus without substantial changes in thepressure-induced level of [Ca²⁺]_i.

     

Inhibition of glutamate-induced delayed calcium deregulation by 2-APB and La3+ in cultured cortical neurones

Exposure of neurones in culture to excitotoxic levels of glutamate results in an initial transient spike in [Ca2+]i followed by a delayed, irreversible [Ca2+]i rise governed by rapid kinetics, with Ca2+ originating from the extracellular medium. The molecular mechanism responsible for the secondary Ca2+ rise is unknown. Here, we report that the delayed Ca2+ entry in cortical neurones is diminished by 2-aminoethoxydiphenyl borate (2-APB: IC50 = 62 +/- 9 microm) and La3+ (IC50 = 7.2 +/- 3 microm), both known to inhibit transient receptor potential (TRP) and store-operated Ca2+ (SOC) channels. Application of thapsigargin, however, failed to exacerbate the delayed Ca2+ deregulation, arguing against a store depletion event as the stimulus for induction of the secondary [Ca2+]i rise. In addition, these neurones did not exhibit SOC entry. Unexpectedly, application of ryanodine or caffeine significantly inhibited glutamate-induced delayed Ca2+ deregulation. In basal Ca2+ entry experiments, La3+ and 2-APB modulated the rapid rise in [Ca2+]i caused by exposure of neurones to Ca2+ after pre-incubating in a calcium-free medium. This basal Ca2+ influx was mitigated by extracellular Mg2+ but not aggravated by thapsigargin, ryanodine or caffeine. These results indicate that 2-APB and La3+ influence non-store-operated Ca2+ influx in cortical neurones and that this route of Ca2+ entry is involved in glutamate-induced delayed Ca2+ deregulation.