Calcium-free Solution Research Articles

TRPA1-Mediated Accumulation of Aminoglycosides in Mouse Cochlear Outer Hair Cells

Aminoglycoside ototoxicity involves the accumulation of antibiotic molecules in the inner ear hair cells and the subsequent degeneration of these cells. The exact route of entry of aminoglycosides into the hair cells in vivo is still unknown. Similar to other small organic cations, aminoglycosides could be brought into the cell by endocytosis or permeate through large non-selective cation channels, such as mechanotransduction channels or ATP-gated P2X channels. Here, we show that the aminoglycoside antibiotic gentamicin can enter mouse outer hair cells (OHCs) via TRPA1, non-selective cation channels activated by certain pungent compounds and by endogenous products of lipid peroxidation. Using conventional and perforated whole-cell patch clamp recordings, we found that application of TRPA1 agonists initiates inward current responses in wild-type OHCs, but not in OHCs of homozygous Trpa1 knockout mice. Similar responses consistent with the activation of non-selective cation channels were observed in heterologous cells transfected with mouse Trpa1. Upon brief activation with TRPA1 agonists, Trpa1-transfected cells become loaded with fluorescent gentamicin-Texas Red conjugate (GTTR). This uptake was not observed in mock-transfected or non-transfected cells. In mouse organ of Corti explants, TRPA1 activation resulted in the rapid entry of GTTR and another small cationic dye, FM1-43, in OHCs and some supporting cells, even when hair cell mechanotransduction was disrupted by pre-incubation in calcium-free solution. This TRPA1-mediated entry of GTTR and FM1-43 into OHCs was observed in wild-type but not in Trpa1 knockout mice and was not blocked by PPADS, a non-selective blocker of P2X channels. Notably, TRPA1 channels in mouse OHCs were activated by 4-hydroxynonenal, an endogenous molecule that is known to be generated during episodes of oxidative stress and accumulate in the cochlea after noise exposure. We concluded that TRPA1 channels may provide a novel pathway for the entry of aminoglycosides into OHCs.

Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent

Levobupivacaine is a long-acting local anesthetic that intrinsically produces vasoconstriction in isolated vessels. The goals of this study were to investigate the calcium-dependent mechanism underlying levobupivacaine-induced contraction of isolated rat aorta in vitro and to elucidate the pathway responsible for the endothelium-dependent attenuation of levobupivacaine-induced contraction. Isolated rat aortic rings were suspended to record isometric tension. Cumulative levobupivacaine concentration-response curves were generated in either the presence or absence of the antagonists verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, Gd(3+), N(W)-nitro-l-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and methylene blue, either alone or in combination. Verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, low calcium concentrations, and calcium-free Krebs solution attenuated levobupivacaine-induced contraction. Gd(3+) had no effect on levobupivacaine-induced contraction. Levobupivacaine increased intracellular calcium levels in vascular smooth muscle cells. L-NAME, ODQ, and methylene blue increased levobupivacaine-induced contraction in endothelium-intact aorta. SKF-96365 attenuated calcium-induced contraction in a previously calcium-free isotonic depolarizing solution containing 100mmol/L KCl. Levobupivacaine-induced contraction of rat aortic smooth muscle is mediated primarily by calcium influx from the extracellular space mainly via voltage-operated calcium channels and, in part, by inositol 1,4,5-trisphosphate receptor-mediated release of calcium from the sarcoplasmic reticulum. The nitric oxide - cyclic guanosine monophosphate pathway is involved in the endothelium-dependent attenuation of levobupivacaine-induced contraction.

Pressure induces intracellular calcium changes in juxtaglomerular cells in perfused afferent arterioles

Calcium (Ca(2+)) has an important role in nearly all types of cellular secretion, with a particularly novel role in the juxtaglomerular (JG) cells in the kidney. In JG cells, Ca(2+) inhibits renin secretion, which is a major regulator of blood pressure and renal hemodynamics. However, whether alterations in afferent arteriolar (Af-Art) pressure change intracellular Ca(2+) concentration ([Ca(2+)](i)) in JG cells and whether [Ca(2+)](i) comes from extracellular or intracellular sources remains unknown. We hypothesize that increases in perfusion pressure in the Af-Art result in elevations in [Ca(2+)](i) in JG cells. We isolated and perfused Af-Art of C57BL6 mice and measured changes in [Ca(2+)](i) in JG cells in response to perfusion pressure changes. The JG cells' [Ca(2+)](i) was 93.3±2.2 nM at 60 mm Hg perfusion pressure and increased to 111.3±13.4, 119.6±7.3, 130.3±2.9 and 140.8±12.1 nM at 80, 100, 120 and 140 mm Hg, respectively. At 120 mm Hg, increases in [Ca(2+)](i) were reduced in mice receiving the following treatments: (1) the mechanosensitive cation channel blocker, gadolinium (94.6±7.5 nM); (2) L-type calcium channel blocker, nifedipine (105.8±7.5 nM); and (3) calcium-free solution plus ethylene glycol tetraacetic acid (96.0±5.8 nM). Meanwhile, the phospholipase C inhibitor, inositol triphosphate receptor inhibitor, T-type calcium channel blocker, N-type calcium channel blocker and Ca(2+)-ATPase inhibitor did not influence changes in [Ca(2+)](i) in JG cells. In summary, JG cell [Ca(2+)](i) rise as perfusion pressure increases; furthermore, the calcium comes from extracellular sources, specifically mechanosensitive cation channels and L-type calcium channels.

Possible Involvement of Calmodulin in the Spasmolytic Effect of Extracts from Sabal serrulata Fruits

The effect of total lipidic and saponifiable extracts from Sabal serrulata fruits on contraction induced by vanadate (0–3 mM) in the uterus of Wistar rats incubated in calcium-free solution have been assayed. The two extracts relaxed the contraction induced by vanadate. The EC50 values were 43.9 ± 8.4 and 11.4 ± 1.4 μg mL−1 for total lipid and saponifiable material, respectively. Total lipid and saponifiable extracts also inhibited calmodulin-dependent cAMP phosphodiesterase activity in a dose-dependent way. The EC50 values were 25.1 ± 4 and 28.6 ± 4 μg mL−1 for total lipid and saponifiable material, respectively. Phosphodiesterase basal activity was only modified with the maximum concentration (100 μg mL−1). These results suggest that the effects of extracts from Sabal serrulata fruits could be due, at least partially, to the inhibition of calmodulin but other mechanisms cannot be excluded.

Heavy metal cations permeate the TRPV6 epithelial cation channel

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca2+, Ba2+, Sr2+, Mn2+, Zn2+, Cd2+, and interestingly also for La3+ and Gd3+. These results were confirmed using the patch clamp technique. 45Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.

Depolarization-Stimulated Contractility of Gastrointestinal Smooth Muscle in Calcium-Free Solution: A Review

The membrane of most gastrointestinal smooth muscles shows slow waves, slow rhythmic changes in membrane potential. Slow waves serve to bring the membrane potential of smooth muscle cells to a threshold level that elicits a second electrical event known as the spike or action potential. The inward current of the spike, in most gastrointestinal smooth muscle preparations, is carried, at least in part, by calcium. Indeed, considering the narrow diameter of smooth muscle cells, some have hypothesized that the influx of calcium during the spike is sufficient for activation of the contractile machinery. Findings consistent with this include marked reduction in contractility during exposure of muscle segments to blockers of L-type calcium channels or following reductions in external calcium levels. However, it has also been observed that following exposure of muscle segments to external bathing solutions containing no added calcium plus 5 mM EGTA to remove any remaining extracellular calcium, contractions can be triggered following membrane depolarization. It is noteworthy that in isolated smooth muscle cells or in small muscle segments, during incubation in calcium-free solution, depolarization does not induce contractions. The present paper discusses the evidence in support of depolarization-mediated contractions occurring in gastrointestinal smooth muscle segments during incubation in solutions devoid of calcium.

Genistein stimulates duodenal HCO3− secretion through PI3K pathway in mice

Genistein has been proposed as a promising pharmacotherapeutic for cystic fibrosis. We recently found that genistein stimulates murine duodenal HCO3− secretion through cystic fibrosis transmembrane conductance regulator (CFTR). The aim of the present study was to determine the intracellular signal pathways involved in genistein-stimulated duodenal HCO3− secretion. Murine duodenal mucosal HCO3− secretion was examined in vitro in Ussing chambers by the pH-stat technique. The results showed that neither cAMP-dependent signal pathway inhibitors MDL-12330A and KT-5720, nor cGMP signal pathway inhibitors NS2028 and KT5823, nor calcium signal pathway inhibitors verapamil and W-13, altered genistein-stimulated duodenal HCO3− secretion. In calcium-free solution, genistein-stimulated duodenal HCO3− secretion was not altered either. Vanadate, an inhibitor of protein tyrosine phosphatase, only partially inhibited genistein-stimulated duodenal HCO3− secretion. However, both wortmannin and LY294002, two structurally and mechanistically distinct phosphatidylinositol 3-kinase (PI3K) inhibitors, markedly inhibited genistein-stimulated duodenal HCO3− secretion. Genistein increased duodenal mucosal PI3K activity and induced the phosphorylation of Akt, a signaling molecule downstream of PI3K, which was again inhibited by wortmannin. Estrogen receptor antagonist, ICI182,780, also markedly inhibited genistein-stimulated duodenal HCO3− secretion and genistein-induced PI3K activity increase in duodenal mucosa. These results demonstrate that genistein stimulates duodenal HCO3− secretion mainly through estrogen receptor and PI3K-dependent pathway. These findings contribute to the understanding of the molecular mechanism of genistein-induced anion secretion and further pharmacotherapeutic development and use of genistein or related substances in the treatment of diseases of epithelial tissues.

Lateral Cell Membranes and Shunt Resistance in Rabbit Esophageal Epithelium

The structures that contribute to shunt resistance (Rs) in esophageal epithelium are incompletely understood, with 35-40% of Rs known to be calcium-dependent, reflecting the role of e-cadherin. Two calcium-independent candidates for the remaining approximately 60% of Rs have been identified: the glycoprotein matrix (GPM) within stratum corneum of esophageal epithelium, and the lateral cell membranes (LCMs) from neighboring cells. To determine the contribution of GPM and LCMs to Rs, rabbit esophageal epithelium was mounted in Ussing chambers so that transepithelial resistance (R(T)), a marker of Rs, could be monitored during luminal exposure to either glycosidases for disruption of the GPM or to hypertonic urea for separation of the LCMs. Glycosidases had no effect on R(T). In contrast, hypertonic urea reduced R(T), increased fluorescein flux and widened the intercellular spaces. That urea reduced R(T), and so Rs, by widening the intercellular spaces, and not by altering the e-cadherin-dependent apical junctional complex, was supported by the ability of: (a) calcium-free solution to reduce R(T) beyond that produced by urea, (b) hypertonic urea to reduce R(T) beyond that produced by calcium free solution, (c) hypertonic sucrose to collapse the intercellular spaces and raise R(T), and (d) empigen, a zwitterionic detergent, to non-osmotically widen the intercellular spaces and reduce R(T). These data indicate that the LCMs from neighboring cells are a major contributor to shunt resistance in esophageal epithelium. As resistor, they are distinguishable from the apical junctional complex by their sensitivity to (luminal) hypertonicity and insensitivity to removal of calcium.

Trafficking and Surface Expression of Hyperpolarization-activated Cyclic Nucleotide-gated Channels in Hippocampal Neurons

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate the hyperpolarization-activated current I(h) and thus play important roles in the regulation of brain excitability. The subcellular distribution pattern of the HCN channels influences the effects that they exert on the properties and activity of neurons. However, little is known about the mechanisms that control HCN channel trafficking to subcellular compartments or that regulate their surface expression. Here we studied the dynamics of HCN channel trafficking in hippocampal neurons using dissociated cultures coupled with time lapse imaging of fluorophore-fused HCN channels. HCN1-green fluorescence protein (HCN1-GFP) channels resided in vesicle-like organelles that moved in distinct patterns along neuronal dendrites, and these properties were isoform-specific. HCN1 trafficking required intact actin and tubulin and was rapidly inhibited by activation of either NMDA or AMPA-type ionotropic glutamate receptors in a calcium-dependent manner. Glutamate-induced inhibition of the movement of HCN1-GFP-expressing puncta was associated with increased surface expression of both native and transfected HCN1 channels, and this surface expression was accompanied by augmented I(h). Taken together, the results reveal the highly dynamic nature of HCN1 channel trafficking in hippocampal neurons and provide a novel potential mechanism for rapid regulation of I(h), and hence of neuronal properties, via alterations of HCN1 trafficking and surface expression.

S1799 Specialized Spinal Afferent Neurons to Detect Mucosal Shear in Guinea-Pig Colon

Background and aims. Extrinsic sensory neurons to the gut mediate both sensation and gastrointestinal reflexes important for gut function in health and disease. Different classes of afferents which convey various types of information about the gut wall and contents can be reliably identified by characterizing the morphology of their physiologically-identified peripheral axons. “Muscular mucosal” afferents have been identified on the basis of their responses to both colorectal distension and to light mucosal stroking. Methods. Extracellular recording from colonic nerves in isolated preparations of guinea pig distal colon were combined with biotinamide filling In Vitro. Results. When all layers of the colon were present in the preparation, a class of afferents could be strongly activated by light von Frey hairs (0.1 10 mN, maximum instantaneous firing: 83+/-18 Hz, n=6). The same units could also be activated by circumferential distension with constant loads (10 100mN) evoking firing of 52+/-30 Hz (n=10). Both types of activity were preserved in preparations where the muscularis externa had been removed prior to recording, suggesting that transduction sites were located in the submucosa or mucosa. Surprisingly, afferents of this class also fired in concert with spontaneous contractions of preparations, measured under isotonic conditions. The action potentials of these units were significantly larger and of shorter duration than action potentials from mechanonociceptors axons recorded from the same nerve trunks (341+/-146 vs 139+/-34μV P<0.01, 0.4+/-0.1 vs 1.1+/-0.8 ms, P<0.05, n=9). None of 9 “muscular mucosal” axons recorded responded to focal application of capsaicin (0.3 μM) and their mechanosensitivity persisted in calcium-free solution (with raised [Mg++]). This suggests that they are intrinsically mechanosensitive rather than relying on release of mediators from other cells. Biotinamide dye filling revealed fine branching axons running in the subepithelial plexus in regions corresponding closely to mechanosensitive receptive fields of these units (n=4). Conclusions. We conclude that “muscular mucosal” afferents comprise a discrete and distinctive class of spinal afferent innervation, well suited to detect physiological levels of mucosal shear in the distal colon of the guinea-pig.

S1800 Characterization of a Reproducible Gastric Pain Model Using Oral Capsaicin Titration: A Placebo-Controlled, Double-Blind Study

Background and aims. Extrinsic sensory neurons to the gut mediate both sensation and gastrointestinal reflexes important for gut function in health and disease. Different classes of afferents which convey various types of information about the gut wall and contents can be reliably identified by characterizing the morphology of their physiologically-identified peripheral axons. “Muscular mucosal” afferents have been identified on the basis of their responses to both colorectal distension and to light mucosal stroking. Methods. Extracellular recording from colonic nerves in isolated preparations of guinea pig distal colon were combined with biotinamide filling In Vitro. Results. When all layers of the colon were present in the preparation, a class of afferents could be strongly activated by light von Frey hairs (0.1 10 mN, maximum instantaneous firing: 83+/-18 Hz, n=6). The same units could also be activated by circumferential distension with constant loads (10 100mN) evoking firing of 52+/-30 Hz (n=10). Both types of activity were preserved in preparations where the muscularis externa had been removed prior to recording, suggesting that transduction sites were located in the submucosa or mucosa. Surprisingly, afferents of this class also fired in concert with spontaneous contractions of preparations, measured under isotonic conditions. The action potentials of these units were significantly larger and of shorter duration than action potentials from mechanonociceptors axons recorded from the same nerve trunks (341+/-146 vs 139+/-34μV P<0.01, 0.4+/-0.1 vs 1.1+/-0.8 ms, P<0.05, n=9). None of 9 “muscular mucosal” axons recorded responded to focal application of capsaicin (0.3 μM) and their mechanosensitivity persisted in calcium-free solution (with raised [Mg++]). This suggests that they are intrinsically mechanosensitive rather than relying on release of mediators from other cells. Biotinamide dye filling revealed fine branching axons running in the subepithelial plexus in regions corresponding closely to mechanosensitive receptive fields of these units (n=4). Conclusions. We conclude that “muscular mucosal” afferents comprise a discrete and distinctive class of spinal afferent innervation, well suited to detect physiological levels of mucosal shear in the distal colon of the guinea-pig.

S1803 Early Life Esophageal Acid Exposure Accentuates the Expression of Nr1 and PSD-95 in ACC Neurons in Response to Acid Rechallenge in Adulthood

Background and aims. Extrinsic sensory neurons to the gut mediate both sensation and gastrointestinal reflexes important for gut function in health and disease. Different classes of afferents which convey various types of information about the gut wall and contents can be reliably identified by characterizing the morphology of their physiologically-identified peripheral axons. “Muscular mucosal” afferents have been identified on the basis of their responses to both colorectal distension and to light mucosal stroking. Methods. Extracellular recording from colonic nerves in isolated preparations of guinea pig distal colon were combined with biotinamide filling In Vitro. Results. When all layers of the colon were present in the preparation, a class of afferents could be strongly activated by light von Frey hairs (0.1 10 mN, maximum instantaneous firing: 83+/-18 Hz, n=6). The same units could also be activated by circumferential distension with constant loads (10 100mN) evoking firing of 52+/-30 Hz (n=10). Both types of activity were preserved in preparations where the muscularis externa had been removed prior to recording, suggesting that transduction sites were located in the submucosa or mucosa. Surprisingly, afferents of this class also fired in concert with spontaneous contractions of preparations, measured under isotonic conditions. The action potentials of these units were significantly larger and of shorter duration than action potentials from mechanonociceptors axons recorded from the same nerve trunks (341+/-146 vs 139+/-34μV P<0.01, 0.4+/-0.1 vs 1.1+/-0.8 ms, P<0.05, n=9). None of 9 “muscular mucosal” axons recorded responded to focal application of capsaicin (0.3 μM) and their mechanosensitivity persisted in calcium-free solution (with raised [Mg++]). This suggests that they are intrinsically mechanosensitive rather than relying on release of mediators from other cells. Biotinamide dye filling revealed fine branching axons running in the subepithelial plexus in regions corresponding closely to mechanosensitive receptive fields of these units (n=4). Conclusions. We conclude that “muscular mucosal” afferents comprise a discrete and distinctive class of spinal afferent innervation, well suited to detect physiological levels of mucosal shear in the distal colon of the guinea-pig.

Evidence of sternal artery vasoconstriction in Crayfish, Procambarus clarkii (Pc) but not Pacifastacus leniusculus (Pl)

Procambarus clarkii (Pc) are known to re‐distribute arterial flows when exposed to hypoxic water. In response to hypoxia, flow to the anterior aorta increases while flows to the posterior dorsal aorta and sternal artery decrease, causing an increase of hemolymph flow to the animal's head. For this study we compared isolated blood vessel function from Pc and another crayfish, Pacifastacus leniusculus (Pl). Both crayfish survive for some time out of water, but Pl is more active physically and has narrower arteries. Sternal arteries from both species were isolated. Compliance was measured in cannulated pressurized blood vessels and isometric force was measured on a wire myograph with bicarbonate‐buffered crayfish saline. After relaxation in a calcium‐free solution containing 0.1 mM EGTA, KCl depolarization produced a small transient contraction for Pc but this response has not been observed for Pl. We are now investigating compliance curves in bicarbonate‐buffered crayfish saline in the presence or absence of calcium for Pc and Pl. Vasoconstriction in arteries may have effects on blood flow resistance in Pc but not in Pl.

Evidence for a protein tether involved in somatic touch

The gating of ion channels by mechanical force underlies the sense of touch and pain. The mode of gating of mechanosensitive ion channels in vertebrate touch receptors is unknown. Here we show that the presence of a protein link is necessary for the gating of mechanosensitive currents in all low-threshold mechanoreceptors and some nociceptors of the dorsal root ganglia (DRG). Using TEM, we demonstrate that a protein filament with of length ∼100 nm is synthesized by sensory neurons and may link mechanosensitive ion channels in sensory neurons to the extracellular matrix. Brief treatment of sensory neurons with non-specific and site-specific endopeptidases destroys the protein tether and abolishes mechanosensitive currents in sensory neurons without affecting electrical excitability. Protease-sensitive tethers are also required for touch-receptor function in vivo. Thus, unlike the majority of nociceptors, cutaneous mechanoreceptors require a distinct protein tether to transduce mechanical stimuli.

Hydrostatic Pressure–Induced Release of Stored Calcium in Cultured Rat Optic Nerve Head Astrocytes

Elevated intraocular pressure is associated with glaucomatous optic nerve damage. Other investigators have shown functional changes in optic nerve head astrocytes subjected to elevated hydrostatic pressure (HP) for 1 to 5 days. Recently, the authors reported ERK1/2, p90(RSK) and NHE1 phosphorylation after 2 hours. Here they examine calcium responses at the onset of HP to determine what precedes ERK1/2 phosphorylation. Cytoplasmic calcium concentration ([Ca(2+)](i)) was measured in cultured rat optic nerve astrocytes loaded with fura-2. The cells were placed in a closed imaging chamber and subjected to an HP increase of 15 mm Hg. Protein phosphorylation was detected by Western blot analysis. The increase of HP caused an immediate slow increase in [Ca(2+)](i). The response persisted in calcium-free solution and when nickel chloride (4 mM) was added to suppress channel-mediated calcium entry. Previous depletion of the ER calcium stores by cyclopiazonic acid abolished the HP-induced calcium level increase. The HP-induced increase persisted in cells exposed to xestospongin C, an inhibitor of IP3R-mediated calcium release. In contrast, ryanodine receptor (RyR) antagonist ruthenium red (10 microM) or dantrolene (25 microM) inhibited the HP-induced calcium increase. The HP-induced calcium increase was abolished when ryanodine-sensitive calcium stores were pre-depleted with caffeine (3 mM). HP caused ERK1/2 phosphorylation. The magnitude of the ERK1/2 phosphorylation response was reduced by ruthenium red and dantrolene. Increasing HP causes calcium release from a ryanodine-sensitive cytoplasmic store and subsequent ERK1/2 activation. Calcium store release appears to be a required early step in the initial astrocyte response to an HP increase.

Rhythmic contraction generates adjustable passive stiffness in rabbit detrusor

The length-tension (L-T) relationships in airway and vascular smooth muscles have been shown to adapt with length changes over time. Our prior studies have shown that the active and passive L-T relationships in rabbit detrusor smooth muscle (DSM) can adapt and that DSM exhibits adjustable passive stiffness (APS) characterized by a passive L-T curve that is a function of strain and activation history. The present study demonstrates that passive tension due to APS can represent a substantial fraction of total tension over a broad length range. Our previous studies have shown that maximal KCl-induced contractions at short muscle lengths generate APS that is revealed by increased pseudo-steady-state passive tension at longer lengths compared with previous measurements at those lengths. The objective of the present study was to determine the mechanisms involved in APS generation. Increasing the number of KCl-induced contractions or the duration of a contraction increased the amount of APS generated. Furthermore, a fraction of APS was restored in calcium-free solution and was sensitive to the general serine and threonine protein kinase inhibitor staurosporine. Most importantly, rhythmic contraction (RC) generated APS, and because RC occurs spontaneously in human bladder, a physiological role for RC was potentially identified.

The Effect of Calcium Ions and Peptide Ligands on the Relative Stabilities of the Calmodulin Dumbbell and Compact Structures

A combination of ion mobility and mass spectrometry methods was used to characterize the molecular shape of the protein calmodulin (CaM) and its complexes with calcium and a number of peptide ligands. CaM, a calcium-binding protein composed of 148 amino acid residues, was found by X-ray crystallography to occur both in a globular shape and in the shape of an extended dumbbell. Here, it was found, as solutions of CaM and CaM complexes were sprayed into the solvent-free environment of the mass spectrometer, that major structural features of the molecule and the stoichiometry of the units constituting a complex in solution were preserved in the desolvation process. Two types of CaM structures were observed in our experiments: a compact and an extended form of CaM with measured cross sections in near-perfect agreement with those calculated for the known globular and extended dumbbell X-ray geometries. Calcium-free solutions yielded predominantly an extended CaM conformation. Ca(n)(2+)-CaM complexes were observed in calcium-containing solutions, n = 0-4, with the population of the compact conformation increasing relative to the elongated conformation as n increases. For n = 4, a predominantly compact globular conformation was observed. Solutions containing the peptide CaMKII(290-309), the CaM target domain of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) enzyme, yielded predominantly globular Ca(4)(2+)-CaM-CaMKII(290-309) complexes. Similar results were obtained with the 26-residue peptide melittin. For the 14-residue C-terminal melittin fragment, on the other hand, formation of both a 1:1 and a 1:2 CaM-peptide complex was detected. On the basis of the entirety of our results, we conclude that the collapse of extended (dumbbell-like) CaM structures into more compact globular structures occurs upon specific binding of four calcium ions. Furthermore, this calcium-induced structural collapse of CaM appears to be a prerequisite for formation of a particularly stable CaM-peptide complex involving peptides long enough to be engaged in interactions with both lobes of CaM.

The Direct Effect of Levobupivacaine in Isolated Rat Aorta Involves Lipoxygenase Pathway Activation and Endothelial Nitric Oxide Release

Levobupivacaine is a long-acting local anesthetic with a clinical profile similar to that of racemic bupivacaine but with a greater margin of safety. Levobupivacaine produces dose-dependent vasoconstriction in vivo. Our goal in this in vitro study was to investigate the role of pathways involved in arachidonic acid metabolism in the levobupivacaine-induced contraction of isolated rat aorta and to determine which endothelium-derived vasodilators are involved in the modulation of levobupivacaine-induced contraction. Rat thoracic aortic rings were isolated and suspended for isometric tension recording. Cumulative levobupivacaine dose-response curves over a range of 10(-6) to 3 x 10(-4) M were constructed in 1) aortic rings with no drug pretreatment; 2) endothelium-denuded rings pretreated with quinacrine dihydrochloride (nonspecific phospholipase A(2) inhibitor: 2 x 10(-5), 4 x 10(-5) M), nordihydroguaiaretic acid (NDGA) (lipoxygenase inhibitor: 10(-5), 3 x 10(-5) M), indomethacin (nonspecific cyclooxygenase inhibitor: 10(-5) M), AA-861 (5-lipoxygenase inhibitor: 10(-5), 5 x 10(-5) M), fluconazole (cytochrome P450 epoxygenase inhibitor: 10(-5) M), verapamil (10(-5) M), or calcium-free solution; and 3) endothelium-intact rings pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME) (nitric oxide synthase inhibitor: 5 x 10(-5) M), indomethacin, or fluconazole. Levobupivacaine-induced contractile response at each concentration (10(-4), 3 x 10(-4) M) was assessed in endothelium-denuded rings. Dose-response curves for potassium chloride in endothelium-denuded rings were generated in the presence or absence of NDGA and AA-861. Intracellular Ca(2+) levels were monitored by Ca(2+) image analysis using Fluo-4 fluorescence in vascular smooth muscle cells treated with levobupivacaine alone or AA-861 plus levobupivacaine. Levobupivacaine produced a tonic contraction in isolated rat aorta rings; this response was maximal at 10(-4) M levobupivacaine and gradually attenuated at 3 x 10(-4) M levobupivacaine. Levobupivacaine-induced contractions of endothelium-denuded rings were larger than those of endothelium-intact rings. Levobupivacaine-induced contraction of endothelium-denuded rings was attenuated by quinacrine dihydrochloride, NDGA, AA-861, verapamil, and calcium-free solution and, to a lesser extent, by indomethacin. L-NAME enhanced levobupivacaine-induced contraction of endothelium-intact rings and indomethacin slightly attenuated this contraction. NDGA and AA-861 attenuated the potassium chloride-induced contraction. AA-861 attenuated the levobupivacaine-induced intracellular calcium increase in vascular smooth muscle cells. Our data indicate that levobupivacaine-induced contraction of rat aortic smooth muscle is mediated mainly by activation of the lipoxygenase pathway and in part by activation of the cyclooxygenase pathway. In addition, activation of the lipoxygenase pathway seems to facilitate calcium influx via L-type calcium channels. Endothelial nitric oxide attenuates levobupivacaine-induced contraction.

Impaired cellular contractile function in thiamine‐deficient rat cardiomyocytes

Background Thiamine (vitamin B1) is an essential cofactor of important metabolic enzymes. 1,2 Studies have demonstrated that thiamine deficiency (TD) leads to changes in cardiac morphological, electrophysiological and mechanical properties, as well as in cardiac metabolism. 3 - 9 Clinical studies have indicated that the use of loop diuretics can induce TD and eventually cause heart dysfunction. The most important reported clinical manifestation of TD involves high-output heart failure. In humans, TD damages the cardiovascular system causing heart failure. 10-12 The altered excitation-contraction coupling that characterizes cardiac failure occurs due to changes in Ca 2+ dynamics, action potential duration, global Ca 2+ transient, and contraction and relaxation rates. 13 Oliveira et al. 8 found that thiamine deprivation decreased the global Ca 2+ release from the sarcoplasmic reticulum (SR) due to the lower SR Ca 2+ load without changes in Ca 2+ current density. Aim According to data in the literature, TD may elicit deregulation of SR Ca 2+ handling in the heart, with implications on cellular cardiac contractility. However, it is unknown whether TD would cause contractile dysfunction at the cellular level. In this study, we sought to determine if TD affects cellular contractility. Methods Male Wistar rats (200-250 g) were randomly allocated to receive a diet containing thiamine (control, n = 6) or a TD diet (TD, n = 6) for 35 days as described previously by our group. 8,9 All experimental procedures were conducted in accordance with international guidelines (Declaration of Helsinki). Cell isolation Ventricular cardiomyocytes from age-matched control and TD rats were enzymatically isolated as described previously. 8,9 Briefly, the heart was mounted on a custom-designed Langendorff system, perfused for ∼3-5 min with calcium-free solution following perfusion with 1 mg/mL collagenase type II (Worthington, USA). After 10-15 min, single cells were isolated by mechanical dispersion and stored in Dulbecco's modified Eagle's medium. The average cell yield under these conditions is 60-70%. Measurement of cellular contractility Cellular contractility was measured as described previously. 14 Briefly, isolated cells were placed in an experimental chamber with a glass cover-slip base mounted on the stage of an inverted microscope. Cells were perfused with Tyrodes solution containing 1.8 mM CaCl 2 and field stimulated at frequencies of 1 and 3 Hz (20 V, 5 ms duration square pulses). Cells were imaged using a NTSC camera in a partial scanning mode. Cell shortening in response to electrical stimulation was measured with a video-edge detection system at 240 Hz frame rate (lonoptix). All parameters were evaluated using customized software developed in MatLab ® platform. 14 Experiments were performed at room temperature (∼25°C). Only calcium-tolerant, quiescent, rod-shaped myocytes showing clear cross striations were studied. Statistical analysis All results are expressed as mean ± standard error of the mean. For statistical analysis, we used Student's t-test and ANOVA for repeated measurements with Bonferroni post hoc testing. Values of P < 0.05 were considered significant. Results After 35 days of thiamine deprivation, cellular ATP availability decreases, leading to impairment of heart contraction as we reported in a previous study. 8 Thus, we hypothesized that a reduction in circulating thiamine levels would affect cardiomyocyte.

Endothelium-Dependent Vasodilator Effects of Peroxisome Proliferator-Activated Receptor β Agonists via the Phosphatidyl-Inositol-3 Kinase-Akt Pathway

Peroxisome proliferator-activated receptor beta/delta (PPAR-beta) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily that regulates the transcription of many target genes. More recently, acute, nongenomic effects of PPAR-beta agonists have also been described. In the present study, we hypothesized that PPAR-beta agonists might exert acute nongenomic effects on vascular tone. Here, we report that the structurally unrelated PPAR-beta ligands [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxy]acetic acid (L-165041) and 4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl] methyl]thio]-2-methylphenoxy]acetic acid (GW0742) induced vascular relaxation in phenylephrine-precontracted endothelium-intact rat aortic rings, which was significantly inhibited by endothelial denudation or nitric-oxide synthase (NOS) inhibition with N(G)-nitro-l-arginine methylester. These relaxant effects reached steady state within 15 min. The relaxation induced by L-165041 and GW0742 in aortic rings precontracted with the thromboxane A(2) analog 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F2alpha (U-46619) was unaffected either by removal of extracellular calcium or by incubation with calcium-free solution containing the intracellular calcium chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester. However, the phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY-294002) inhibited the endothelium-dependent relaxant responses induced by both PPAR-beta agonists. Blockade of PPAR-beta with 3-[[[2-methoxy-4-(phenylamino)phenyl]amino]sulfonyl]-2-thiophenecarboxylic acid methyl ester (GSK0660) also partially inhibited these relaxant responses, although PPAR-gamma blockade with 2-chloro-5-nitro-N-phenylbenzamide (GW9662) had no effect. In human umbilical vein endothelial cells, L-165041 and GW0742 increased nitric oxide (NO) production and Akt and endothelial NOS (eNOS) phosphorylation, which were sensitive to PI3K inhibition and PPAR-beta blockade. In conclusion, the PPAR-beta agonists acutely caused vasodilatation, which was partially dependent on endothelial-derived NO. The eNOS activation is calcium-independent and seems to be related to activation of the PI3K-Akt-eNOS pathway.