Calcium-free Solution Research Articles

Characterization of voltage‐operated and non‐voltage‐operated calcium channels in endothelin‐induced vasoconstriction of rat cerebral arteries (841.6)

Endothelin‐1 (ET‐1) is a potential mediator of the pathogenesis of cerebral vasospasm. This study analysed the role of voltage‐operated calcium channels (VOCC) and non‐VOCC in ET‐1‐ induced vasoconstriction of rat cerebral arteries.Rat isolated cerebral arteries (320‐430 µm i.d.) (anterior cerebral, middle cerebral, posterior communicating and basilar) were investigated using wire myography. ET‐1 concentration‐contraction response curves were constructed in arteries in calcium‐free medium or in the presence of nifedipine, NNC 55‐0396 or SK&F 96365 to inhibit the L‐type VOCC, T‐type VOCC and non‐VOCC, respectively. The ability of these antagonists to reverse the ET‐1‐induced contraction was also tested.Neither inhibition of calcium channels nor removal of calcium altered the potency (pEC50) of ET‐1, but the maximum effects of ET‐1 (Emax) were variably attenuated. ET‐1 caused a small contraction (11‐22%) in calcium‐free solution. Nifedipine (1 µM) significantly decreased the Emax only in the middle cerebral artery, while NNC 55‐0396 (1 µM) and SK&F 96365 (30‐100 µM) attenuated the Emax in all cerebral arteries. Combination of nifedipine with SK&F 96365 more moderately decreased the Emax. Both nifedipine and SK&F 96365 reversed the ET‐1‐induced pre‐contraction in all arteries.In conclusion, ET‐1 causes potent contraction by differentially activating both VOCC and non‐VOCC in rat cerebral arteries.

Effect of Vitis vinifera Leaf Hydroalcoholic Extract on Rat Urinary Bladder Contractility

Introduction: Several reports have shown the various medicinal effects of grape (Vitis vinifera) seed and skin extracts, such as antioxidant, hypotensive and vasodilatory effects. It has been recently shown the relaxatory effect of grape leaf hydroalcoholic extract on rat ileum, uterus, aorta, trachea and vas deferens as well as on frog isolated heart. The aim of the present study was to investigate the effect of Vitis vinifera leaf hydroalcoholic extract (VLHE) on contractility of urinary bladder smooth muscle in male rats and to study the mechanism(s) of its action. Materials and Methods: The extract of dried grape leaf was prepared by macerated method using 70% ethanol for 72h in room temperature. The solvent was then evaporated. Urinary bladder was removed from male adult wistar rats after induction of deep anaesthetize by di-ethyle-ether. Tissues were then suspended in the organ bath containing physiologic solution (37oC, pH 7.4) bubbled with oxygen. Contractions were recorded isometrically under 1g resting tension. Results: VLHE at 1, 2, 4 and 8 mg/ml significantly reduced the urinary bladder contractions evoked by KCl (60 mM P<0.05) and (0.5, 1, 2 and 4 mg/ml) also reduced the contractions evoked by Acetylcholine (5 µM) dose dependently (P<0.05). In calcium free physiologic solution, KCl-induced (120 mM P<0.05) contractions were occurred only after adding calcium (0.312, 0.625, 1.25, 2.5 and 5 mM) to the organ bath solution. VLHE (2 mg/ml) also reduced different concentration of calcium-induced contraction in the presence of KCl (P<0.05). The VLHE- induced urinary bladder relaxation was unaffected by propranolol (1 µM for 20 mins) while the presence of tetraethylamonium (10 mM for 20 mins) could strongly reduce the relaxatory effect of VLHE. Conclusion: The data suggest that VLHE induces spasmolytic effect in rat urinary bladder, possibly via blocking voltage dependent calcium channels and activation of calcium-activated potassium channels.

Mepivacaine-induced intracellular calcium increase appears to be mediated primarily by calcium influx in rat aorta without endothelium.

BackgroundMepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca2+]i). However, the mechanism responsible for the mepivacaine-evoked [Ca2+]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca2+]i increment in isolated rat aorta.MethodsIsometric tension was measured in isolated rat aorta without endothelium. In addition, fura-2 loaded aortic muscle strips were illuminated alternately (48 Hz) at two excitation wavelengths (340 and 380 nm). The ratio of F340 to F380 (F340/F380) was regarded as an amount of [Ca2+]i. We investigated the effects of nifedipine, 2-aminoethoxydiphenylborate (2-APB), gadolinium chloride hexahydrate (Gd3+), low calcium level and Krebs solution without calcium on the mepivacaine-evoked contraction in isolated rat aorta and on the mepivacaine-evoked [Ca2+]i increment in fura-2 loaded aortic strips. We assessed the effect of verapamil on the mepivacaine-evoked [Ca2+]i increment.ResultsMepivacaine produced vasoconstriction and increased [Ca2+]i. Nifedipine, 2-APB and low calcium attenuated vasoconstriction and the [Ca2+]i increase evoked by mepivacaine. Verapamil attenuated the mepivacaine-induced [Ca2+]i increment. Calcium-free solution almost abolished mepivacaine-induced contraction and strongly attenuated the mepivacaineinduced [Ca2+]i increase. Gd3+ had no effect on either vasoconstriction or the [Ca2+]i increment evoked by mepivacaine.ConclusionsThe mepivacaine-evoked [Ca2+]i increment, which contributes to mepivacaine-evoked contraction, appears to be mediated mainly by calcium influx and partially by calcium released from the sarcoplasmic reticulum.

Connexins form functional hemichannels in porcine ciliary epithelium

The expression of connexins in the ciliary epithelium is consistent with gap junctions between the pigmented (PE) and nonpigmented ciliary epithelium (NPE) that form when connexon hemichannels from adjacent cells pair to form a channel. Here we present evidence that suggests undocked connexons may form functional hemichannels that permit exchange of substances between NPE and the aqueous humor. Intact porcine eyes were perfused via the ciliary artery and propidium iodide (PI) (MW 668) was added to the aqueous humor compartment as a tracer. After calcium-free solution containing PI was introduced into the aqueous humor compartment for 30 min, fluorescence microscopy revealed PI in the NPE cell layer. PI entry into the NPE was inhibited by calcium and by the connexin antagonist 18α-glycyrrhetinic acid (18-AGA). Studies also were carried out with cultured porcine NPE. Under normal conditions, little PI entered the cultured cells but calcium-free medium stimulated PI accumulation and the entry was inhibited by 18-AGA. In cells loaded with calcein (MW 622), calcium-free solution stimulated calcein exit. 18-AGA partially suppressed calcein exit in calcium-free medium. Connexin 43 and connexin 50 proteins were detected by western blot analysis in both native and cultured NPE. In the intact eye, immunolocalization studies revealed connexin 50 at the basolateral, aqueous humor-facing, margin of the NPE. In contrast, connexin 43 was observed at the junction of the PE and NPE layer and on the basolateral membrane of PE. The results point to functional hemichannels at the NPE basolateral surface. It is feasible that hemichannels might contribute to the transfer of substances between the ciliary epithelium cytoplasm and aqueous humor.

Calcium paradox induces apoptosis in the isolated perfusedRana ridibundaheart: involvement of p38-MAPK and calpain

"Calcium paradox" as a term describes the deleterious effects conferred to a heart perfused with a calcium-free solution followed by repletion, including loss of mechanical activity and sarcomere disruption. Given that the signaling mechanisms triggered by calcium paradox remain elusive, in the present study, we tried to investigate them in the isolated perfused heart from Rana ridibunda. Calcium paradox was found to markedly activate members of the MAPKs (p43-ERK, JNKs, p38-MAPK). In addition to lactate dehydrogenase (LDH) release in the perfusate (indicative of necrosis), we also confirmed the occurrence of apoptosis by using the TUNEL assay and identifying poly(ADP-ribose) polymerase (PARP) fragmentation and upregulated Bax expression. Furthermore, using MDL28170 (a selective calpain inhibitor), a role for this protease was revealed. In addition, various divalent cations were shown to exert a protective effect against the calcium paradox. Interestingly, SB203580, a p38-MAPK inhibitor, alleviated calcium-paradox-conferred apoptosis. This result indicates that p38-MAPK plays a pro-apoptotic role, contributing to the resulting myocardial dysfunction and cell death. To our knowledge, this is the first time that the calcium paradox has been shown to induce apoptosis in amphibians, with p38-MAPK and calpain playing significant roles.

Regenerative Glutamate Release by Presynaptic NMDA Receptors Contributes to Spreading Depression

Spreading depression (SD) is a slowly propagating neuronal depolarization that underlies certain neurologic conditions. The wave-like pattern of its propagation suggests that SD arises from an unusual form of neuronal communication. We used enzyme-based glutamate electrodes to show that during SD induced by transiently raising extracellular K(+) concentrations ([K(+)]o) in rat brain slices, there was a rapid increase in the extracellular glutamate concentration that required vesicular exocytosis but unlike fast synaptic transmission, still occurred when voltage-gated sodium and calcium channels (VGSC and VGCC) were blocked. Instead, presynaptic N-methyl-D-aspartate (NMDA) receptors (NMDARs) were activated during SD and could generate substantial glutamate release to support regenerative glutamate release and propagating waves when VGSCs and VGCCs were blocked. In calcium-free solutions, high [K(+)]o still triggered SD-like waves and glutamate efflux. Under such a condition, glutamate release was blocked by mitochondrial Na(+)/Ca(2+) exchanger inhibitors that likely blocked calcium release from mitochondria secondary to NMDA-induced Na(+) influx. Therefore presynaptic NMDA receptor activation is sufficient for triggering vesicular glutamate release during SD via both calcium entry and release from mitochondria by mitochondrial Na(+)/Ca(2+) exchanger. Our observations suggest that presynaptic NMDARs contribute to a cycle of glutamate-induced glutamate release that mediate high [K(+)]o-triggered SD.

The Molecular Basis for Species-specific Activation of Human TRPA1 Protein by Protons Involves Poorly Conserved Residues within Transmembrane Domains 5 and 6

The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons.

A Close Look at Hearing Repair, One Protein at a Time

Hearing is a matter of transducing mechanical forces into electrical signals. Pressure waves coming to the ear drum are transmitted through the middle ear to the mechanosensory hair cells within the fluid-filled inner ear. The “hairs” (more properly called stereocilia) on a hair cell are arranged in semicircular rows, with the shortest at the front and the tallest at the back. An incoming wave deflects the stereocilia backward, causing protein chains called tip links, which link the tip of a short stereocilium to the side of a taller one, to pull on and open up ion channels at the base of the protein chains. The influx of ions from the surrounding fluid causes hair cell depolarization and firing of the attached sensory neurons. The inner ear transduces sound via tip links that open up ion channels; loud sounds can break tip links, but their regeneration is possible through a two-step remodeling process of their molecular components. Exposure to excessively loud noises—a jet engine, a rock concert—leads to temporary hearing loss. One cause of that loss is breakage of those tip links, which is not surprising, given both their central importance and relative fragility. Tip links re-form over the course of several days, restoring normal hearing. While the molecular composition of tip links is known, the details of regeneration have remained unclear. In a new study in PLOS Biology, Artur Indzhykulian, Gregory Frolenkov, and colleagues begin to reveal those details, showing that of the two components of the mature link, one is solely responsible for the initial re-formation of the chain, and that full restoration of normal hearing only occurs once the second protein is back in place. The two proteins are known as protocadherin 15 (PCDH15), which on the mature tip link is found closest to the shorter stereocilium, and cadherin 23 (CDH23), which links to the longer stereocilium (mutations of the genes for both these proteins cause inherited deafness). The authors treated mouse hair cells with calcium-free solution to disrupt all the links connecting the stereocilia, and then observed tip link regeneration, using scanning electron microscopy. Over the first several hours, new links formed only at the top of the stereocilium, rather than forming at the base and migrating, as an early hypothesis of regeneration had suggested. Mechanotransduction, measured by deflecting stereocilia with a rigid probe and measuring the properties of the resulting current, was abolished by the calcium-free solution. Current flow gradually returned over the next 24 hours, accompanied by restoration of the full complement of tip links. But a property of the mechanotransduction response called fast adaptation (in which activation is followed by rapid deactivation) remained aberrant, before gradually normalizing beginning at about 36 hours. This normalization appeared to be connected to changes in the molecular composition of the restored tip links. The authors found that immediately after disruption, PCDH15 was redistributed along the surface of the stereocilia. In contrast, CDH23 largely disappeared from them. As the links were reformed, the total amount of PCDH15 in the tip link area increased, while the amount of CDH23 remained relatively low compared to untreated controls. Furthermore, PCDH15 was found at both ends of the links, not just the lower end, as in controls. This suggested that PCDH15 was serving, at least temporarily, in place of CDH23 as links reformed. Then, after the links were in place, CDH23 was gradually restored, coincident with recovery of normal adaptation. Some physiologists have speculated that the repair process recapitulates the developmental formation of tip links. In support of that idea, the authors found that in normal development in postnatal in mice, PCDH15 was localized at both ends of the tip link at postnatal day 1, and 5 days later, it was concentrated mainly at the lower end. Over the same period, CDH23 remained only at the upper end. One outcome of this study is to resolve a small dispute over the composition of tip links, in which one camp argued for a PCDH15-only structure, and the other for a combination of the two proteins. Apparently, both are right, depending on when one looks. More importantly, it reveals the details of a process vital for the development, maintenance, and restoration of normal hearing. Indzhykulian AA, Stepanyan R, Nelina A, Spinelli KJ, Ahmed ZM, et al. (2013) Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells. doi:10.1371/journal.pbio.1001583

Rheb (Ras Homologue Enriched in Brain)-dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Becomes Indispensable for Cardiac Hypertrophic Growth after Early Postnatal Period

Cardiomyocytes proliferate during fetal life but lose their ability to proliferate soon after birth and further increases in cardiac mass are achieved through an increase in cell size or hypertrophy. Mammalian target of rapamycin complex 1 (mTORC1) is critical for cell growth and proliferation. Rheb (Ras homologue enriched in brain) is one of the most important upstream regulators of mTORC1. Here, we attempted to clarify the role of Rheb in the heart using cardiac-specific Rheb-deficient mice (Rheb(-/-)). Rheb(-/-) mice died from postnatal day 8 to 10. The heart-to-body weight ratio, an index of cardiomyocyte hypertrophy, in Rheb(-/-) was lower than that in the control (Rheb(+/+)) at postnatal day 8. The cell surface area of cardiomyocytes isolated from the mouse hearts increased from postnatal days 5 to 8 in Rheb(+/+) mice but not in Rheb(-/-) mice. Ultrastructural analysis indicated that sarcomere maturation was impaired in Rheb(-/-) hearts during the neonatal period. Rheb(-/-) hearts exhibited no difference in the phosphorylation level of S6 or 4E-BP1, downstream of mTORC1 at postnatal day 3 but showed attenuation at postnatal day 5 or 8 compared with the control. Polysome analysis revealed that the mRNA translation activity decreased in Rheb(-/-) hearts at postnatal day 8. Furthermore, ablation of eukaryotic initiation factor 4E-binding protein 1 in Rheb(-/-) mice improved mRNA translation, cardiac hypertrophic growth, sarcomere maturation, and survival. Thus, Rheb-dependent mTORC1 activation becomes essential for cardiomyocyte hypertrophic growth after early postnatal period.

Propranolol-induced relaxation in the rat basilar artery

Propranolol is a non-selective beta-adrenergic receptor blocker used in the treatment of cardiovascular diseases and migraine prophylaxis. Although it has been shown that propranolol dilates the peripheral arteries of rat, its action in the central nervous system vasculature has not been investigated. In this study, the effects of propranolol in rat basilar artery were investigated. Basilar arteries from male Wistar rats were examined in a myograph system. The relaxant effects of propranolol, pindolol, atenolol, pizotifen and methysergide were examined in basilar arteries precontracted by serotonin or PGF2α. Only propranolol and pizotifen induced vasorelaxations; the pD2 values were 5.23±0.13 and 5.94±0.03; respectively. The vasorelaxation induced by propranolol and pizotifen was not affected by endothelium or the presence of l-NOARG and/or indomethacin. The calcium channel blocking activity of propranolol and pizotifen was compared with that of nifedipine in a calcium free solution with high K+ (60mM) concentration. These drugs shifted the concentration–response curves of calcium induced contractions with pA2 values of 5.45±0.04; 7.14±0.09; and 9.22±0.06 respectively. The P2Y receptor agonist UTP was used to induce sustained and stable contractions in basilar artery segments. Nifedipine caused a marked, but an incomplete relaxation. Cyclopiazonic acid, an inhibitor of sarcoplasmic reticulum calcium channels, but not propranolol or pizotifen abolished the remaining tonus after partial relaxations obtained with nifedipine.These results suggest that propranolol causes vasorelaxation by blocking the L-type voltage-gated calcium channels in the rat basilar artery.

Interaction Sites between Voltage-Sensor and Gate in BK Channels

BK channels are modulated by membrane voltage as well as intracellular calcium and participate in many physiologically important processes. To study the molecular basis and mechanism of voltage sensor/gate interactions in BK channels we performed an alanine-scan of the S4-S5 linker and the C-terminal end of S6 in mSlo1. Our results show: First, mutations Q222A, I233A, K234A, V319A shift activation to more positive voltages in both calcium and calcium free solutions and decrease the channel open probability (PO) 10 to 100-fold compared to the WT at negative voltages where the voltage sensors are in the resting state. SinceQ222 and I233, K234 are at the ends of the S4∼S5 linker and V319 in S6 is in a region (YVP) corresponding to the PXP motif in other KV channels, these results suggest the flexibility of “hinge” regions may play an important role in voltage-sensor/gate interaction and gate stability.Second,both F223A(in the S4∼S5 linker) and K330A(near the end of S6) produce an approximate 3 fold increase in PO when the voltage sensors are in the resting state, and F223A/K330A shows a non-additive effect compared to the single mutants, suggesting intrasubunit interaction between these two sites may stabilize the closed gate and mediate voltage-sensor/gate coupling. N237A produced a similar increase in PO suggesting it might also interact with F223A or K330A in an adjacent subunit. Third, mutations in S6 (P320A, E321A, E324A) that produce the largest increases in PO when the voltage sensors are in the resting state may alter the stability of the gate directly since no obvious interaction partner was identified in the S4∼S5 linker.

AlGaN/GaN-based biosensor for label-free detection of biological activity

Abstract In this work, detection of live cell biological activity has been confirmed for label-free AlGaN/GaN based biosensors. Live cells were seeded in solution onto the exposed gate region of packaged AlGaN/GaN field effect transistor (FET) devices and subsequently subjected to various chemical changes in the solution. Cell depolarisation with KCl was used to induce biological signals. Significant optimisation of experimental parameters was performed, including environmental conditions and cell seeding concentration. Selectivity towards biological activity was improved by buffering with 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Reproducible calcium concentration-dependent responses were observed with saturation at the normal physiological level of around 2.5 mM. Cells in a calcium-free solution were pre-treated in separate experiments with calcium ion channel inhibitors Mibefradil, Nisoldipine and HC-030031, and the calcium channel activator S-BayK(−)8644, before calcium dosing. The resulting responses were compared to depolarisation-induced calcium intake and demonstrated physiologically relevant behaviour of calcium ion channels.

Potent inhibition of human neutrophil activations by bractelactone, a novel chalcone from Fissistigma bracteolatum

Fissistigma bracteolatum is widely used in traditional medicine to treat inflammatory diseases. However, its active components and mechanisms of action remain unclear. In this study, (3Z)-6,7-dihydroxy-4-methoxy-3-(phenylmethylidene)-5-(3-phenylpropanoyl)-1-benzofuran-2(3H) (bractelactone), a novel chalcone from F. bracteolatum, showed potent inhibitory effects against superoxide anion (O2−) production, elastase release, and CD11b expression in formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-induced human neutrophils. However, bractelactone showed only weak inhibition of phorbol myristate acetate-caused O2− production. The peak cytosolic calcium concentration ([Ca2+]i) was unaltered by bractelactone in FMLP-induced neutrophils, but the decay time of [Ca2+]i was significantly shortened. In a calcium-free solution, changes in [Ca2+]i caused by the addition of extracellular Ca2+ were inhibited by bractelactone in FMLP-activated cells. In addition, bractelactone did not alter the phosphorylation of p38 MAPK, ERK, JNK, or AKT or the concentration of cAMP. These results suggest that bractelactone selectively inhibits store-operated calcium entry (SOCE). In agreement with this concept, bractelactone suppressed sustained [Ca2+]i changes in thapsigargin-activated neutrophils. Furthermore, bractelactone did not alter FMLP-induced formation of inositol 1,4,5-triphosphate. Taken together, our results demonstrate that the anti-inflammatory effects of bractelactone, an active ingredient of F. bracteolatum, in human neutrophils are through the selective inhibition of SOCE.

Isolated small uterine arteries from non-pregnant and term pregnant rats exhibit regulatory responses to elevations in extravascular pressure

An adequate blood supply is essential for the maintenance of uterine function and fetal health during parturition. However, labouring uterine contractions will impart compressive forces on small uterine arteries (SUA). We demonstrate that isolated, pressurised rat SUA arteries, pre-constricted with arginine vasopressin or high potassium solution, exhibit regulatory responses to elevations in extravascular pressure (EVP) which maintain internal diameter constant at EVPs of 0–40 mm Hg. This response is endothelium independent and is not modulated by pregnancy. No regulation was observed in calcium free solution. SUA myogenic responses to elevated EVP likely represents a mechanism for limiting reductions in uterine blood flow during uterine contraction.

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

Changes of cytosolic calcium and contractility of young rat vas deferens by acute treatment with amphetamine, fluoxetine or sibutramine

Previous studies conducted in our laboratory indicated that administration of amphetamine, fluoxetine or sibutramine affects the sympathetic nervous system of the rat vas deferens. Therefore, our goal was to verify the role of calcium in vasa deferentia from young rats pretreated with a single dose of these drugs. Young 40-day-old male Wistar rats were pretreated with amphetamine 3mg/kg, fluoxetine 10mg/kg or sibutramine 6mg/kg for 4h before the experiments. CaCl2 (10mM) was used to induce contraction through time–effect curves in calcium-free solution to measure phasic and tonic components. We also evaluated the calcium-induced fluorescence of vas deferens cut into thin slices. In rats pretreated with amphetamine, we found an increase of the tonic contraction component which was reduced by verapamil. The phasic and tonic responses were increased in the group treated with fluoxetine, but only the tonic response was more sensitive to the antagonism by verapamil. The group treated with sibutramine showed an increase of phasic response whereas the tonic component was decreased. In this group an increase of the affinity for verapamil antagonism was found. In the calcium fluorescence study it was observed that the group treated with amphetamine, fluoxetine or sibutramine showed higher basal Ca2+ fluorescence after stimulus with KCl (70mM), noradrenaline (10−4M) or acetylcholine (10−4M). In all pretreated groups the calcium fluorescence was diminished by nifedipine 10−7M. Therefore, the pretreatment with amphetamine, fluoxetine or sibutramine seems to affect the calcium contractility and homeostasis in young rat vas deferens.

Mepivacaine-induced contraction is attenuated by endothelial nitric oxide release in isolated rat aorta

Mepivacaine is an aminoamide-linked local anesthetic with an intermediate duration that intrinsically produces vasoconstriction both in vivo and in vitro. The aims of this in-vitro study were to examine the direct effect of mepivacaine in isolated rat aortic rings and to determine the associated cellular mechanism with a particular focus on endothelium-derived vasodilators, which modulate vascular tone. In the aortic rings with or without endothelium, cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following antagonists: N(ω)-nitro-L-arginine methyl ester [L-NAME], indomethacin, fluconazole, methylene blue, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one [ODQ], verapamil, and calcium-free Krebs solution. Mepivacaine produced vasoconstriction at low concentrations (1 × 10(-3) and 3 × 10(-3) mol/L) followed by vasodilation at a high concentration (1 × 10(-2) mol/L). The mepivacaine-induced contraction was higher in endothelium-denuded aortae than in endothelium-intact aortae. Pretreatment with L-NAME, ODQ, and methylene blue enhanced mepivacaine-induced contraction in the endothelium-intact rings, whereas fluconazole had no effect. Indomethacin slightly attenuated mepivacaine-induced contraction, whereas verapamil and calcium-free Krebs solution more strongly attenuated this contraction. The vasoconstriction induced by mepivacaine is attenuated mainly by the endothelial nitric oxide - cyclic guanosine monophosphate pathway. In addition, mepivacaine-induced contraction involves cyclooxygenase pathway activation and extracellular calcium influx via voltage-operated calcium channels.

Adjustable passive stiffness in mouse bladder: regulated by Rho kinase and elevated following partial bladder outlet obstruction

Detrusor smooth muscle (DSM) contributes to bladder wall tension during filling, and bladder wall deformation affects the signaling system that leads to urgency. The length-passive tension (L-T(p)) relationship in rabbit DSM can adapt with length changes over time and exhibits adjustable passive stiffness (APS) characterized by a L-T(p) curve that is a function of both activation and strain history. Muscle activation with KCl, carbachol (CCh), or prostaglandin E(2) at short muscle lengths can increase APS that is revealed by elevated pseudo-steady-state T(p) at longer lengths compared with prior T(p) measurements at those lengths, and APS generation is inhibited by the Rho Kinase (ROCK) inhibitor H-1152. In the current study, mouse bladder strips exhibited both KCl- and CCh-induced APS. Whole mouse bladders demonstrated APS which was measured as an increase in pressure during passive filling in calcium-free solution following CCh precontraction compared with pressure during filling without precontraction. In addition, CCh-induced APS in whole mouse bladder was inhibited by H-1152, indicating that ROCK activity may regulate bladder compliance during filling. Furthermore, APS in whole mouse bladder was elevated 2 wk after partial bladder outlet obstruction, suggesting that APS may be relevant in diseases affecting bladder mechanics. The presence of APS in mouse bladder will permit future studies of APS regulatory pathways and potential alterations of APS in disease models using knockout transgenetic mice.

A Comparison of the Reproductive Performance of the Wild Pink Shrimp SpeciesFarfantepenaeus paulensisandFarfantepenaeus brasiliensisin Captivity

ABSTRACT The aim of this study was to compare the reproductive performance of the wild shrimp species Farfantepenaeus paulensis and Farfantepenaeus brasiliensis in captivity. The broodstocks were collected in offshore waters in Santa Catarina and transferred to the Marine Station of Aquaculture of the Federal University of Rio Grande. Broodstocks with a similar body weight between the two species were selected to measure reproductive performance. Females with mature ovaries were identified each day to obtain measures of reproductive performance. After spawning, fertilization rates were determined by microscopic examination. In addition, three samples of 100 eggs were collected to determine hatching rates, and three samples of 100 nauplii were collected to evaluate the rate of metamorphosis to protozoa. For males, spermatophores were manually extruded, weighed, and homogenized in 2 mL calcium-free saline solution, and 0.1 mL trypan blue was added. Sperm counts, abnormal cells (malformations of the main bod...

Vascular Smooth Muscle Relaxation by a Lectin from Pisum arvense: Evidences of Endothelial NOS Pathway

The vasorelaxant effect of the lectin of Pisum arvense (PAL) seeds was investigated in rat aorta. PAL (10-100 µg/ml) was applied on aorta rings, with or without endothelium, pre-contracted with phenylephrine (Phe; 0.1 µM). Participation of endothelium derived relaxant factors was evaluated incubating the tissue with indomethacin (10 µM), L-nitro arginine methyl ester (L-NAME, 100 µM) and tetraethylammonium (TEA, 5 mM) before addition of PAL. The role of the lectin domain was investigated by addition of PAL into tissue in presence of glucose (3x 10⁻⁵ M), or N-acetyl Dglucosamine (GlcNAc; 3 x 10⁻⁴ M). The importance of extracellular calcium (Ca²⁺e) or interaction with muscarinic receptors in the relaxant effect was evaluated by addition of PAL into aorta rings containing calcium free solution (OCa) and atropine (1 µ M), respectively. PAL induced concentration-dependent relaxation in endothelized aorta (IC50 =58.38 ± 1.87 µg/ml), which was reversed by L-NAME and glucose. The lectin effect was totally inhibited when the preparation was inserted in OCa, but not in presence of atropine. Summarizing, our data showed a relaxant effect of PAL in isolated rat aorta rings in presence of endothelium, suggestive of interaction between the lectin carbohydrate binding sites with specific receptors located in vascular endothelial cells leading to nitric oxide synthase activation. This effect seems to require Ca²⁺e but is independent on muscarinic receptors interaction.