Functional serotonin 5-HT4 receptors in porcine and human ventricular myocardium with increased 5-HT4 mRNA in heart failure

Serotonin (5-hydroxytryptamine, 5-HT) increases contractile force and elicits arrhythmias through 5-HT4 receptors in porcine and human atrium, but its ventricular effects are unknown. We now report functional 5-HT4 receptors in porcine and human ventricle. 5-HT4 mRNA levels were determined in porcine and human ventricles and contractility studied in ventricular trabeculae. Cyclic AMP-dependent protein kinase (PKA) activity was measured in porcine ventricle. Porcine and human ventricles expressed 5-HT4 receptor mRNA. Ventricular 5-HT4(b) mRNA was increased by four times in 20 failing human hearts compared with five donor hearts. 5-HT increased contractile force maximally by 16% (EC50=890 nM) and PKA activity by 20% of the effects of (-)-isoproterenol (200 muM) in ventricular trabeculae from new-born piglets in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine. In ventricular trabeculae from adult pigs (3-isobutyl-1-methylxanthine present) 5-HT increased force by 32% (EC50=60 nM) and PKA activity by 39% of (-)-iso-proterenol. In right and left ventricular trabeculae from failing hearts, exposed to modified Krebs solution, 5-HT produced variable increases in contractile force in right ventricular trabeculae from 4 out of 6 hearts and in left ventricular trabeculae from 3 out of 3 hearts- range 1-39% of (-)-isoproterenol, average 8%. In 11 left ventricular trabeculae from the failing hearts of four beta-blocker-treated patients, pre-exposed to a relaxant solution with 0.5 mM Ca2+ and 1.2 mM Mg2+ followed by a switch to 2.5 mM Ca2+ and 1 mM Mg2+, 5-HT (1-100 muM, 3-isobutyl-1-melhylxanthine present) consistently increased contractile force and hastened relaxation by 46% and 25% of (-)-isoproterenol respectively. 5-HT caused arrhythmias in three trabeculae from 3 out of I I patients. In the absence of phosphodiesterase inhibitor, 5-HT increased force in two trabeculae, but not in another six trabeculae from 4 patients. All 5-HT responses were blocked by 5-HT4 receptor antagonists. We conclude that phosphodiesterase inhibition uncovers functional ventricular 5-HT4 receptors, coupled to a PKA pathway, through which 5-HT enhances contractility, hastens relaxation and can potentially cause arrhythmias.

Direct electrochemistry of porcine purple acid phosphatase (uteroferrin)

Cyclic voltammetry of the non-heme diiron enzyme porcine purple acid phosphatase (uteroferrin, Uf) has been reported for the first time. Totally reversible one-electron oxidation responses (Fe-III-Fe-II --> Fe-III-Fe-III) are seen both in the absence and in the presence of weak competitive inhibitors phosphate and arsenate, and dissociation constants of these oxoanion complexes formed with uteroferrin in its oxidized state (Uf(o)) have been determined. The effect of pH on the redox potentials has been investigated in the range 3 < pH < 6.5, enabling acid dissociation constants for Uf(o) and its phosphate and arsenate complexes to be calculated.

Increased site 1 affinity improves biopotency of porcine growth hormone - Evidence against diffusion dependent receptor dimerization

Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp(104) and Trp(169) in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys(165)), which in addition to His(169), restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.

Changes to porcine blastocyst vitrification methods and improved litter size after transfer

The objective was to improve the protocol that was used to obtain the first reported piglets from transferred vitrified and warmed zona-intact blastocysts. Blastocysts were collected from superovulated sows and gilts, centrifuged to polarize lipid, vitrified, warmed and cultured for 24 h or transferred immediately. Removing the zona pellucida after warming increased the number of cells in the surviving blastocysts (zona-free 60.8 +/- 4.3, zona-intact 39.1 +/- 2.8; P < 0.05). Thinning the zona pellucida produced similar results to zona removal. Changing the basal medium of the vitrification and warming solutions from modified PBS to phosphate buffered NCSU-23 increased the number of cells (44.7 +/- 2.2 versus 56.0 +/- 3.9, respectively; P < 0.05). Reducing the plunge temperature of the liquid nitrogen from - 196 degrees C to less than -204 degrees C improved the embryo survival rate (61.9% versus 82.9%, respectively; P < 0.05). These modifications were incorporated into the vitrification protocol that was used to vitrify and warm 105 blastocysts (that were subsequently transferred into four recipients). Three recipients became pregnant, farrowing three litters (average litter size, 5.3; 18.8% embryo survival in farrowing sows). Changing the warming protocol to using sucrose rather than ethylene glycol resulted in a trend towards improved embryo survival (73.5% versus 91.2%) but this was not statistically significant. Incorporating this modification, 203 blastocysts were vitrified, warmed and transferred into seven recipients. Five became pregnant and 36 fetuses were recovered (average litter size 7.2; 24.8% embryo survival in pregnant sows) at Day 40 of pregnancy. In conclusion, changes made to the vitrification protocol improved pregnancy rate and in vivo embryo survival compared to an earlier study using the original protocol. (c) 2005 Elsevier Inc. All rights reserved.

Dissolution of porcine incisor pulps in sodium hypochlorite solutions of varying compositions and concentrations

Background: The solubility of dental pulp tissue in sodium hypochlorite has been extensively investigated but results have been inconsistent; due most likely to variations in experimental design, the volume and/or rate of replenishment of the solutions used and the nature of the tissues assessed. Traditionally, the sodium hypochlorite solutions used for endodontic irrigation in Australia have been either Milton or commercial bleach, with Milton being the most common. Recently, a range of Therapeutic Goods Administration (TGA) approved proprietary sodium hypochlorite solutions, which contain surfactant, has become available. Some domestic chlorine bleaches now also contain surfactants. The purpose of this study was to perform new solubility assessments, comparing Milton with new TGA approved products, Hypochlor 1% and Hypochlor 4% forte, and with a domestic bleach containing surfactant (White King). Methods: Ten randomly assigned pulp samples of porcine dental pulp of approximately equal dimensions were immersed in the above solutions, as well as representative concentrations of sodium hydroxide. Time to complete dissolution was measured and assessed statistically. Results: White King 4% showed the shortest dissolution time, closely followed by Hypochlor 4% forte. White King 1% and Hypochlor 1% each took around three times as long to completely dissolve the samples of pulp as their respective 4% concentrations, while Milton took nearly 10 times as long. The sodium hydroxide solutions showed no noticeable dissolution of the pulp samples. Conclusions: The composition and content of sodium hypochlorite solutions had a profound effect on the ability of these solutions to dissolve pulp tissue in vitro. Greater concentrations provided more rapid dissolution of tissue. One per cent solutions with added surfactant and which contained higher concentrations of sodium hydroxide were significantly more effective in dissolution of pulp tissue than Milton.

Resistance of porcine blood clots to lysis relates to poor activation of porcine plasminogen by tissue plasminogen activator

In-vitro experimentation was performed on porcine and human blood to determine their comparative responsiveness to a novel fibrinolytic inhibitor and thereby assess whether the pig is a suitable animal model for subsequent in-vivo testing of this inhibitor. Thromboelastography showed the clots formed from porcine whole blood to be highly resistant to tissue plasminogen activator (t-PA)-catalyzed lysis, and this communication offers the resistance of porcine plasminogen to activation by t-PA as an explanation. Porcine blood containing 100 and 1500 IU/ml added t-PA lysed very slowly, having LY30 values of 1.9 +/- 1.4 and 2.9 +/- 1.9%, respectively. In contrast, the LY30 values for the human clots containing 100 and 1500 IU/ml t-PA were 77.1 +/- 6.3 and 93.3 +/- 1.3%, respectively. Moreover, purified porcine plasminogen was activated very slowly by added t-PA in the presence of both human and porcine fibrin. Activation of plasminogen by the endogenous activators, as measured by the euglobulin clot lysis time, was greatly prolonged for the pig (22 +/- 3 h) compared with the human (3.5 +/- 1.5 h). These results suggest caution in using the pig as an experimental model when studying the effects of various agents on fibrinolysis.

Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines

Receptor-mediated endocytosis is a constitutive high capacity pathway for the reabsorption of proteins from the glomerular filtrate by the renal proximal tubule. ClC-5 is a voltage-gated chloride channel found in the proximal tubule where it has been shown to be essential for protein uptake, based on evidence from patients with Dent's disease and studies in ClC-5 knockout mice. To further delineate the role of ClC-5 in albumin uptake, we performed a yeast two-hybrid screen with the C-terminal tail of ClC-5 to identify any interactions of the channel with proteins involved in endocytosis. We found that the C-terminal tail of ClC-5 bound the actin depolymerizing protein, cofilin, a result that was confirmed by GST-fusion pulldown assays. In cultured proximal tubule cells, cofilin was distributed in nuclear, cytoplasmic, and microsomal fractions and co-localized with ClC-5. Phosphorylation of cofilin by overexpressing LIM kinase 1 resulted in a stabilization of the actin cytoskeleton. Phosphorylation of cofilin in two proximal tubule cell models (porcine renal proximal tubule and opossum kidney) was also accompanied by a pronounced inhibition of albumin uptake. This study identifies a novel interaction between the C-terminal tail of ClC-5 and cofilin, an actin-associated protein that is crucial in the regulation of albumin uptake by the proximal tubule.