Voltage-gated potassium currents in rat vas deferens smooth muscle cells

Voltage-gated components of the outward current in single smooth muscle cells isolated from the epididymal part of the rat vas deferens were studied using amphotericin B perforated patch-clamp techniques. The complex kinetics of the net outward current elicited by positive voltage steps from -80 mV to +40 mV suggested the presence of several components. Bath application of 200 nM charybdotoxin, a potent blocker of large-conductance, Ca(2+)-dependent K(+) channels (BK(Ca)), reduced the current amplitude significantly. When BK(Ca) channels were suppressed, fast-inactivating (I(K,f)) and delayed rectifying (I(K,dr)) components of the outward current were identified. I(K,f) was characterized by fast kinetics of current decay, negative steady-state activation and inactivation dependencies and sensitivity to 4-aminopyridine with an apparent K(d) of 0.32 mM, properties similar to those of the A-type K(+) current. In contrast, I(K,dr) activated and inactivated at more positive potentials. The time constant of activation of I(K,dr) was voltage dependent with an e-fold decrease per 21 mV depolarization. I(K,dr) was inhibited by clofilium, a blocker of voltage-gated K(+) channels, with an IC(50) of 12 micro M and was not blocked by 5 mM 4-aminopyridine. The possible significance of the voltage-gated currents is discussed.     

Inhibition of cardiac inward-rectifier K current by terodiline

The antispasmodic agent terodiline has cardiotoxic effects that include QT lengthening. To determine whether inhibition of inwardly-rectifying K⁺ current (I_K1) might be a factor in the cardiotoxicity, we measured I_K1 in guinea pig ventricular myocytes. Terodiline reduced outward I_K1 with an IC₅₀ of 7 μM; maximal reduction was 60% with 100–300 μM concentration. Inhibition was independent of current direction, and persisted after removal of the drug. Terodiline (3–5 μM) lengthened action potentials in guinea pig papillary muscles by ca. 10%, primarily by slowing phase 3 repolarization; higher concentrations abbreviated the plateau and markedly slowed late repolarization. Terodiline washout provoked an extra lengthening, consistent with persistent inhibition of I_K1 and rapid recovery of net inward plateau current. The results suggest that inhibition of I_K1 is a likely factor in the cardiotoxicity of the drug.     

Functionalization of Polycaprolactone Electrospun Osteoplastic Scaffolds with Fluorapatite and Hydroxyapatite Nanoparticles: Biocompatibility Comparison of Human Versus Mouse Mesenchymal Stem Cells

A capability for effective tissue reparation is a living requirement for all multicellular organisms. Bone exits as a precisely orchestrated balance of bioactivities of bone forming osteoblasts and bone resorbing osteoclasts. The main feature of osteoblasts is their capability to produce massive extracellular matrix enriched with calcium phosphate minerals. Hydroxyapatite and its composites represent the most common form of bone mineral providing mechanical strength and significant osteoinductive properties. Herein, hydroxyapatite and fluorapatite functionalized composite scaffolds based on electrospun polycaprolactone have been successfully fabricated. Physicochemical properties, biocompatibility and osteoinductivity of generated matrices have been validated. Both the hydroxyapatite and fluorapatite containing polycaprolactone composite scaffolds demonstrated good biocompatibility towards mesenchymal stem cells. Moreover, the presence of both hydroxyapatite and fluorapatite nanoparticles increased scaffolds’ wettability. Furthermore, incorporation of fluorapatite nanoparticles enhanced the ability of the composite scaffolds to interact and support the mesenchymal stem cells attachment to their surfaces as compared to hydroxyapatite enriched composite scaffolds. The study of osteoinductive properties showed the capacity of fluorapatite and hydroxyapatite containing composite scaffolds to potentiate the stimulation of early stages of mesenchymal stem cells’ osteoblast differentiation. Therefore, polycaprolactone based composite scaffolds functionalized with fluorapatite nanoparticles generates a promising platform for future bone tissue engineering applications.     

Retinal ganglion cell topography and spatial resolution in the Japanese smelt Hypomesus nipponensis (McAllister, 1963)

Vision plays a crucial role in the life of the vast majority of vertebrate species. The spatial arrangement of retinal ganglion cells has been reported to be related to a species’ visual behavior. There are many studies focusing on the ganglion cell topography in bony fish species. However, there are still large gaps in our knowledge on the subject. We studied the topography of retinal ganglion cells (GCs) in the Japanese smelt Hypomesus nipponensis, a highly visual teleostean fish with a complex life cycle. DAPI labeling was used to visualize cell nuclei in the ganglion cell and inner plexiform layers. The ganglion cell layer was relatively thin (about 6-8 μm), even in areas of increased cell density (area retinae temporalis), and was normally composed of a single layer of cells. In all retinal regions, rare cells occurred in the inner plexiform layer. Nissl-stained retinae were used to estimate the proportion of displaced amacrine cells and glia in different retinal regions. In all retinal regions, about 84.5% of cells in the GC layer were found to be ganglion cells. The density of GCs varied across the retina in a regular way. It was minimum (3990 and 2380 cells/mm² in the smaller and larger fish, respectively) in the dorsal and ventral periphery. It gradually increased centripetally and reached a maximum of 14,275 and 10,960 cells/mm² (in the smaller and larger fish, respectively) in the temporal retina, where a pronounced area retinae temporalis was detected. The total number of GCs varied from 177 × 10³ (smaller fish) to 212 × 10³ cells (larger fish). The theoretical anatomical spatial resolution (the anatomical estimate of the upper limit of visual acuity calculated from the density of GCs and eye geometry and expressed in cycles per degree) was minimum in the ventral periphery (smaller fish, 1.46 cpd; larger fish, 1.26 cpd) and maximum in area retinae temporalis (smaller fish, 2.83 cpd; larger fish, 2.75 cpd). The relatively high density of GCs and the presence of area retinae temporalis in the Japanese smelt are consistent with its highly visual behavior. The present findings contribute to our understanding of the factors affecting the topography of retinal ganglion cells and visual acuity in fish.     

Chitosan-g-oligo/polylactide copolymer non-woven fibrous mats containing protein: from solid-state synthesis to electrospinning

Graft-copolymers based on bioresorbable synthetic (oligo-/polylactide) and natural (chitosan and collagen/gelatin) components were synthesized through solid-state reactive co-extrusion and used for fabrication of fibrous non-woven mats via the electrospinning technique. The effect of the macromolecular features of the initial components on the copolymer characteristics was evaluated using FTIR-spectroscopy, differential scanning calorimetry and elemental analysis. Dynamic light scattering analysis showed that the copolymers have a tendency to form stable ultra-fine dispersions with a mean size of macromolecular aggregates of 150 nm within chlorinated solvents. The copolymer-containing non-woven fibrous mats were fabricated via an electrospinning procedure using chloroform as a solvent. An effect of the copolymer composition on the casting solution''s viscosity, conductivity and surface tension was evaluated. Scanning electron microscopy showed that the obtained mats consist of randomly distributed fibers with a mean size of ∼5 μm and a more complex morphology than mats fabricated from neat polylactide. The proposed mechanochemical approach to obtain hybrid copolymeric compositions differs from typical liquid-phase methods in terms of high efficiency, simplicity and cleanness.

Amphiphilic chitosan-g-oligo/polylactide graft-copolymers were synthesized through solid-state reactive co-extrusion and used for fabrication of fibrous non-woven mats via the electrospinning technique using chloroform as a solvent.     

Regulation of cardiac sodium-calcium exchanger by beta-adrenergic agonists.

Na+-Ca2+ exchanger and Ca2+ channel are two major sarcolemmal Ca2+-transporting proteins of cardiac myocytes. Although the Ca2+ channel is effectively regulated by protein kinase A-dependent phosphorylation, no enzymatic regulation of the exchanger protein has been identified as yet. Here we report that in frog ventricular myocytes, isoproterenol down-regulates the Na+-Ca2+ exchanger, independent of intracellular Ca2+ and membrane potential, by activation of the beta-receptor/adenylate-cyclase/cAMP-dependent cascade, resulting in suppression of transmembrane Ca2+ transport via the exchanger and providing for the well-documented contracture-suppressant effect of the hormone on frog heart. The beta-blocker propranolol blocks the isoproterenol effect, whereas forskolin, cAMP, and theophylline mimic it. In the frog heart where contractile Ca2+ is transported primarily by the Na+-Ca2+ exchanger, the beta-agonists' simultaneous enhancement of Ca2+ current, ICa, and suppression of Na+-Ca2+ exchanger current, INa-Ca would enable the myocyte to develop force rapidly at the onset of depolarization (enhancement of ICa) and to decrease Ca2+ influx (suppression of INa-Ca) later in the action potential. This unique adrenergically induced shift in the Ca2+ influx pathways may have evolved in response to paucity of the sarcoplasmic reticulum Ca2+-ATPase/phospholamban complex and absence of significant intracellular Ca2+ release pools in the frog heart.