NS309 restores EDHF-type relaxation in mesenteric small arteries from type 2 diabetic ZDF rats.

Background And Purpose: The endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in mesenteric small arteries from 21 week old Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats was investigated using (6,7-dichloro-1H-indole-2,3-dione 3-oxime) (NS309), a potent activator of small-conductance, calcium-activated potassium channel (SK(Ca)) and intermediate-conductance, calcium-activated potassium channel (IK(Ca)).

Experimental Approach: In the presence of inhibitors of cyclooxygenase and nitric oxide synthase [indomethacin and N(omega)-nitro-L-arginine methyl ester (l-NAME), respectively], acetylcholine (ACh)-induced hyperpolarization and EDHF-type relaxation were investigated under isometric conditions in the wire myograph using 0.5 and 1 microM NS309 and/or selective blockers of SK(Ca) and IK(Ca) channels.

Resting membrane potential and Na+,K+-ATPase of rat fast and slow muscles during modeling of hypogravity.

Antiorthostatic hindlimb suspension (unloading) decreased the resting membrane potential (RMP) of skeletal muscle fibers in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle of the rat by about 10% within 7 days and more. Inactivation of the membrane Na+,K+-pump by ouabain brought about similar depolarization as unloading. The increased sodium permeability of the membrane was excluded as the major cause of this depolarization by experiments in which TRIS was substituted for Na+ in the medium.

Jugular venous pooling during lowering of the head affects blood pressure of the anesthetized giraffe.

How blood flow and pressure to the giraffe's brain are regulated when drinking remains debated. We measured simultaneous blood flow, pressure, and cross-sectional area in the carotid artery and jugular vein of five anesthetized and spontaneously breathing giraffes. The giraffes were suspended in the upright position so that we could lower the head.

Role of potassium and chlorine channels in the regulation of thymocyte volume in rats.

Regulatory decrease in thymocyte volume under conditions of osmotic stress was abolished by potassium and chlorine channel blockers. Osmotic stress-activated chlorine channels belong to 2 pharmacological types. The maxi-anion channel is sensitive to Gd(3+).

Insulin inhibits Na+/H+ exchange in vascular smooth muscle and endothelial cells in situ: involvement of H2O2 and tyrosine phosphatase SHP-2.

Insulin signals through several intracellular pathways. Here, we tested the hypothesis that insulin modulates Na+/H+ exchange (NHE) activity in vascular cells through H2O2-mediated inhibition of tyrosine phosphatase Src homology 2 domain containing tyrosine phosphatase 2 (SHP-2). We measured intracellular pH (pHi) in isolated mouse mesenteric arteries using fluorescence confocal and wide-field microscopy.

Imaging of Ca2+ responses mediated by presynaptic L-type channels on GABAergic boutons of cultured hippocampal neurons.

We have previously demonstrated that L-type Ca(2+) channels are involved in post-tetanic potentiation (PTP) of GABAergic IPSCs in cultured hippocampal neurons. Here we have used intracellular Fluo-3 to detect [Ca(2+)](i) in single GABAergic boutons in response to stimulation that evokes PTP. During control stimulation of the presynaptic GABAergic neuron at 40 Hz for 1-2 s, DeltaF/F(0) increased rapidly to a peak value and started to decline shortly after the train ended, returning to baseline within 10-20 s.

Increased contractility to noradrenaline and normal endothelial function in mesenteric small arteries from the Goto-Kakizaki rat model of type 2 diabetes.

Unlabelled: Type 2 diabetes is associated with many circulatory manifestations, including alteration in endothelial function and hypertension. In this study we investigate the morphology and contractile response as well as the endothelial function of resistance arteries from the spontaneously diabetic Goto-Kakizaki (GK) rat, a model of lean type 2 diabetes expressing glucose intolerance.

Methods: Isolated mesenteric small arteries were investigated under isometric conditions in a wire myograph system using noradrenaline (NA) and the endothelium-dependent vasorelaxant acetylcholine (ACh).

Bestrophin-3 (vitelliform macular dystrophy 2-like 3 protein) is essential for the cGMP-dependent calcium-activated chloride conductance in vascular smooth muscle cells.

Although the biophysical fingerprints (ion selectivity, voltage-dependence, kinetics, etc) of Ca(2+)-activated Cl(-) currents are well established, their molecular identity is still controversial. Several molecular candidates have been suggested; however, none of them has been fully accepted. We have recently characterized a cGMP-dependent Ca(2+)-activated Cl(-) current with unique characteristics in smooth muscle cells.

SSADH deficiency leads to elevated extracellular GABA levels and increased GABAergic neurotransmission in the mouse cerebral cortex.

Succinic semialdehyde dehydrogenase (SSADH) deficiency is an inherited disorder in which patients display neurodevelopmental retardation, ataxia, and epileptic seizures. The recently engineered SSADH knock-out (KO) mouse models the severe form of the human disorder. The SSADH enzyme participates in the breakdown of the inhibitory neurotransmitter GABA, and studies have shown increases in brain GABA and downregulation of GABA(A) receptor beta(2) subunits in the cerebral cortex of these mice.

Identification and function of a cytoplasmic K+ site of the Na+, K+ -ATPase.

A cytoplasmic nontransport K(+)/Rb(+) site in the P-domain of the Na(+), K(+)-ATPase has been identified by anomalous difference Fourier map analysis of crystals of the [Rb(2)].E(2).MgF(4)(2-) form of the enzyme.

Effects of calcitonin gene-related peptide on rat soleus muscle excitability: mechanisms and physiological significance.

Intense exercise causes a large loss of K(+) from contracting muscles. The ensuing elevation of extracellular K(+) ([K(+)](o)) has been suggested to cause fatigue by depressing muscle fiber excitability. In isolated muscles, however, repeated contractions confer some protection against this effect of elevated K(+).

Aldosterone increases KCa1.1 (BK) channel-mediated colonic K+ secretion.

Mammalian K(+) homeostasis results from highly regulated renal and intestinal absorption and secretion, which balances the unregulated K(+) intake. Aldosterone is known to enhance both renal and colonic K(+) secretion. In mouse distal colon K(+) secretion occurs exclusively via luminal K(Ca)1.

Capsaicin stimulates uncoupled ATP hydrolysis by the sarcoplasmic reticulum calcium pump.

In muscle cells the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) couples the free energy of ATP hydrolysis to pump Ca(2+) ions from the cytoplasm to the SR lumen. In addition, SERCA plays a key role in non-shivering thermogenesis through uncoupled reactions, where ATP hydrolysis takes place without active Ca(2+) translocation. Capsaicin (CPS) is a naturally occurring vanilloid, the consumption of which is linked with increased metabolic rate and core body temperature.

Vasomotion has chloride-dependency in rat mesenteric small arteries.

The possibility that Ca(2+)-activated Cl(-) conductances (CaCCs) contribute to oscillations in vascular tone (vasomotion) is tested in isolated mesenteric small arteries from rats where cGMP independent (I (Cl(Ca))) and cGMP-dependent (I (Cl(Ca,cGMP))) chloride conductances are important. The effect of anion substitution and Cl(-) channel blockers on noradrenaline (NA)-stimulated tension in isometrically mounted mesenteric arteries and for chloride conductance of smooth muscle cells isolated from these arteries were assessed electrophysiologically. Cl(-) (o) replacement with aspartate blocked vasomotion while 36mM SCN(-) (o) (substituted for Cl(-)) was sufficient to inhibit vasomotion.

Cell type-specific GABA A receptor-mediated tonic inhibition in mouse neocortex.

Activity of extrasynaptic GABA A receptors mediating tonic inhibition is thought to play an important role for the excitability of the mammalian cerebral cortex. However, little is known about the cell type-specific expression of tonic inhibition in particular types of cortical interneurons. Here, we used transgenic mice expressing green fluorescent protein (GFP) in somatostatin-positive (SOM) interneurons and investigated tonic inhibition in SOM interneurons versus pyramidal cells in neocortical layers 2/3.

Clearance of extracellular K+ during muscle contraction--roles of membrane transport and diffusion.

Excitation of muscle often leads to a net loss of cellular K + and a rise in extracellular K+([K+]o), which in turn inhibits excitability and contractility. It is important, therefore, to determine how this K+ is cleared by diffusion into the surroundings or by reaccumulation into the muscle cells. The inhibitory effects of the rise in [K+] o may be assessed from the time course of changes in tetanic force in isolated muscles where diffusional clearance of K+ is eliminated by removing the incubation medium and allowing the muscles to contract in air.

Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase.

Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of K(+). Studies with purified membranes revealed that CPZ reduced Na(+)-dependent phosphorylation by interference with Na(+) binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K(+).

Cholesterol-dependent interaction of polyunsaturated phospholipids with Na,K-ATPase.

Polyunsaturated phospholipids such as 16:0-22:6 PC and 22:6 PC both stabilized the E1 conformation and inhibited turnover of Na,K-ATPase reconstituted into 18:1 PC or 18:1 PC/cholesterol liposomes. The inhibition increases in the order 22:6 PC > 16:0-22:6 PC both in the presence and in the absence of cholesterol, but is most pronounced in the absence of cholesterol. The inhibition of Na,K-ATPase turnover may thus correlate with the capability of polyunsaturated phospholipids and cholesterol to induce liquid-disordered and liquid-ordered lipid phases, respectively.

Regulation of Na+-K+ homeostasis and excitability in contracting muscles: implications for fatigue.

The performance of skeletal muscles depends on their ability to initiate and propagate action potentials along their outer membranes in response to motor signals from the central nervous system. This excitability of muscle fibres is related to the function of Na+ and K+ and Cl- channels and to steep chemical gradients for the ions across the cell membranes, i.e.

Osmotic stress and viscous retardation of the Na,K-ATPase ion pump.

The transport function of the Na pump (Na,K-ATPase) in cellular ion homeostasis involves both nucleotide binding reactions in the cytoplasm and alternating aqueous exposure of inward- and outward-facing ion binding sites. An osmotically active, nonpenetrating polymer (poly(ethyleneglycol); PEG) and a modifier of the aqueous viscosity (glycerol) were used to probe the overall and partial enzymatic reactions of membranous Na,K-ATPase from shark salt glands. Both inhibit the steady-state Na,K-ATPase as well as Na-ATPase activity, whereas the K(+)-dependent phosphatase activity is little affected by up to 50% of either.

Slow spontaneous [Ca2+] i oscillations reflect nucleotide release from renal epithelia.

Renal epithelia can be provoked mechanically to release nucleotides, which subsequently increases the intracellular Ca(2+) concentration [Ca(2+)](i) through activation of purinergic (P2) receptors. Cultured cells often show spontaneous [Ca(2+)](i) oscillations, a feature suggested to involve nucleotide signalling. In this study, fluo-4 loaded Madin-Darby canine kidney (MDCK) cells are used as a model for quantification and characterisation of spontaneous [Ca(2+)](i) increases in renal epithelia.

Role of Na+,K+-pumps and transmembrane Na+,K+-distribution in muscle function. The FEPS lecture - Bratislava 2007.

Na(+),K(+)-ATPase situated in the plasma membrane mediates active extrusion of Na(+) and intracellular accumulation of K(+). This transport system the Na(+),K(+)-pump is the major regulator of the transmembrane distribution of Na(+) and K(+), and is itself subject to regulation by a wide variety of factors in skeletal muscles. The excitation of skeletal muscles is elicited by a rapid influx of Na(+), followed by an equivalent efflux of K(+) across sarcolemmal and t-tubular membranes.

Potassium, Na+,K+-pumps and fatigue in rat muscle.

During contractile activity, skeletal muscles undergo a net loss of cytoplasmic K(+) to the interstitial space. During intense exercise, plasma K(+) in human arterial blood may reach 8 mm, and interstitial K(+) 10-12 mm. This leads to depolarization, loss of excitability and contractile force.

Nucleotide binding to Na,K-ATPase: pK values of the groups affecting the high affinity site.

Investigation of the ionic strength effect on the interactions between nucleotides (ATP and ADP) and Na,K-ATPase in a broad pH range was aimed at revealing pK values of the charged groups of the interacting species. Ionic strength experiments suggested that an amino acid residue with a pK > 8.0 is part of the protein binding site.