14-3-3 proteins: a family of versatile molecular regulators.

The 14-3-3 proteins are a family of acidic regulatory molecules found in all eukaryotes. 14-3-3 proteins function as molecular scaffolds by modulating the conformation of their binding partners. Through the functional modulation of a wide range of binding partners, 14-3-3 proteins are involved in many processes, including cell cycle regulation, metabolism control, apoptosis, and control of gene transcription. This minireview includes a short overview of 14-3-3 proteins and then focuses on their role in the regulation of two important binding partners: FOXO forkhead transcription factors and an enzyme tyrosine hydroxylase.

Subcellular redistribution of trimeric G-proteins--potential mechanism of desensitization of hormone response: internalization, solubilization, down-regulation.

Agonist-induced subcellular redistribution of G-protein coupled receptors (GPCR) and of trimeric guanine-nucleotide binding regulatory proteins (G-proteins) represent mechanisms of desensitization of hormone response, which have been studied in our laboratory since 1989. This review brings a short summary of these results and also presents information about related literature data covering at least small part of research carried out in this area. We have also mentioned sodium plus potassium dependent adenosine triphosphatase (Na, K-ATPase) and 3H-ouabain binding as useful reference standard of plasma membrane purity in the brain.

Functional changes in the vanilloid receptor subtype 1 channel during and after acute desensitization.

Agonist-induced desensitization of the transient receptor potential vanilloid receptor-1 (TRPV1) is one of the key strategies that offer a way to alleviate neuropathic and inflammatory pain. This process is initiated by TRPV1 receptor activation and the subsequent entry of extracellular Ca(2+) through the channel into sensory neurones. One of the prominent mechanisms responsible for TRPV1 desensitization is dephosphorylation of the TRPV1 protein by the Ca(2+)/calmodulin-dependent enzyme, phosphatase 2B (calcineurin). Of several consensus phosphorylation sites identified so far, the most notable are two sites for Ca(2+)/calmodulin dependent kinase II (CaMKII) at which the dynamic equilibrium between the phosphorylated and dephosphorylated states presumably regulates agonist binding. We examined the mechanisms of acute Ca(2+)-dependent desensitization using whole-cell patch-clamp techniques in human embryonic kidney (HEK) 293T cells expressing the wild type or CaMKII phosphorylation site mutants of rat TRPV1. The nonphosphorylatable mutant S502A/T704I was capsaicin-insensitive but the S502A/T704A construct was fully functional, indicating a requirement for a specific residue at position 704. A point mutation at the nearby conserved residue R701 strongly affected the heat, capsaicin and pH-evoked currents. As this residue constitutes a stringent CaMKII consensus site but is also predicted to be involved in the interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), these data suggest that in addition to dephosphorylation, or as its consequence, a short C-terminal juxtamembrane segment adjacent to the transient receptor potential box composed of R701 and T704 might be involved in the decelerated gating kinetics of the desensitized TRPV1 channel.

An upstream enhancer regulating brown-fat-specific expression of the mitochondrial uncoupling protein gene.

Previous studies on the regulation of a Ucp minigene in transgenic mice demonstrated that the sequences necessary for brown-fat-specific expression and inducibility by norepinephrine were located in the 5' flanking region between 1 and 2.8 kb from the transcriptional start site. We have investigated this region in more detail in cultured mouse brown adipocyte tumor cells. Deletion analysis of two types of chloramphenicol acetyltransferase reporter gene constructs under control of either the Ucp promoter or a heterologous herpes simplex virus-tk promoter defined an enhancer in a 220-bp HindIII-XbaI fragment which was essential for both brown fat specificity and norepinephrine inducibility. Site-directed mutagenesis of the reporter gene constructs established that independent mutations to a cyclic AMP-responsive element (CRE-2) or one of two TTCC motifs (BRE [brown fat regulatory element]), all within 17 bp, eliminated transient expression. Competitive DNA mobility shift assays with probes of the CRE and BRE motifs indicate that nuclear proteins interact with these motifs in a cooperative, synergistic manner. While these CRE-BRE probes do not show changes in binding which is dependent on norepinephrine treatment, a probe containing a third TTCC motif located 130 bp downstream of BRE-1 does show this dependency. The results indicate that a complex interaction of the CRE and BRE motifs, which cannot be functionally separated, control Ucp expression.

Hyperpolarization of mouse skeletal muscle plasma membrane induced by extracellular NADH.

Extracellularly applied NADH, but not NAD or NADPH, increases the resting membrane potential from -74.1 to -76.6 mV in freshly isolated muscles in the presence of K+ in the incubation medium and from -64.6 to -72.9 mV in muscles equilibrated for 4-6 h in a K+-free solution. The NADH-induced hyperpolarization is blocked by pretreatment of muscles with ouabain, and the inhibitors of plasma membrane NADH dehydrogenase (adriamycin, azide, PCMB, atebrine, DIDS and bleomycin). The effect of NADH is accompanied by the disappearance of NADH from the incubation medium and by decreased membrane resistance. We conclude that NADH hyperpolarization is due to the enhancement of passive membrane permeability, apparently for K+, which might result from the conformational changes in the plasma membrane during the NADH dehydrogenase reaction. The possibility is discussed that NADH dehydrogenase mediates transport of K+ out from the cell using a pathway connected with the transmembrane Na+/K+ pump.

Mg2+-induced changes of lipid order and conformation of (Na+ + K+)-ATPase.

The effect of magnesium on the phospholipid order parameter and not the conformation of purified pig kidney outer medulla (Na+ + K+)-ATPase was investigated by fluorescence techniques. Measurements with a fluorescent probe TMA-DPH and its sensitized fluorescence with tryptophan residues as donors revealed that magnesium increased the order of the membrane phospholipids both in the lipid annulus and in the bulk phase. Changes in the lipid order induced by Mg2+ can be closely referred to the protein arrangement followed by the steady-state anisotropy of FITC-labeled (Na+ + K+)-ATPase.

The comparison of vanadyl (IV) and insulin-induced hyperpolarization of the mammalian muscle cell.

Extracellularly applied vanadyl (IV) hyperpolarized the membrane potential of mouse diaphragm muscle from about -74.0 mV up to -81.7 mV. The hyperpolarizing effect of 10(-4) mol.I-1 vanadyl (IV) is comparable with hyperpolarization induced by 100 mU.ml-1 insulin. Both compounds increased the intracellular K+ concentration, the hyperpolarizing effect of vanadyl (IV) and insulin is blocked by ouabain and is unaffected by removal of K+ from the external medium. Triggering of the release of intracellular K+ associated with cellular proteins is proposed as the mechanism of vanadyl (IV) and insulin-induced hyperpolarization.

The role of carboxyl groups of Na+/K+-ATPase in the interaction with divalent cations.

Mg2+-induced subconformational changes of the E1 conformation of partly purified pig kidney Na+/K+-ATPase were studied by fluorescence techniques. In the enzyme with carboxyl groups modified by carbodiimide in the presence of an exogenous nucleophile the efficiency to pass through conformational substates was substantially lower than in the unmodified enzyme. Magnesium could form bridges between carboxyl groups near the membrane/water interface and negatively charged phospholipid polar heads.

Vanadyl ions increase the order parameter of plasma membranes without changing the rotational relaxation time.

Differential polarized phase fluorometry of 1,6-diphenyl-1,3,5-hexatriene showed that vanadyl ions (VO2+) increased its limiting anisotropy (order parameter) in crude plasma membranes from brown adipose tissue of the golden hamster (Mesocricetus auratus). This was about 10(3) times larger than the effect of Ca2+ and was several times greater than the action of Co2+. Vanadate anions were without any effect. During the membrane treatment with VO2+, the rotational relaxational time of diphenylhexatriene did not change. This results suggest a possible positive influence of tetravalent vanadium on the stability of cell membranes.

The effect of anion channel blockers on enzymatic activity of Na+/K+-ATPase and the electrogenic Na+/K+ pump.

Disulfonic stilbenes which block the anion-transport in red blood cells were found to inhibit the brain microsomal Na+/K+-ATPase but not the electrogenic Na+/K+ pump in intact muscle cells. In contrast to the anion-transport system, the Na+/K+-ATPase is inhibited by disulfonic stilbenes, apparently from the cytoplasmic side of the membrane. The pathways for anion and active cation transport are thus different but similar groups of sulfhydryl and/or amino acid residues must play an important role in both systems.

Different cation binding to the I domains of alpha1 and alpha2 integrins: implication of the binding site structure.

In the present work, we studied the interactions of recombinant alpha1 and alpha2 integrin I domains with cations Tb(3+), Mn(2+), Mg(2+) and Ca(2+). We observed that alpha1 and alpha2 I domains bind these cations with significantly different characteristics. The binding of Mg(2+) by the alpha1 I domain was accompanied by significant changes of tryptophan fluorescence which could be interpreted as a conformational change. Comparison of the alpha1 integrin I domain structure obtained by comparative modeling with a known structure of the alpha2 integrin I domain shows distinct differences in the metal ion binding sites which could explain the differences in cation binding.

Oxidizing reagent copper-o-phenanthroline is an open channel blocker of the vanilloid receptor TRPV1.

The TRPV1 channel plays an important role in generating nociceptive signals in mammalian primary sensory neurons. It consists of 838 amino acids with six transmembrane segments (TM1-TM6), a pore-forming loop between TM5 and TM6 and N- and C- terminals located intracellularly. It is a homotetramer and forms a nonselective cationic channel that can be opened by capsaicin, weak acids and noxious heat. There are 18 cysteines (Cys), three of which are located on the extracellular side of the receptor in and around the region of the pore-forming loop. We report that the TRPV1 channel in transfected HEK293T cells and in cultured rat DRG neurons is blocked in the open state by an oxidizing agent Cu-o-phenanthroline complex (Cu:Phe). The effects of Cu:Phe are concentration dependent ( IC50 = 5.2 : 20.8 microm ) and fully reversible. Cu:Phe applied immediately before exposure to an acidic solution, capsaicin or noxious heat is without effect. Substitutions of the extracellular Cys residues (616, 621, 634) by glycine individually or together do not alter the blocking effects of Cu:Phe suggesting that disulfide cross-linking does not represent the underlying mechanism. It is suggested that the complex Cu:Phe, a bulky, positively charged molecule, represents a very effective and reversible open channel blocker of TRPV1.

Reducing agent dithiothreitol facilitates activity of the capsaicin receptor VR-1.

The vanilloid receptor subtype 1 (VR1) is expressed in a sub-population of small dorsal root ganglion (DRG) neurones in mammals and serves as the common transducer of the pain-producing signals, such as noxious heat, acids and capsaicin [Caterina et al., Nature 389 (1997) 816-824; Tominaga et al., Neuron 21 (1998) 531-543]. On the extracellular side, VR1 has three cysteine residues at positions 616, 621 and 634. Here we report that dithiothreitol (DTT) (2-60 mM), an agent that maintains -SH groups of cysteines in a reduced state, greatly facilitates membrane currents induced by noxious heat or capsaicin (1 microM) in cultured DRG neurones from the rat and in VR1-transfected HEK293 cells. The effects of DTT are concentration-dependent and fully reversible. We suggest that the ratio between free sulfhydryl groups and disulfide bonds of the cysteine residues of VR1 pre-sets sensitivity of primary nociceptors to algogens and may represent a new target for treating some pain states in humans.

Vanadyl (VO2+) induced lipoperoxidation in the brain microsomal fraction is not related to VO2+ inhibition of Na,K-ATPase.

Vanadyl (VO2+) is a potent inductor of the lipid peroxidation in brain microsomes. This effect, however, is obtained at concentrations by two orders of magnitude higher (10(-4)-10(-3)M) than those which effectively inhibit the brain microsomal Na,K-ATPase. At 10(-6)M VO2+ which inhibits 50% of the Na,K-ATPase activity there is no measurable malonyldialdehyde production. Vanadate (VO-3) which is an equally potent inhibitor of Na,K-ATPase as VO2+ has almost no capacity to induce the lipoperoxidation. The addition of 10(-4)M ascorbate to the brain microsomes stimulates the lipoperoxidation to the maximum level regardless of the presence or absence of exogenous vanadium ions. Ascorbate-induced inhibition of brain Na,K-ATPase which is known to be associated with lipoperoxidation is strictly additive with the vanadyl (VO2+) inhibition of this enzyme. Even at submaximal concentrations there is no indication for any potentiation between these two inhibitory systems. The disparity between the mechanisms of ascorbate and vanadyl-induced inhibition of Na,K-ATPase is also documented by the effect of EDTA which inhibits the former type only. It is concluded, that the vanadium-induced inhibition of brain microsomal Na,K-ATPase is not related to induction of lipoperoxidative capacity of the brain.

Vanadyl (VO2+) and vanadate (VO-3) ions inhibit the brain microsomal Na,K-ATPase with similar affinities. Protection by transferrin and noradrenaline.

The activity of Na,K-ATPase was measured in brain microsomes as the function of increasing concentrations of vanadyl (VOSO4, V4+) and the vanadate (NaVO3, V5+) ions. Both forms of vanadium inhibited the Na,K-ATPase activity with high affinity -Ki (vanadate) = 3 X 10(-7)M and Ki (vanadyl = 1 X 10(-6)M. The stability of V4+ in ATPase reaction media (Tris buffers) was measured by electron spin resonance spectroscopy. Without any reducing agent, V4+ was quickly oxidised by atmospheric oxygen. When a reducing agent such as dithiothreitol was added, the V4+ was stable for at least 30 min and the inhibition pattern of Na,K-ATPase by V4+ was not changed. The blocking effect of V4+ in the presence of dithiothreitol was counteracted by pre-incubation with equimolar concentrations of transferrin or 100 times excess of noradrenaline. The regulation of brain Na,K-ATPase by vanadate may be represented by competition between low-capacity inhibitory binding sites localized on the enzyme molecule and high-capacity sites of intracellular proteins. Preferential binding of vanadyl to the latter type of sites will decrease the intracellular concentration of the free metal and thus eliminate the enzyme inhibition.

Na(+), K(+) ATPase activity in membrane fractions of rat central and peripheral nervous tissues modified by opioids.

The inhibition by ouabain of Na(+), K(+) ATPase activity in lumbar spinal cord membranes followed a sigmoid curve and in sciatic nerve membranes an exponential curve. The degree of inhibition in spinal cord depended on the presence of opioid ligands; fractions became more sensitive to ouabain after removal of bound opioids. The saturation of receptors by specific ligands reversed the effect of preincubation. Opioids had no effect on Na(+), K(+) ATPase in sciatic nerve. Membranes isolated from spinal cord were highly sensitive to oubain inhibition and their Na(+), K(+) ATPase resembled that containing the ? (+) subunit; membranes from peripheral nerve were less sensitive to ouabain and their enzyme reacted as that containing the ? subunit.

Effect of cycloheximide administered to rats in early postnatal life: prolonged inhibition of DNA synthesis in the developing brain.

Administration of cycloheximide in a single dose of 0.6 mg/kg to 7-day-old rats was used to induce short-term inhibition of protein synthesis at the period of brain 'growth spurt'. Measurement of the rate of [14C]lysine incorporation indicated that the initial inhibition of protein synthesis in the brain (by 75%) was released within about 12 h. The normal rate of protein synthesis was attained by 48 h after cycloheximide administration; there was no sign of protein synthesis stimulation. The estimation of [14C]thymidine incorporation into brain DNA showed that inhibition of DNA synthesis was greater and longer lasting in the forebrain and olfactory bulbs (by about 80%) than in the cerebellum (by about 40%). Similar differential inhibition of thymidine kinase activity was observed in the olfactory bulbs (by 75%) and cerebellum (by 30%) at 24 h after cycloheximide, suggesting that the formation of [14C]thymidine nucleotides may have been impaired. However, a retardation of DNA accumulation was found in the forebrain and cerebellum at 72 h after cycloheximide. Thus, the short-term inhibition of protein synthesis produced prolonged inhibition of DNA synthesis and altered cell proliferation in the developing brain.

Arachidonate activates muscle electrogenic sodium pump and brain microsome Na+,K+-ATPase under suboptimal conditions.

Arachidonate 5 x 10(-5) mol.l-1 increased the rate of hyperpolarization induced in Na+-loaded mouse diaphragm fibers by 5 mmol.l-1 K+. When applied to Na+-loaded muscles without potassium, arachidonate 1 x 10(-6) and 5 x 10(-5) mol.l-1 induced a ouabain-sensitive hyperpolarization of the muscle fibers. The activity of rat brain microsomal Na+,K+-ATPase was stimulated by 1 x 10(-7)-5 x 10(-6) mol.l-1 arachidonate in reaction media with reduced amounts of ATP or K+ and after short-lasting sonication of the samples. It was concluded that, under particular conditions, arachidonate might serve as a Na+,K+-ATPase activator or inhibitor regulating its ion transport and electrogenicity.

The effect of opioids and of naloxone on Na+,K+-adenosine triphosphatase activity in frog spinal cord membrane fractions.

The effects of opioids and of naloxone on ouabain-sensitive Na+,K+-adenosine triphosphatase (ATPase) activity were studied in vitro on membrane fractions from frog spinal cords. The addition of morphine and of the stable enkephalin analogue, D-Ala2,D-Leu5-enkephalin, in concentrations from 10(-7) to 10(-4) M significantly increased Na+,K+-ATPase activity. No effect was found with methionine enkephalin (Met-Enk). However, the addition of two peptidase inhibitors, captopril and phosphoramidon (10(-5) M each), significantly increased Na+,K+-ATPase activity. A further increase in enzyme activity was found when Met-Enk (10(-4) or 10(-7) M) was added simultaneously with peptidase inhibitors. On the other hand, the addition of the opiate antagonist, naloxone, at low concentration (10(-7) M) decreased the activity of Na+,K+-ATPase. These results are discussed with respect to the effect of synthetic and endogenous opioids on the activity of Na+,K+-ATPase.