Horizontal connections are a principal component of intrinsic cortical circuitry. They arise mainly from pyramidal cells and course parallel to the brain's surface for distances as long as 8 mm, linking columns with shared orientation preference and allowing cells to integrate visual information from outside their receptive fields. We examined the synaptic physiology of the horizontal pathway in slices of the cat's striate cortex and found that activating lateral fibers produced both excitation and inhibition. We recorded the postsynaptic responses of identified pyramidal cells in layer 2 + 3 of area 17 to electrical shocks applied at three sites: in the home column of the impaled neuron either in layer 2 + 3 or 4, or at a lateral distance of 0.9-3 mm in layer 2 + 3. Within the home column, suprathreshold stimuli produced compound EPSPs with action potentials, followed by fast, GABAAergic IPSPs and a slower, GABABergic IPSP. For the distant stimulating site, the threshold response was an EPSP. Stronger shocks frequently evoked a disynaptic, GABAAergic IPSP that truncated the EPSP and could dominate the postsynaptic response. At the resting potential, the horizontally evoked EPSP was too small to elicit spikes. With depolarization of the membrane, however, it grew several hundred-fold. This amplification was blocked by N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314), but not by 2-amino-5-phosphonovalerate (APV), indicating that it was mediated by Na+ channels, rather than by NMDA receptors. We propose that the horizontal connections provide the means for stimuli outside the receptive field to modulate activity elicited within its confines. The voltage-dependent enhancement of the laterally evoked EPSP may explain why stimulating the surround by itself fails to drive cells but can facilitate their response to stimuli within the receptive field. The ability to initiate disynaptic inhibition from lateral sites shows that recruiting appropriate groups of horizontal fibers can also have a suppressive effect. Thus, the effect of horizontal input is state dependent, with the size and sign of the laterally evoked response changing according to the balance of converging inputs.

Four isoforms of the Na+/H+ exchanger (NHE6-NHE9) are distributed to intracellular compartments in human cells. They are localized to Golgi and post-Golgi endocytic compartments as follows: mid- to trans-Golgi, NHE8; trans-Golgi network, NHE7; early recycling endosomes, NHE6; and late recycling endosomes, NHE9. No significant localization of these NHEs was observed in lysosomes. The distribution of these NHEs is not discrete in the cells, and there is partial overlap with other isoforms, suggesting that the intracellular localization of the NHEs is established by the balance of transport in and out of the post-Golgi compartments as the dynamic membrane trafficking. The overexpression of NHE isoforms increased the luminal pH of the compartments in which the protein resided from the mildly acidic pH to the cytosolic pH, suggesting that their in vivo function is to regulate the pH and monovalent cation concentration in these organelles. We propose that the specific NHE isoforms contribute to the maintenance of the unique acidic pH values of the Golgi and post-Golgi compartments in the cell.

Increased diastolic SR Ca2+ leak (J(leak)) could depress contractility in heart failure, but there are conflicting reports regarding the J(leak) magnitude even in normal, intact myocytes. We have developed a novel approach to measure SR Ca2+ leak in intact, isolated ventricular myocytes. After stimulation, myocytes were exposed to 0 Na+, 0 Ca2+ solution +/-1 mmol/L tetracaine (to block resting leak). Total cell [Ca2+] does not change under these conditions with Na+-Ca2+ exchange inhibited. Resting [Ca2+]i declined 25% after tetracaine addition (126+/-6 versus 94+/-6 nmol/L; P<0.05). At the same time, SR [Ca2+] ([Ca2+](SRT)) increased 20% (93+/-8 versus 108+/-6 micromol/L). From this Ca2+ shift, we calculate J(leak) to be 12 micromol/L per second or 30% of the SR diastolic efflux. The remaining 70% is SR pump unidirectional reverse flux (backflux). The sum of these Ca2+ effluxes is counterbalanced by unidirectional forward Ca2+ pump flux. J(leak) also increased nonlinearly with [Ca2+](SRT) with a steeper increase at higher load. We conclude that J(leak) is 4 to 15 micromol/L cytosol per second at physiological [Ca2+](SRT). The data suggest that the leak is steeply [Ca2+](SRT)-dependent, perhaps because of increased [Ca2+]i sensitivity of the ryanodine receptor at higher [Ca2+](SRT). Key factors that determine [Ca2+](SRT) in intact ventricular myocytes include (1) the thermodynamically limited Ca2+ gradient that the SR can develop (which depends on forward flux and backflux through the SR Ca2+ ATPase) and (2) diastolic SR Ca2+ leak (ryanodine receptor mediated).

Chronic hyperglycemia underlies microvascular complications in patients with type 1 diabetes. The mechanisms leading to these vascular complications are not fully understood. Recently, we observed that acute hyperglycemia results in endothelial glycocalyx damage. To establish whether glycocalyx is associated with microvascular damage, we performed glycocalyx perturbation volume measurements in type 1 diabetic patients with microalbuminuria (DM1-MA group; n = 7), without microalbuminuria (DM1-NA group; n = 7), and in age-matched control subjects (CON; n = 7). Systemic glycocalyx volume was determined comparing intravascular distribution volume of a glycocalyx-permeable tracer (dextran 40) to that of a glycocalyx-impermeable tracer (labeled erythrocytes). Sublingual capillaries were visualized using orthogonal polarization spectral microscopy to estimate microvascular glycocalyx. Patients and control subjects were matched according to age and BMI. Glycocalyx volume decreased in a stepwise fashion from CON, DM1-NA, and finally DM1-MA subjects (1.5 +/- 0.1, 0.8 +/- 0.4, and 0.2 +/- 0.1 l, respectively, P < 0.05). Microvascular glycocalyx in sublingual capillaries was also decreased in type 1 diabetes versus the control group (0.5 +/- 0.1 vs. 0.9 +/- 0.1 microm, P < 0.05). Plasma hyaluronan, a principal glycocalyx constituent, and hyaluronidase were increased in type 1 diabetes. In conclusion, type 1 diabetic patients are characterized by endothelial glycocalyx damage, the severity of which is increased in presence of microalbuminuria.

Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.

The salt tolerance of rice (Oryza sativa) correlates with the ability to exclude Na+ from the shoot and to maintain a low cellular Na+/K+ ratio. We have identified a rice plasma membrane Na+/H+ exchanger that, on the basis of genetic and biochemical criteria, is the functional homolog of the Arabidopsis (Arabidopsis thaliana) salt overly sensitive 1 (SOS1) protein. The rice transporter, denoted by OsSOS1, demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (Saccharomyces cerevisiae) cells and reduced their net cellular Na+ content. The Arabidopsis protein kinase complex SOS2/SOS3, which positively controls the activity of AtSOS1, phosphorylated OsSOS1 and stimulated its activity in vivo and in vitro. Moreover, OsSOS1 suppressed the salt sensitivity of a sos1-1 mutant of Arabidopsis. These results represent the first molecular and biochemical characterization of a Na+ efflux protein from monocots. Putative rice homologs of the Arabidopsis protein kinase SOS2 and its Ca2+-dependent activator SOS3 were identified also. OsCIPK24 and OsCBL4 acted coordinately to activate OsSOS1 in yeast cells and they could be exchanged with their Arabidopsis counterpart to form heterologous protein kinase modules that activated both OsSOS1 and AtSOS1 and suppressed the salt sensitivity of sos2 and sos3 mutants of Arabidopsis. These results demonstrate that the SOS salt tolerance pathway operates in cereals and evidences a high degree of structural conservation among the SOS proteins from dicots and monocots.

The observations summarized in this review indicate immunity to infection with type A influenza viruses is subtype specific since little or none is conveyed to subtypes possessing immunologically distinct HA and NA proteins. However, within a subtype, a prior antigenic experience with one variant may prevent or modify illness to another. The resulting degree of subtype immunity depends on the extent of relatedness between variants. Observations with H3N2 viruses indicate that homotypic resistance to subsequent infection and illness with the same virus is potent and of relatively long duration. The long lasting durability of such immunity was indicated by the epidemiologic pattern following the reappearance of H1N1 virus. Knowledge of the duration and specificity of immunity aids considerably in assessing mechanisms that account for host resistance to influenza. Recovery from influenza virus infection must involve a variety of humoral and cell-mediated immune mechanisms, and conclusions regarding the relative importance of each one are not possible at present. To prevent infection, involved immune mechanism(s) must account for: (a) subtype specificity, (b) reduced cross-reactivity of immunity for succeeding antigenic variants, (c) a long duration of immunity, and (d) immunity at the mucosal surface. Only antibody directed toward the HA molecule presently satisfies these properties and thus should be considered the major mediator of resistance to infection. Study of naturally occurring infection is needed for determining the duration and specificity of secretory IgA in nasal and lower respiratory secretions so as to establish its relative importance as a mediator of immunity. However, the described duration, specificity, and consistent relationship to immunity suggest that IgG antibody in respiratory secretions, derived entirely or partly from serum, is the most likely mediator of resistance to natural influenza.

Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV) infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants) to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus) with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na⁺/H⁺ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH.

Recent studies suggest that the human primary motor cortex (M1) is involved in motor learning, but the nature of that involvement is not clear. Here, learning-related changes in M1 excitability were studied with transcranial magnetic stimulation (TMS) while na subjects practiced either a ballistic or a ramp pinch task to the 0.5-Hz beat of a metronome. Subjects rapidly learned to optimize ballistic contractions as indicated by a significant increase in peak pinch acceleration and peak force after the 60-min practice epoch. The increase in force and acceleration was associated with an increase in motor evoked potential (MEP) amplitude in a muscle involved in the training (flexor policis brevis) but not in a muscle unrelated to the task (abductor digiti minimi). MEPs returned to their baseline amplitude after subjects had acquired the new skill, whereas no practice-induced changes in MEP amplitude were observed after subjects had overlearned the task, or after practicing slow ramp pinches. Since the changes in MEP amplitude were observed only after TMS of M1 but not after direct stimulation of the corticospinal tract, these findings indicate task- and effector-specific involvement of human M1 in rapid motor learning.

Electrophysiological and pharmacological studies of a cloned human dopamine transporter (hDAT) were undertaken to investigate the mechanisms of transporter function and the actions of drugs at this target. Using two-electrode voltage-clamp techniques with hDAT-expressing Xenopus laevis oocytes, we show that hDAT can be considered electrogenic by two criteria. (1) Uptake of hDAT substrates gives rise to a pharmacologically appropriate "transport-associated" current. (2) The velocity of DA uptake measured in oocytes clamped at various membrane potentials was voltage-dependent, increasing with hyperpolarization. Concurrent measurement of transport-associated current and substrate flux in individual oocytes revealed that charge movement during substrate translocation was greater than would be expected for a transport mechanism with fixed stoichiometry of 2 Na+ and 1 Cl- per DA+ molecule. In addition to the transport-associated current, hDAT also mediates a constitutive leak current, the voltage and ionic dependencies of which differ markedly from those of the transport-associated current. Ion substitution experiments suggest that alkali cations and protons are carried by the hDAT leak conductance. In contrast to the transport-associated functions, the leak does not require Na+ or Cl-, and DAT ligands readily interact with the transporter even in the absence of these ions. The currents that hDAT mediates provide a functional assay that readily distinguishes the modes of action of amphetamine-like "DA-releasing" drugs from cocaine-like translocation blockers. In addition, the voltage dependence of DA uptake suggests a mechanism through which presynaptic DA autoreceptor activation may accelerate the termination of dopaminergic neurotransmission in vivo.

The epithelial cells of the choroid plexuses secrete cerebrospinal fluid (CSF), by a process which involves the transport of Na(+), Cl(-) and HCO(3)(-) from the blood to the ventricles of the brain. The unidirectional transport of ions is achieved due to the polarity of the epithelium, i.e. the ion transport proteins in the blood-facing (basolateral) membrane are different to those in the ventricular (apical) membrane. The movement of ions creates an osmotic gradient which drives the secretion of H(2)O. A variety of methods (e.g. isotope flux studies, electrophysiological, RT-PCR, in situ hybridization and immunocytochemistry) have been used to determine the expression of ion transporters and channels in the choroid plexus epithelium. Most of these transporters have now been localized to specific membranes. For example, Na(+)-K(+)ATPase, K(+) channels and Na(+)-2Cl(-)-K(+) cotransporters are expressed in the apical membrane. By contrast the basolateral membrane contains Cl(-)- HCO(3) exchangers, a variety of Na(+) coupled HCO(3)(-) transporters and K(+)-Cl(-) cotransporters. Aquaporin 1 mediates water transport at the apical membrane, but the route across the basolateral membrane is unknown. A model of CSF secretion by the mammalian choroid plexus is proposed which accommodates these proteins. The model also explains the mechanisms by which K(+) is transported from the CSF to the blood.

Micropuncture studies have shown that glomerular filtration rate (GFR) falls in response to a rise in Na(+) or Cl(-) concentrations in the loop of Henle, whereas studies in isolated kidneys have shown that GFR falls in response to osmotic diuresis. To define the separate effects of an acute increase in plasma sodium (P(Na)), chloride (P(Cl)) or osmolality (P(osmol)), changes in renal blood flow (RBF) and GFR were measured during intrarenal infusions of hypertonic NaCl, NaHCO(3), Na acetate, dextrose, NH(4)Cl or NH(4)acetate to denervated kidneys. The infusions raised P(osmol) at the experimental kidney by 30-45 mosmol. RBF increased abruptly by 10-30% with all hypertonic infusions indicating that an acute increase in plasma tonicity causes renal vasodilatation. Renal vasodilatation persisted or increased further during infusion of dextrose, NaHCO(3) and Na acetate, but GFR was unchanged. In contrast, during infusion of the two Cl-containing solutions, vasodilatation was reversed after 1-5 min and RBF and GFR decreased (P < 0.01) below preinfusion levels. Prior salt depletion doubled the vasoconstriction seen with hypertonic NaCl infusions. Overall, changes in RBF were unrelated to changes in P(Na) or fractional Na or fluid reabsorption but correlated with changes in P(Cl) (r = -0.91) and fractional Cl(-) reabsorption (r = 0.94). The intrafemoral arterial infusion of the two Cl-containing solutions did not increase femoral vascular resistance. In conclusion, hyperchloremia produces a progressive renal vasoconstriction and fall in GFR that is independent of the renal nerves, is potentiated by prior salt depletion and is related to tubular Cl(-) reabsorption. Chloride-induced vasoconstriction appears specific for the renal vessels.

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca(2+) sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca(2+) for channels composed primarily of Orai1, Orai2, and Orai3, by co-expressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (I(crac)), but Orai3 failed to produce any detectable Ca(2+)-selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca(2+) and Ba(2+) or Mg(2+) solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na(+) currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na(+) currents in HEK293 cells, were all inhibited by extracellular Ca(2+) with a half-maximal concentration of approximately 20 mum. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca(2+), indicating similar affinities for Ca(2+) within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca(2+) depotentiation.

Klotho mutant mouse (kl-/-), a mouse model for human aging, exhibits various phenotypes in a wide range of organs including arteriosclerosis, neural degeneration, skin and gonadal atrophy, pulmonary emphysema, calcification of soft tissues, and cognition impairment. Klotho mRNA, however, is expressed only in brain, kidney, reproductive organs, pituitary gland, and parathyroid gland. Therefore it remains to be elucidated how lack of Klotho protein in these limited organs leads to the variety of phenotypes. To shed light on mechanisms by which Klotho protein acts on distant targets, we examined localization of Klotho protein in brain, kidney, and reproductive organs, and analyzed brain and kidney in kl-/- mice searching for changes in target regions in these organs. In brain, Klotho proteins were localized at choroid plexus, where the proteins were dominantly localized at the apical plasma membrane of ependymal cells. In kl-/- brain, reduction of synapses was evident in hippocampus, suggesting a role of Klotho as a humoral factor in cerebrospinal fluid. Klotho proteins in kidney localized at distal renal tubules. Interestingly, in kl-/-mice, type IIa Na/phosphate (Pi) cotransporters, which function at the proximal renal tubules in reabsorption of phosphate ions, were translocated. This suggests that Klotho protein in kidney is implicated in calcium homeostasis which regulates localization of type IIa Na/Pi cotransporters via parathyroid hormone (PTH). Klotho proteins in reproductive organs were expressed only in mature germ cells, although in kl-/- mice germ cell maturation was arrested at earlier stages. Thus, Klotho proteins not only function as a humoral factor, but also are implicated in hormonal regulation, which may explain why mutation of klotho gene results in a variety of phenotypes.

Electrogenic Na-Ca exchange has been known to act in the cardiac sarcolemma as a major mechanism for extruding Ca ions. Ionic flux measurements in cardiac vesicles have recently suggested that the exchange ratio is probably 3 Na:1 Ca, although a membrane current generated by such a process has not been isolated. Using the intracellular perfusion technique combined with the whole-cell voltage clamp, we were able to load Na+ inside and Ca2+ outside the single ventricular cells of the guinea pig and have succeeded in recording an outward Na-Ca exchange current while blocking most other membrane currents. The current is voltage-dependent, blocked by La3+ and does not develop in the absence of intracellular free Ca2+. This report presents the first direct measurement of the cardiac Na-Ca exchange current, and should facilitate the study of Ca2+ fluxes during cardiac activity, together with various electrical changes attributable to the Na-Ca exchange and the testing of proposed models.

We have recently disrupted Slc12a2, the gene encoding the secretory Na-K-2Cl cotransporter in mice (NKCC1) (Delpire et al., 1999). Gramicidin perforated-patch and whole-cell recordings were performed to study GABA-induced currents in dorsal root ganglion (DRG) neurons isolated from wild-type and homozygote NKCC1 knock-out mice. In wild-type DRG neurons, strong GABA-evoked inward current was observed at the resting membrane potential, suggesting active accumulation of Cl(-) in these cells. This GABA-induced current was blocked by picrotoxin, a GABA(A) receptor blocker. The strong Cl(-) accumulation that gives rise to depolarizing GABA responses is caused by Na-K-2Cl cotransport because reduction of external Cl(-) or application of bumetanide induced a decrease in [Cl(-)](i), whereas an increase in external K(+) caused an apparent [Cl(-)](i) accumulation. In contrast to control neurons, little or no net current was observed at the resting membrane potential in homozygote NKCC1 mutant DRG neurons. E(GABA) was significantly more negative, demonstrating the absence of Cl(-) accumulation in these cells. Application of bumetanide induced a positive shift of E(GABA), suggesting the presence of an outward Cl(-) transport mechanism. In agreement with an absence of GABA depolarization in DRG neurons, behavioral analysis revealed significant alterations in locomotion and pain perception in the knock-out mouse. Our results clearly demonstrate that the Na-K-2Cl cotransporter is responsible for [Cl(-)](i) accumulation in DRG neurons and that via regulation of intracellular Cl(-), the Na-K-2Cl cotransporter participates in the modulation of GABA neurotransmission and sensory perception.

The effects of 0.1-100 microM riluzole, a neuroprotective agent with anticonvulsant properties, were studied on neurons from rat brain cortex. Patch-clamp whole-cell recordings in voltage-clamp mode were performed on thin slices to examine the effects of the drug on a noninactivating (persistent) Na+ current (INa,p). INa,p was selected because it enhances neuronal excitability near firing threshold, which makes it a potential target for anticonvulsant drugs. When added to the external solution, riluzole dose-dependently inhibited INa,p up to a complete blocking of the current (EC50 2 microM), showing a significant effect at therapeutic drug concentrations. A comparative dose-effect study was carried out in the same cells for the other main known action of riluzole, the inhibitory effect on the fast transient sodium current. This effect was confirmed in our experiments, but we found that it was achieved at levels much higher than putative therapeutic concentrations. Only the effect on INa,p, and not that on fast sodium current, can account for the reduction in neuronal excitability observed in cortical neurons following riluzole treatment at therapeutic concentrations, and this might represent a novel mechanism accounting for the anticonvulsant and neuroprotective properties of riluzole.

The orphan receptor APJ and its recently identified endogenous ligand, apelin, exhibit high levels of mRNA expression in the heart. However, the functional importance of apelin in the cardiovascular system is not known. In isolated perfused rat hearts, infusion of apelin (0.01 to 10 nmol/L) induced a dose-dependent positive inotropic effect (EC50: 33.1+/-1.5 pmol/L). Moreover, preload-induced increase in dP/dt(max) was significantly augmented (P<0.05) in the presence of apelin. Inhibition of phospholipase C (PLC) with U-73122 and suppression of protein kinase C (PKC) with staurosporine and GF-109203X markedly attenuated the apelin-induced inotropic effect (P<0.001). In addition, zoniporide, a selective inhibitor of Na+-H+ exchange (NHE) isoform-1, and KB-R7943, a potent inhibitor of the reverse mode Na+-Ca2+ exchange (NCX), significantly suppressed the response to apelin (P<0.001). Perforated patch-clamp recordings showed that apelin did not modulate L-type Ca2+ current or voltage-activated K+ currents in isolated adult rat ventricular myocytes. Apelin mRNA was markedly downregulated in cultured neonatal rat ventricular myocytes subjected to mechanical stretch and in vivo in two models of chronic ventricular pressure overload. The present study provides the first evidence for the physiological significance of apelin in the heart. Our results show that apelin is one of the most potent endogenous positive inotropic substances yet identified and that the inotropic response to apelin may involve activation of PLC, PKC, and sarcolemmal NHE and NCX.

Ruthenium red is a well known inhibitor of Ca2+ uptake into mitochondria in vitro. However, its utility as an inhibitor of Ca2+ uptake into mitochondria in vivo or in situ in intact cells is limited because of its inhibitory effects on sarcoplasmic reticulum Ca2+ release channel and other cellular processes. We have synthesized a ruthenium derivative and found it to be an oxygen-bridged dinuclear ruthenium amine complex. It has the same chemical structure as Ru360 reported previously (Emerson, J., Clarke, M. J., Ying, W-L., and Sanadi, D. R. (1993) J. Am. Chem. Soc. 115, 11799-11805). Ru360 has been shown to be a potent inhibitor of Ca2+-stimulated respiration of liver mitochondria in vitro. However, the specificity of Ru360 on Ca2+ uptake into mitochondria in vitro or in intact cells has not been determined. The present study reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specificity in vitro in isolated mitochondria and in situ in isolated cardiac myocytes. Ru360 was more potent (IC50 = 0.184 nM) than ruthenium red (IC50 = 6.85 nM) in inhibiting Ca2+ uptake into mitochondria. 103Ru360 was found to bind to isolated mitochondria with high affinity (Kd = 0.34 nM, Bmax = 80 fmol/mg of mitochondrial protein). The IC50 of 103Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nM, indicating that saturation of a specific binding site is responsible for the inhibition of Ca2+ uptake. Ru360, as high as 10 microM, produced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sarcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ channel current, cytosolic Ca2+ transients, or cell shortening. 103Ru360 was taken up by isolated myocytes in a time-dependent biphasic manner. Ru360 (10 microM) applied outside intact voltage-clamped ventricular myocytes prevented Ca2+ uptake into mitochondria in situ where the cells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exchange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells.

Mutations in the kinase WNK4 cause pseudohypoaldosteronism type II (PHAII), a syndrome featuring hypertension and high serum K(+) levels (hyperkalemia). WNK4 has distinct functional states that regulate the balance between renal salt reabsorption and K(+) secretion by modulating the activities of renal transporters and channels, including the Na-Cl cotransporter NCC and the K(+) channel ROMK. WNK4's functions could enable differential responses to intravascular volume depletion (hypovolemia) and hyperkalemia. Because hypovolemia is uniquely associated with high angiotensin II (AngII) levels, AngII signaling might modulate WNK4 activity. We show that AngII signaling in Xenopus oocytes increases NCC activity by abrogating WNK4's inhibition of NCC but does not alter WNK4's inhibition of ROMK. This effect requires AngII, its receptor AT1R, and WNK4, and is prevented by the AT1R inhibitor losartan. NCC activity is also increased by WNK4 harboring mutations found in PHAII, and this activity cannot be further augmented by AngII signaling, consistent with PHAII mutations providing constitutive activation of the signaling pathway between AT1R and NCC. AngII's effect on NCC is also dependent on the kinase SPAK because dominant-negative SPAK or elimination of the SPAK binding motif in NCC prevent activation of NCC by AngII signaling. These effects extend to mammalian cells. AngII increases phosphorylation of specific sites on SPAK and NCC that are necessary for activation of each in mpkDCT cells. These findings place WNK4 in the signaling pathway between AngII and NCC, and provide a mechanism by which hypovolemia maximizes renal salt reabsoprtion without concomitantly increasing K(+) secretion.

Accumulation of Na(+) channels at the nodes of Ranvier is a prerequisite for saltatory conduction. In peripheral nerves, clustering of these channels along the axolemma is regulated by myelinating Schwann cells through a yet unknown mechanism. We report the identification of gliomedin, a glial ligand for neurofascin and NrCAM, two axonal immunoglobulin cell adhesion molecules that are associated with Na+ channels at the nodes of Ranvier. Gliomedin is expressed by myelinating Schwann cells and accumulates at the edges of each myelin segment during development, where it aligns with the forming nodes. Eliminating the expression of gliomedin by RNAi, or the addition of a soluble extracellular domain of neurofascin to myelinating cultures, which caused the redistribution of gliomedin along the internodes, abolished node formation. Furthermore, a soluble gliomedin induced nodal-like clusters of Na+ channels in the absence of Schwann cells. We propose that gliomedin provides a glial cue for the formation of peripheral nodes of Ranvier.

Bile salts are the major organic solutes in bile and undergo extensive enterohepatic circulation. Hepatocellular bile salt uptake is mediated predominantly by the Na(+)-taurocholate cotransport proteins Ntcp (rodents) and NTCP (humans) and by the Na(+)-independent organic anion-transporting polypeptides Oatp1, Oatp2, and Oatp4 (rodents) and OATP-C (humans). After diffusion (bound by intracellular bile salt-binding proteins) to the canalicular membrane, monoanionic bile salts are secreted into bile canaliculi by the bile salt export pump Bsep (rodents) or BSEP (humans). Both belong to the ATP-binding cassette (ABC) transporter superfamily. Dianionic conjugated bile salts are secreted into bile by the multidrug-resistance-associated proteins Mrp2/MRP2. In bile ductules, a minor portion of protonated bile acids and monomeric bile salts are reabsorbed by non-ionic diffusion and the apical sodium-dependent bile salt transporter Asbt/ASBT, transported back into the periductular capillary plexus by Mrp3/MRP3 [and/or a truncated form of Asbt (tAsbt)], and subjected to cholehepatic shunting. The major portion of biliary bile salts is aggregated into mixed micelles and transported into the intestine, where they are reabsorbed by apical Oatp3, the apical sodium-dependent bile salt transporter (ASBT), cytosolic intestinal bile acid-binding protein (IBABP), and basolateral Mrp3/MRP3 and tAsbt. Transcriptional and posttranscriptional regulation of these enterohepatic bile salt transporters is closely related to the regulation of lipid and cholesterol homeostasis. Furthermore, defective expression and function of bile salt transporters have been recognized as important causes for various cholestatic liver diseases.

Malignant gliomas have been shown to release glutamate, which kills surrounding brain cells, creating room for tumor expansion. This glutamate release occurs primarily via system xC, a Na+-independent cystine-glutamate exchanger. We show here, in addition, that the released glutamate acts as an essential autocrine/paracrine signal that promotes cell invasion. Specifically, chemotactic invasion and scrape motility assays each show dose-dependent inhibition of cell migration when glutamate release was inhibited using either S-(4)-CPG or sulfasalazine, both potent blockers of system xC. This inhibition could be overcome by the addition of exogenous glutamate (100 micromol/L) in the continued presence of the inhibitors. Migration/invasion was also inhibited when Ca2+-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPA-R) were blocked using GYKI or Joro spider toxin, whereas CNQX was ineffective. Ca2+ imaging experiments show that the released glutamate activates Ca2+-permeable AMPA-R and induces intracellular Ca2+ oscillations that are essential for cell migration. Importantly, glioma cells release glutamate in sufficient quantities to activate AMPA-Rs on themselves or neighboring cells, thus acting in an autocrine and/or paracrine fashion. System xC and the appropriate AMPA-R subunits are expressed in all glioma cell lines, patient-derived glioma cells, and acute patient biopsies investigated. Furthermore, animal studies in which human gliomas were xenographed into scid mice show that chronic inhibition of system xC-mediated glutamate release leads to smaller and less invasive tumors compared with saline-treated controls. These data suggest that glioma invasion is effectively disrupted by inhibiting an autocrine glutamate signaling loop with a clinically approved candidate drug, sulfasalazine, already in hand.