Regulation of vascular function by RCAN1 (ADAPT78)

RCAN1 (Adapt78) functions mainly, if not exclusively, as a regulator of calcineurin, a phosphatase that mediates many cellular responses to calcium. Identification of this regulatory activity has led to a surge of interest in RCAN1, since calcineurin is involved in many cellular and tissue functions, and its abnormal expression is associated with multiple pathologies. Recent studies have implicated RCAN1 as a regulator of angiogenesis. To more fully investigate the role of RCAN1 in vascular function, we first extended previous studies by assessing RCAN1 response in cultured endothelial cells to various vascular agonists. Strong induction of isoform 4 but not isoform 1 was observed in human umbilical vein- and bovine pulmonary aortic-endothelial cells in response to VEGF, thrombin, and ATP but not other agonists. Inductions were both calcium and calcineurin dependent, with the relative effect of each agonist cell-type dependent. Ectopic RCAN1 expression also inhibited calcineurin signaling in the HUVEC cells. Based on these strong RCAN1 responses and a lack of RCAN1-associated vascular studies beyond angiogenesis, we investigated the potential role of RCAN1 in vascular tone using whole mounted mesenteric artery. RCAN1 knockout mice exhibited an attenuated mesenteric vasoconstriction to phenylephrine as compared with wild-type. Overall contractility was unaffected, suggesting that this component of smooth muscle action is similar in the two mouse strains. Constriction in the knockout artery appeared to be potentiated by the addition of the nitric oxide synthase (NOS) inhibitor l-NAME, suggesting that elevated nitric oxide (NO) production occurs in the knockout vasculature and contributes to the weakened vasoconstriction. Our results reveal a newly identified vascular role for RCAN1, and a potential new target for treating vascular- and calcineurin-related disorders.     

Characterization of junctate-SERCA2a interaction in murine cardiomyocyte

Junctate is a newly identified sarcoplasmic reticulum (SR) Ca²⁺ binding protein, but its function in cardiac muscle has remained unclear. Our previous study showed that chronic over-expression of junctate in transgenic mice led to altered SR functions and development of severe hypertrophy. In this study, we identified the interaction of junctate with SERCA2a by co-immunoprecipitation and GST-pull-down assay. This interaction was inhibited by higher Ca²⁺ concentration. Immunolocalization assays also showed that junctate and SERCA2a were co-localized in the SR of cardiomyocytes. Direct binding of the C-terminal region of junctate (amino acids 79-270) and luminal domain of SERCA2a (amino acids 70-89) was observed by deletion mutation experiments. Adenovirus-mediated transient over-expression of junctate in cardiomyocytes showed a reduced decay time of Ca²⁺ transients and increased oxalate-supported SERCA2 Ca²⁺ uptake, suggesting an increased activity of SERCA2a. Taken together, according to our data, junctate may play an important role in the regulation of SR Ca²⁺ cycling through the interaction with SERCA2a in the murine heart.     

The inositol 1,4,5-trisphosphate (InsP3) receptor

Conclusion In this review, we have described the functional properties and regulation of the InsP₃R. Not all aspects of InsP₃R function and regulation were covered, the main focus was on the most recent and physiologically important data. Information about the structure, heterogeneity, functional properties, and regulation of the InsP₃R is useful for understanding the spatiotemporal aspects of Ca signaling. The combination of biochemical, biophysical and molecular biological techniques has revealed the intricacies of the InsP₃R over the past decade. However, questions about the functional differences between various isoforms and splice variants of the InsP₃R, the structural determinants responsible for regulation of InsP₃R by Ca and ATP, the functional effects of InsP₃R phosphorylation and many others remain to be elucidated. Future investigations can be expected to provide answers to these important questions.We thank S. Bezprozvannaya for expert technical assistance. This work was supported by National Institutes of Health grants HL 33026 and GM 39029, and a Grant-in-Aid from the Patrick and Catherine Weldon Donaghue Medical Research Foundation.     

Extracellular pH Regulates Zinc Signaling via an Asp Residue of the Zinc-sensing Receptor (ZnR/GPR39)

Zinc activates a specific Zn²⁺-sensing receptor, ZnR/GPR39, and thereby triggers cellular signaling leading to epithelial cell proliferation and survival. Epithelial cells that express ZnR, particularly colonocytes, face frequent changes in extracellular pH that are of physiological and pathological implication. Here we show that the ZnR/GPR39-dependent Ca²⁺ responses in HT29 colonocytes were maximal at pH 7.4 but were reduced by about 50% at pH 7.7 and by about 62% at pH 7.1 and were completely abolished at pH 6.5. Intracellular acidification did not attenuate ZnR/GPR39 activity, indicating that the pH sensor of this protein is located on an extracellular domain. ZnR/GPR39-dependent activation of extracellular-regulated kinase (ERK)1/2 or AKT pathways was abolished at acidic extracellular pH of 6.5. A similar inhibitory effect was monitored for the ZnR/GPR39-dependent up-regulation of Na⁺/H⁺ exchange activity at pH 6.5. Focusing on residues putatively facing the extracellular domain, we sought to identify the pH sensor of ZnR/GPR39. Replacing the histidine residues forming the Zn²⁺ binding site, His¹⁷ or His¹⁹, or other extracellular-facing histidines to alanine residues did not abolish the pH dependence of ZnR/GPR39. In contrast, replacing Asp³¹³ with alanine resulted in similar Ca²⁺ responses triggered by ZnR/GPR39 at pH 7.4 or 6.5. This mutant also showed similar activation of ERK1/2 and AKT pathways, and ZnR-dependent up-regulation of Na⁺/H⁺ exchange at pH 7.4 and pH 6.5. Substitution of Asp³¹³ to His or Glu residues restored pH sensitivity of the receptor. This indicates that Asp³¹³, which was shown to modulate Zn²⁺ binding, is an essential residue of the pH sensor of GPR39. In conclusion, ZnR/GPR39 is tuned to sense physiologically relevant changes in extracellular pH that thus regulate ZnR-dependent signaling and ion transport activity.     

Brain-derived Neurotrophic Factor (BDNF) Induces Sustained Intracellular Ca2+ Elevation through the Up-regulation of Surface Transient Receptor Potential 3 (TRPC3) Channels in Rodent Microglia

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca²⁺]_i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca²⁺ elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca²⁺ elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.     

Osmotic Sensitivity of Ca2+ and H+ Transporters in Corn Roots: Effect on Fluxes and Their Oscillations in the Elongation Region

Seedling roots of corn were treated with different concentrations of mannitol-containing solution for 1 to 1.5 hr, and net fluxes of Ca²⁺ and H⁺ were measured in the elongation region. H⁺ fluxes were much more sensitive to osmotic pressure than were Ca²⁺ fluxes. Oscillations of 7-min period in H⁺ flux, normally observed in the control, were almost fully suppressed at high osmotic concentrations. Net H⁺ flux was shifted from average efflux of 25 ± 3 nmol m⁻² sec⁻¹ to average influx of 10 ± 5 nmol m⁻² sec⁻¹ after the incubation in 100 mm mannitol. The larger the osmotic concentration, the larger was the H⁺ influx. This flux caused the unbuffered solution of pH 4.85 to change to pH 5.3 after mannitol application. It appears that the osmoticum suppresses oscillatory H⁺ extrusion at the plasma membrane. Discrete Fourier Transforms of the H⁺ flux data showed that, apart from suppression of the 7-min oscillations in H⁺ flux, mannitol also promoted the appearance of faster 2-min oscillations. Ca²⁺ influx slightly increased after mannitol treatment. In addition the 7-min oscillatory component of Ca²⁺ flux remained apparent thereby showing independence of H⁺ flux. Received: 25 April 1997/Revised: 11 August 1997     

Calcium Effects on the Solubilization of Membrane-Bound Histidine Decarboxylase in the Rat Brain

In a previous work we have shown that histidine decarboxylase (HD) activity is found in a soluble and a membrane-bound form. A major part (82%) of the membrane-bound HD activity in the crude mitochondrial fraction (P2) was present in the synaptic plasma membrane-containing subfraction. Physiological concentrations of Ca2+ had no direct effect on HD activity but caused a solubilization of approximately 50% of membrane-bound HD in the P2 fraction. Mg2+ had similar but lower effects (20% solubilization) than Ca2+. Incubation with depolarizing concentrations of K+ in the presence of 1 mM CaCl2 caused a significant (30%) solubilization of HD.     

Electrostatic basis of valence selectivity in cationic channels

We examine how a variety of cationic channels discriminate between ions of differing charge. We construct models of the KcsA potassium channel, voltage gated sodium channel and L-type calcium channel, and show that they all conduct monovalent cations, but that only the calcium channel conducts divalent cations. In the KcsA and sodium channels divalent ions block the channel and prevent any further conduction. We demonstrate that in each case, this discrimination and some of the more complex conductance properties of the channels is a consequence of the electrostatic interaction of the ions with the charges in the channel protein. The KcsA and sodium channels bind divalent ions strongly enough that they cannot be displaced by other ions and thereby block the channel. On the other hand, the calcium channel binds them less strongly such that they can be destabilized by the repulsion of another incoming divalent ion, but not by the lesser repulsion from monovalent ions.     

Chitosan derivatives alter release profiles of model compounds from calcium phosphate implants

The aim of the current study was to evaluate the impact of chitosan derivatives, namely N-octyl-chitosan and N-octyl-O-sulfate chitosan, incorporated in calcium phosphate implants to the release profiles of model drugs. The rate and extent of calcein (on M.W. 650 Da) ED, and FITC-dextran (M.W. 40 kDa) on in vitro release were monitored by fluorescence spectroscopy. Results show that calcein release is affected by the type of chitosan derivative used. A higher percentage of model drug was released when the hydrophilic polymer N-octyl-sulfated chitosan was present in the tablets compared with the tablets containing the hydrophobic polymer N-octyl-chitosan. The release profiles of calcein or FD from tablets containing N-octyl-O-sulfate revealed a complete release for FD after 120 h compared with calcein where 20% of the drug was released over the same time period. These results suggest that the difference in the release profiles observed from the implants is dependent on the molecular weight of the model drugs. These data indicate the potential of chitosan derivatives in controlling the release profile of active compounds from calcium phosphate implants.     

Time scales of circulation and mixing processes of San Francisco Bay waters

Conceptual models for tidal period and low-frequency variations in sea level, currents, and mixing processes in the northern and southern reaches of San Francisco Bay describe the contrasting characteristics and dissimilar processes and rates in these embayments: The northern reach is a partially mixed estuary whereas the southern reach (South Bay) is a tidally oscillating lagoon with density-driven exchanges with the northern reach.The mixed semidiurnal tides are mixtures of progressive and standing waves. The relatively simple oscillations in South Bay are nearly standing waves, with energy propagating down the channels and dispersing into the broad shoal areas. The tides of the northern reach have the general properties of a progressive wave but are altered at the constriction of the embayments and gradually change in an upstream direction to a mixture of progressive and standing waves. The spring and neap variations of the tides are pronounced and cause fortnightly varying tidal currents that affect mixing and salinity stratification in the water column.Wind stress on the water surface, freshwater inflow, and tidal currents interacting with the complex bay configuration are the major local forcing mechanisms creating low-frequency variations in sea level and currents. These local forcing mechanisms drive the residual flows which, with tidal diffusion, control the water-replacement rates in the estuary. In the northern reach, the longitudinal density gradient drives an estuarine circulation in the channels, and the spatial variation in tidal amplitude creates a tidally-driven residual circulation. In contrast, South Bay exhibits a balance between wind-driven circulation and tidally-driven residual circulation for most of the year. During winter, however, there can be sufficient density variations to drive multilayer (2 to 3) flows in the channel of South Bay.Mixing models (that include both diffusive and dispersive processes) are based on time scales associated with salt variations at the boundaries and those associated with the local forcing mechanisms, while the spatial scales of variations are dependent upon the configuration of the embayments. In the northern reach, where the estuarine circulation is strong, the salt flux is carried by the mean advection of the mean salt field. Where large salinity gradients are present, the tidal correlation part of the salt flux is of the same order as the advective part. Our knowledge of mixing and exchange rates in South Bay is poor. As this embayment is nearly isohaline, the salt flux is dominated entirely by the mean advection of the mean salt field. During and after peaks in river discharge, water mixing becomes more dynamic, with a strong density-driven current creating a net exchange of both water mass and salt. These exchanges are stronger during neap tides.Residence times of the water masses vary seasonally and differ between reaches. In the northern reach, residence times are on the order of days for high winter river discharge and of months for summer periods. The residence times for South Bay are fairly long (on the order of several months) during summer, and typically shorter (less than a month) during winter when density-driven exchanges occur.