Victor Babich

Selective release of molecules from weibel-palade bodies during a lingering kiss

Exocytosis of specialized endothelial cell secretory organelles, Weibel-Palade bodies (WPBs), is thought to play an important role in regulating hemostasis and intravascular inflammation. The major WPB core proteins are Von Willebrand factor (VWF) and its propolypeptide (Proregion), constituting more than 95% of the content. Although the composition of the WPBs can be fine-tuned to include cytokines and chemokines (eg, interleukin-8 [IL-8] and eotaxin-3), it is generally assumed that WPB exocytosis is inextricably associated with secretion of VWF. Here we show that WPBs can undergo a form of exocytosis during which VWF and Proregion are retained while smaller molecules, such as IL-8, are released. Imaging individual WPBs containing fluorescent cargo molecules revealed that during weak stimulation approximately 25% of fusion events result in a failure to release VWF or Proregion. The WPB membrane protein P-selectin was also retained; however, the membrane tetraspannin CD63 was released. Accumulation or exclusion of extracellular fluorescent dextran molecules ranging from 3 kDa to 2 mDa show that these events arise due to the formation of a fusion pore approximately 12 nm in diameter. The pore behaves as a molecular filter, allowing selective release of WPB core and membrane proteins. WPB exocytosis is not inextricably associated with secretion of VWF.

Rate, extent and concentration dependence of histamine-evoked Weibel-Palade body exocytosis determined from individual fusion events in human endothelial cells

The rate, concentration dependence and extent of histamine‐evoked Weibel–Palade body (WPB) exocytosis were investigated with time‐resolved fluorescence microscopy in cultured human umbilical vein endothelial cells expressing WPB‐targeted chimeras of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence, morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra‐WPB pH from resting levels, estimated as pH 5.45 ± 0.26 (s.d., n= 144), to pH 7.40. It coincided with uptake of extracellular Alexa‐647, indicating the formation of a fusion pore, prior to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine and the first fusion event were 10.0 ± 4.42 s (n= 9 cells) at 0.3 μm histamine and 1.57 ± 0.21 s (n= 15 cells) at 100 μm histamine, indicating the existence of a slow process or processes in histamine‐evoked WPB exocytosis. The maximum rates of exocytosis were 1.20 ± 0.16 WPB s−1 (n= 9) at 0.3 μm and 3.66 ± 0.45 WPB s−1 at 100 μm histamine (n= 15). These occurred 2–5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca2+ indicating that the initial burst of secretion is driven by Ca2+ release from internal stores, but sustained exocytosis required external Ca2+. Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 μm), for which there was a delay between calcium elevation and secretion of 1.67 ± 0.24 s (n= 6), and a peak fusion rate of ∼10 WPB s−1.

Acute Regulation of Na/H Exchanger NHE3 by Adenosine A1 Receptors Is Mediated by Calcineurin Homologous Protein

Adenosine is an autacoid that regulates renal Na+ transport. Activation of adenosine A1 receptor (A1R) by N6-cyclopentidyladenosine (CPA) inhibits the Na+/H+ exchanger 3 (NHE3) via phospholipase C/Ca2+/protein kinase C (PKC) signaling pathway. Mutation of PKC phosphorylation sites on NHE3 does not affected regulation of NHE3 by CPA, but amino acid residues 462 and 552 are essential for A1R-dependent control of NHE3 activity. One binding partner of the NHE family is calcineurin homologous protein (CHP). We tested the role of NHE3-CHP interaction in mediating CPA-induced inhibition of NHE3 in opossum kidney (OK) and Xenopus laevis uroepithelial (A6) cells. Both native and transfected NHE3 and CHP are present in the same immuno-complex by co-immunoprecipitation. CPA (10-6 M) increases CHP-NHE3 interaction by 30 - 60% (native and transfected proteins). Direct CHP-NHE3 interaction is evident by yeast two-hybrid assay (bait, NHE3C terminus; prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3C terminus (amino acids 462-552 of opossum NHE3). The yeast data were confirmed in OK cells where truncated NHE3 (NHE3Δ552) still shows CPA-stimulated CHP interaction. Overexpression of the polypeptide from the CHP binding region (NHE3462-552) interferes with the ability of CPA to inhibit NHE3 activity and to increase CHPNHE3Full-length interaction. Reduction of native CHP expression by small interference RNA abolishes the ability of CPA to inhibit NHE3 activity. We conclude that CHPNHE3 interaction is regulated by A1R activation and this interaction is a necessary and integral part of the signaling pathway between adenosine and NHE3.

Calcineurin homologous protein: A multifunctional Ca2+-binding protein family

The calcineurin homologous protein (CHP) belongs to an evolutionarily conserved Ca(2+)-binding protein subfamily. The CHP subfamily is composed of CHP1, CHP2, and CHP3, which in vertebrates share significant homology at the protein level with each other and between other Ca(2+)-binding proteins. The CHP structure consists of two globular domains containing from one to four EF-hand structural motifs (calcium-binding regions composed of two helixes, E and F, joined by a loop), the myristoylation, and nuclear export signals. These structural features are essential for the function of the three members of the CHP subfamily. Indeed, CHP1-CHP3 have multiple and diverse essential functions, ranging from the regulation of the plasma membrane Na(+)/H(+) exchanger protein function, to carrier vesicle trafficking and gene transcription. The diverse functions attributed to the CHP subfamily rendered an understanding of its action highly complex and often controversial. This review provides a comprehensive and organized examination of the properties and physiological roles of the CHP subfamily with a view to revealing a link between CHP diverse functions.

The Calcineurin Homologous Protein-1 Increases Na+/H+-Exchanger 3 Trafficking via Ezrin Phosphorylation

The Na(+)/H(+)-exchanger 3 (NHE3) is essential for regulation of Na(+) transport in the renal and intestinal epithelium. Although changes in cell surface abundance control NHE3 function, the molecular signals that regulate NHE3 surface expression are not well defined. We found that overexpression of the calcineurin homologous protein-1 (CHP1) in opossum kidney cells increased NHE3 transport activity, surface protein abundance, and ezrin phosphorylation. CHP1 knockdown by small interfering RNA had the opposite effects. Overexpression of wild-type ezrin increased both NHE3 transport activity and surface protein abundance, confirming that NHE3 is downstream of ezrin. Expression of a pseudophosphorylated ezrin enhanced these effects, whereas expression of an ezrin variant that could not be phosphorylated prevented the downstream effects on NHE3. Furthermore, CHP1 knockdown reversed the activation of NHE3 by wild-type ezrin but not by the pseudophosphorylated ezrin. Taken together, these results demonstrate that CHP1 increases NHE3 abundance and constitutive function in a manner dependent on ezrin phosphorylation.

Differential Effect of Extracellular Acidosis on the Release and Dispersal of Soluble and Membrane Proteins Secreted from the Weibel-Palade Body

Proteins secreted from Weibel-Palade bodies (WPBs) play important roles in regulating inflammatory and hemostatic responses. Inflammation is associated with the extracellular acidification of tissues and blood, conditions that can alter the behavior of secreted proteins. The effect of extracellular pH (pHo) on the release of von Willebrand factor (VWF), the VWF-propolypeptide (Proregion), interleukin-8, eotaxin-3, P-selectin, and CD63 from WPBs was investigated using biochemical approaches and by direct optical analysis of individual WPB fusion events in human endothelial cells expressing green or red fluorescent fusions of these different cargo proteins. Between pHo 7.4 and 7.0, ionomycin-evoked WPB exocytosis was characterized by the adhesion of VWF to the cell surface and the formation of long filamentous strands. The rapid dispersal of Proregion, interleukin-8, and eotaxin-3 into solution, and of P-selectin and CD63 into the plasma membrane, was unaltered over this pHo range. At pHo 6.8 or lower, Proregion remained associated with VWF, in many cases WPB failed to collapse fully and VWF failed to form filamentous strands. At pHo 6.5 dispersal of interleukin-8, eotaxin-3, and the membrane protein CD63 remained unaltered compared with that at pHo 7.4; however, P-selectin dispersal into the plasma membrane was significantly slowed. Thus, extracellular acidification to levels of pHo 6.8 or lower significantly alters the behavior of secreted VWF, Proregion, and P-selectin while rapid release of the small pro-inflammatory mediators IL-8 and eotaxin-3 is essentially unaltered. Together, these data suggest that WPB exocytosis during extracellular acidosis may favor the control of inflammatory processes.

Protein mobilities and P-selectin storage in Weibel–Palade bodies

Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel–Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P-selectin became immobilised, whereas small proteins (ssEGFP, eotaxin-3) became less mobile. WPB maturation led to further decreases in mobility of small proteins and CD63. Acute alkalinisation of mature WPBs selectively increased the mobilities of small soluble proteins without affecting larger molecules and the membrane proteins. Disruption of the Proregion–VWF paracrystalline core by prolonged incubation with NH4Cl rendered P-selectin mobile while VWF remained immobile. FRAP of P-selectin mutants revealed that immobilisation most probably involves steric entrapment of the P-selectin extracellular domain by the Proregion–VWF paracrystal. Significantly, immobilisation contributed to the enrichment of P-selectin in WPBs; a mutation of P-selectin preventing immobilisation led to a failure of enrichment. Together these data shed new light on the transitions that occur for soluble and membrane proteins following their entry and storage into post-Golgi-regulated secretory organelles.

The reduction of Na/H exchanger-3 protein and transcript expression in acute ischemia–reperfusion injury is mediated by extractable tissue factor(s)

Ischemic renal injury is a formidable clinical problem, the pathophysiology of which is incompletely understood. As the Na/H exchanger-3 (NHE3) mediates the bulk of apical sodium transport and a significant fraction of oxygen consumption in the proximal tubule, we examined mechanisms by which ischemia-reperfusion affects the expression of NHE3. Ischemia-reperfusion dramatically decreased NHE3 protein and mRNA (immunohistochemistry, immunoblot, and RNA blot) in rat kidney cortex and medulla. The decrease in NHE3 protein was uniform throughout all tubules, including those appearing morphologically intact. In the kidney cortex, a decrease in NHE3 surface protein preceded that of NHE3 total protein and mRNA. Kidney homogenates from rats exposed to mild renal ischemia-reduced cell surface NHE3 protein expression in opossum kidney cells in vitro, whereas homogenates from animals with moderate-to-severe ischemia reduced both total NHE3 protein and mRNA. The decrease in total NHE3 protein was dependent on the proteasomal degradation associated with NHE3 ubiquitylation measured by coimmunoprecipitation. The transferable factor(s) from the ischemic homogenate that reduce NHE3 expression were found to be heat sensitive and to be associated with a lipid-enriched fraction, and did not include regulatory RNAs. Thus, transferable factor(s) mediate the ischemia-reperfusion injury-induced decrease in NHE3 of the kidney.

Short‐ and long‐term A3 adenosine receptor activation inhibits the Na+/H+ exchanger NHE3 activity and expression in opossum kidney cells

The renal function of the A3 adenosine receptor (A3AR) is poorly characterized. In this study, we report that the A3AR‐selective agonist, 1‐[2‐chloro‐6‐[[(3‐iodophenyl)methyl]amino]‐9H‐purine‐9‐yl]‐1‐deoxy‐N‐methyl‐b‐D‐ribofuranuronamide (2‐Cl‐IBMECA) regulates the Na+/H+ exchanger‐3 (NHE3) in a dose‐ and time‐dependent fashion. In opossum kidney (OK) cells, 2‐Cl‐IBMECA at high (10−6 M) and low (10−8 M) dose inhibits NHE3 by a multiphasic time course with an acute phase of NHE3 inhibition from 15 min to 1 h, followed by a chronic phase of NHE3 inhibition from 24 to 48 h. Pre‐incubation with either the selective A3AR‐antagonist MRS1523 (10−7 M) or the protein kinase C inhibitor, Calphostin C (10−8 M) completely blocked 10−6 M 2‐Cl‐IBMECA‐induced acute (15 min) and chronic (24 h) phases of NHE3 inhibition. In contrast, the acute inhibitory phase (15 min) of 10−8 M 2‐Cl‐IBMECA was completely prevented only when Calphostin C (10−8 M) was added in conjunction with the protein kinase A inhibitor, H89 (10−7 M). Acute (15 or 30 min depending on the A3AR‐agonist concentration) A3AR‐dependent inhibition of NHE3 activity was accompanied by decrease in cell surface NHE3 protein with no change in total NHE3 antigen. Chronic (24 h) A3AR‐mediated down‐regulation of NHE3 was associated with reduction of surface NHE3, decreased total NHE3 protein (70%) and a paradoxical rise of NHE3 RNA (40%). In summary, these results indicate that A3AR directly regulates NHE3 at multiple levels in a complex pattern. A3AR‐dependent short‐ and long‐term inhibition of NHE3 may be a fundamental mechanism of net sodium and fluid balance. J. Cell. Physiol. 216: 221–233, 2008.

The biophysical and molecular basis of intracellular pH sensing by Na+/H+ exchanger-3

Epithelial Na+/H+ exchanger‐3 (NHE3) transport is fundamental for renal and intestinal sodium reabsorption. Cytoplasmic protons are thought to serve as allosteric modifiers of the exchanger and to trigger its transport through protein conformational change. This effect presupposes an intracellular pH (pHi) dependence of NHE3 activity, although the biophysical and molecular basis of NHE3 pHi sensitivity have not been defined. NHE3, when complexed with the calcineurin homologous protein‐1 (CHP1), had a shift in pHi sensitivity (0.4 units) toward the acidic side in comparison with NHE3 alone, as measured by oscillating pH electrodes combined with whole‐cell patch clamping. Indeed, CHP1 interaction with NHE3 inhibited NHE3 transport in a pHi ‐dependent manner. CHP1 binding to NHE3 also affected its acute regulation. Intracellular perfusion of peptide from the CHP1 binding region (or pHi modification to reduce the CHP1 amount bound to NHE3) was permissive and cooperative for dopamine inhibition of NHE3 but reversed that of adenosine. Thus, CHP1 interaction with NHE3 apparently establishes the exchanger set point for pHi, and modification in this set point is effective in the hormonal stimuli‐mediated regulation of NHE3. CHP1 may serve as a regulatory cofactor for NHE3 conformational change, dependent on intracellular protonation.—Babich V., Vadnagara K., Di Sole, F. The biophysical and molecular basis of intracellular pH sensing by the Na+/H+ exchanger‐3. FASEB J. 27, 4646–4658 (2013). www.fasebj.org