Cellular actions of cAMP on HCO3(-)-secreting cells of rabbit CCD: dependence on in vivo acid-base status

1994 ◽  
Vol 266 (4) ◽  
pp. F528-F535 ◽  
Author(s):  
C. Emmons ◽  
J. B. Stokes
Keyword(s):  
Anion Exchanger ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Acid Base Status

HCO3- secretion by cortical collecting duct (CCD) occurs via beta-intercalated cells. In vitro CCD HCO3- secretion is modulated by both the in vivo acid-base status of the animal and by adenosine 3',5'-cyclic monophosphate (cAMP). To investigate the mechanism of cAMP-induced HCO3- secretion, we measured intracellular pH (pHi) of individual beta-intercalated cells of CCDs dissected from alkali-loaded rabbits perfused in vitro. beta-Intercalated cells were identified by demonstrating the presence of an apical anion exchanger (cell alkalinization in response to removal of lumen Cl-). After 180 min of perfusion to permit decrease of endogenous cAMP, acute addition of 0.1 mM 8-bromo-cAMP or 1 microM isoproterenol to the bath caused a transient cellular alkalinization (> 0.20 pH units). In the symmetrical absence of either Na+, HCO3-, or Cl-, cAMP produced no change in pHi. Basolateral dihydrogen 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (0.1 mM) for 15 min before cAMP addition also prevented this alkalinization. In contrast to the response of cells from alkali-loaded rabbits, addition of basolateral cAMP to CCDs dissected from normal rabbits resulted in an acidification of beta-intercalated cells (approximately 0.20 pH units). The present studies demonstrate the importance of the in vivo acid-base status of the animal in the regulation of CCD HCO3- secretion by beta-intercalated cells. The results identify the possible existence of a previously unrecognized Na(+)-dependent Cl-/HCO3- exchanger on the basolateral membrane of beta-intercalated cells in alkali-loaded rabbits.

AE4 is a DIDS-sensitive Cl−/HCO 3 − exchanger in the basolateral membrane of the renal CCD and the SMG duct

2002 ◽  
Vol 283 (4) ◽  
pp. C1206-C1218 ◽  
Author(s):  
Shigeru B. H. Ko ◽  
Xiang Luo ◽  
Henrik Hager ◽  
Alexandra Rojek ◽  
Joo Young Choi ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Basolateral Membrane ◽  
Rabbit Kidney ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Specific Expression ◽  
Hek 293 ◽  
Acid Base Status ◽  
Species Specific

The renal cortical collecting duct (CCD) plays an important role in systemic acid-base homeostasis. The β-intercalated cells secrete most of the HCO[Formula: see text], which is mediated by a luminal, DIDS-insensitive, Cl−/HCO[Formula: see text] exchange. The identity of the luminal exchanger is a matter of debate. Anion exchanger isoform 4 (AE4) cloned from the rabbit kidney was proposed to perform this function (Tsuganezawa H et al. J Biol Chem 276: 8180–8189, 2001). By contrast, it was proposed (Royaux IE et al. Proc Natl Acad Sci USA 98: 4221–4226, 2001) that pendrin accomplishes this function in the mouse CCD. In the present work, we cloned, localized, and characterized the function of the rat AE4. Northern blot and RT-PCR showed high levels of AE4 mRNA in the CCD. Expression in HEK-293 and LLC-PK1 cells showed that AE4 is targeted to the plasma membrane. Measurement of intracellular pH (pHi) revealed that AE4 indeed functions as a Cl−/HCO[Formula: see text] exchanger. However, AE4 activity was inhibited by DIDS. Immunolocalization revealed species-specific expression of AE4. In the rat and mouse CCD and the mouse SMG duct AE4 was in the basolateral membrane. By contrast, in the rabbit, AE4 was in the luminal and lateral membranes. In both, the rat and rabbit CCD AE4 was in α-intercalated cells. Importantly, localization of AE4 was not affected by the systemic acid-base status of the rats. Therefore, we conclude that expression and possibly function of AE4 is species specific. In the rat and mouse AE4 functions as a Cl−/HCO[Formula: see text] exchanger in the basolateral membrane of α-intercalated cells and may participate in HCO[Formula: see text] absorption. In the rabbit AE4 may contribute to HCO[Formula: see text] secretion.

scholarly journals Characterization of anion exchangers in an inner medullary collecting duct cell line.

1998 ◽  
Vol 9 (5) ◽  
pp. 746-754
Author(s):  
G Obrador ◽  
H Yuan ◽  
T M Shih ◽  
Y H Wang ◽  
M A Shia ◽  
...  
Keyword(s):  
Cell Line ◽  
Anion Exchanger ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Kidney Cortex ◽  
Intercalated Cells ◽  
Rat Stomach ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

Although the inner medullary collecting duct (IMCD) plays a major role in urinary acidification, the molecular identification of many of the specific components of the transport system in this nephron segment are lacking. A cultured line of rat IMCD cells was used to characterize the mediators of cellular HCO3 exit. This cell line functionally resembles alpha-intercalated cells. Physiologic experiments document that HCO3- transport is a reversible, electroneutral, Cl dependent, Na+-independent process. It can be driven by Cl-gradients and inhibited by stilbenes such as 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid. Immunohistochemical analysis, using a rabbit polyclonal antibody against the carboxy-terminal 12 amino acids of anion exchanger 1 (AE1), revealed a distribution of immunoreactive protein that is consistent with a basolateral localization of AE in cultured cells and in alpha-intercalated cells identified in sections of rat kidney cortex. Immunoblot revealed two immunoreactive bands (approximately 100 and 180 kD in size) in membranes from cultured IMCD cells, rat renal medulla, and freshly isolated IMCD cells. The mobility of the lower molecular weight band was similar to that of AE1 in red blood cell ghosts and kidney homogenate and therefore probably represents AE1. The mobility of the 180-kD band is similar to that for rat stomach and kidney AE2 and therefore probably represents AE2. Selective biotinylation of the apical or basolateral membrane proteins in cultured IMCD cells revealed that both AE1 and AE2 are polarized to the basolateral membrane. Northern blot analysis documented the expression of mRNA for AE1 and AE2 but not AE3. Furthermore, the cDNA sequence of AE1 and AE2 expressed by these cells was found to be virtually identical to that reported for kidney AE1 and rat stomach AE2. It is concluded that this cultured line of rat IMCD cells expresses two members of the anion exchanger gene family, AE1 and AE2, and both of these exchangers probably mediate the electroneutral Cl--dependent HCO3-transport observed in this cell line.

Functional characterization of alpha- and beta-intercalated cell types in rabbit cortical collecting duct

1991 ◽  
Vol 261 (3) ◽  
pp. F377-F385 ◽  
Author(s):  
H. Furuya ◽  
M. D. Breyer ◽  
H. R. Jacobson
Keyword(s):  
Collecting Duct ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Cell Types ◽  
Disulfonic Acid ◽  
Cortical Collecting Duct ◽  
Electrical Measurements ◽  
Collecting Ducts

Single-cell electrical measurements and spectrophotometric determinations of intracellular pH were used to determine unique features of alpha- and beta-intercalated cells (alpha-IC, beta-IC) in in vitro perfused rabbit cortical collecting ducts (CCD). pHi rose in alpha-IC and fell in beta-IC after bath Cl- removal. Luminal Cl- removal did not change pHi of alpha-IC, but pHi of beta-IC rose by 0.36 +/- 0.01 pH units. Cl- concentration-dependent recovery of beta-IC pHi revealed a Cl- Km of 18.7 mM for the luminal Cl(-) -HCO3- exchanger. Measurements of basolateral membrane voltage (Vbl) also showed two IC cell types. Removal of luminal Cl- did not change Vbl in alpha-IC, whereas Vbl hyperpolarized by a mean of 73.2 +/- 3.5 mV in beta-IC. Reducing bath Cl- depolarized both alpha- and beta-IC Vbl. In alpha-IC a large repolarization of 39.8 +/- 5.2 mV followed acute depolarization after bath Cl- removal. Reducing bath HCO3- (constant CO2) had little effect on beta-IC Vbl, whereas alpha-IC Vbl depolarized by 5.2 +/- 0.7 mV. Reducing luminal HCO3- in the absence of luminal Cl- produced a 17.6 +/- 1.8 mV depolarization in beta-IC. This change was independent of luminal Na+ and was not blocked by luminal 10(-4) M 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In beta-IC, Vbl was not altered by either bath or lumen DIDS in the presence of luminal Cl-. However, when luminal Cl- was removed, luminal DIDS reversibly depolarized Vbl by 9.6 +/- 2.9 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

H(+)-K(+)-ATPase activity in rat collecting duct segments

1992 ◽  
Vol 262 (4) ◽  
pp. F692-F695 ◽  
Author(s):  
J. D. Gifford ◽  
L. Rome ◽  
J. H. Galla
Keyword(s):  
Atpase Activity ◽  
Marked Decrease ◽  
Collecting Duct ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Medullary Collecting Duct ◽  
Gastric Proton Pump

Previous studies have suggested the presence of an H(+)-K(+)-ATPase in rat cortical and medullary intercalated cells with similar properties to the gastric proton pump. The purpose of this study was to determine the functional contribution of an H(+)-K(+)-adenosinetriphosphatase(ATPase) to total CO2 (tCO2) transport along the rat collecting duct. After baseline determination of tCO2 transport in isolated perfused collecting duct segments, Sch 28080 (10 microM) was added to either the perfusate or bath. When Sch 28080 was added to the perfusate, there was no effect in the cortical collecting duct (CCD, 20.8 +/- 6.7 vs. 25.3 + 3.0 pmol.mm-1.min-1), but a marked decrease in tCO2 absorption was effected in both the outer medullary (OMCD, 37.6 + 6.2 vs. 10.7 +/- 4.1 pmol.mm-1.min-1) and initial inner medullary collecting duct (IMCD1, 34.4 +/- 8.1 vs. 16.2 +/- 5.6 pmol.mm-1.min-1). In the CCD from rats with acute alkalosis in vivo, Sch 28080 added to the bath inhibited tCO2 secretion in the CCD (-17.1 +/- 4.4 vs 3.5 + 3.3 pmol.mm-1.min-1). These findings suggest that 1) H(+)-K(+)-ATPase is important in tCO2 absorption in the OMCD and IMCD1 and in tCO2 secretion in the CCD, 2) HCO3(-)-absorbing intercalated cells differ functionally in the cortex and medulla, 3) HCO3- secretion is not the reverse process of HCO3- absorption in the CCD, and 4) H(+)-K(+)-ATPase is important in distal acidification under normal and altered acid-base conditions.

Effects of calcitonin on function of intercalated cells of rat cortical collecting duct

1993 ◽  
Vol 264 (2) ◽  
pp. F221-F227 ◽  
Author(s):  
E. Siga ◽  
B. Mandon ◽  
N. Roinel ◽  
C. de Rouffignac
Keyword(s):  
Collecting Duct ◽  
Disulfonic Acid ◽  
Na Channel ◽  
Adenylate Cyclase System ◽  
Cortical Collecting Duct ◽  
Osmotic Water ◽  
Acid Base Status ◽  
Hormonal Action ◽  
Specific Receptors

In the rat cortical collecting duct (CCD), the presence of highly specific receptors to calcitonin (CT) coupled to a sensitive adenylate cyclase system suggests that this segment is a target site for CT. Our aim was to explore the effects of CT on the rat CCD microperfused in vitro. The hormone failed to alter the osmotic water permeability and did not affect net Na+ transport but generated a lumen-positive transepithelial potential difference (PDte), which under control conditions was close to zero. This response was dose dependent and was still observed in the presence of luminal amiloride, despite the luminal positivity generated by the Na+ channel blocker (PDte increased from 4.0 +/- 0.8 to 9.5 +/- 1.1 mV). In contrast, the nominal absence of CO2/HCO3- or the use of a low-Cl- solution totally prevented the PDte changes caused by CT. The CT-induced lumen-positive PDte was reduced by 2.3 +/- 0.8 mV after the basolateral addition of the Cl- channel inhibitor diphenylamine-2-carboxylate. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and acetazolamide, which inhibit Cl-/HCO3- exchangers and carbonic anhydrase activities, respectively, also inhibited the CT-induced PDte by 4.6 +/- 0.5 and 5.0 +/- 0.9 mV. To test whether the acid-base status of the animals influences the response to CT, rats underwent an acid or alkali load. CCD dissected from acid-loaded rats responded to CT to the same extent as control animals, but the hormonal action was significantly attenuated when the CCD was harvested from alkali-loaded rats (PDte increases: acid 4.0 +/- 0.3 vs. alkali 1.6 +/- 0.6 mV, P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals TRPV4 as a flow sensor in flow-dependent K+ secretion from the cortical collecting duct

2007 ◽  
Vol 292 (2) ◽  
pp. F667-F673 ◽  
Author(s):  
Junichi Taniguchi ◽  
Shuichi Tsuruoka ◽  
Atsuko Mizuno ◽  
Jun-ichi Sato ◽  
Akio Fujimura ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Basolateral Membrane ◽  
Cortical Collecting Duct ◽  
Urine Production ◽  
K Secretion ◽  
Distal Tubules ◽  
Transient Receptor

The transient receptor vanilloid-4 (TRPV4) is a mechanosensitive, swell-activated cation channel that is abundant in the renal distal tubules. Immunolocalization studies, however, present conflicting data as to whether TRPV4 is expressed along the apical and/or basolateral membranes. To disclose the role of TRPV4 in flow-dependent K+ secretion in distal tubules in vivo, urinary K+ excretion and net transports of K+ and Na+ in the cortical collecting duct (CCD) were measured with an in vitro microperfusion technique in TRPV4 +/+ and TRPV4 −/− mice. Both net K+ secretion and Na+ reabsorption were flow dependently increased in the CCDs isolated from TRPV4 +/+mice, which were significantly enhanced by a luminal application of 50 μM 4α-phorbol-12,13-didecanoate (4αPDD), an agonist of TRPV4. No flow dependence of net K+ and Na+ transports or effects of 4αPDD on CCDs were observed in TRPV4 −/− mice. A basolateral application of 4αPDD had little effect on these ion transports in the TRPV4 +/+ CCDs, while the luminal application did. Urinary K+ excretion was significantly smaller in TRPV4 −/− than in TRPV4 +/+ mice when urine production was stimulated by a venous application of furosemide. These observations suggested an essential role of the TRPV4 channels in the luminal or basolateral membrane as flow sensors in the mechanism underlying the flow-dependent K+ secretion in mouse CCDs.

Mechanisms of HCO 3 − secretion in the rabbit connecting segment

1999 ◽  
Vol 277 (4) ◽  
pp. F567-F574 ◽  
Author(s):  
Shuichi Tsuruoka ◽  
George J. Schwartz
Keyword(s):  
Cell Function ◽  
Collecting Duct ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base ◽  
Intercalated Cell ◽  
Atpase Inhibitors ◽  
Transepithelial Voltage ◽  
Β Adrenergic Receptors

The connecting tubule (CNT) contains α-(H+-secreting) and β-([Formula: see text]-secreting) intercalated cells and is therefore likely to contribute to acid-base homeostasis. To characterize the mechanisms of [Formula: see text]transport in the rabbit CNT, in which there is little definitive data presently available, we microdissected the segments from the superficial cortical labyrinth, perfused them in vitro, measured net[Formula: see text] transport ( J [Formula: see text] ) by microcalorimetry, and examined the effects of several experimental maneuvers. Mean ± SE basal J [Formula: see text] was −3.4 ± 0.1 pmol ⋅ min−1 ⋅ mm−1(net [Formula: see text] secretion), and transepithelial voltage was −13 ± 1 mV ( n = 47). Net[Formula: see text] secretion was markedly inhibited by removal of luminal Cl− or application of basolateral H+-ATPase inhibitors (bafilomycin or concanamycin), maneuvers that inhibit β-intercalated cell function. Net [Formula: see text] secretion was not affected by inhibitors of α-intercalated cell function (basolateral Cl− removal, basolateral DIDS, or luminal H+-ATPase inhibitors). Net [Formula: see text] secretion was stimulated by isoproterenol and inhibited by acetazolamide. These data indicate that 1) CNTs secrete[Formula: see text] via an apical DIDS-insensitive Cl−/[Formula: see text]exchanger, mediated by a basolateral bafilomycin- and concanamycin-sensitive H+-ATPase; 2) inhibition of cytosolic carbonic anhydrase decreases [Formula: see text] secretion; and 3) stimulation of β-adrenergic receptors increases [Formula: see text] secretion. The failure to influence net [Formula: see text]transport by inhibiting α-intercalated cell apical H+-ATPases or basolateral Cl−/[Formula: see text]exchange suggests that the CNT has fewer functioning α-intercalated cells than the cortical collecting duct. These are the first studies to examine the rate and mechanisms of[Formula: see text] secretion by the rabbit CNT; this is clearly an important segment in mediating acid-base homeostasis.

H-K-ATPase activity in PNA-binding intercalated cells of newborn rabbit cortical collecting duct

1997 ◽  
Vol 272 (2) ◽  
pp. F167-F177 ◽  
Author(s):  
A. Constantinescu ◽  
R. B. Silver ◽  
L. M. Satlin
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Adult Rabbit ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Load ◽  
Ethanesulfonic Acid

Functional and immunocytochemical studies indicate that intercalated cells in the adult rabbit cortical collecting duct (CCD) possess an H-K-adenosinetriphosphatase (H-K-ATPase). Because growing subjects must retain K+ and excrete H+, we sought to determine whether H-K-ATPase is present in the CCD early in life and, if so, to assess its activity and polarity. H-K-ATPase activity was defined as the initial rate of Sch-28080-inhibitable K+-dependent cell pH (pHi) recovery observed, in the absence of Na+, in response to an in vitro acid load. Transporter activity was assayed in intercalated cells labeled with the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and apical cell surface marker rhodamine peanut lectin (PNA) in split-open CCDs isolated from neonatal and adult New Zealand White rabbits. In Na+-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered solutions (nominal absence of CO2/HCO3-), the rate of K+-dependent pH(i) recovery from a NH4Cl-induced acid load was similar in newborn (0.056 +/- 0.015 pH U/min, n = 9) and adult (0.060 +/- 0.019 pH U/min; n = 9, P = not significant) cells. This rate of K+-dependent pH(i) recovery was significantly reduced by 10-20 pM Sch-28080, an inhibitor of gastric H-K-ATPase, in both newborns (0.009 +/- 0.003 pH U/min, n = 7) and adults (0.013 +/- 0.007 pH U/min, n = 9) (P < 0.05 compared with rates in absence of inhibitor). To determine whether the location of the transporter is consistent with a role in K+ absorption and H+ secretion, pH(i) recovery of acutely acid-loaded intercalated cells in neonatal CCDs (n = 7) microperfused and bathed in the absence of Na+ and K+ was monitored after selective addition of K+ to either the luminal or basolateral membrane. Addition of 5 mM K+ led to a significantly greater rate of pH(i) recovery when it was added to the luminal rather than the peritubular solution (0.049 +/- 0.005 vs. 0.018 +/- 0.005 pH U/min, P < 0.05). We conclude that PNA-binding intercalated cells of the neonatal CCD possess H-K-ATPase activity, predominantly located in the apical membrane. This provides a mechanism for H secretion and K+ retention, processes required for growth.

Expression of rat kidney anion exchanger 1 in type A intercalated cells in metabolic acidosis and alkalosis

1999 ◽  
Vol 277 (6) ◽  
pp. F841-F849 ◽  
Author(s):  
Saskia Huber ◽  
Esther Asan ◽  
Thomas Jöns ◽  
Christiane Kerscher ◽  
Bernd Püschel ◽  
...  
Keyword(s):  
Anion Exchanger ◽  
Direct Evidence ◽  
Rat Kidney ◽  
Enzyme Reaction ◽  
Type A ◽  
Mrna Levels ◽  
Intercalated Cells ◽  
Acid Base ◽  
Anion Exchanger 1 ◽  
Acid Base Status

By enzyme-linked in situ hybridization (ISH), direct evidence is provided that acid-secreting intercalated cells (type A IC) of both the cortical and medullary collecting ducts of the rat kidney selectively express the mRNA of the kidney splice variant of anion exchanger 1 (kAE1) and no detectable levels of the erythrocyte AE1 (eAE1) mRNA. Using single-cell quantification by microphotometry of ISH enzyme reaction, medullary type A IC were found to contain twofold higher kAE1 mRNA levels compared with cortical type A IC. These differences correspond to the higher intensity of immunostaining in medullary versus cortical type A IC. Chronic changes of acid-base status induced by addition of NH4Cl (acidosis) or NaHCO3 (alkalosis) to the drinking water resulted in up to 35% changes of kAE1 mRNA levels in both cortical and medullary type A IC. These experiments provide direct evidence at the cellular level of kAE1 expression in type A IC and show moderate capacity of type A IC to respond to changes of acid-base status by modulation of kAE1 mRNA levels.

Regulation of AE2 mRNA expression in the cortical collecting duct by acid/base balance

1998 ◽  
Vol 274 (3) ◽  
pp. F596-F601 ◽  
Author(s):  
Géza Fejes-Tóth ◽  
Erzsébet Rusvai ◽  
Emily S. Cleaveland ◽  
Anikó Náray-Fejes-Tóth
Keyword(s):  
Mrna Expression ◽  
Collecting Duct ◽  
Cell Types ◽  
Alkaline Media ◽  
Mrna Levels ◽  
Intercalated Cells ◽  
Cortical Collecting Duct ◽  
Acid Base Balance ◽  
Acid Base ◽  
Base Balance

AE2 mRNA and protein is expressed in several nephron segments, one of which is the cortical collecting duct (CCD). However, the distribution of AE2 among the different cell types of the CCD and the function of AE2 in the kidney are not known. The purpose of this study was to determine the distribution of AE2 mRNA among the three CCD cell types and to examine the effects of changes in acid/base balance on its expression. Following NH4Cl (acid) or NaHCO3 (base) loading of rabbits for ∼18 h, CCD cells were isolated by immunodissection. AE2 mRNA levels were determined by RT-PCR and were normalized for β-actin levels. We found that CCD cells express high levels of AE2 mRNA (∼500 copies/cell). AE2 mRNA levels were significantly higher in CCD cells originating from base-loaded than acid-loaded rabbits, with an average increase of 3.7 ± 1.07-fold. The effect of pH on AE2 mRNA levels was also tested directly using primary cultures of CCD cells. CCD cells incubated in acidic media expressed significantly lower levels of AE2 mRNA than those in normal or alkaline media. Experiments with isolated principal cells, α-intercalated cells, and β-intercalated cells (separated by fluorescence-activated cell sorting) demonstrated that AE2 mRNA levels are comparable in the three collecting duct cell subtypes and are similarly regulated by changes in acid/base balance. Based on these results, we conclude that adaptation to changes in extracellular H+ concentration is accompanied by opposite changes in AE2 mRNA expression. The observations that AE2 mRNA is not expressed in a cell-type-specific manner and that changes in acid/base balance have similar effects on each CCD cell subtype suggest that AE2 might serve a housekeeping function rather than being the apical anion exchanger of β-intercalated cells.

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