Intracellular pH regulation in rabbit renal medullary collecting duct cells. Role of chloride-bicarbonate exchange.

122

Osteoclasts resorb bone by first attaching to the bone surface and then secreting protons into an isolated extracellular compartment formed at the cell-bone attachment site. This secretion of protons (local acidification) is required to solubilize bone hydroxyapatite crystals and for activity of bone collagendegrading acid proteases. However, the large quantity of protons required, 2 mol/mol of calcium, would result in an equal accumulation of cytosolic base equivalents. This alkaline load must be corrected to maintain cytosolic pH within physiologic limits. In this study, we have measured cytoplasmic pH with pH-sensitive fluorescent compounds, while varying the extracellular ionic composition of the medium, to determine the nature of the compensatory mechanism used by osteoclasts during bone resorption.Our data show that osteoclasts possess a chloride/bicarbonate exchanger that enables them to maintain normal intracellular pH in the face of a significant proton efflux. This conclusion follows from the demonstration of a dramatic cytoplasmic acidification when osteoclasts that have been incubated in bicarbonate-containing medium are transferred into bicarbonate-free medium. This acidification is absolutely dependent on and proportional to medium [CI-]. Furthermore, acidification is inhibited by the classic inhibitor of red cell anion exchange, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, and by diphenylamine-2-carboxylate, an inhibitor of chloride specific channels. However, the acidification process is neither energy nor sodium dependent. The physiologic importance of chloride/bicarbonate exchange is demonstrated by the chloride dependence of recovery from an endogenous or exogenous alkaline load in osteoclasts. We conclude that chloride/bicarbonate exchange is in large part responsible for cytoplasmic pH homeostasis of active osteoclasts, showing that these cells are similar to renal tubular epithelial cells in their regulation of intracellular pH.

Section: Resultsmentioning

confidence: 94%

Cytoplasmic pH regulation and chloride/bicarbonate exchange in avian osteoclasts.

Teti¹,

Blair²,

Teitelbaum³

et al. 1989

122

“…The method used in the present study to analyze the bicarbonate transport mechanism of the rat MTAL, i.e., dilution of HCO3 /CO2-loaded cells into a low-HCO3 /CO2 medium, has been previously documented ( 18 ). Because outward-directed gradients were established simultaneously for HCO-and CO2, dissipation of the HCO-gradient could occur by direct efflux of HCO-or by conversion of HCO-to H2CO3 and subsequent diffusion of CO2 out of the cell.…”

Section: Discussionmentioning

confidence: 99%

“…The selectivity of K+/Na+ was 104:1. Rat MTAL tubules were isolated as described above and abruptly diluted into 500 ,1 of various experimental media at a final density of 1.0-2.4 mg ofcell protein/ ml in the electrodes-containing chamber; the experimental media contained agents (ouabain, barium, furosemide) that inhibit known potassium transport mechanisms, which is derived from protocols previously described by others ( 16,17) of HCO-efflux, the general approach was to study the evolution of pHi after transferring these HCO--loaded cells into a nominally HCO3/CO2-free medium (solution B in Table I), which was derived from the method first described by Zeidel et al ( 18 ). After abruptly diluting 200 ul ofthe bicarbonate-solution containing HCO--loaded cells into 3 ml of HCO -/CO2-free solution in the spectrofluorometer cuvette, extracellular…”

Section: Methodsmentioning

confidence: 99%

Electroneutral K+/HCO3- cotransport in cells of medullary thick ascending limb of rat kidney.

Leviel

Borensztein²,

Houillier³

et al. 1992

The renal medullary thick ascending limb (MTAL) of the rat absorbs bicarbonate through luminal H + secretion and basolateral HCO3 transport into the peritubular space. To characterize HCO3 transport, intracellular pH (pHi) was monitored by use of the pH-sensitive fluorescent probe (2',7')-bis-(carboxyethyl)-(5,6)-carboxyfluorescein in fresh suspensions of rat MTAL tubules. When cells were preincubated in HCO3 /C02-containing solutions and then abruptly diluted into HCO3/ C02-free media, the pHi response was an initial alkalinization due to CO2 efflux, followed by an acidification (pHi recovery). The pHi recovery required intracellular HCO3, was inhibited by i0' M diisothiocyanostilbene-2-2'-disulphonic acid (DIDS), and was not dependent on Cl -or Na+. As assessed by use of the cell membrane potential-sensitive fluorescent probe 3,3'-dipropylthiadicarbocyanine, cell depolarization by abrupt Cl -removal from or addition of 2 mM barium into the external medium did not affect HCO3-dependent pHi recovery, and the latter was not associated per se with any change in potential difference, which indicated that HCO3 transport was electroneutral. The HCO3-dependent pHi recovery was inhibited by raising extracellular potassium concentration and by intracellular potassium depletion.

“…Na+-independent Cl-/ HCO-exchange appears an unlikely mediator, first because this exchanger normally functions in the direction of HCO3 extrusion (15)(16)(17)(18)(19) and secondly because most of the HCOdependent recovery is also Na+ dependent (Figs. 8 and 9).…”

Section: Discussionmentioning

confidence: 99%

“…Received for publication 19 CL-/HCO-exchange (15)(16)(17)(18)(19), and (c) Na'-HCO-cotransport (20)(21)(22)(23)(24)(25).…”

Section: Introductionmentioning

confidence: 99%

Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na+-HCO3- cotransport.

Gleeson¹,

Smith²,

Boyer³

1989

103

Using the pH-sensitive dye 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein and a continuously perfused subconfluent hepatocyte monolayer cell culture system, we studied rat hepatocyte intracellular pH (pH,) regulation in the presence (+HCO3) and absence (-HCO3) of bicarbonate. Baseline pH, was higher (7.28±09) in +HCO3 than in -HCO3 (7.16±0.14). Blocking Na+/H' exchange with amiloride had no effect on pH1 in +HCO3 but caused reversible 0.1-0.2-U acidification in -HCO3 or in +HCO3 after preincubation in the anion transport inhibitor 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). Acute Na' replacement in +HCO3 also caused acidification which was amiloride independent but DIDS inhibitible.The recovery of pH, from an intracellular acid load (maximum H+ efilux rate) was 50% higher in +HCO3 than in -HCO3. Amiloride inhibited H+ efflux..M by 75% in -HCOj but by only 27% in +HCO3. The amiloride-independent pH, recovery in +HCO3 was inhibited 50-63% by DIDS and 79% by Na+ replacement but was unaffected by depletion of intracellular Cl-, suggesting that Cl-/HCO3 exchange is not involved. Depolarization of hepatocytes (raising external K+ from 5 to 25 mM) caused reversible 0.05-0.1-U alkalinization, which, however, was neither Na+ nor HCO3 dependent, nor DIDS inhibitible, findings consistent with electroneutral HCO3 transport.We conclude that Na'-HCO3 cotransport, in addition to Na+/H+ exchange, is an important regulator of pH1 in rat hepatocytes.