A depolarization‐stimulated, bafilomycin‐inhibitable H⁺ pump in hippocampal astrocytes

Abstract

Relatively little is known about the mechanisms of pHi regulation in mammalian glial cells. We analyzed pHi regulation in rat hippocampal astrocytes in vitro using the pH-sensitive dye BCECF. All experiments were carried out in CO2/HCO3(-)-free solutions. Recovery from NH4(+)-induced acid loads was strongly dependent on the presence of extracellular Na+ and was inhibited by amiloride and its more specific analog EIPA, indicating the presence of Na(+)-H+ exchange in these cells. Removing bath Na+ or adding amiloride caused resting pHi to shift in the acid direction. Even in the absence of bath Na+ or presence of Na+/H+ inhibitors, however, these astrocytes continued to show significant recovery from acid loads. The mechanism of this amiloride-insensitive and Na(+)-independent pHi recovery process was sought and appeared to be a proton pump. In the absence of Na+, recovery from an acid load was completely blocked by the highly specific blocker of vacuolar-type (v-type) H+ ATPase, bafilomycin A1 (BA1). In normal Na+ containing solutions, exposure to BA1 caused a small acid shift in baseline pHi and slowed recovery rate from NH4(+)-induced acid loads by about 32%. The rate of Na(+)-independent pHi recovery was increased by depolarization with 50 mM [K+] solution, and this effect was rapidly reversible and blocked by BA1. These results indicate that, in CO2/HCO3(-)-free solution, pHi regulation in hippocampal astrocytes was mediated by Na(+)-H+ exchange and by a BA1-inhibitable proton pump. Because the proton pump's activity was influenced by membrane potential, this acid exporting mechanism could contribute to the depolarization-induced alkalinization that is seen in astrocytes. Although v-type H(+)-ATPase had been previously isolated from the brain, this is the first report indicating that it has a role in regulating pHi in brain cells.

A vacuolar-type H+-ATPase and a Na+/H+exchanger contribute to intracellular pH regulation in cockroach salivary ducts

“…In human eccrine sweat ducts, a V-ATPase is involved in pH i recovery after NH 4 Cl-induced acidification and probably acidifies the sweat in the duct lumen (Granger et al, 2002). An important role of V-ATPase in pH i regulation has also been suggested for Drosophila Malpighian tubules (Bertram and Wessing, 1994), collecting ducts in rabbit kidneys (Yip et al, 2002), guinea pig inner ear (Stankovic et al, 1997) and rat hippocampal astrocytes (Pappas and Ransom, 1993).…”

“…They also energise fluid secretion in insect Malpighian tubules and salivary glands (Wieczorek et al, 1999;Nishi and Forgac, 2002;Zimmermann et al, 2003;Beyenbach and Wieczorek, 2006). In addition, a contribution of the V-ATPase to pH i regulation has been described in some vertebrate epithelia (Pappas and Ransom, 1993;Stankovic et al, 1997;Granger et al, 2002;Yip et al, 2002). Although the V-ATPase has been found in a number of insect tissues (Beyenbach and Wieczorek, 2006), an involvement of the V-ATPase in pH i regulation in insects has only been suggested for Drosophila Malpighian tubules (Bertram and Wessing, 1994).…”

Excessive Na+/H+ Exchange in Disruption of Dendritic Na+ and Ca2+ Homeostasis and Mitochondrial Dysfunction following in Vitro Ischemia

“…The H ϩ pumps are highly expressed in the vesicles of synaptic terminals and responsible for acid loading and accumulation of neurotransmitters (23). Although the H ϩ pumps are typically expressed in membranes of organelles, they have been detected in the plasma membrane of hippocampal astrocytes and are active in pH i regulation under Na ϩ -and HCO 3 Ϫ -free conditions (24,25). Our findings of H ϩ pump activity in the dendritic plasma membrane suggest that dendrites are equipped with multiple H ϩ extrusion mechanisms to counteract the robust Ca 2ϩ -dependent intracellular acidification during synchronous neural activity (26).…”

Effects of acute hypoxia on intracellular-pH regulation in astrocytes cultured from rat hippocampus

“…Subsequently, pH i recovered (de) to a value similar to that prevailing before the prepulse. The segment-de pH i recovery is primarily due to an amiloride-sensitive Na-H exchanger (Bevensee et al, 1997a), although a bafilomycin A1-sensitive H + pump may also make a small contribution (Pappas and Ransom, 1993).…”

“…It is important to note that other acid-extrusion mechanisms in astrocytes such as an H + pump (Pappas and Ransom, 1993) and an H-Lactate cotransporter (Wuttke and Walz, 1990) may also contribute to the hypoxia-induced increase in acid extrusion. The high glucose concentrations of our cultured medium (i.e., 28 mM) and experimental solutions (e.g., ∼10 mM) likely promotes an elevated glycogen content and increased lactate release from our cells (Abe et al, 2006).…”