Video microscopy of intracellular pH in primary cultures of rabbit proximal and early distal tubules

Abstract

The purpose of this study was to investigate intracytoplasmic pH (pHi) regulation in primary cultures of proximal (PCT) and distal bright (DCTb) convoluted tubules. PCT and DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The cultured epithelia were grown on semi-transparent permeable supports. The pHi was determined by video microscopy and digital image processing using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and measuring the ratio of BCECF fluorescence excited by two successive wavelengths (490 nm and 450 nm). Resting pHi values, determined in bicarbonate-free medium (extracellular pH: 7.40), were 7.25 +/- 0.02 (n = 23) and 7.17 +/- 0.04 (n = 30) for cultured PCT and DCTb respectively. After the acid-loading procedure, cultured proximal cells recovered their pHi by means of the classic Na+/H+ antiporter, sensitive to amiloride and located in the apical membrane only. In cultured DCTb part of the pHi recovery was mediated by a Na+/H+ exchange present in the basolateral side. Moreover, at physiological initial pHi values, chloride removal from the apical solution caused the pHi to increase in the presence of bicarbonate. In acidified cultured DCTb cells, a partial pHi recovery was induced in sodium-free media by 15 mM HCO(-3) in the presence of an outward chloride gradient. This pHi change was completely abolished by 4,4'-diisothiocyanostilbene 2,2'-disulfonic acid (1 mM). These data suggest that DCTb cells possess in apical anion/base exchanger that resembles the Na(+)-independent Cl-/HCO(-3) exchanger.

Biological significance of agmatine, an endogenous ligand at imidazoline binding sites

“…At concentrations that induce no changes in haemo‐dynamics or creatinine clearance, agmatine as well as the I 1 ‐ligands moxonidine, clonidine and 2,6‐dimethyl‐clonidine increase urinary flow‐rate, whereby agmatine first shows significant changes at ∼10 fold higher doses ( Allan et al ., 1993 ; Li et al ., 1994b ; Ernsberger et al ., 1995 ; Penner & Smyth, 1996 ). The increase in urinary flow under moxonidine, 2,6‐dimethylclonidine and agmatine results from an increased osmotic clearance, while under clonidine it is due to an increased clearance of free water, consistent with the imidazoline‐mediated inhibition of the Na + /H + ‐exchange in isolated renal tubular cells ( Bidet et al ., 1990 ). This suggests that the osmotic clearance is possibly an imidazoline binding site‐mediated effect, but that free water clearance might be due to an α 2 ‐receptor‐related mechanism ( Smyth & Penner, 1995 ), since compared to clonidine the relative affinities of agmatine, moxonidine and dimethyl clonidine are higher for imidazoline binding sites than they are for α 2 ‐adrenoceptors ( Ernsberger et al ., 1995 ).…”

“…The negative results of agmatine on isolated vessels ( Pinthong et al ., 1995a ; Schäfer et al ., 1999a ), however, contradict such a participation. The simultaneous increase in APR could be directly mediated, a supposition supported by the finding that agmatine increases the Na + /K + ATPase in renal membranes ( Bidet et al ., 1990 ). The fact that agmatine increases SNGFR after denervation to the same extent as that seen in innervated kidneys, excludes the possibility of a presynaptic mechanism.…”

Na+/H+ and Cl–/HCO3 –-antiporters of bovine pigmented ciliary epithelial cells

“…Intracellular pH (pH c ) was measured using the pH-sensitive fluorescent dye BCECF [6,24]. Cells grown in Petri dishes were loaded for ≅10 min with 1 µM of the pentaacetoxy-methyl ester of BCECF at 37°C.…”

Principal and intercalated cells in primary cultures of rabbit renal collecting tubules revelaed by monoclonal antibodies

“…The primary cell culture technique has been described in detail in previous papers [3,11,24]. The cortical collecting tubules were carefully microdissected under sterile conditions from 4-5-weekold male New Zealand rabbit kidneys.…”