Extracellular Ca2+ regulates the stimulation of Na+ transport in A6 renal epithelia

We investigated the involvement of intracellular and extracellular Ca2+ in the stimulation of Na+ transport during hyposmotic treatment of A6 renal epithelia. A sudden osmotic decrease elicits a biphasic stimulation of Na+ transport, recorded as increase in amiloride-sensitive short-circuit current ( Isc) from 3.4 ± 0.4 to 24.0 ± 1.3 μA/cm2 ( n = 6). Changes in intracellular Ca2+ concentration ([Ca2+]i) were prevented by blocking basolateral Ca2+ entry with Mg2+ and emptying the intracellular Ca2+ stores before the hyposmotic challenge. This treatment did not noticeably affect the hypotonicity-induced stimulation of Isc. However, the absence of extracellular Ca2+ severely attenuated Na+ transport stimulation by the hyposmotic shock, and Isc merely increased from 2.2 ± 0.3 to 4.8 ± 0.7 μA/cm2. Interestingly, several agonists of the Ca2+-sensing receptor, Mg2+ (2 mM), Gd3+ (0.1 mM), neomycin (0.1 mM), and spermine (1 mM) were able to substitute for extracellular Ca2+. When added to the basolateral solution, these agents restored the stimulatory effect of the hyposmotic solutions on Isc in the absence of extracellular Ca2+ to levels that were comparable to control conditions. None of the above-mentioned agonists induced a change in [Ca2+]i. Quinacrine, an inhibitor of PLA2, overruled the effect of the agonists on Na+ transport. In conclusion, we suggest that a Ca2+-sensing receptor in A6 epithelia mediates the stimulation of Na+ transport without the interference of changes in [Ca2+]i.

Role of PKCα in feedback regulation of Na+transport in an electrically tight epithelium

It has long been known that Na+ channels in electrically tight epithelia are regulated by homeostatic mechanisms that maintain a steady state and allow new levels of transport to be sustained in hormonally challenged cells. Little is known about the potential pathways involved in these processes. In addition to short-term effect, recent evidence also indicates the involvement of PKC in the long-term regulation of the epithelial Na+ channel (ENaC) at the protein level (40). To determine whether stimulation of ENaC involves feedback regulation of PKC levels, we utilized Western blot analysis to determine the distribution of PKC isoforms in polarized A6 epithelia. We found the presence of PKC isoforms in the conventional (α and γ), novel (δ, η, and ε), and atypical (ι, λ, and ζ) groups. Steady-state stimulation of Na+ transport with aldosterone was accompanied by a specific decrease of PKCα protein levels in both the cytoplasmic and membrane fractions. Similarly, overnight treatment with an uncharged amiloride analog (CDPC), a procedure that through feedback regulation causes a stimulation of Na+ transport, also decreased PKCα levels. These effects were additive, indicating separate mechanisms that converge at the level of PKCα. These effects were not accompanied by changes of PKCα mRNA levels as determined by Northern blot analysis. We propose that this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na+ transport.

cAMP-sensitive endocytic trafficking in A6 epithelia

Blocker-induced noise analysis and laser scanning confocal microscopy were used to test the idea that cAMP-mediated vesicle exocytosis/endocytosis may be a mechanism for regulation of functional epithelial Na+ channels (ENaCs) at apical membranes of A6 epithelia. After forskolin stimulation of Na+ transport and labeling apical membranes with the fluorescent dye N-(3-triethylammoniumpropyl)4-(6-4 diethylaminophenyl) hexatrienyl pyridinium dibromide (FM 4-64), ENaC densities ( N T) decreased exponentially (time constant ∼20 min) from mean values of 320 to 98 channels/cell within 55 min during washout of forskolin. Two populations of apical membrane-labeled vesicles appeared in the cytosol within 55 min, reaching mean values near 18 vesicles/cell, compared with five vesicles per cell in control, unstimulated tissues. The majority of cAMP-dependent endocytosed vesicles remained within a few micrometers of the apical membranes for the duration of the experiments. A minority of vesicles migrated to >5 μm below the apical membrane. Because steady states require identical rates of endocytosis and exocytosis, and because forskolin increased endocytic rates by fivefold or more, cAMP/protein kinase A acts kinetically not only to increase rates of cycling of vesicles at the apical membranes, but also principally to increase exocytic rates. These observations are consistent with and support, but do not prove, that vesicle trafficking is a mechanism for cAMP-mediated regulation of apical membrane channel densities in A6 epithelia.

Regulation of the epithelial Na+ channel by extracellular acidification

The effect of extracellular acidification was tested on the native epithelial Na+ channel (ENaC) in A6 epithelia and on the cloned ENaC expressed in Xenopusoocytes. Channel activity was determined utilizing blocker-induced fluctuation analysis in A6 epithelia and dual electrode voltage clamp in oocytes. In A6 cells, a decrease of extracellular pH (pHo) from 7.4 to 6.4 caused a slow stimulation of the amiloride-sensitive short-circuit current ( I Na) by 68.4 ± 11% ( n = 9) at 60 min. This increase of I Na was attributed to an increase of open channel and total channel ( N T) densities. Similar changes were observed with pHo 5.4. The effects of pHo were blocked by buffering intracellular Ca2+ with 5 μM 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid. In oocytes, pHo 6.4 elicited a small transient increase of the slope conductance of the cloned ENaC (11.4 ± 2.2% at 2 min) followed by a decrease to 83.7 ± 11.7% of control at 60 min ( n = 6). Thus small decreases of pHostimulate the native ENaC by increasing N T but do not appreciably affect ENaC expressed in Xenopus oocytes. These effects are distinct from those observed with decreasing intracellular pH with permeant buffers that are known to inhibit ENaC.

Aldosterone induces Ras methylation in A6 epithelia

Aldosterone increases Na+ reabsorption by renal epithelial cells: the acute actions (<4 h) appear to be promoted by protein methylation. This paper describes the relationship between protein methylation and aldosterone's action and describes aldosterone-mediated targets for methylation in cultured renal cells (A6). Aldosterone increases protein methylation from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/μg protein. Aldosterone stimulates protein methylation by increasing methyltransferase activity from 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/μg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce the aldosterone-induced increase in methyltransferase activity. One of these inhibitors, the isoprenyl-cysteine methyltransferase-specific inhibitor, S- trans, trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone induces protein methylation in two molecular weight ranges: near 90 kDa and around 20 kDa. The lower molecular weight range is the weight of small G proteins, and aldosterone does increase both Ras protein 1.6-fold and Ras methylation almost 12-fold. Also, Ras antisense oligonucleotides reduce the activity of Na+ channels by about fivefold. We conclude that 1) protein methylation is essential for aldosterone-induced increases in Na+ transport; 2) one target for methylation is p21ras; and 3) inhibition of Ras expression or Ras methylation inhibits Na+ channel activity.

LY-294002-inhibitable PI 3-kinase and regulation of baseline rates of Na+ transport in A6 epithelia

Blocker-induced noise analysis of epithelial Na+ channels (ENaCs) was used to investigate how inhibition of an LY-294002-sensitive phosphatidylinositol 3-kinase (PI 3-kinase) alters Na+transport in unstimulated and aldosterone-prestimulated A6 epithelia. From baseline Na+ transport rates ( I Na) of 4.0 ± 0.1 (unstimulated) and 9.1 ± 0.9 μA/cm2 (aldosterone), 10 μM LY-294002 caused, following a relatively small initial increase of transport, a completely reversible inhibition of transport within 90 min to 33 ± 6% and 38 ± 2% of respective baseline values. Initial increases of transport could be attributed to increases of channel open probability ( P o) within 5 min to 143 ± 17% (unstimulated) and 142 ± 10% of control (aldosterone) from baseline P o averaging near 0.5. Inhibition of transport was due to much slower decreases of functional channel densities ( N T) to 28 ± 4% (unstimulated) and 35 ± 3% (aldosterone) of control at 90 min. LY-294002 (50 μM) caused larger but completely reversible increases of P o (215 ± 38% of control at 5 min) and more rapid but only slightly larger decreases of N T. Basolateral exposure to LY-294002 induced no detectable effect on transport, P o or N T. We conclude that an LY-294002-sensitive PI 3-kinase plays an important role in regulation of transport by modulating N T and P o of ENaCs, but only when presented to apical surfaces of the cells.

Aldosterone action: induction of p21rasandfra-2 and transcription-independent decrease inmyc,jun, andfos

Adrenal steroids induce an increase in transcellular Na+reabsorption across Xenopus laevis A6 cell epithelia that requires the action of transcriptionally regulated gene products. In a previous study we identified K- ras2 as an aldosterone-upregulated mRNA in A6 epithelia. Here, we show that in vivo injection of aldosterone in Xenopus (2.5 h) increases K- ras2 mRNA specifically in the kidney (2.5-fold) and that in A6 epithelia aldosterone (2.5 h) increases Ras protein synthesis (∼6-fold). Xl- ras, another ras mRNA expressed at a low level in A6 cells, was also induced (2-fold). Aldosterone was shown to regulate the mRNA levels of several transcription factors as well. After 2 h of aldosterone treatment, fra-2 mRNA was upregulated by 130%, whereas c- myc, c- jun, c- fos, and glucocorticoid receptor mRNAs were downregulated by 23–43%. After 16 h, c- fos and GR mRNAs were further decreased, whereas levels of fra-2, c- jun, and c- myc began to return to control levels. Interestingly, the downregulation of the protooncogene mRNAs was independent of transcription. These results support the view that aldosterone exerts complex pleiotropic transcriptional and nontranscriptional actions that involve the regulation of signaling cascade elements (i.e., K-Ras2) as well as that of transcription factors.