Effects Of Aldosterone On Sodium Hydrogen Exchangers Biology Essay

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Aldosterone is a mineralcorticoid that is secreted by cells of the adrenal cortex. Aldosterone has multiple effects on multiple organ systems, including maintaining sodium, potassium, and acid-base balance in the kidney (Good 2006) and colon (Harvey 2002). Aldosterone is traditionally known to mediate its effects by genomic mechanisms, which consists of altering RNA transcription and protein synthesis. However, aldosterone can also mediate rapid, non-genomic effects by activation of multiple second messenger systems. Aldosterone can mediate a wide variety of responses, depending on which second messenger system is activated.

Sodium-hydrogen exchangers (NHE) are antiports found on the plasma membrane of a variety of cell types. There are at least eight different NHE isoforms (Watts III 2005) that show variable expression. Studies have shown that aldosterone regulates NHE expression and activity by genomic as well as non-genomic mechanisms. The purpose of this review is to provide a systematic overview of the non-genomic effects of aldosterone on NHE reported in the last twenty years. First, aldosterone binding to receptors that mediate rapid cellular effects will be described. Second, signal transduction pathways activated by these receptors will be discussed in detail. And lastly, the direct interaction with NHE by kinases activated by aldosterone-induced signaling pathways will be described.

Aldosterone receptor

It has been thoroughly shown that the genomic effects of aldosterone are mediated through aldosterone binding the mineralcorticoid receptor (MR). The mineralcorticoid receptor is an intracellular receptor that, once activated, binds to a specific DNA sequence called a hormone response element (HRE) (Hadley 2007). The activated MR binds to a specific HRE, leading to alteration in the amount of mRNA that is transcribed and thus the amount of protein that is translated. Therefore, aldosterone, acting through the MR, has genomic effects by altering the amount of protein that is made (Hadley 2007). Numerous studies have shown that the non-genomic effects of aldosterone are not mediated through the mineralcorticoid receptor (Ebata 1999; Gekle 1997; Gekle 2001; Sheader 2002; Winter 1999). Therefore, aldosterone must bind a different receptor or multiple different receptors in order to mediate its non-genomic effects. At this point, there is no definitive nongenomic aldosterone receptor. However, Wehling et al reported high affinity binding of aldosterone to the membrane of human mononuclear leukocytes in a time span that is consistent with rapid non-genomic effects (Wehling 1992). Gekle et al also reported aldosterone binding to a membrane receptor and mediating non-genomic effects in kidney cells (Gekle 2002). Specifically, Gekle et al used Madin Darby Canine Kidney cells (MDCK cells), which showed similar properties as cells of the kidney collecting duct. They reported that aldosterone activated the epidermal growth factor (EGF) receptor, resulting in increased levels of phosphorylated ERK 1/2 and intracellular calcium (Gekle 2002), both of which can enhance NHE activity. Furthermore, Gekle et al showed that inhibiting the EGF receptor kinase (which is stimulated by activation of the EGF receptor) with c56 or tyrphostin resulted in a similar, significant decrease in the levels of phosphorylated ERK 1/2, regardless if aldosterone or EGF was added (Gekle 2002). Therefore, since the effect of aldosterone on ERK 1/2 was inhibited by inhibition of the EGF signaling pathway, it is a logical conclusion that aldosterone mediated its effects on ERK 1/2 through the EGF receptor. The finding of a steroid hormone binding and activating a peptide receptor is not a novel finding. Progesterone bound the oxytocin G-protein coupled receptor and inhibited signaling in rat uterine cells (Grazzini 1998). Progesterone acted as a competitive inhibitor by not allowing oxytocin to bind its receptor (Grazzini 1998). Also, progesterone decreased inositol-triphosphate (IP3) production and calcium release mediated by oxytocin binding (Grazzini 1998). Other steroid hormones mediated their non-genomic effects by following signal transduction pathways very similar to peptide and catecholamine signaling pathways, involving phospholipase C, protein kinase C, alterations in pH and calcium levels, and tyrosine kinases (Wehling 1997). This leads to the possibility of crosstalk between rapid steroid, peptide, and catecholamine signaling. More research needs to be conducted in order to determine whether aldosterone and other steroid hormones mediate their non-genomic effects through interaction with other peptide as well as catecholamine receptors, if there is indeed a unique non-genomic aldosterone receptor, or both.

Protein Kinases A and C

Protein kinases are enzymes that catalyze the removal of phosphate groups from adenosine tri-phosphate (ATP) and then add them to other molecules. Phosphorylation by kinases serves as a very common mechanism to activate signaling components. Protein kinases A (PKA) and C (PKC) represent two distinct signaling pathways. Multiple studies have shown that aldosterone activates second messenger systems involving PKC (Christ 1993; Christ 1995; Ebata 1999; Harvey 2002). Furthermore, the activation of PKC has been shown to lead to the activation of NHE (Winter 1999; Harvey 2002). In mammalian colonic crypt cells, aldosterone induced a cellular pH recovery after acid loading due to enhanced NHE activity (Harvey 2002; Winter 1999). This effect was inhibited by the addition of the NHE inhibitor ethyl-isopropylamiloride (EIPA) and occured within ten minutes of aldosterone addition (Harvey 2002; Winter 1999). One study reported that this was due specifically to activation of basolateral NHE 1 (Harvey 2002) while another reported simply NHE activation (Winter 1999). Aldosterone activated PKC (Christ 1995; Ebata 1999) and subsequently NHE in vascular smooth muscle cells as well, showing rapid cellular pH recovery after acid loading (Ebata 1999). This pH recovery was inhibited by the addition of NHE and PKC inhibitors (Ebata 1999). The researchers hypothesized that aldosterone-activated PKC led to a signaling pathway that resulted in exocytotic insertion of NHE into the plasma membrane (Ebata 1999). This is because they reported that inhibiting microtubule formation by adding colchicine or inhibiting F-actin polymerization abolished the aldosterone-mediated pH recovery after acid loading (Ebata 1999). Lastly, aldosterone was found to increase IP3 levels in human mononuclear leukocytes, which led to activation of PKC (Christ 1993). This finding is contradictory to a report from kidney cells (MDCK cells), where aldosterone had no effect on IP3 levels (Gekle 2002). Since aldosterone did not have an effect on IP3 levels in the kidney, but increased IP3 levels in leukocytes, this may represent a tissue-specific difference in signaling mediated by aldosterone; however, a study specifically looking at the alterations in IP3 levels due to aldosterone in multiple tissues, including the kidney and leukocytes, would have to be conducted in order to substantiate this hypothesis.

Protein kinase A represents another signaling pathway and is mediated by increasing levels of cAMP. Studies have shown that aldosterone rapidly increased cAMP levels in colonic crypt cells (Winter 1999) and in inner medullary collecting duct (IMCD) cells of the kidney (Sheader 2002). However, unlike PKC, this subsequent activation of PKA was reported to inhibit NHE, resulting in cell acidification and insensitivity to activation of NHE by aldosterone (Winter 1999).


Calcium is a cofactor that is necessary for the function of a variety of proteins in multiple cell types, including NHE (Gekle 1996; Gekle 1997; Gekle 2002). Calcium works by directly binding the NHE and other proteins and modulating their activity. Aldosterone has been shown to modulate calcium levels, which subsequently affects NHE (Gekle 2002, Harvey 2002). In MDCK cells of the kidney, aldosterone was found to increase calcium entry by signaling through the epidermal growth factor (EGF) receptor, resulting in subsequent NHE activation (Gekle 2002). These effects on calcium entry were found to occur within minutes of aldosterone binding (Gekle 1996). The kidney is not the only organ in which binding of aldosterone leads to rapid increases in intracellular calcium. In colonic crypt cells, aldosterone induced cell alkalization that required PKC activation and calcium entry (Harvey 2002). The addition of aldosterone led to cell alkalization that was inhibited by basolateral amiloride or the absence of extracellular sodium, which the authors reported was due to the inhibition of specifically basolateral NHE 1. This cell alkalization was also inhibited by adding HBDDE, which inhibited PKC activity. PKC is needed to activate calcium entry through calcium channels. Therefore, inhibiting PKC and subsequently calcium entry resulted in the inhibition of aldosterone-mediated cell alkalization via basolateral NHE 1 (Harvey 2002).

MAP-Kinase Pathway

Traditionally, the Mitogen-activated protein kinase (MAP-K) pathway is associated with activation of transcription factors, i.e. genomic effects. However, in multiple cells of the kidney, aldosterone was shown to rapidly activate this signaling pathway (Gekle 2001; Gekle 2002; Markos 2005). In both MDCK cells and M-1 cortical collecting ducts cells, aldosterone was shown to mediate a rapid pH recovery that was abolished by specific NHE 1 inhibitors in one study (Markos 2005), general NHE inhibitors in a second study (Gekle 2001), and by inhibiting phosphorylation of MAP-K proteins ERK 1/2 in both (Gekle 2001; Markos 2005). Also, Gekle et al reported increased phosphorylation of ERK 1/2 as well as an increase in intracellular calcium after the addition of aldosterone (Gekle 2002); although he did not show specifically that these events led to activation of NHE, others have shown that these two events can result in the activation of NHE (Gekle 2001, Markos 2005). These effects of aldosterone were unaffected when the mineralcorticoid receptor (MR) antagonist spironolactone was added (Gekle 2001; Markos 2005). Interestingly, one study found that aldosterone not only led to ERK 1/2 phosphorylation, but also activation of PKC isoform α (Markos 2005). Inhibitors of PKC isoform α (HBDDE) and MAP-K (PD98059) both led to decreased pH recovery (mediated by NHE 1) after the addition of aldosterone (Markos 2005). Furthermore, these researchers found that PKC isoform α had to be activated prior to MAP-K, since adding PKC antagonist chelerythrine first, then adding aldosterone resulted in a decreased amount of phosphorylated ERK (part of MAP-K pathway) versus the control (Markos 2005). Therefore, PKC may serve as a connection between aldosterone-mediated signal transduction at the cell membrane and the MAP-K pathway. However, what is unknown is whether PKC directly phosphorylated the first MAP-K protein, or if other intermediates were involved. The effect of aldosterone on the rapid activation of the MAP-K pathway in tissues other than the kidney is uncertain.

Proton Conductance

Proton conductance is the transport of hydrogen ions into the cell through specific protein transporters. This influx of protons would serve to activate NHE because it acidifies the cell interior, providing more protons to be secreted in exchange for sodium. Enhancing proton conductance can therefore serve to activate NHE (Gekle 1996; Gekle 1997). This enhancement of proton conductance has been shown to be due to aldosterone-mediated signaling (Gekle 1996; Gekle 1997). In MDCK cells of the kidney, aldosterone was shown to stimulate rapid cell acidification that was inhibited by zinc (a known inhibitor of proton conductance) (Gekle 1996; Gekle 1997). Furthermore, it was shown that PKC was involved in the activation of this proton conductance, since stimulating PKC without aldosterone showed similar cell acidification as was found with aldosterone addition (Gekle 1997). This demonstrated that aldosterone must activate PKC, which then enhanced proton conductance (Gekle 1997). It is believed that this proton conductance, rather than drastically acidifying the entire cell, forms an acidic microenvironment that serves to activate NHE (Gekle 1997). Interestingly, some believe that such a microenvironment is due to an intracellular sub domain surrounding NHE that is impermeable to protons (Dagher 1996). At this point, this is simply a theory and requires further substantiation.

Arachidonic acid, Prostaglandins, and G proteins

Arachidonic acid is another signaling molecule that connects membrane signaling with interior cytoplasmic signaling. Arachidonic acid is released from membrane phospholipids by activation of the enzyme phospholipase A2. At present, the involvement of arachidonic acid in aldosterone-mediated NHE activation is not clear, but certain researchers have reported a coorelation between arachidonic acid stimulation and NHE activity after aldosterone addition (Harvey 2002; Maguire 1999 Winter 1999). Harvey et al reported that an increase in intracellular calcium mediated by the addition of aldosterone involved a phospholipase A2 signal transduction pathway in colonic cells. As stated above, calcium can act to enhance NHE activity (Harvey 2002). Arachidonic acid signaling may be involved in aldosterone’s effects on NHE 1 in colonic cells, since blocking phospholipase A2 activity with quaracrine inhibited NHE 1-mediated cell alkalization in the presence of aldosterone (Winter 1999). Inhibition of prostaglandins and heterotrimeric G proteins negated aldosterone-mediated effects on NHE activity, whereas stimulation of heterotrimeric G proteins resulted in normal aldosterone-mediated cell alkalization due to NHE activation (Winter 1999). The effects of G protein stimulation after aldosterone addition is not surprising, since this is a common mechanism that connects receptor activation to second messenger systems, such as PKC, MAP-K, and calcium entry.

Direct interaction with NHE

For the aldosterone-activated secondary messenger systems described above, none of these proteins directly interact with NHE; rather, these messenger systems activate other regulatory proteins that can bind to or alter regulatory domains on the cytoplasmic tail of NHE (Lin 1996; Takahashi 1999). One such protein is p90RSK, a NHE 1 kinase that is activated directly by phosphorylation by ERK 1/2 of the MAP-K pathway. p90RSK phosphorylated a serine residue on the NHE 1 cytoplasmic tail at position 703 (this location termed P5). This phophorylation action was not necessary for NHE activity, but was required for enhanced NHE activity when stimulated by aldosterone (Takahashi 1999). It is believed that this phosphorylation event altered a calcium-calmodulin binding site, which, when bound, down regulated NHE 1 activity. Findings by Lin et al supported this hypothesis. Lin et al found that calcineurin B homologous protein (CHP) was constitutively bound to the cytoplasmic region of NHE 1 at AA 567-637. Upon stimulation to enhance NHE 1 activity (such as the addition of aldosterone), CHP was dephosphorylated and released from this binding site (Lin 1996). This would explain how p90RSK phosphorylation is coupled to NHE activation. As stated above, p90RSK phosphorylates a serine at position 703 in the amino acid sequence of NHE 1, which is very close to the 567-637 sequence bound by CHP. It is very plausible that phosphorylation at serine 703 altered the binding domain of CHP at 567-637, thus not allowing CHP to bind and allowing for activation of NHE. P5 is central to a short phosphorylation motif that was found in multiple locations on the cytoplasmic tail of NHE 1 (Takahashi 1999). Specifically, Takahashi et al cited five potential locations of p90RSK phosphorylation (termed P1-5). Of these, two sites, P5 and P4, showed increased phosphorylation when incubated with serum (serum stimulates the activity of p90RSK). However, a later experiment showed that MAP-K inhibition of p90RSK led only to decreased phosphorylation of P5, and so their experiments focused on this site (Takahashi 1999). The signaling pathway that leads to p90RSK phosphorylation of P4 is unknown. What is also unknown is the consequences of the P4 phosphorylation state on NHE 1 activity. Does phosphorylation at this site simply exert a supporting role to phosphorylation at P5, or can phosphorylation of P4 lead to alterations in NHE 1 activity? Further research needs to be conducted in order to answer these questions, as well as to explain why p90RSK activity led to increased phosphorylation only at two of five potential sites. Does a different stimulus lead to phosphorylation at these sites by p90RSK or does a different kinase phosphorylate these sites? Also, are these results NHE isoform- or tissue-specific, or can these results be applied to different NHE isoforms in different tissues?


Of the questions that one may have about non-genomic activation of NHE by aldosterone, we do have some answers. First, aldosterone does activate non-genomic pathways that subsequently lead to NHE activation, such as PKC (Christ 1993; Christ 1995; Ebata 1999; Harvey 2002) and MAP-K pathways (Gekle 2001; Gekle 2002; Markos 2005). Second, aldosterone activates cellular mechanisms that may enhance NHE activation, such as increased intracellular calcium (Gekle 1996; Gekle 1997; Gekle 2002) and enhanced proton conductance (Gekle 1996; Gekle 1997). Third, aldosterone may lead to direct phosphorylation of NHE by p90RSK, mediated through the MAP-K pathway (Takahashi 1999). However, as always occurs, finding these answers leads to new questions. What are not clearly defined are the differences in activation of these signaling pathways between different cellular tissues and between different NHE isoforms. Does p90RSK only phosphorylate NHE 1, or can it phosphorylate some or all of the other eight known NHE isoforms? Does PKC activation occur in the kidney, or is this a novel tissue where aldosterone addition does not lead to PKC activation and subsequent NHE activation. Most studies do not even consider NHE isoform-specific effects on these pathways. Also, the role of arachidonic acid and prostaglandin signaling needs to be more clearly defined. Most importantly, the nature of the aldosterone non-genomic receptor needs to be worked out. Is the membrane receptor that mediated the non-genomic effects in the Wehling et al study the EGF receptor cited by Gekle et al, or a different synonymous peptide receptor? Or do these two studies cite two distinct receptors that can mediate the effects of aldosterone? It may be a possibility that aldosterone may bind a different non-genomic receptor in each tissue type, since the EGF receptor is not going to be expressed in smooth muscle cells. A study similar to the Gekle et al study needs to be conducted with a different cell type that expresses this EGF receptor to see if aldosterone activates the EGF receptor in this tissue. Even if a ubiquitously expressed non-genomic aldosterone receptor is found, the question of why aldosterone can bind the EGF receptor still needs to be answered, as well as a study to determine whether receptors similar to the EGF receptor can mediate the same aldosterone-mediated non-genomic effects.