CFTR-mediated anion secretion in parathyroid hormone-treated Caco-2 cells is associated with PKA and PI3K phosphorylation but not intracellular pH changes or Na+/K+-ATPase abundance

Parathyroid hormone (PTH) has previously been shown to enhance the transepithelial secretion of Cl^? and HCO₃^? across the intestinal epithelia including Caco-2 monolayer, but the underlying cellular mechanisms are not completely understood. Herein, we identified the major signaling pathways that possibly mediated the PTH action to its known target anion channel, i.e., cystic fibrosis transmembrane conductance regulator anion channel (CFTR). Specifically, PTH was able to induce phosphorylation of protein kinase A and phosphoinositide 3-kinase. Since the apical HCO₃^? efflux through CFTR often required the intracellular H⁺/HCO₃^? production and/or the Na⁺-dependent basolateral HCO₃^? uptake, the intracellular pH (pH_i) balance might be disturbed, especially as a consequence of increased endogenous H⁺ and HCO₃^? production. However, measurement of pH_i by a pH-sensitive dye suggested that the PTH-exposed Caco-2 cells were able to maintain normal pH despite robust HCO₃^? transport. In addition, although the plasma membrane Na⁺/K⁺-ATPase (NKA) is normally essential for basolateral HCO₃^? uptake and other transporters (e.g., NHE1), PTH did not induce insertion of new NKA molecules into the basolateral membrane as determined by membrane protein biotinylation technique. Thus, together with our previous data, we concluded that the PTH action on Caco-2 cells is dependent on PKA and PI3K with no detectable change in pH_i or NKA abundance on cell membrane.     

PKA Regulates Vacuolar H+-ATPase Localization and Activity via Direct Phosphorylation of the A Subunit in Kidney Cells

The vacuolar H⁺-ATPase (V-ATPase) is a major contributor to luminal acidification in epithelia of Wolffian duct origin. In both kidney-intercalated cells and epididymal clear cells, cAMP induces V-ATPase apical membrane accumulation, which is linked to proton secretion. We have shown previously that the A subunit in the cytoplasmic V₁ sector of the V-ATPase is phosphorylated by protein kinase A (PKA). Here we have identified by mass spectrometry and mutagenesis that Ser-175 is the major PKA phosphorylation site in the A subunit. Overexpression in HEK-293T cells of either a wild-type (WT) or phosphomimic Ser-175 to Asp (S175D) A subunit mutant caused increased acidification of HCO₃⁻-containing culture medium compared with cells expressing vector alone or a PKA phosphorylation-deficient Ser-175 to Ala (S175A) mutant. Moreover, localization of the S175A A subunit mutant expressed in HEK-293T cells was more diffusely cytosolic than that of WT or S175D A subunit. Acute V-ATPase-mediated, bafilomycin-sensitive H⁺ secretion was up-regulated by a specific PKA activator in HEK-293T cells expressing WT A subunit in HCO₃⁻-free buffer. In cells expressing the S175D mutant, V-ATPase activity at the membrane was constitutively up-regulated and unresponsive to PKA activators, whereas cells expressing the S175A mutant had decreased V-ATPase activity that was unresponsive to PKA activation. Finally, Ser-175 was necessary for PKA-stimulated apical accumulation of the V-ATPase in a polarized rabbit cell line of collecting duct A-type intercalated cell characteristics (Clone C). In summary, these results indicate a novel mechanism for the regulation of V-ATPase localization and activity in kidney cells via direct PKA-dependent phosphorylation of the A subunit at Ser-175.     

Yeast 3-phosphoinositide-dependent protein kinase-1 (PDK1) orthologs Pkh1-3 differentially regulate phosphorylation of protein kinase A (PKA) and the protein kinase B (PKB)/S6K ortholog Sch9

Pkh1, -2, and -3 are the yeast orthologs of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated that Pkh1 phosphorylates Sch9 both in vitro and in vivo on its PDK1 site and that this phosphorylation is essential for a wild type cell size. In vivo phosphorylation on this site disappeared during nitrogen deprivation and rapidly increased again upon nitrogen resupplementation. In addition, we have shown here for the first time that the PDK1 site in protein kinase A is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkh-dependent in vivo and occurs during or shortly after synthesis of the protein kinase A catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolished binding of the regulatory subunit and cAMP dependence. As opposed to PDK1 site phosphorylation of Sch9, phosphorylation of the PDK1 site in Tpk1 was not regulated by nitrogen availability. These results bring new insight into the control and prevalence of PDK1 site phosphorylation in yeast by Pkh protein kinases.     

Na+/K+-ATPase resistance and cardenolide sequestration: basal adaptations to host plant toxins in the milkweed bugs (Hemiptera: Lygaeidae: Lygaeinae)

Despite sequestration of toxins being a common coevolutionary response to plant defence in phytophagous insects, the macroevolution of the traits involved is largely unaddressed. Using a phylogenetic approach comprising species from four continents, we analysed the ability to sequester toxic cardenolides in the hemipteran subfamily Lygaeinae, which is widely associated with cardenolide-producing Apocynaceae. In addition, we analysed cardenolide resistance of their Na⁺/K⁺-ATPases, the molecular target of cardenolides. Our data indicate that cardenolide sequestration and cardenolide-resistant Na⁺/K⁺-ATPase are basal adaptations in the Lygaeinae. In two species that shifted to non-apocynaceous hosts, the ability to sequester was secondarily reduced, yet Na⁺/K⁺-ATPase resistance was maintained. We suggest that both traits evolved together and represent major coevolutionary adaptations responsible for the evolutionary success of lygaeine bugs. Moreover, specialization on cardenolides was not an evolutionary dead end, but enabled this insect lineage to host shift to cardenolide-producing plants from distantly related families.     

Nitric Oxide Induces Ca2+-independent Activity of the Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)

Both signaling by nitric oxide (NO) and by the Ca²⁺/calmodulin (CaM)-dependent protein kinase II α isoform (CaMKIIα) are implicated in two opposing forms of synaptic plasticity underlying learning and memory, as well as in excitotoxic/ischemic neuronal cell death. For CaMKIIα, these functions specifically involve also Ca²⁺-independent autonomous activity, traditionally generated by Thr-286 autophosphorylation. Here, we demonstrate that NO-induced S-nitrosylation of CaMKIIα also directly generated autonomous activity, and that CaMKII inhibition protected from NO-induced neuronal cell death. NO induced S-nitrosylation at Cys-280/289, and mutation of either site abolished autonomy, indicating that simultaneous nitrosylation at both sites was required. Additionally, autonomy was generated only when Ca²⁺/CaM was present during NO exposure. Thus, generation of this form of CaMKIIα autonomy requires simultaneous signaling by NO and Ca²⁺. Nitrosylation also significantly reduced subsequent CaMKIIα autophosphorylation specifically at Thr-286, but not at Thr-305. A previously described reduction of CaMKII activity by S-nitrosylation at Cys-6 was also observed here, but only after prolonged (>5 min) exposure to NO donors. These results demonstrate a novel regulation of CaMKII by another second messenger system and indicate its involvement in excitotoxic neuronal cell death.     

A Single Amino Acid Tunes Ca2+ Inhibition of Brain Liver Intestine Na+ Channel (BLINaC)

Ion channels of the degenerin/epithelial Na⁺ channel gene family are Na⁺ channels that are blocked by the diuretic amiloride and are implicated in several human diseases. The brain liver intestine Na⁺ channel (BLINaC) is an ion channel of the degenerin/epithelial Na⁺ channel gene family with unknown function. In rodents, it is expressed mainly in brain, liver, and intestine, and to a lesser extent in kidney and lung. Expression of rat BLINaC (rBLINaC) in Xenopus oocytes leads to small unselective currents that are only weakly sensitive to amiloride. Here, we show that rBLINaC is inhibited by micromolar concentrations of extracellular Ca²⁺. Removal of Ca²⁺ leads to robust currents and increases Na⁺ selectivity of the ion pore. Strikingly, the species ortholog from mouse (mBLINaC) has an almost 250-fold lower Ca²⁺ affinity than rBLINaC, rendering mBLINaC constitutively active at physiological concentrations of extracellular Ca²⁺. In addition, mBLINaC is more selective for Na⁺ and has a 700-fold higher amiloride affinity than rBLINaC. We show that a single amino acid in the extracellular domain determines these profound species differences. Collectively, our results suggest that rBLINaC is opened by an unknown ligand whereas mBLINaC is a constitutively open epithelial Na⁺ channel.     

Sodium and ATP affinities of the cardiac (Na,K)-ATPase in relation to nitric oxide synthesis in spontaneously hypertensive rats

The (Na,K)-ATPase is hypothesized to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and cardiac contractility. Investigating the kinetic properties of the above enzyme we tried to assess the molecular basis of alterations in transmembraneous efflux of Na(+) from cardiac cells in spontaneously hypertensive rats (SHR) with increased synthesis of nitric oxide (NO). In the investigated group of SHR the systolic blood pressure was increased by 64% and the synthesis of NO was increased by 60% in the heart. When activating the cardiac (Na,K)-ATPase with substrate, its activity was higher in SHR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed an increase of the V(max) (by 37%) probably due to increased affinity of the ATP-binding site as indicated by the lowered K(m) value (by 38%) in SHR. During activation with Na(+), we observed no change in the enzyme activity below 10 mmol/l of NaCl whereas in the presence of higher concentrations of NaCl the (Na,K)-ATPase was stimulated. The value of V(max) increased (by 64%), however the K(Na) increased (by 106%), indicating an adaptation of the Na(+)-binding site of the enzyme to increased [Na(+)](i). Thus the (Na,K)-ATPase in our SHR group is able to extrude the excessive Na(+) from myocardial cells more effectively also at higher [Na(+)](i), while the enzyme from controls is unable to increase its activity further. This improvement of the (Na,K)-ATPase function is supported also by increased affinity of its ATP-binding site probably due to enhanced NO-synthesis.     

Optimal metabolic regulation of the mammalian heart Na(+)/Ca(2+) exchanger requires a spacial arrangements with a PtdIns(4)-5kinase

In inside-out bovine heart sarcolemmal vesicles, p-chloromercuribenzenesulfonate (PCMBS) and n-ethylmaleimide (NEM) fully inhibited MgATP up-regulation of the Na⁺/Ca²⁺ exchanger (NCX1) and abolished the MgATP-dependent PtdIns-4,5P2 increase in the NCX1-PtdIns-4,5P2 complex; in addition, these compounds markedly reduced the activity of the PtdIns(4)-5kinase. After PCMBS or NEM treatment, addition of dithiothreitol (DTT) restored a large fraction of the MgATP stimulation of the exchange fluxes and almost fully restored PtdIns(4)-5kinase activity; however, in contrast to PCMBS, the effects of NEM did not seem related to the alkylation of protein SH groups. By itself DTT had no effect on the synthesis of PtdIns-4,5P2 but affected MgATP stimulation of NCX1: moderate inhibition at 1 mM MgATP and 1 μM Ca²⁺ and full inhibition at 0.25 mM MgATP and 0.2 μM Ca²⁺. In addition, DDT prevented coimmunoprecipitation of NCX1 and PtdIns(4)-5kinase. These results indicate that, for a proper MgATP up-regulation of NCX1, the enzyme responsible for PtdIns-4,5P2 synthesis must be (i) functionally competent and (ii) set in the NCX1 microenvironment closely associated to the exchanger. This kind of supramolecular structure is needed to optimize binding of the newly synthesized PtdIns-4,5P2 to its target region in the exchanger protein.     

Position 170 of Rabbit Na+/glucose cotransporter (rSGLT1) lies in the Na+ pathway; modulation of polarity/charge at this site regulates charge transfer and carrier turnover

Positions 163, 166, and 173, within the putative external loop joining transmembrane segments IV and V of rabbit Na(+)/glucose cotransporter, form part of its Na(+) interaction and voltage-sensing domain. Since a Q170C mutation within this region exhibits anomalous behavior, its function was further investigated. We used Xenopus oocytes coinjected with mouse T-antigen to enhance Q170C expression, and the two-microelectrode voltage-clamp technique. For Q170C, alpha-methyl D-glucopyranoside, phloridzin, and Na(+) affinity values are equivalent to those of wild-type; but turnover is reduced approximately 50%. Decreased [Na(+)] reduces Q170C, but not wild-type, charge transfer. Q170C presteady-state currents exhibit three time constants, tau, identical to wild-type. MTSES decreases maximal alpha-methyl D-glucopyranoside-induced currents by approximately 64% and Na(+) leak by approximately 55%; phloridzin and Na(+) affinity are unchanged. MTSES also reduces charge transfer (dithiothreitol-reversible) and Q170C turnover by approximately 60-70%. MTSEA and MTSET protect against MTSES, but neither affect Q170C function. MTSES has no obvious effect on the tau-values. Q170A behaves the same as Q170C. The mutation Q170E affects voltage sensitivity and reduces turnover, but also appears to influence Na(+) interaction. We conclude that 1), glutamine 170 lies in the Na(+) pathway in rabbit Na(+)/glucose cotransporter and 2), altered polarity and charge at position 170 affect a cotransporter conformational state and transition, which is rate-limiting, but probably not associated with empty carrier reorientation.