The Na,K-ATPase receptor complex: its organization and membership

A major difference between the Na,K-ATPase ion pump and other P-type ATPases is its ability to bind cardiotonic steroids such as ouabain. Na,K-ATPase also interacts with many membrane and cytosolic proteins. In addition to their role in Na,K-ATPase regulation, it became apparent that some of the newly identified interactions are capable of organizing the Na,K-ATPase into various signaling complexes. This new function confers a ligand-like effect to cardiotonic steroids on cellular signal transduction. This article reviews these new developments and provides a comparison of Na,K-ATPase-mediated signal transduction with other receptors and ion transporters.     

An ion-transporting ATPase encodes multiple apical localization signals

Epithelial cells accumulate distinct populations of membrane proteins at their two plasmalemmal domains. We have examined the molecular signals which specify the differential subcellular distributions of two closely related ion pumps. The Na,K-ATPase is normally restricted to the basolateral membranes of numerous epithelial cell types, whereas the H,K-ATPase is a component of the apical surfaces of the parietal cells of the gastric epithelium. We have expressed full length and chimeric H,K-ATPase/Na,K-ATPase cDNAs in polarized renal proximal tubular epithelial cells (LLC-PK1). We find that both the alpha and beta subunits of the H,K-ATPase encode independent signals that specify apical localization. Furthermore, the H,K-ATPase beta-subunit possesses a sequence which mediates its participation in the endocytic pathway. The interrelationship between epithelial sorting and endocytosis signals suggested by these studies supports the redefinition of apical and basolateral as functional, rather than simply topographic domains.     

Novel role for Na,K-ATPase in phosphatidylinositol 3-kinase signaling and suppression of cell motility

The Na,K-ATPase, consisting of alpha- and beta-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase beta1-subunit (Na,K-beta) in highly motile Moloney sarcoma virus-transformed Madin-Darby canine kidney (MSV-MDCK) cells suppressed their motility. However, until now, the mechanism by which Na,K-beta reduces cell motility remained elusive. Here, we demonstrate that Na,K-beta localizes to lamellipodia and suppresses cell motility by a novel signaling mechanism involving a cross-talk between Na,K-ATPase alpha1-subunit (Na,K-alpha) and Na,K-beta with proteins involved in phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway. We show that Na,K-alpha associates with the regulatory subunit of PI3-kinase and Na,K-beta binds to annexin II. These molecular interactions locally activate PI3-kinase at the lamellipodia and suppress cell motility in MSV-MDCK cells, independent of Na,K-ATPase ion transport activity. Thus, these results demonstrate a new role for Na,K-ATPase in regulating carcinoma cell motility.     

Conformational Coupling: The Moving Parts of an Ion Pump

The Na,K-ATPase carries out the coupled functions of ATP hydrolysis and cation transport. These functions are performed by two distinct regions of the protein. ATP binding and hydrolysis is mediated by the large central cytoplasmic loop of about 430 amino-acids. Transmembrane cation transport is accomplished via coordination of the Na and K ions by side-chains of the amino-acids of several of the transmembrane segments. The way in which these two protein domains interact lies at the heart of the molecular mechanism of active transport, or ion pumping. We summarize evidence obtained from protein chemistry studies of the purified renal Na,K-ATPase and from bacterially expressed polypeptides which characterize these separate functions and point to various movements which may occur as the protein transits through its reaction cycle. We then describe recent work using heterologous expression of renal Na,K-ATPase in baculovirus-infected insect cells which provides a suitable system to characterize such protein motions and which can be employed to test specific models arising from recently acquired high resolution structural information on related ion pumps.     

The conformation of H,K-ATPase determines the nucleoside triphosphate (NTP) selectivity for active proton transport

The gastric H,K-ATPase is related to other cation transport ATPases, for example, Na,K-ATPase and Ca-ATPase, which are called E1-E2 ATPases in recognition of conformational transitions during their respective transport and catalytic cycles. Generally, these ATPases cannot utilize NTPs other than ATP for net ion transport activity. For example, under standard assay conditions, rates of NTP hydrolysis and H+ pumping by the H,K-ATPase for CTP are about 10% of those for ATP and undetectable with GTP, ITP, and UTP. However, we observed that H,K-ATPase will catalyze NTP/ADP phosphate exchange at similar rates for all of these NTPs, suggesting that a common phosphoenzyme intermediate is formed. The present study was undertaken to evaluate the specificity of nucleotides to power the H,K-ATPase and several of its partial reactions, including NTP/ADP exchange, K+-catalyzed phosphatase activity, and proton pumping. Results demonstrate that under conditions that promote the conformational change of the K+ bound form of the enzyme, K.E2, to E1, all NTPs tested support K+-stimulated NTPase activity and H+ pumping up to 30-50% of that with ATP. These conditions include (1) the presence of ADP as well as the NTP energy source and (2) reduced K+ concentration on the cytoplasmic side to approximately 0. These data conform to structural models for E1-E2 ATPases whereby adenosine binding promotes the K.E2 to E1 conformational change and K+ deocclusion.     

The amino acid sequence of an active site peptide from the H,K-ATPase of gastric mucosa

The gastric H,K-ATPase is an active transport protein that is responsible for the maintenance of a large pH gradient across the secretory canaliculus of the mammalian parietal cell. Acid secretion across these epithelial cell membranes is coupled to the potassium-stimulated hydrolysis of ATP catalyzed by H,K-ATPase, but the mechanism of coupling between ion transport and ATP hydrolysis is unknown. In order to investigate the enzymatic mechanism of this coupling, a peptide derived from the ATP binding site of H,K-ATPase has been purified and its amino acid sequence has been determined. The peptide was identified by the incorporation of a fluorescent probe, fluorescein 5'-isothiocyanate (FITC), into the active site before trypsin digestion of the protein. The labeling of the enzyme by FITC was associated with the irreversible inhibition of enzymatic activity, and both the labeling of the tryptic peptide and inhibition of activity were prevented when the reaction was performed in the presence of ATP. At 100% inhibition of activity, 3.5 +/- 1.6 nmol of FITC were incorporated per mg of protein. The amino acid sequence of the active site peptide is His-Val-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Gln-Leu-Ser-Ile-Arg, and FITC reacts with the lysine. This sequence is very similar to sequences of fluorescein-labeled peptides from the ATP binding sites of Na,K-ATPase and Ca2+-ATPase, and suggests that the active site structures of these ion transport ATPases are similar.     

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.     

Oxidative Resistance of Na/K-ATPase

1. Oxidative modification of Na/K-ATPase from brain and kidney has been studied. Brain enzyme has been found to be more sensitive than kidney enzyme to inhibition by both H₂O₂ and NaOCl.2. The inhibition of Na/K-ATPase correlates well with the decrease in a number of SH groups, suggesting that the latter belong mainly to ATPase protein and are essential for the enzyme activity. We suggest that the differences in the number, location, and accessibility of SH groups in Na/K-ATPase isozymes predict their oxidative stability.3. The hydrophilic natural antioxidant carnosine, the hydrophobic natural antioxidant -tocopherol, and the synthetic antioxidant ionol as well as the ferrous ion chelating agent deferoxamine were found to protect Na/K-ATPase from oxidation by different concentrations of H₂O₂. The data suggest that these antioxidants are effective due to their ability to neutralize or to prevent formation of hydroxyl radicals.     

The sodium pump and cardiotonic steroids-induced signal transduction protein kinases and calcium-signaling microdomain in regulation of transporter trafficking

The Na/K-ATPase was discovered as an energy transducing ion pump. A major difference between the Na/K-ATPase and other P-type ATPases is its ability to bind a group of chemicals called cardiotonic steroids (CTS). The plant-derived CTS such as digoxin are valuable drugs for the management of cardiac diseases, whereas ouabain and marinobufagenin (MBG) have been identified as a new class of endogenous hormones. Recent studies have demonstrated that the endogenous CTS are important regulators of renal Na⁺ excretion and blood pressure. The Na/K-ATPase is not only an ion pump, but also an important receptor that can transduce the ligand-like effect of CTS on intracellular protein kinases and Ca²⁺ signaling. Significantly, these CTS-provoked signaling events are capable of reducing the surface expression of apical NHE3 (Na/H exchanger isoform 3) and basolateral Na/K-ATPase in renal proximal tubular cells. These findings suggest that endogenous CTS may play an important role in regulation of tubular Na⁺ excretion under physiological conditions; conversely, a defect at either the receptor level (Na/K-ATPase) or receptor–effector coupling would reduce the ability of renal proximal tubular cells to excrete Na⁺, thus culminating/resulting in salt-sensitive hypertension.     

Structure–function relationship in P-type ATPases—a biophysical approach

P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes. In these proteins the energy-providing ATP hydrolysis is coupled to ion-transport that builds up or maintains the electrochemical potential gradients of one or two ion species across the membrane. P-type ATPases are found in virtually all eukaryotic cells and also in bacteria, and they are transporters of a broad variety of ions. So far, a crystal structure with atomic resolution is available only for one species, the SR Ca-ATPase. However, biochemical and biophysical studies provide an abundance of details on the function of this class of ion pumps. The aim of this review is to summarize the results of preferentially biophysical investigations of the three best-studied ion pumps, the Na,K-ATPase, the gastric H,K-ATPase, and the SR Ca-ATPase, and to compare functional properties to recent structural insights with the aim of contributing to the understanding of their structure–function relationship.     

Do electromagnetic fields interact with electrons in the Na,K‐ATPase?

The effects of low frequency electric and magnetic fields on several biochemical systems, including the Na,K-ATPase, indicate that electromagnetic (EM) fields interact with electrons. The frequency optima for two enzymes in response to EM fields are very close to their turnover numbers, suggesting that these interactions directly affect reaction rates. Nevertheless, generally accepted ideas about Na,K-ATPase function and ion transport mechanisms do not consider interactions with electrons. To resolve the clash of paradigms, we hypothesize interaction with transient electrons and protons that arise from flickering of H-bonds in the hydrated protein. These transient charges in the enzyme could provide a trigger for the sequence of conformation changes that are part of the ion transport mechanism. If the distributions of transient electrons and protons in the membrane are affected by their concentration and the membrane potential, as expected from electric double layer theory, this can account for the different effects of low frequency electric and magnetic fields, as well as for the observation that membrane hyperpolarization reverses the ATPase reaction to generate ATP.     

Extracellular domains,transmembrane segments,and intracellular domains interact to determine the cation selectivity of Na,K- and gastric H,K-ATPase

We have previously reported that three residues of the fourth transmembrane segment (TM4) of the Na,K- and gastric H,K-ATPase alpha-subunits appear to play a major role in the distinct cation selectivities of these pumps [Mense, M., et al. (2000) J. Biol. Chem. 275, 1749-1756]. Substituting these three residues in the Na,K-ATPase sequence with their H,K-ATPase counterparts (L319F, N326Y, T340S) and replacing the TM3-TM4 ectodomain sequence with that of the H,K-ATPase alpha-subunit result in a pump that exhibits 50% of its maximal ATPase activity in the absence of Na(+) when the assay is performed at pH 6.0. This effect is not seen when the ectodomain alone is replaced. To gain more insight into the contributions of the three residues to establishing the selectivity of these pumps for Na(+) ions versus protons, we generated Na,K-ATPase constructs in which these residues are replaced by their H,K-ATPase counterparts either singly or in combinations. Surprisingly, none of the point mutants nor even the triple mutant was able to hydrolyze ATP at pH 6.0 at a rate greater than 20% of their respective V(max)s. For the point mutants L319F and N326Y, protons seem to competitively inhibit ATP hydrolysis at pH 6.0, based on the low apparent affinity for Na(+) ions at pH 6.0 compared to pH 7.5. It would appear, therefore, that the cation selectivity of Na,K- and H,K-ATPase is generated through a cooperative effort between residues of transmembrane segments and the flanking loops that connect these transmembrane domains. This view is further supported by homology modeling of the Na,K-ATPase based on the crystal structure of the SERCA pump.     

Physico-chemical principles for discrimination of sodium and potassium ions by membrane Na pumps. I. Effect of organic compounds on the ionic specificity of Na, K-ATPase

Effects of some organic compounds of different hydrophobicity on the structure and ion specificity of the sodium-potassium adenosine triphosphatase (Na,K-ATPase) membrane preparation were studied. Inhibition abilities of these compounds correlate well with their lipophilic properties. High hydrophobic compounds change mainly the enzyme activation by potassium ions and the spin label mobilities in hydrophobic regions of the membrane preparation. Polar species, in contrast, influence the enzyme activation by sodium ions and the surface polar properties of the membrane preparation. It is supposed, that the Na,K-ATPase activations by potassium and sodium ions are correspondingly related to hydrophobic regions of the lipoprotein enzyme complex and to the polar regions stabilized by hydrogen bonds.     

Characterization of ouabain-sensitive phosphatase activity in the absence of potassium ion in purified pig kidney Na,K-ATPase

The ouabain-sensitive phosphatase activity of purified pig kidney Na,K-ATPase preparation in the absence of potassium ion ((-K)phosphatase) was examined precisely. During the preparation procedures, the (-K)3-O-methylfluoresceinphosphatase ((-K)3-OMFPase) activity or the (-K)p-nitrophenylphosphatase ((-K)pNPPase) activity appeared to be purified in parallel with the Na,K-ATPase activity. The (-K)phosphatase activity was competitively inhibited by ATP and by ADP, with the K1 values of 0.25 microM and 1.4 microM, respectively. These values are consistent with their Kd values for the high-affinity ATP binding site of the Na,K-ATPase (Hegyvary, C. & Post, R.L. (1971) J. Biol. Chem. 246, 5234-5240). The substrate, pNPP, apparently competed with covalently bound fluorescein-5'-isothiocyanate (FITC), which is known to bind in the neighborhood of the high-affinity ATP binding site of the Na,K-ATPase, in both the (-K)phosphatase and the (+K)phosphatase reactions. The FITC-fluorescence intensity of FITC-labeled enzyme at the maximal steady-state activity of the (-K)phosphatase reaction was at a similar level to that of the E2 species. However, the FITC-labeled enzyme in the presence of only magnesium ion or only pNPP gave a fluorescence level similar to that of the E1 species. Oligomycin inhibited the (-K)phosphatase activity by at most 46%. On the basis of these results, it is strongly suggested that the (-K)phosphatase reaction is catalyzed at the high-affinity ATP binding site of Na,K-ATPase, and the (-K)phosphatase reaction proceeds in a cyclic manner (E1----E2----E1).     

A new approach for determination of Na,K-ATPase activity: application to intact pancreatic β-cells

It has been postulated that a decrease in Na,K-ATPase-mediated ion gradients may be a contributing mechanism to insulin secretion. However, the precise role of the Na,K-ATPase in pancreatic β-cell membrane depolarization and insulin secretion signalling have been difficult to evaluate, mostly because data reporting changes in enzymatic activity have been obtained in cell homogenates or membrane preparations, lacking intact intracellular signalling pathways. The aim of this work was to develop a method to characterize Na,K-ATPase activity in intact pancreatic β-cells that will allow the investigation of putative Na,K-ATPase activity regulation by glucose and its possible role in insulin secretion signalling. This work demonstrates for the first time that it is possible to determine Na,K-ATPase activity in intact pancreatic β-cells and that this is a suitable method for the study of the mechanisms involved in the Na,K-ATPase regulation and eventually its relevance for insulin secretion signalling.     

A novel cholesterol-producing Pichia pastoris strain is an ideal host for functional expression of human Na,K-ATPase α3β1 isoform

The heterologous expression of mammalian membrane proteins in lower eukaryotes is often hampered by aberrant protein localization, structure, and function, leading to enhanced degradation and, thus, low expression levels. Substantial quantities of functional membrane proteins are necessary to elucidate their structure–function relationships. Na,K-ATPases are integral, human membrane proteins that specifically interact with cholesterol and phospholipids, ensuring protein stability and enhancing ion transport activity. In this study, we present a Pichia pastoris strain which was engineered in its sterol pathway towards the synthesis of cholesterol instead of ergosterol to foster the functional expression of human membrane proteins. Western blot analyses revealed that cholesterol-producing yeast formed enhanced and stable levels of human Na,K-ATPase α3β1 isoform. ATPase activity assays suggested that this Na,K-ATPase isoform was functionally expressed in the plasma membrane. Moreover, [³H]-ouabain cell surface-binding studies underscored that the Na,K-ATPase was present in high numbers at the cell surface, surpassing reported expression strains severalfold. This provides evidence that the humanized sterol composition positively influenced Na,K-ATPase α3β1 stability, activity, and localization to the yeast plasma membrane. Prospectively, cholesterol-producing yeast will have high potential for functional expression of many mammalian membrane proteins.     

Beta1-integrins co-localize with Na,K-ATPase,epithelial sodium channels (ENaC) and voltage activated calcium channels (VACC) in mechanoreceptor complexes of mouse limb-bud chondrocytes

Interactions between chondrocytes and their extracellular matrix are partly mediated by beta1-integrin receptors. Recent studies have shown that beta1-integrins co-localize with a variety of cytoskeletal complexes, signaling proteins and growth factor receptors. Since mechanosensitive ion channels and integrins have been proposed to participate in skeletal mechanotransduction, in this study, we investigated the possible co-localization of beta1-integrins with two ion channels and a P-type ATPase in mouse limb-bud chondrocytes. The alpha subunits of Na, K-ATPase, the epithelial sodium channel (ENaC) and the voltage activated calcium channel (VACC) were immunostained in organoid cultures derived from limb-buds of 12-day-old mice using well-characterized antibodies. Indirect immunofluorescence revealed abundant expression of beta1-integrins and each of the selected systems in limb-bud chondrocytes. Two-fluorochrome immunostaining demonstrated that beta1-integrin, Na, K-ATPase, ENaC and VACC co-localize in chondrocytes. Co-imunoprecipitation experiments revealed co-localization and association of integrins with ENaC, VACC and Na, K-ATPase. Cellular responses and signaling cascades initiated by the influx of calcium or sodium through putative mechanosensitive channels may be regulated more effectively if such channels were organized around integrins with receptors, kinases and cytoskeletal complexes clustered about them. The close proximity of ATPase ion pumps such as Na, K-ATPase to chondrocyte mechanoreceptor complexes could facilitate rapid homeostatic responses to the ionic perturbations brought about by activation of mechanically gated cation channels and efficiently regulate the intracellular milieu of chondrocytes.     

An extracellular loop of the human non-gastric H,K-ATPase alpha-subunit is involved in apical plasma membrane polarization.

The human non-gastric H,K-ATPase, ATP1AL1, belongs to the gene family of P-type ATPases. Consistent with their physiological roles in ion transport, members of this group, including the Na,KATPase and the gastric and non-gastric H,K-ATPases, are differentially polarized to either the basolateral or apical plasma membrane in epithelial cells. However, their polarized distribution is highly complex and depends on specific sorting signals or motifs which are recognized by the subcellular targeting machinery. For the gastric H,K-ATPase it has been suggested that the 4(th) transmembrane spanning domain (TM4) and its flanking regions induce conformational sorting motifs which direct the ion pump exclusively to the epithelial apical membrane. Here, we show in transfected Madin-Darby canine kidney (MDCK) cells that the related non-gastric H,KATPase, ATP1AL1, does contain similar sorting motifs in close proximity to TM4. A short extracellular loop between TM3 and TM4 is critical for this pump's apical delivery. A single point mutation in the corresponding region redirects ATP1AL1 to the basolateral membrane. In conclusion, our work provides further evidence that the cellular distribution of P-type ATPases is determined by conformational sorting motifs.     

The isoform-specific region of the Na,K-ATPase catalytic subunit: role in enzyme kinetics and regulation by protein kinase C

Comparisons of the primary structures of the Na,K-ATPase alpha-isoforms reveal the existence of regions of structural divergence, suggesting that they are involved in unique functions. One of these regions is the isoform-specific region (ISR), located near the ATP binding site in the major cytoplasmic loop. To evaluate its importance, we constructed mutants of the rodent wild-type alpha1 and alpha3 isoforms in which the ISR was replaced with irrelevant sequences, i.e., the analogous region from the rat gastric H,K-ATPase catalytic subunit or a region from the human c-myc oncogene. Opossum kidney (OK) cells were transfected with wild-type rat alpha1, alpha3, or their corresponding chimeras and selected in ouabain. Introduction of either mutant produced ouabain-resistant colonies, consistent with functional expression of the chimeric protein and indicating that the ISR is not essential for overall Na,K-ATPase function. The introduced chimeras were then characterized enzymatically by measuring the relative rate of K(+) and Li(+) deocclusions. Results showed that exchanges of both alpha1 and alpha3 ISRs significantly modified the sensitivity for the enzyme to either K(+) or Li(+). Subsequent treatment of the cells with phorbol esters revealed an altered Na,K-ATPase transport in response to protein kinase C activation for the alpha1 chimeras. No changes were observed for the alpha3 isoform, suggesting that it is not sensitive to PKC regulation. These results demonstrated that the ISR plays an important role in ion deocclusion and in the response to PKC (only for the alpha1 isoform).     

Modulation of Na, K-ATPase activity by prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin

Adenylyl cyclase is activated by prostaglandin E and inhibited by mu-opioids. Since cAMP-related events influence the activity of the Na Pump and its biochemical correlate Na,K-ATPase in many systems, we tested the hypothesis that prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), a mu-opioid agonist, have opposing actions on Na,K-ATPase activity. Studies were conducted with alamethicin-permeabilized SH-SY5Y human neuroblastoma cells. Prostaglandin E1 (1 microM) transiently inhibited Na,K-ATPase activity for 10-15 min. A direct activator of protein kinase A, 8-Br-cAMP (150 and 500 microM), also inhibited, but more rapidly and for a shorter duration. Both DAMGO (1 microM) and Rp-adenosine 3',5'-cyclic monophosphorothioate (500 microM), a protein kinase A-inhibitor, reversed the inhibitory effect of prostaglandin E1. DAMGO alone (1 microM) stimulated Na,K-ATPase activity up to nearly three-fold control activity. The stimulatory action of DAMGO was blocked by cyclosporine A (2 microM), an inhibitor of calcineurin, and was dependent on Ca2+ entry through nifedipine-sensitive Ca2+ channels. In the presence of 1 mM EGTA, DAMGO inhibited Na,K-ATPase activity. DAMGO-induced inhibition was blocked by the inositol 1,4,5-trisphosphate receptor antagonist xestospongin C (1 microM). Na,K-ATPase is poised to modulate neuronal excitability through its roles in maintaining the membrane potential and transmembrane ion gradients. The differential effects of prostaglandin E1 and opioids on Na,K-ATPase activity may be related to their actions in hyperalgesia.