Morphological and biochemical evidence for the evolution of salt glands in snakes

Vertebrate salt glands have evolved independently multiple times, yet there are few hypotheses about the processes underlying the convergent evolution of salt glands across taxa. Here, we compare the morphology and molecular biology of specialized salt-secreting glands from a marine snake (Laticauda semifasciata) with the cephalic glands from semi-marine (Nerodia clarkii clarkii) and freshwater (N. fasciata) watersnakes to look for evidence of a salt gland in the former and to develop hypotheses about the evolution of snake salt glands. Like the salt gland of L. semifasciata, the nasal and anterior/posterior sublingual glands in both species of Nerodia exhibit a compound tubular shape, and express basolateral Na(+)/K(+)-ATPase (NKA) and Na(+)/K(+)/2Cl(-)cotransporter (NKCC); however, the abundance of NKA and NKCC in N. fasciata appears lower than in N. c. clarkii. Aquaporin 3 (AQP3) is also basolateral in the sublingual glands of both species of Nerodia, as is abundant neutral mucin; both AQP3 and mucin are absent from the salt gland in L. semifasciata. Thus, we propose that the evolution of the snake salt gland by co-option of an existing gland involved at least two steps: (i) an increase in the abundance of NKA and NKCC in the basolateral membranes of the secretory epithelia, and (ii) loss of AQP3/mucus secretion from these epithelia.     

Distinct Na+/K+/2Cl- cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish

The kidney is an organ playing an important role in ion regulation in both freshwater (FW) and seawater (SW) fish. The mechanisms of ion regulation in the fish kidney are less well studied than that of their gills, especially at the level of transporter proteins. We have found striking differences in the pattern of Na⁺/K⁺/2Cl^- cotransporter (NKCC) expression between species. In the killifish kidney, NKCC is apically localized in the distal and collecting tubules and basolaterally localized in the proximal tubules. However, in the SW killifish gill, NKCC is basolaterally co-localized with Na⁺/K⁺-ATPase, whereas in FW, NKCC immunoreactivity is primarily apical, although still colocalized within the same mitochondria-rich cell with basolateral Na⁺/K⁺-ATPase. Rainbow trout kidney has NKCC only in the apical membrane of the distal and collecting tubules in both environments, with no signal being detected in the proximal tubule. On the other hand, in the trout gill, NKCC is found basolaterally in both FW and SW environments. An important observation is that, in the gills of rainbow trout, the trailing edge of the filament possesses mostly Na⁺/K⁺-ATPase-positive but NKCC-negative mitochondria-rich cells, whereas in the region between and at the roots of the gill lamellae, most mitochondria-rich cells exhibit both Na⁺/K⁺-ATPase- and NKCC-positive immunoreactivity. These results suggest that the differential localization of transporters between the two species represents differences in function between these two euryhaline fishes with different life histories and strategies. Funding for this research was provided by NSERC Discovery Grants to G.G.G. and W.S.M., an Alberta Ingenuity Fund PDF, and a fellowship from the NSERC Research Capacity Development Grant to F.K.     

Identification and reconstitution of a Na/K/Cl cotransporter and K channel from luminal membranes of renal red outer medulla

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na⁺/K⁺/Cl⁻ cotransport system and a K⁺ channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of ⁸⁶Rb⁺ uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two ⁸⁶Rb⁺ fluxes. (A) A furosemide-inhibited ⁸⁶Rb⁺ flux in the absence of Na⁺ (K⁺-K⁺ exchange). This flux is stimulated by an inward Na⁺ gradient (Na⁺/K⁺ cotransport) and is inhibited also by bumetanide. (B) A Ba²⁺-inhibited ⁸⁶Rb⁺ flux, through the K⁺ channel. Luminal membranes containing the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channels, and basolateral membranes containing the Na⁺/K⁺ pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.     

Effects of anions on cellular volume and transepithelial Na+ transport across toad urinary bladder

Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl^– on transepithelial Na⁺ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V _T) decreased rapidly, transepithelial resistance (R _T) increased, and calculated short-circuit current (I _sc) decreased. CalculatedE _Na was unaffected, indicating that the inhibition of Na⁺ transport was a consequence of a decreased apical membrane Na⁺ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV _T decreased,R _T increased andI _sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE _Na fell substantially (from 130 to 68 mV on average). Ba²⁺ (3mm) reduced calculatedE _Na in Cl^– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl^– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE _Na did not fall. Replacement of serosal isosmotic Cl^– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na⁺ transport. Thus the inhibition of Na⁺ transport can be correlated with changes in cell volume rather than with the change in Cl^– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K⁺ gluconate mucosa, Na⁺ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na⁺/K⁺ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl^– results in a loss of highly specific Ba²⁺-sensitive K⁺ conductance channels from the basolateral plasma membrane. It is possible that the number of Na⁺ pump sites in this membrane is also decreased.     

Active and passive Na+ fluxes across the basolateral membrane of rabbit urinary bladder

Summary The apical membrane of rabbit urinary bladder can be functionally removed by application of nystatin at high concentration if the mucosal surface of the tissue is bathed in a saline which mimics intracellular ion concentrations. Under these conditions, the tissue is as far as the movement of univalent ions no more than a sheet of basolateral membrane with some tight junctional membrane in parallel. In this manner the Na⁺ concentration at the inner surface of the basolateral membrane can be varied by altering the concentration in the mucosal bulk solution. When this was done both mucosal-to-serosal²²Na flux and net change in basolateral current were measured. The flux and the current could be further divided into the components of each that were either blocked by ouabain or insensitive to ouabain. Ouabain-insensitive mucosal-to-serosal Na⁺ flux was a linear function of mucosal Na⁺ concentration. Ouabain-sensitive Na⁺ flux and ouabain-sensitive, Na⁺-induced current both display a saturating relationship which cannot be accounted for by the presence of unstirred layers. If the interaction of Na⁺ with the basolateral transport process is assumed to involve the interaction of some number of Na⁺ ions,n, with a maximal flux,M _max, then the data can be fit by assuming 3.2 equivalent sites for interaction and a value forM _max of 287.8pm cm^–2 sec^–1 with an intracellular Na concentration of 2.0mm Na⁺ at half-maximal saturation. By comparing these values with the ouabain-sensitive, Na⁺-induced current, we calculate a Na⁺ to K⁺ coupling ratio of 1.40±0.07 for the transport process.     

Immunocytochemical localization of Na+/K+-ATPase and V-H+-ATPase in the salivary glands of the cockroach,Periplaneta americana

The acinous salivary glands of the cockroach (Periplaneta americana) consist of four morphologically different cell types with different functions: the peripheral cells are thought to produce the fluid component of the primary saliva, the central cells secrete the proteinaceous components, the inner acinar duct cells stabilize the acini and secrete a cuticular, intima, whereas the distal duct cells modify the primary saliva via the transport of water and electrolytes. Because there is no direct information available on the distribution of ion transporting enzymes in the salivary glands, we have mapped the distribution of two key transport enzymes, the Na⁺/K⁺-ATPase (sodium pump) and a vacuolar-type H⁺-ATPase, by immunocytochemical techniques. In the peripheral cells, the Na⁺/K⁺-ATPase is localized to the highly infolded apical membrane surface. The distal duct cells show large numbers of sodium pumps localized to the basolateral part of their plasma membrane, whereas their highly folded apical membranes have a vacuolar-type H⁺-ATPase. Our immunocytochemical data are supported by conventional electron microscopy, which shows electrondense 10-nm particles (portasomes) on the cytoplasmic surface of the infoldings of the apical membranes of the distal duct cells. The apically localized Na⁺/K⁺-ATPase in the peripheral cells is probably directly involved in the formation of the Na⁺-rich primary saliva. The latter is modified by the distal duct cells by transport mechanisms energized by the proton motive force of the apically localized V-H⁺-ATPase.     

Na+/Na+ exchange and Na+/H+ antiport in rabbit erythrocytes: Two distinct transport systems

Summary Rabbit erythrocytes are well known for possessing highly active Na⁺/Na⁺ and Na⁺/H⁺ countertransport systems. Since these two transport systems share many similar properties, the possibility exists that they represent different transport modes of a single transport molecule. Therefore, we evaluated this hypothesis by measuring Na⁺ transport through these exchangers in acid-loaded cells. In addition, selective inhibitors of these transport systems such as ethylisopropyl-amiloride (EIPA) and N-ethylmaleimide (NEM) were used. Na⁺/Na⁺ exchange activity, determined as the Na _o ⁺ -dependent²²Na efflux or Na _i ⁺ -induced²²Na entry was completely abolished by NEM. This inhibitor, however, did not affect the H _i ⁺ -induced Na⁺ entry sensitive to amiloride (Na⁺/H⁺ exchange activity). Similarly, EIPA, a strong inhibitor of the Na⁺/H⁺ exchanger, did not inhibit Na⁺/Na^– countertransport, suggesting the independent nature of both transport systems. The possibility that the NEM-sensitive Na⁺/Na⁺ exchanger could be involved in Na⁺/H⁺ countertransport was suggested by studies in which the net Na⁺ transport sensitive to NEM was determined. As expected, net Na⁺ transport through this transport system was zero at different [Na⁺] _i /[Na⁺] _o ratios when intracellular pH was 7.2. However, at pH _i =6.1, net Na⁺ influx occurred when [Na⁺] _i was lower than 39mm. Valinomycin, which at low [K⁺] _o was lower than 39mm. Valinomycin, which at low [K⁺] _o clamps the membrane potential close to the K⁺ equilibrium potential, did not affect the net NEM-sensitive Na⁺ entry but markedly stimulated, the EIPA-and NEM-resistant Na⁺ uptake. This suggest that the net Na⁺ entry through the NEM-sensitive pathway at low pH _i , is mediated by an electroneutral process possibly involving Na⁺/H⁺ exchange. In contrast, the EIPA-sensitive Na⁺/H⁺ exchanger is not involved in Na⁺/Na⁺ countertransport, because Na⁺ transport through this mechanism is not affected by an increase in cell Na^– from 0.4 to 39mm. Altogether, these findings indicate that both transport systems: the Na⁺/Na⁺ and Na⁺/H⁺ exchangers, are mediated by distinct transport proteins.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

The Detailed Localization Pattern of Na+/K+/2Cl? Cotransporter Type 2 and Its Related Ion Transport System in the Rat Endolymphatic Sac

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na⁺/K⁺/2Cl⁻ cotransporter type 2 (NKCC-2) and Na⁺/K⁺-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na⁺/K⁺-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na⁺/K⁺-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules. (J Histochem Cytochem 58:759–763, 2010)     

Enhancement of the salt tolerance of Triticum turgidum L. by the Kna1 locus transferred from the Triticum aestivum L. chromosome 4D by homoeologous recombination

Durum wheat, Triticum turgidum L. (2n= 4x=28, genome formula AABB) is inferior to bread wheat, T. aestivum L. (2n=6x=42, genome formula AABBDD), in the ability to exclude Na⁺ under salt strees, in the ratio of the accumulated K⁺ to Na⁺ in the leaves under salt stress, and in tolerance of salt stress. Previous work showed that chromosome 4D has a major effect on Na⁺ and K⁺ accumulation in the leaves of bread wheat. The 4D chromosome was recombined with chromosome 4B in the genetic background of durum wheat. The recombinants showed that Na⁺ exclusion and enhanced K⁺/Na⁺ ratio in the shoots were controlled by a single locus, Kna1, in the long arm of chromosome 4D. The recombinant families were grown in the field under non-saline conditions and two levels of salinity to determine whether Kna1 confers salt tolerance. Under salt stress, the Kna1 families had higher K⁺/Na⁺ ratios in the flag leaves and higher yields of grain and biomass than the Kna1 ^- families and the parental cultivars. Kna1 is, therefore, one of the factors responsible for the higher salt tolerance of bread wheat relative to durum wheat. The present work provides conceptual evidence that tolerance of salt stress can be transferred between species in the tribe Triticeae.     

Differential expression of branchial Na+/K(+)-ATPase of two medaka species, Oryzias latipes and Oryzias dancena, with different salinity tolerances acclimated to fresh water, brackish water and seawater

Previous studies on non-diadromous euryhaline teleosts introduced a hypothesis that the lowest level of gill Na⁺/K⁺-ATPase (NKA) activity occurs in the environments with salinity close to the primary natural habitats of the studied species. To provide more evidence of the hypothesis, two medaka species, Oryzias latipes and O. dancena, whose primary natural habitats are fresh water (FW) and brackish water (BW) environments, respectively, were compared from levels of mRNA to cells in this study. The plasma osmolalities of O. latipes and O. dancena were lowest in the FW individuals. The muscle water contents of O. latipes decreased with elevated external salinities, but were constant among FW-, BW-, and seawater (SW)-acclimated O. dancena. Expression of NKA, the primary driving force of ion transporters in gill ionocytes, revealed different patterns in the two Oryzias species. The highest NKA α-subunit mRNA abundances were found in the gills of the SW O. latipes and the FW O. dancena, respectively. The pattern of NKA activity and α-subunit protein abundance in the gills of O. latipes revealed that the FW group was the lowest, while the pattern in O. dancena revealed that the BW group was the lowest. Immunohistochemical staining showed similar profiles of NKA immunoreactive (NKIR) cell activities (NKIR cell number × cell size) in the gills of these two species among FW, BW, and SW groups. Taken together, O. latipes exhibited better hyposmoregulatory ability, while O. dancena exhibited better hyperosmoregulatory ability. Our results corresponding to the hypothesis indicated that the lowest branchial NKA activities of these two medaka species were found in the environments with salinities similar to their natural habitats.     

Cross-reactivity of an antiserum to the α-subunit of the Na+, K+-ATPase of toad (Bufo marinus) kidney with basal and apical membranes of transporting epithelia of the rat

Summary An antibody to the 96 kD -subunit of the Na⁺, K⁺ -ATPase from Bufo marinus has been used in immunostaining rat kidney and salivary glands. Intense staining was observed on basolateral membranes of distal tubules of the kidney and striated ducts of the three major salivary glands. Less intense staining was seen on the basolateral membranes of parotid acinar cells, but no staining was seen on the acinar cells of submandibular or sublingual glands. These sites of staining have been shown, by other methods, to posses substantial Na⁺, K⁺ -ATPase, indicating that the antibody recognizes antigenic determinants of the sodium pump highly conserved in the course of evolution. In addition, staining with this antibody was observed at the apical region of cells of the proximal straight tubule and of the papillary collecting duct in the kidney. Absorption studies suggest that the apical antigenic determinants are the same or closely related to each other but are distinct from basolateral antigenic determinants.     

Mechanistic distinction between activation and inhibition of (Na+K)-ATPase-mediated Ca influx in cardiomyocytes

(Na⁺+K⁺)-ATPase (NKA) mediates positive inotropy in the heart. Extensive studies have demonstrated that the reverse-mode Na⁺/Ca²⁺-exchanger (NCX) plays a critical role in increasing intracellular Ca²⁺ concentration through the inhibition of NKA-induced positive inotropy by cardiac glycosides. Little is known about the nature of the NCX functional mode in the activation of NKA-induced positive inotropy. Here, we examined the effect of an NKA activator SSA412 antibody on ⁴⁵Ca influx in isolated rat myocytes and found that KB-R7943, a NCX reverse-mode inhibitor, fails to inhibit the activation of NKA-induced ⁴⁵Ca influx, suggesting that the Ca²⁺ influx via the reverse-mode NCX does not mediate this process. Nifedipine, an L-type Ca²⁺ channel (LTCC) inhibitor, completely blocks the activation of NKA-induced ⁴⁵Ca influx, suggesting that the LTCC is responsible for the moderate increase in intracellular Ca²⁺. In contrast, the inhibition of NKA by ouabain induces 4.7-fold ⁴⁵Ca influx compared with the condition of activation of NKA. Moreover, approximately 70% of ouabain-induced ⁴⁵Ca influx was obstructed by KB-R7943 and only 30% was impeded by nifedipine, indicating that both the LTCC and the NCX contribute to the rise in intracellular Ca²⁺ and that the NCX reverse-mode is the major source for the ⁴⁵Ca influx induced by the inhibition of NKA. This study provides direct evidence to demonstrate that the activation of NKA-induced Ca²⁺ increase is independent of the reverse-mode NCX and pinpoints a mechanistic distinction between the activation and inhibition of the NKA-mediated Ca²⁺ influx path ways in cardiomyocytes.     

Histological and functional renal alterations caused by Bothrops alternatus snake venom: Expression and activity of Na/K-ATPase

Background

Acute renal failure is a serious complication of human envenoming by Bothrops snakes. The ion pump Na⁺/K⁺-ATPase has an important role in renal tubule function, where it modulates sodium reabsorption and homeostasis of the extracellular compartment. Here, we investigated the morphological and functional renal alterations and changes in Na⁺/K⁺-ATPase expression and activity in rats injected with Bothrops alternatus snake venom.

Methods

Male Wistar rats were injected with venom (0.8 mg/kg, i.v.) and renal function was assessed 6, 24, 48 and 72 h and 7 days post-venom. The rats were then killed and renal Na⁺/K⁺-ATPase activity was assayed based on phosphate release from ATP; gene and protein expressions were assessed by real time PCR and immunofluorescence microscopy, respectively.

Results

Venom caused lobulation of the capillary tufts, dilation of Bowman's capsular space, F-actin disruption in Bowman's capsule and renal tubule brush border, and deposition of collagen around glomeruli and proximal tubules that persisted seven days after envenoming. Enhanced sodium and potassium excretion, reduced proximal sodium reabsorption, and proteinuria were observed 6 h post-venom, followed by a transient decrease in the glomerular filtration rate. Gene and protein expressions of the Na⁺/K⁺-ATPase α₁ subunit were increased 6 h post-venom, whereas Na⁺/K⁺-ATPase activity increased 6 h and 24 h post-venom.

Conclusions

Bothrops alternatus venom caused marked morphological and functional renal alterations with enhanced Na⁺/K⁺-ATPase expression and activity in the early phase of renal damage.

General significance

Enhanced Na⁺/K⁺-ATPase activity in the early hours after envenoming may attenuate the renal dysfunction associated with venom-induced damage.     

Salinity-dependent changes in Na+/K+-ATPase content of mitochondria-rich cells contribute to differences in thermal tolerance of Mozambique tilapia

The Mozambique tilapia (Oreochromis mossambicus) is prone to osmoregulatory disturbances when faced with fluctuating ambient temperatures. To investigate the underlying causes of this phenomenon, freshwater (FW)- and seawater (SW)-acclimated tilapia were transferred to 15, 25, or 35°C for 2 weeks, and along with typically used indicators of osmoregulatory status [plasma osmolality and branchial and intestinal specific Na⁺, K⁺-ATPase (NKA) activity], we used tissue microarrays (TMA) and laser-scanning cytometry (LSC) to characterize the effects of temperature acclimation. Tissue microarrays were stained with fluorescently labeled anti-Na⁺, K⁺-ATPase antibodies that allowed for the quantification of NKA abundance per unit area within individual branchial mitochondria-rich cells (MRCs) as well as sections of renal tissue. Mitochondria-rich cell counts and estimates of size were carried out for each treatment by the detection of DASPMI fluorescence. The combined analyses showed that SW fish have larger but fewer MRCs that contain more NKA per unit area. After a 2-week acclimation to 15°C tilapia experienced osmotic imbalances in both FW and SW that were likely due to low NKA activity. SW-acclimated fish compensated for the low activity by increasing MRC size and subsequently the concentration of NKA within MRCs. Although there were no signs of osmotic stress in FW-acclimated tilapia at 25°C, there was an increased NKA capacity that was most likely mediated by a higher MRC count. We conclude on the basis of the different responses to temperature acclimation that salinity-induced changes in the NKA concentration of MRCs alter thermal tolerance limits of tilapia.     

短期NaCl胁迫对不同小麦品种幼苗K+吸收和Na+、K+积累的影响

为研究盐胁迫下小麦幼苗生长及Na⁺、K⁺的吸收和积累规律,以中国春、洲元9369和长武134等3种耐盐性不同小麦品种为材料,采用非损伤微测技术检测盐胁迫2 d后的根系K⁺离子流变化,并对植株体内的Na⁺、K⁺含量进行测定。结果表明:短期(2d)盐胁迫对小麦生长有抑制作用,且对根系的抑制大于地上部,耐盐品种下降幅度小于盐敏感品种。盐胁迫下,小麦根际的 K⁺大量外流,盐敏感品种中国春K⁺流速显著高于耐盐品种长武134,最高可达15倍。小麦幼苗地上部分和根系均表现为Na⁺积累增加,K⁺积累减少,Na⁺/K⁺比随盐浓度增加而上升。中国春限Na⁺能力显著低于长武134,Na⁺/K⁺则显著高于长武134。综上所述,盐胁迫下造成小麦组织器官中Na⁺/K⁺比上升的主要原因是根系K⁺大量外流和Na⁺的过量积累,耐盐性不同的小麦品种间差异显著,并认为根系对K⁺的保有能力可能是作物耐盐性评价的一个重要指标。     

Intracellular calcium content of human erythrocytes: Relation to sodium transport systems

Summary To study the possible role of intracellular Ca (Ca _i ) in controlling the activities of the Na⁺–K⁺ pump, the Na⁺–K⁺ cotransport and the Na⁺/Li⁺ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO₄ in Teflon^® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca _i of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca _i could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na⁺–K⁺ pump, of Na⁺–K⁺ cotransport and Na⁺/Li⁺ exchange and the mean cellular hemoglobin content fell with rising Ca _i ; the red cell Na⁺ and K⁺ contents rose with Ca _i . Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca²⁺ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca _i of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

Allosteric property of the (Na+K)-ATPase β1 subunit

(Na⁺+K⁺)-ATPase (NKA) comprises two basic α and β subunits: The larger α subunit catalyzes the hydrolysis of ATP for active transport of Na⁺ and K⁺ ions across the plasma membrane; the smaller β subunit does not take part in the catalytic process of the enzyme. Little is known about allosteric regulation of the NKA β subunit. Here, we report a surprising finding that extracellular stimuli on the native β₁ subunit can generate a significant impact on the catalytic function of NKA. By using a β₁ subunit-specific monoclonal antibody JY2948, we found that the JY2948–β₁ subunit interaction markedly enhances the catalytic activity of the enzyme and increases the apparent affinity of Na⁺ and K⁺ ions for both ouabain-resistant rat NKA and ouabain-sensitive dog NKA. This study provides the first evidence to identify an allosteric binding site residing on the NKA β₁ subunit and uncovers the latent allosteric property of the β₁ subunit, which remotely controls the NKA catalytic function.