Fluid and cation secretion by the Malpighian tubules of Locusta

In vitro preparations of Locusta Malpighian tubules are able to transport K⁺ against its concentration gradient. The ‘urine’ is slightly hyper-osmotic with respect to the bathing solution and the rate of secretion is inversely dependent on the osmotic pressure of the latter. The rate of fluid secretion increases with increasing temperature; being maximal at approx 40°C. The ionic composition of the secreted fluid, as indicated by Na⁺/K⁺ ratios, is altered by the presence of 1 mM ouabain in the bathing solution. Fluid secretion is inhibited by 1 mM ouabain. In addition, oxygen consumption by the Malpighian tubules is inhibited by either the presence of 1 mM ouabain or the absence of K⁺ in the bathing solution. The relationship between respiration, active transport and the Na⁺K⁺-activated ATPase is discussed.     

Secretion of Na+, K+ and fluid by the Malpighian (renal) tubule of the larval cabbage looper Trichoplusia ni (Lepidoptera: Noctuidae)

The Malpighian (renal) tubules play important roles in ionic and osmotic homeostasis in insects. In Lepidoptera, the Malpighian tubules are structurally regionalized and the concentration of Na⁺ and K⁺ in the secreted fluid varies depending on the segment of tubule analyzed. In this work, we have characterized fluid and ion (Na⁺, K⁺, H⁺) transport by tubules of the larval stage of the cabbage looper Trichoplusia ni; we have also evaluated the effects of fluid secretion inhibitors and stimulants on fluid and ion transport. Ramsay assays showed that fluid was secreted by the iliac plexus but not by the yellow and white regions of the tubule. K⁺ and Na⁺ were secreted by the distal iliac plexus (DIP) and K⁺ was reabsorbed in downstream regions. The fluid secretion rate decreased > 50% after 25 μM bafilomycin A1, 500 μM amiloride or 50 μM bumetanide was added to the bath. The concentration of K⁺ in the secreted fluid did not change, whereas the concentration of Na⁺ in the secreted fluid decreased significantly when tubules were exposed to bafilomycin A1 or amiloride. Addition of 500 μM cAMP or 1 μM 5-HT to the bath stimulated fluid secretion and resulted in a decrease in K⁺ concentration in the secreted fluid. An increase in Na⁺ concentration in the secreted fluid was observed only in cAMP-stimulated tubules. Secreted fluid pH and the transepithelial electrical potential (TEP) did not change when tubules were stimulated. Taken together, our results show that the secretion of fluid is carried out by the upper regions (DIP) in T. ni Malpighian tubules. Upper regions of the tubules secrete K⁺, whereas lower regions reabsorb it. Stimulation of fluid secretion is correlated with a decrease in the K⁺/Na⁺ ratio.     

5-hydroxytryptamine regulates the (Na++K+)ATPase activity in malpighian tubules of Rhodnius prolixus: Evidence for involvement of G-protein and cAMP-dependent protein kinase

5-Hydroxytryptamine (5-HT, serotonin) acts as a diuretic hormone in Rhodnius prolixus, where it increases to 0.1 μM in the haemolymph during feeding and stimulates the fluid secretion in isolated Malpighian tubules. The ouabain-sensitive (Na⁺+K⁺)ATPase activity present in homogenates of Malpighian tubules from unfed Rhodnius prolixus is inhibited 60% by 0.01 μM 5-HT. This inhibition is reversed by ketanserin, a 5-HT₂ receptor antagonist in mammals, and also by GDPβS, a competitive inhibitor of G-protein GTPase activity. GTPγS, a nonhydrolysable analog of GTP, and cholera toxin, a G_s-protein activator, also inhibit the ouabain-sensitive (Na⁺+K⁺)ATPase activity, while pertussis toxin, a G_i-protein inhibitor, has no effect. The (Na⁺+K⁺)ATPase activity is inhibited 55% by 0.4–100 μM dibutyryl-cAMP in the presence of IBMX, a phosphodiesterase inhibitor, which also potentiates the effect of a low concentration of 5-HT. The cAMP-dependent protein kinase inhibitor peptide abolishes the 5-HT effect. These data suggest that the (Na⁺+K⁺)ATPase activity in Malpighian tubules is inhibited by 5-HT through activation of G_s-protein and a cAMP-dependent protein kinase. Inhibition of the Na⁺+K⁺ pump would contribute to the diuretic effect of 5-HT. Arch. Insect Biochem. Physiol. 36:203–214, 1997. © 1997 Wiley- Liss, Inc.     

Effect of corpus cardiacum extracts on the ATPase activity of locust rectum

The Mg²⁺ dependent and Na⁺K⁺-activated ATPase activities of microsomal preparations from the rectum of Locusta migratoria were both stimulated, to varying extents, by crude extracts of the corpora cardiaca of this species. Mg²⁺ ATPase activity increased by approximately 549% whereas the hormonal stimulation of Na⁺K⁺-activated ATPase depended upon the concentration of sodium and potassium ions. At 100 mM Na⁺ and 20 mM K⁺, conditions which approximate to optimum for this enzyme system, Na⁺K⁺-activated ATPase activity increased by about 14%. At sub-optimum concentrations of these ions, i.e. 50 and 5 mM Na⁺ and K⁺ respectively, the increase in Na⁺K⁺-activated ATPase activity was about 205%. Ouabain at a concentration of 10^?3 M completely abolished this stimulated activity and was consistently effective in partially reducing the stimulation of Mg²⁺ ATPase activity by corpora cardiaca extracts.     

Effect of Na+, K+, ouabain,amiloride and ethacrynic acid on the transepithelial potential across Malpighian tubules of Locusta

A transepithelial potential of +8.74±0.29 mV (n = 85) has been recorded across the Malpighian tubules of Locusta. The effect of varying the Na⁺ and K⁺ concentration in the bathing medium on the transepithelial potential has been determined. The data show that the transepithelial potential does not obey the Nernst equation for K⁺. Ouabain, ethacrynic acid and amiloride all inhibit the transepithelial potential. The results are discussed in relation to the nature of the mechanisms of cation transport across the Malpighian tubules.     

Direct stimulation of human erythrocyte membrane (Na+ + k+)-mg ATPase activity in vitro by physiological concentrations of d-aldosterone.

The effect of d-aldosterone on human erythrocyte ghost (Na⁺ + K⁺)-Mg ATPase has been studied. Aldosterone at 3.225 × 10^?10M caused a 450% activation of (Na⁺ + K⁺)-Mg ATPase activity whilst inhibiting (Na⁺ + Na⁺)-Mg ATPase activity. Aldosterone acts by reducing the affinity of the external K⁺ site of (Na⁺ + K⁺)Mg ATPase for Na⁺ thereby resulting in improved efficiency of Na⁺ ? K⁺ transfer. Aldosterone was additionally found to modify both the Na⁺ and K⁺ activation of (Na⁺ + K⁺)Mg ATPase incubated in the presence of commercial ATP containing orthovanadate. Aldosterone was found to reverse the inhibitory effects of orthovanadate at high Na⁺ and K⁺ concentrations. The physiological significance of orthovanadate and aldosterone are discussed.     

Influence of ouabain on Na+ and K+ concentration in haemolymph of Drosophila hydei and appearance of Malpighian tubules

After injection of ouabain into the body cavity, the Malpighian tubules of Drosophila larvae show a characteristic appearance which differs from the normal. The primary urine, which is mainly found in particles, is diluted and the concretions are washed away through the proximal part of the tubule and the ureter into the hindgut. In the haemolymph the Na⁺ and K⁺ concentrations change significantly. The K⁺ concentration increases rapidly to double the normal, while later the Na⁺ concentration rises up to 2·3 times the normal. Water movements are not the cause of the concentration changes because the quotient $\text{Na}\text{K}$ varies widely. Thus primary ion regulation mechanisms are influenced in the insect body by application of g-strophanthin. This is evidence for the existence of a ouabain-sensitive ATPase, which is decisively involved in the ion transport mechanisms in the insect body.     

Ageing in the honey-bee, Apis mellifera, as related to brain ATPases and their DDT sensitivity

The adenosine triphosphatase (ATPase) system in worker honey-bee brains showed an increased activity of 57 per cent in Na⁺K⁺ATPase and 63 per cent in Mg²⁺ATPase from adult emergence to 7 days post-emergence. Mg²⁺ATPase activity remained about the same throughout the remainder of adult life, while Na⁺K⁺ATPase remained the same until the sixth week, when a decline occurred. The percentage mortality of the bees exceeded 90 per cent at the time of decline of Na⁺K⁺ATPase. The in vitro inhibition of Mg²⁺ATPase and Na⁺K⁺ATPase by 10 μM DDT was between 40 and 50 per cent and about 20 per cent, respectively. A somewhat greater sensitivity to DDT was determined in brains of older honey-bees.     

Studies on the subcellular distribution of (Na+ + K+)-ATPase,K+-stimulated ATPase and HCO3−-stimulated ATPase activities in malpighian tubules of Locusta migratoria L.

The present study confirms previous reports of the presence of (Na⁺ + K⁺)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K⁺-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na⁺ + K⁺)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K⁺-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na⁺ + K⁺)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K⁺-stimulated ATPase was not associated with the basolateral plasma membrane is discussed.Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction contaning mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na⁺ + K⁺)-ATPase but do not support the K⁺-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.     

Sensitivity of ATPase activities to fire ant venom and abdomen preparations.

The sensitivity of fire ant, $\text{Solenopsis richteri}$ (Forel), head homogenate ATPase to its venom and to a cyclohexane extract of whole fire ants were investigated. Na⁺K⁺ and oligomycin-sensitive Mg²⁺ ATPase activities were inhibited by both preparations. Oligomycin-insensitive Mg²⁺ ATPase activity was inhibited by low concentrations but showed strong stimulation at high concentrations of the venom preparations. Lineweaver-Burk plots of enzyme data in the presence or absence of inhibitor indicated that the inhibitor action was non-competitive with ATP for Na⁺K⁺ and oligomycin-sensitive Mg²⁺ ATPase activities. However, the oligomycin-insensitive Mg²⁺ ATPase activity showed a mixed type response to the inhibitor. Tests on pure samples of known venom components indicate that they cause the observed effects on the ATPase activities.     

Na+ K+ ATPase activity in synaptosomes and myelin of developing control and intra-uterine growth retarded rats: effects of lead and serotonin

An intrauterine growth retarded (IUGR) model based on restriction of blood supply to fetuses at 17 days of pregnancy in rats was studied. We investigated in vitro the effects of lead on Na⁺K⁺ ATPase activity in synaptosomes and myelin of IUGR and control rats from 6 to 60 days after birth. In both groups an age-dependent effects existed in synaptosomes for the lowest doses of lead. The experimental group tended to be more sensitive to the metal than the control group and the Na⁺K⁺ATPase activity was less inhibited in the younger rats as compared to mature rats. Serotonin (5-HT) added to the subcellular preparations produced different changes in Na⁺K⁺ATPase activity. In synaptosomes, 5-HT stimulated the enzyme activity in a dose-related manner and apparently reversed the inhibiton induced by lead up to 22 days after birth in the control group. This action was less marked in the IUGR group. In myelin fractions, the Na⁺K⁺ATPase activity was inhibited by lead in both groups but the “protective effect of monoamines” was never observed. The Na⁺K⁺ ATPase activity was modulated by monoamines in synaptosomes and not in myelin, perhaps through a mechanism involving soluble factor(s).     

K+-stimulated ATPase in purified microsomes of bullfrog oxyntic cells

Differential centrifugation of oxyntic cell homogenates yielded microsomal fractions which contained large amounts of mitochondrial membrane. The presence of marker enzymes (succinate dehydrogenase and cytochrome c oxidase) indicated that mitochondrial contamination of crude microsomes ranged from 20 to 60% in different preparations. A discontinuous sucrose density gradient procedure was developed for the routine preparation of purified oxyntic cell microsomes. A K⁺-stimulated, Mg²⁺-requiring ATPase was localized in these purified membranes and coincided with the presence of a K⁺-stimulated p-nitrophenylphosphatase. Na⁺ and ouabain had no effect on the K⁺ stimulation of the microsomal ATPase. The apparent activation constant for K⁺ was approximately 1 mM at pH 7.5, the optimal pH for stimulation.An anion-sensitive ATPase has been widely studied in gastric microsomal preparations. We found that the basal microsomal ATPase (i.e. without K⁺) and the mitochondrial ATPase were inhibited by SCN^? and enhanced by HCO₃^?, however, the K⁺-stimulated component of the microsomal ATPase was virtually unaffected by these anions.     

ATPases in rabbit erythrocytes: stimulation by HCO3-anion and by Na-cation-plus-K-cation.

A Mg²⁺Na⁺K⁺ATPase was found in a ghost preparation from rabbit erythrocytes, a finding in conflict with previous reports, but in agreement with the known kinetics of cation movements in these cells. However the Mg²⁺Na⁺K⁺ATPase was not inhibited by 10⁻⁴M ouabain, nor by 10⁻⁴M Ca²⁺. The physiological status of this enzyme is discussed. The basic Mg²⁺-ATPase activity in this preparation is also stimulated by HCO₃⁻; it is suggested that the HCO₃⁻-stimulated ATPases reported in a variety of other preparations are not necessarily due to mitochondrial contamination but could well originate from the plasma membrane.     

C-peptide,Na+,K+-ATPase,and Diabetes

Na⁺,K⁺-ATPase is an ubiquitous membrane enzyme that allows the extrusion of three sodium ions from the cell and two potassium ions from the extracellular fluid. Its activity is decreased in many tissues of streptozotocin-induced diabetic animals. This impairment could be at least partly responsible for the development of diabetic complications. Na⁺,K⁺-ATPase activity is decreased in the red blood cell membranes of type 1 diabetic individuals, irrespective of the degree of diabetic control. It is less impaired or even normal in those of type 2 diabetic patients. The authors have shown that in the red blood cells of type 2 diabetic patients, Na⁺,K⁺-ATPase activity was strongly related to blood C-peptide levels in non–insulin-treated patients (in whom C-peptide concentration reflects that of insulin) as well as in insulin-treated patients. Furthermore, a gene-environment relationship has been observed. The alpha-1 isoform of the enzyme predominant in red blood cells and nerve tissue is encoded by the ATP1A1 gene.Apolymorphism in the intron 1 of this gene is associated with lower enzyme activity in patients with C-peptide deficiency either with type 1 or type 2 diabetes, but not in normal individuals. There are several lines of evidence for a low C-peptide level being responsible for low Na⁺,K⁺-ATPase activity in the red blood cells. Short-term C-peptide infusion to type 1 diabetic patients restores normal Na⁺,K⁺-ATPase activity. Islet transplantation, which restores endogenous C-peptide secretion, enhances Na⁺,K⁺-ATPase activity proportionally to the rise in C-peptide. This C-peptide effect is not indirect. In fact, incubation of diabetic red blood cells with C-peptide at physiological concentration leads to an increase of Na⁺,K⁺-ATPase activity. In isolated proximal tubules of rats or in the medullary thick ascending limb of the kidney, C-peptide stimulates in a dose-dependent manner Na⁺,K⁺-ATPase activity. This impairment in Na⁺,K⁺-ATPase activity, mainly secondary to the lack of C-peptide, plays probably a role in the development of diabetic complications. Arguments have been developed showing that the diabetesinduced decrease in Na⁺,K⁺-ATPase activity compromises microvascular blood flow by two mechanisms: by affecting microvascular regulation and by decreasing red blood cell deformability, which leads to an increase in blood viscosity. C-peptide infusion restores red blood cell deformability and microvascular blood flow concomitantly with Na⁺,K⁺-ATPase activity. The defect in ATPase is strongly related to diabetic neuropathy. Patients with neuropathy have lower ATPase activity than those without. The diabetes-induced impairment in Na⁺,K⁺-ATPase activity is identical in red blood cells and neural tissue. Red blood cell ATPase activity is related to nerve conduction velocity in the peroneal and the tibial nerve of diabetic patients. C-peptide infusion to diabetic rats increases endoneural ATPase activity in rat. Because the defect in Na⁺,K⁺-ATPase activity is also probably involved in the development of diabetic nephropathy and cardiomyopathy, physiological C-peptide infusion could be beneficial for the prevention of diabetic complications.     

Stimulated fluid secretion is sodium dependent in the Malpighian tubules of Locusta migratoria

The ionic dependencies of stimulated and unstimulated Locusta tubules have been studied. K⁺, Na⁺, Cl^? are essential to both basal and stimulated secretion. K⁺ is secreted against a concentration gradient in unstimulated tubules. In response to diuretic hormone or cAMP application, there is a dramatic influx of K⁺ into the lumen. A high level of Na⁺ and Cl^? in the bathing medium is required to allow maximal fluid secretion. The tubules show an apparent impermeability to Na⁺; its concentration in the secreted fluid is always much less than in the bathing medium. If Na⁺ is omitted from the medium and excess K⁺ added (80 mM K), then although basal secretion occurs (2.5 nl/min), the tubules fail to respond to stimulation. Clearly Na⁺ has an important indirect role to play in stimulated fluid secretion.     

The disparity between effects of vanadate (V) and vanadyl (IV) ions on (Na+-K+)-ATPase and K+-phosphatase in skeletal muscle

On crude membrane fractions of skeletal musccle, vanadyl (IV) and vanadate (V) compounds inhibited the membrane (Na⁺K⁺)-ATPase and neutral (K⁺-)p-nitrophenylphosphatase equally with K_i 4×10^?8 mol.1^?1. Only vanadate (V) inhibited significantly the muscle (Na⁺K⁺)ATPase with K_i 1×10^?6 mol.1^?1, whereas vanadyl (IV) ions were almost without effect. Extracellular application of both forms of vanadium failed to inhibit the electrogenic (Na⁺K⁺) pump in intact mouse diaphragm fibres.     

Preliminary studies on rat olfactory tissue: effects of odorants on NA+-K+ atpase activity

Nerve ending particle (B) fractions were prepared from three different rat olfactory endoturbinals. This fraction was tested for Na⁺K⁺ ATPase activity and its response $\text{in vitro}$to low levels of odorants. Quite different differential responses of the enzyme activity were noted between the three tissue preparations for a given odorant and between the three odorants for a given tissue. A possible relationship between enzyme perturbation and initiation of odor sensing mechanism is discussed.     

Effects of Na+ and/or K+ on the Mg2+-dependent ATPase activities in shrimp (Macrobrachium amazonicum) gill homogenates

• 1.1. Homogenates of gills from the freshwater shrimp M. amazonicum exhibit the following ATPase activities: (i) a basal, Mg²⁺-dependent ATPase; (ii) an ouabain-sensitive, Na⁺ + K⁺-stimulated ATPase; (iii) an ouabain-insensitive, Na⁺-stimulated ATPase; and (iv) an ouabain-insensitive, K⁺-stimulated ATPase.
• 2.2. K⁺ suppresses the Na⁺-stimulated ATPase activity in a mixed-type kind of inhibition, whereas Na⁺ does not exert any noticeable effect on the K⁺-stimulated ATPase activity.
• 3.3. The Na⁺- and the K⁺-stimulated ATPase activities are totally inhibited by 5 mM ethacrynic acid in the incubation medium.
• 4.4. The Na⁺- and the K⁺-stimulated ATPase activities are not expressions of the activation of a Ca-ATPase.
• 5.5. The possible localization and roles of the described ATPases within the gill epithelium are briefly discussed and evaluated.

     

A comparison of the subcellular distribution of 5′-nucleotidase, (Na+K+)-ATPase and adenyl cyclase in beef thyroid gland

The validity of 5′-nucleotidase as a plasma membrane marker enzyme in beef thyroid has been tested by comparing the subcellular distribution of its activity to that of (Na⁺K⁺)-activated ATPase and adenyl cyclase. The specific activity and total activity of (Na⁺K⁺)-ATPase and adenyl cyclase were greatest in the 1000 × g (“nuclear”) and 33 000 × g (“mitochondrial and lysosomal”) fractions. In contrast, 5′-nucleotidase activity was concentrated in the 165 000 × g (“microsomal”) pellet and supernatant. Partially purified plasma membranes were separated from the 1000 (N₂), 30 000 (M₂) and 165 000 × g (P₂) pellets by discontinuous sucrose gradient centrifugation. Again a discordant distribution of these enzyme activities was observed. (Na⁺K⁺)-ATPase specific activity was increased approximately 30-fold over the homogenate in Fractions N₂ and M₂. Basal, thyroid-stimulating hormone-and fluoride-stimulated adenyl cyclase activities were concentrated in the same fractions. 5′-Nucleotidase activity was preferentially located in M₂ and P₂. These differences in distribution pattern suggest that 5′-nucleotidase activity is not uniquely located in the plasma membrane in the thyroid.