Increased blood pressure and loss of anp-induced natriuresis in mice lacking DARPP-32 gene

Atrial natriuretic peptide (ANP) is an important regulator of sodium metabolism and indirectly of blood pressure. Evidence has accumulated that ANP regulates sodium metabolism through a cascade of steps involving an increase in the level of cGMP, activation of cGMP-dependent protein kinase (PKG), and inhibition of renal tubular Na+, K+-ATPase activity. One of the major substrates for PKG is DARPP-32. In the present study we observed that ANP does not induce natriuresis in mice that lack DARPP-32. In contrast, there was a 4-fold increase in urinary sodium excretion following ANP administration to wild type mice. ANP as well as Zaprinast, a selective inhibitor of cGMP phosophodiesterase, inhibited renal Na+, K+-ATPase activity in wild type mice but had no such effect in mice lacking DARPP-32. Mean arterial blood pressure, measured in conscious animals, was significantly increased in DARPP-32 deficient mice as compared to wild type mice. The results confirm that DARPP-32 acts as a third messenger in the ANP signaling pathway in renal tissue and suggest an important role of DARPP-32 in the maintenance of normal blood pressure.     

Dopamine- and cAMP-regulated phosphoprotein (DARPP-32) and dopamine DA1 agonist-sensitive Na+,K+-ATPase in renal tubule cells.

The cellular localization of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 that appears to mediate certain actions of dopamine in the mammalian brain by acting as an inhibitor of protein phosphatase 1, was studied in the kidney of several species. DARPP-32 mRNA and DARPP-32-like immunoreactivity were found in the cytoplasm of cells in the thick ascending limb of the loop of Henle. The specific dopamine DA1 agonist SKF 82526 caused a dose-dependent inhibition of Na+,K+-ATPase activity, which could be blocked by SCH 23390, a specific DA1 antagonist, and by PKI-(5-24) amide, a specific inhibitor of cAMP-dependent protein kinase. The results indicate that DA1 dopamine receptors and DARPP-32, an intracellular third messenger for dopamine, are part of the signal-transduction process for dopamine acting on renal tubule cells.     

Hypertension-linked mutation in the adducin alpha-subunit leads to higher AP2-mu2 phosphorylation and impaired Na+,K+-ATPase trafficking in response to GPCR signals and intracellular sodium

Alpha-adducin polymorphism in humans is associated with abnormal renal sodium handling and high blood pressure. The mechanisms by which mutations in adducin affect the renal set point for sodium excretion are not known. Decreases in Na+,K+-ATPase activity attributable to endocytosis of active units in renal tubule cells by dopamine regulates sodium excretion during high-salt diet. Milan rats carrying the hypertensive adducin phenotype have a higher renal tubule Na+,K+-ATPase activity, and their Na+,K+-ATPase molecules do not undergo endocytosis in response to dopamine as do those of the normotensive strain. Dopamine fails to promote the interaction between adaptins and the Na+,K+-ATPase because of adaptin-mu2 subunit hyperphosphorylation. Expression of the hypertensive rat or human variant of adducin into normal renal epithelial cells recreates the hypertensive phenotype with higher Na+,K+-ATPase activity, mu2-subunit hyperphosphorylation, and impaired Na+,K+-ATPase endocytosis. Thus, increased renal Na+,K+-ATPase activity and altered sodium reabsorption in certain forms of hypertension could be attributed to a mutant form of adducin that impairs the dynamic regulation of renal Na+,K+-ATPase endocytosis in response to natriuretic signals.     

Bidirectional regulation of Na+,K(+)-ATPase activity by dopamine and an alpha-adrenergic agonist.

Catecholamines have pronounced effects on the renal handling of sodium and water, dopamine-promoting sodium and water excretion, and norepinephrine-promoting sodium and water retention. In the present study, using isolated permeabilized renal tubule cells and intact rats, we have shown that these effects can be attributed to opposing actions of these transmitters on renal tubular Na+,K(+)-ATPase activity. The ability of each of these catecholamines to regulate Na+,K(+)-ATPase activity is affected by the concentration of Na+ as well as by the absence or presence of the opposing catecholamine.     

Regression of renal hypertrophy and elevated renal Na+,K+-ATPase activity after insulin treatment in streptozotocin-diabetic rats

The effect of insulin treatment on the renal hypertrophy and elevated renal Na+,K+-ATPase activity in rats with streptozotocin (STZ)-induced diabetes was examined. Rats with STZ-diabetes of 6- to 8-week duration had significantly lower body weights, higher plasma and urinary glucose concentrations, greater urinary volumes, increased kidney weights, and increased kidney/body weights and protein/kidney weight ratios compared to those in saline-citrate-injected controls. Specific Na+,K+-ATPase activity per mg protein in both cortical and outer medullary kidney homogenates was significantly elevated in diabetic vs. control animals, as was total renal Na+,K+-ATPase activity. One week of insulin treatment returned elevated plasma glucose, urinary volume, the protein/kidney weight ratio, and cortical and outer medullary Na+,K+-ATPase activity per mg protein to control values. Kidney weights and kidney/body weight ratios of diabetic animals remained elevated, as did absolute total renal Na+,K+-ATPase activity. After 3 weeks of insulin treatment, kidney weight and total renal Na+,K+-ATPase activity in diabetic animals returned to control values, but body weights remained lower than those in the controls, resulting in continued elevation of kidney/body weight ratios in the diabetic animals. The concurrent regression of both renal hypertrophy and elevated Na+,K+-ATPase activity to normal levels after insulin treatment of STZ-diabetic animals implicates renal growth rather than a direct effect of insulin as the primary factor controlling elevation and regression of Na+,K+-ATPase activity in the diabetic kidney. This finding demonstrates that the effect of renal hypertrophy can outweigh the intrinsic effects of insulin on an important renal transport system and that this effect may be as important as lack of hormone in determining the renal physiological responses in the disease. It is suggested that the increased renal tubular Na+,K+-ATPase activity is a key component of the renal hypertrophy and hyperfunction seen in diabetes.     

Renal aging in WKY rats: changes in Na+,K+ -ATPase function and oxidative stress

It has been suggested that alterations in Na(+),K(+)-ATPase mediate the development of several aging-related pathologies, such as hypertension and diabetes. Thus, we evaluated Na(+),K(+)-ATPase function and H(2)O(2) production in the renal cortex and medulla of Wistar Kyoto (WKY) rats at 13, 52 and 91 weeks of age. Creatinine clearance, proteinuria, urinary excretion of Na(+) and K(+) and fractional excretion of Na(+) were also determined. The results show that at 91 weeks old WKY rats had increased creatinine clearance and did not have proteinuria. Despite aging having had no effect on urinary Na(+) excretion, urinary K(+) excretion was increased and fractional Na(+) excretion was decreased with age. In renal proximal tubules and isolated renal cortical cells, 91 week old rats had decreased Na(+),K(+)-ATPase activity when compared to 13 and 52 week old rats. In renal medulla, 91 week old rats had increased Na(+),K(+)-ATPase activity, paralleled by an increase in protein expression of α(1)-subunit of Na(+),K(+)-ATPase. In addition, renal H(2)O(2) production increased with age and at 91 weeks of age renal medulla H(2)O(2) production was significantly higher than renal cortex production. The present work demonstrates that although at 91 weeks of age WKY rats were able to maintain Na(+) homeostasis, aging was accompanied by alterations in renal Na(+),K(+)-ATPase function. The observed increase in oxidative stress may account, in part, for the observed changes. Possibly, altered Na(+),K(+)-ATPase renal function may precede the development of age-related pathologies and loss of renal function.     

Calcineurin mediates alpha-adrenergic stimulation of Na+,K(+)-ATPase activity in renal tubule cells.

The alpha-adrenergic agonist oxymetazoline increased Na+,K(+)-ATPase activity of single proximal convoluted tubules dissected from rat kidney. Activation of the enzyme by oxymetazoline was prevented by either the alpha 1-adrenergic antagonist prazosin or the alpha 2-adrenergic antagonist yohimbine and was mimicked by the calcium ionophore A23187. The effect of oxymetazoline on Na+,K(+)-ATPase activity was prevented by a specific peptide inhibitor of calcineurin, as well as by FK 506, an immunosuppressant agent known to inhibit calcineurin; these results indicate that the action of oxymetazoline is mediated via activation of calcineurin (a calcium/calmodulin-dependent protein phosphatase). Activation of the Na+,K(+)-ATPase by either oxymetazoline or A23187 was associated with a greater than 2-fold increase in its affinity for Na+. The results provide a biochemical mechanism by which norepinephrine, released from renal nerve terminals, stimulates Na+ retention.     

Phosphorylation of the catalytic subunit of Na+,K(+)-ATPase inhibits the activity of the enzyme.

We have examined two distinct protein kinases, cAMP-dependent protein kinase and protein kinase C, for their ability to phosphorylate and regulate the activity of three different types of Na+,K(+)-ATPase preparation. cAMP-dependent protein kinase phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 1 mol of phosphate per mol of alpha subunit. Protein kinase C phosphorylated purified shark rectal gland Na+,K(+)-ATPase to a stoichiometry of approximately 2 mol of phosphate per mol of alpha subunit. The phosphorylation by each of the kinases was associated with an inhibition of Na+,K(+)-ATPase activity of about 40-50%. These two protein kinases also inhibited the activity of a partially purified preparation of Na+,K(+)-ATPase from rat renal cortex and the activity of Na+,K(+)-ATPase present in preparations of basolateral membrane vesicles from rat renal cortex.     

Na+, K(+)-adenosine triphosphatase regulation in hypertrophied vascular smooth muscle cells.

Vascular smooth muscle cell hypertrophy is a normal compensatory state that may play a pathogenic role in hypertension. Angiotensin II stimulates a hypertrophic response in cultured vascular smooth muscle cells. As part of the growth response, angiotensin II rapidly activates the Na(+)-H+ exchanger, increasing Na+ influx. Because Na+, K(+)-ATPase is the major cellular mechanism for regulating intracellular Na+, we studied the effects of angiotensin II-induced hypertrophy on Na+, K(+)-ATPase expression and activity. Angiotensin II caused rapid increases in both steady-state Na+, K(+)-ATPase activity (ouabain-sensitive 86Rb uptake) and intracellular [Na+]. Angiotensin II also caused a sustained increase in Na+, K(+)-ATPase at 24 hours with a 73% increase in maximal 86Rb uptake per milligram protein and a fourfold increase in Na+, K(+)-ATPase alpha-1 messenger RNA levels. Thus, angiotensin II hypertrophy was associated with rapid increases in Na+, K(+)-ATPase activity due to increased Na+ entry and sustained increases due to a specific increase in Na+, K(+)-ATPase expression. These data demonstrate dynamic regulation of Na+, K(+)-ATPase at the functional and molecular level and suggest that similar compensatory mechanisms should be present in vivo. Alterations in such compensatory pathways may be fundamental to the pathogenesis of hypertension.     

Theodore Cooper Lecture: Renal dopamine system: paracrine regulator of sodium homeostasis and blood pressure

All of the components of a complete dopamine system are present within the kidney, where dopamine acts as a paracrine substance in the control of sodium excretion. Dopamine receptors can be divided into D(1)-like (D(1) and D(5)) receptors that stimulate adenylyl cyclase and D(2)-like (D(2), D(3), and D(4)) receptors that inhibit adenylyl cyclase. All 5 receptor subtypes are expressed in the kidney, albeit in low copy. Dopamine is synthesized extraneuronally in proximal tubule cells, exported from these cells largely into the tubule lumen, and interacts with D(1)-like receptors to inhibit the Na(+)-H(+) exchanger and Na(+),K(+)-ATPase, decreasing tubule sodium reabsorption. During moderate sodium surfeit, dopamine tone at D(1)-like receptors accounts for approximately 50% of sodium excretion. In experimental and human hypertension, 2 renal dopaminergic defects have been described: (1) decreased renal generation of dopamine and (2) a D(1) receptor-G protein coupling defect. Both defects lead to renal sodium retention, and each may play an important role in the pathophysiology of essential hypertension.     

Leptin and the Regulation of Renal Sodium Handling and Renal Na-Transporting ATPases: Role in the Pathogenesis of Arterial Hypertension

Leptin, an adipose tissue hormone which regulates food intake, is also involved in the pathogenesis of arterial hypertension. Plasma leptin concentration is increased in obese individuals. Chronic leptin administration or transgenic overexpression increases blood pressure in experimental animals, and some studies indicate that plasma leptin is elevated in hypertensive subjects independently of body weight. Leptin has a dose- and time-dependent effect on urinary sodium excretion. High doses of leptin increase Na(+) excretion in the short run; partially by decreasing renal Na(+),K(+)-ATPase (sodium pump) activity. This effect is mediated by phosphatidylinositol 3-kinase (PI3K) and is impaired in animals with dietary-induced obesity. In contrast to acute, chronic elevation of plasma leptin to the level observed in patients with the metabolic syndrome impairs renal Na(+) excretion, which is associated with the increase in renal Na(+),K(+)-ATPase activity. This effect results from oxidative stress-induced deficiency of nitric oxide and/or transactivation of epidermal growth factor receptor and subsequent stimulation of extracellular signal-regulated kinases. Ameliorating "renal leptin resistance" or reducing leptin level and/or leptin signaling in states of chronic hyperleptinemia may be a novel strategy for the treatment of arterial hypertension associated with the metabolic syndrome.     

INCREASED BLOOD PRESSURE AND LOSS OF ANP-INDUCED NATRIURESIS IN MICE LACKING DARPP-32 GENE

Atrial natriuretic peptide (ANP) is an important regulator of sodium metabolism and indirectly of blood pressure. Evidence has accumulated that ANP regulates sodium metabolism through a cascade of steps involving an increase in the level of cGMP, activation of cGMP-dependent protein kinase (PKG), and inhibition of renal tubular Na⁺,K⁺ -ATPase activity.

One of the major substrates for PKG is DARPP-32. In the present study we observed that ANP does not induce natriuresis in mice that lack DARPP- 32. In contrast, there was a 4-fold increase in urinary sodium excretion following ANP administration to wild type mice. ANP as well as Zaprinast, a selective inhibitor of cGMP phosophodiesterase, inhibited renal Na⁺,K⁺-ATPase activity in wild type mice but had no such effect in mice lacking DARPP-32. Mean arterial blood pressure, measured in conscious animals, was significantly increased in DARPP-32 deficient mice as compared to wild type mice.

The results confirm that DARPP-32 acts as a third messenger in the ANP signaling pathway in renal tissue and suggest an important role of DARPP-32 in the maintenance of normal blood pressure.     

Biphasic effects of dopamine on 86rubidium uptake in rat renal proximal tubules

The mechanism(s) by which dopamine inhibits Na+-K+-ATPase activity in the renal proximal tubule is still controversial. We studied the short-term effects of dopamine on the sodium pump in rat renal proximal tubule suspensions with the 86Rb uptake method. Dopamine and the D1-like agonist, SKF81297, initially stimulated Na+-K+-ATPase activity at 5 min and subsequently inhibited it at 10 min and 20 min; the inhibition by 10 microM dopamine at 20 min was 21.3 +/- 4.5%. The inhibitory effect of dopamine on Na+-K+-ATPase activity was mimicked by thymeleatoxin (a classical protein kinase C [PKC] agonist) while Sp-8-CPT-cAMPS (a protein kinase A [PKA] agonist) had no effect. However, the combination of the PKC and PKA agonists mimicked the biphasic effects of dopamine and SKF81297. Rp-8-CPT-cAMPS (a PKA inhibitor), U-73122 (a phospholipase C inhibitor), or calphostin C (a PKC inhibitor), blocked the dopamine-mediated biphasic effects on Na+-K+-ATPase activity. It is suggested that the biphasic effects of dopamine on Na+-K+-ATPase activity (an initial stimulation and a subsequent inhibition) are transduced by activating both PKA and PKC through a D1-like receptor.     

The paradox of dopamine and angiotensin II-mediated Na(+), K(+)-ATPase regulation in renal proximal tubules

Accumulated studies reported that the natruretic dopamine (DA) and the anti-natruretic angiotensin II (Ang II) represent an important mechanism to regulate renal Na(+) and water excretion through intracellular secondary messengers to inhibit or activate renal proximal tubule (PT) Na(+), K(+)-ATPase (NKA). The antagonistic actions were mediated by the phosphorylation of different position of NKA α?-subunit and different Pals-associated tight junction protein (PATJ) PDZ domains, the different protein kinase C (PKC) isoforms (PKC-β, PKC-ζ), the common adenylyl cyclase (AC) pathway, and the crosstalk and balance between DA and Ang II to NKA regulation. Besides, Ang II-mediated NKA modulation has bi-phasic effects.     

Sodium-independent in vitro induction of Na+,K+-ATPase by aldosterone in renal target cells: permissive effect of triiodothyronine.

The aim of this study was to develop an in vitro system in which we could study the causal relationship between short-term stimulation of Na+,K+-ATPase in the collecting tubule by aldosterone on the one hand and protein synthesis and changes in intracellular Na+ concentration on the other hand. Previous in vivo studies suggested that triiodothyronine might facilitate aldosterone-induced stimulation of Na+,K+-ATPase. Results show that when segments of cortical collecting tubules microdissected from collagenase-treated kidneys of adrenalectomized rats were incubated for 3 hr in the presence of either 10(-8) M aldosterone or 10(-8) M triiodothyronine alone Na+,K+-ATPase activity was not altered, whereas the addition of both hormones markedly stimulated the activity and the number of catalytic sites of Na+,K+-ATPase. This stimulation was abolished by actinomycin D and cycloheximide, whereas it was not altered in the absence of extracellular sodium or in the presence of the luminal Na+-channel blocker amiloride. Thus, triiodothyronine facilitates the in vitro induction of Na+,K+-ATPase synthesis by aldosterone. Aldosterone action on Na+,K+-ATPase is independent of Na+ availability.     

Na(+)-, ouabain-, Ca(2+)-, and thapsigargin-sensitive ATPase activity expressed in chimeras between the calcium and the sodium pump alpha subunits.

Using the chicken sarcoplasmic/endoplasmic reticulum Ca2+ (SERCA)-ATPase as a parental molecule and replacing various portions with the corresponding portions of the chicken Na+,K(+)-ATPase alpha 1 subunit, Ca2+/thapsigargin- and Na+/ouabain-sensitive domains critical for these P-type ATPase activities were identified. In the chimera, [n/c]CC, the amino-terminal amino acids Met-1 to Asp-162 of the SERCA (isoform 1) (SERCA1) ATPase were replaced with the corresponding portion (Met-1-Asp-200) of the Na+,K(+)-ATPase alpha 1 subunit. In the chimera CC[c/n], the carboxyl-terminal amino acids (Ser-830 to COOH) of the SERCA1 ATPase were replaced with the corresponding segment (Leu-861 to COOH) of the Na+,K(+)-ATPase alpha 1 subunit, and in the chimera CNC, the middle part (Gly-354-Lys-712) of the SERCA1 ATPase was exchanged with the Na+,K(+)-ATPase alpha 1 subunit (Gly-378-Lys-724). None of the chimeric molecules exhibited any detectable ouabain-sensitive Na+,K(+)-ATPase activity, but they did exhibit thapsigargin-sensitive Ca(2+)-ATPase activity. Therefore, the segments Ile-163-Gly-354 and Lys-712-Ser-830 of the SERCA1 ATPase are sufficient for Ca2+ and thapsigargin sensitivity. The SERCA1-ATPase activity of [n/c]CC, but not of CCC, CNC, or CC[c/n], was further stimulated by addition of Na+ in the assay medium containing Ca2+. This additional stimulation of SERCA1-ATPase activity by Na+ was abolished when the amino-terminal region (Met-1-Leu-69) of [n/c]CC was deleted ([delta n/c]CC). In the absence of Na+, the SERCA1-ATPase activity of [n/c]CC was inhibited by ouabain, and, in the presence of Na+, its activity was stimulated by this drug. On the other hand, the ATPase activity of [delta n/c]CC was not affected by ouabain, although [delta n/c]CC can still bind [3H]ouabain. These results suggest that a distinct Na(+)-sensitive domain (Na+ sensor) located within the restricted amino-terminal region (Met-1-Leu-69) of the Na+,K(+)-ATPase alpha 1 subunit regulates ATPase activity. The Na+ sensor also controls ouabain action in concert with the major ouabain-binding region between Ala-70 and Asp-200 of alpha 1 subunit.     

Erythrocyte membrane lipids and cationic transport systems in men.

OBJECTIVE: The relationship between erythrocyte membrane and plasma lipids and various transmembrane erythrocyte cationic fluxes was examined in 53 normal men. DESIGN: Different measurements of erythrocyte transport systems were obtained: Na(+)-Li+ countertransport activity; Na+, K+ cotransport activity; Na+, K(+)-ATPase pump activity and the ground membrane permeability for Na+ and K+ as well as the intra-erythrocyte Na+, K+ and Mg2+ concentrations. Plasma cholesterol, triglycerides, phospholipids, free fatty acids, low- and high-density lipoprotein cholesterol levels and the erythrocyte membrane contents of cholesterol, phospholipids and free fatty acids were obtained from fasting subjects. RESULTS: In single regression analysis the erythrocyte Na(+)-Li+ countertransport and Na+, K+ cotransport activities were negatively related to the erythrocyte membrane cholesterol, phospholipids and free fatty acids contents. The Na+, K(+)-ATPase pump activity as assessed by the ouabain-sensitive Na+ efflux was also inversely related to the membrane cholesterol and phospholipids contents. In multiple regression analysis the red blood cell Na(+)-Li+ countertransport activity was independently and negatively related to the membrane cholesterol and free fatty acids contents. CONCLUSION: Our data show that an elevated level of erythrocyte membrane lipids in normal men is accompanied by lower Na(+)-Li+ countertransport, Na+, K+ cotransport and Na+, K(+)-ATPase pump activities.     

Physiology of renal sodium transport

A wealth of studies performed with a spectrum of methods spanning simple clearance studies to the molecular identification of ion transporters has increased our understanding of how approximately 1.7 kg of NaCl and 180 L of H2O are absorbed by renal tubules in man and how the urinary excretion is fine-tuned to meet homeostatic requirements. This review will summarize our current understanding. In the proximal nephron, approximately 60 to 70% of the filtered Na+ and H2O is absorbed together with approximately 90% of the filtered HCO3-. The exact quantities are determined by many regulatory factors, such as glomerulotubular balance, angiotensin II, endothelin, sympathetic innervation, parathyroid hormone, dopamine, acid base status and others. The essential components of absorption are luminal membrane Na+/H+ exchange and the basolateral (Na+ + K+)-ATPase. In the thick ascending limb of the loop of Henle, 20 to 30% of the filtered NaCl is absorbed via Na+2Cl-K+ cotransport driven by the basolateral (Na+ + K+)-ATPase. No H2O is absorbed at this nephron site. The transport rate is determined by the Na+ load and by several hormones and neurotransmitters, including prostaglandins, parathyroid hormone, glucagon, calcitonin, arginine vasopressin (AVP), and adrenaline. In the distal tubule, some 5 to 10% of the filtered load is absorbed via Na+Cl- cotransport in the luminal membrane driven by the basolateral (Na+ + K+)-ATPase. The rate of transport is again determined by the delivered load and by several hormones and neurotransmitters. One of the tasks of the collecting duct is to control the absorption of approximately 10 to 15% of the filtered H2O, regulated by AVP, and just a few percent of the filtered Na+, controlled by aldosterone and natriuretic hormone. The water absorption proceeds through the luminal membrane via aquaporin 2 and through the basolateral membrane via aquaporin 3 channels and is driven by the osmotic gradient built up by the counter current concentrating system. The Na+ absorption occurs via Na+ channels present in the luminal membrane driven by the basolateral (Na+ + K+)-ATPase. With no pharmacological interference, urinary excretion of Na+ can vary between less than 0.1% and no more than 3% of the filtered load, and that of H2O can vary between 0.3 and 15%.     

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

Previous work suggested that the structural gene for the A system transporter and the mRNA for the alpha subunit of the Na+,K(+)-ATPase in Chinese hamster ovary cells CHO-K1 [wild type (WT)] are coordinately controlled by regulatory gene R1. This conclusion was based on analysis of a mutant for the A system, alar4. This mutant had a constitutive level of A system transport activity equal to the level found in derepressed WT cells and a 4 times increase in abundance of the alpha 1 subunit of Na+,K(+)-ATPase mRNA over that found in repressed WT. The level of Na+ per cell in alar4 was not significantly greater than that found in the WT. To further characterize the likely coregulation of both genes, we have studied the A system activity and Na+,K(+)-ATPase mRNA alpha 1-subunit levels in cells grown under various conditions that result in repression or derepression of the A system in the WT. System A activity increased up to 2-3 times the basal transport rate (repressed state) and Na+,K(+)-ATPase mRNA alpha 1-subunit levels showed a 3-fold increase after amino acid starvation (derepressed state). These changes occurred along with a decrease in intracellular Na+ levels. N-Methyl-alpha-aminoisobutyric acid and beta-alanine, previously shown to be corepressors for the A system, prevented to a similar extent A system derepression and Na+,K(+)-ATPase mRNA alpha 1-subunit accumulation. On the other hand, phenylalanine and lysine, amino acids that are not corepressors of the A system, failed to significantly prevent derepression of both genes. Hybrids between the WT and alar4 have the phenotype of the WT when grown under repressed conditions. These results give further support to the proposition that both the A system transporter and mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase are coordinately controlled by regulatory gene R1 and elevated Na+ concentrations are not involved. No Na+,K(+)-ATPase activity was detected in derepressed cells. Activity was restored by the addition of monensin. However, this activity was no greater than that obtained in repressed cells. Indications are that the reduced Na+ content in derepressed cells inhibits Na+,K(+)-ATPase activity and that conditions that favored derepression do not allow for de novo synthesis of the Na+,K(+)-ATPase.     

C-Peptide stimulates Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity via PKC alpha in rat medullary thick ascending limb

AIMS/HYPOTHESIS: C-peptide, the cleavage product of proinsulin processing exerts physiological effects including stimulation of Na(+),K(+)-ATPase in erythrocytes and renal proximal tubules. This study was undertaken to assess the physiological effects of connecting peptide on Na(+),K(+)-ATPase activity in the medullary thick ascending limb of Henle's loop. METHODS: Na(+),K(+)-ATPase activity was measured as the ouabain-sensitive generation of (32)Pi from gamma[(32)P]-ATP and (86)Rb uptake on isolated rat medullary thick ascending limbs. The cell-surface expression of Na(+),K(+)-ATPase was evaluated by Western blotting of biotinylated proteins, and its phosphorylation amount was measured by autoradiography. The membrane-associated fraction of protein kinase C isoforms was evaluated by Western blotting. RESULTS: Rat connecting peptide concentration-dependently stimulated Na(+),K(+)-ATPase activity with a threshold at 10(-9) mol/l and a maximal effect at 10(-7) mol/l. C-peptide (10(-7) mol/l) already stimulates Na(+),K(+)-ATPase activity after 5 min with a plateau from 15 to 60 min. C-peptide (10(-7) mol/l) stimulated Na(+),K(+)-ATPase activity and (86)Rb uptake to the same extent, but did not alter Na(+),K(+)-ATPase cell surface expression. The stimulation of Na(+),K(+)-ATPase activity was associated with an increase in Na(+),K(+)-ATPase alpha-subunit phosphorylation and both effects were abolished by a specific protein kinase C inhibitor. Furthermore, connecting peptide induced selective membrane translocation of PKC-alpha. CONCLUSION/INTERPRETATION: This study provides evidence that in rat medullary thick ascending limb, C-peptide stimulates Na(+),K(+)-ATPase activity within a physiological concentration range. This effect is due to an increase in Na(+),K(+)-ATPase turnover rate that is most likely mediated by protein kinase C-alpha phosphorylation of the Na(+),K(+)-ATPase alpha-subunit, suggesting that C-peptide could control Na(+) reabsorption during non-fasting periods.