Regucalcin modulates hormonal effect on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes

The interaction of various hormones and regucalcin on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10^–6–10^–4 M), and insulin (10^–8–10^– M) in the reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10^–8–3×10^–6 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10^–4 M) which can inhibit the Ca²⁺-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 M), isolated from rat liver cytosol, elevated significantly (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10^–4 M). the epinephrine (10^–5 M) or phenylephrine (10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10^–6 M) was not weakened by the presence of regucalcin (0.5 M). Moreover, GTP (10^–5 and 10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was not seen in the presence of regucalcin (0.25 M). The present finding suggests that the activating mechanism of regucalcin on (Ca²⁺–Mg²⁺)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.     

Age-related responses of hepatic ATPases to starvation and cold stress in male garden lizards

Hepatic Na⁺-K⁺-ATPase and Mg²⁺-ATPase activities of male green lizards declined during the maturation phase (juvenile to 1-year-old) and stabilized thereafter. On the other hand, the Ca²⁺-ATPase activity of the liver declined during the later half of the life span (1-year-old to 2–4-year-old). Starvation stress induced a decline in hepatic Na⁺-K⁺-ATPase and Mg²⁺-ATPase activities of juvenile lizards and caused an increase in 1-year-old and 2–4-year-old counterparts. The Ca²⁺-ATPase activity declined only in starved 1-year-old lizards. Following cold stress, the hepatic Na⁺-K⁺-ATPase activity of juvenile lizards showed a higher degree of decline than 2–4-year-old counterparts. The Mg²⁺-ATPase activity declined in cold-stressed juvenile lizards, but the parameter was elevated in similarly treated 1-year-old lizards. On the other hand, the increase in Ca²⁺-ATPase activity in response to cold stress was confined only to 2–4-year-old lizards.     

Activating effect of regucalcin on (Ca2+-Mg2+)-ATPase in rat liver plasma membranes: relation to sulfhydryl group

The activating mechanism of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase in the plasma membranes of rat liver was investigated. (Ca²⁺–Mg²⁺)-ATPase activity was markedly increased by a sulfhydryl (SH) group protecting reagent dithiothreitol (DTT; 2.5 and 5 mM as a final concentration), while the enzyme activity was significantly decreased by a SH group modifying reagent N-ethylmaleimide (NEM; 0.5–5 mM). The effect of DTT (5 mM) to increase the enzyme activity was clearly blocked by NEM (5 mM). Regucalcin (0.25–1.0 M) significantly increased (Ca²⁺-Mg²⁺)-ATPase activity. This increase was completely blocked by NEM (5 mM). Meanwhile, digitonin (0.04%), which can solubilize the membranous lipids, significantly decreased (Ca²⁺–Mg²⁺)-ATPase activity. Digitonin did not have an effect on the DTT (5 mM)-increased enzyme activity. However, the effect of regucalcin (0.25 M) increasing (Ca²⁺–Mg²⁺)-ATPase activity was entirely blocked by the presence of digitonin. The present results suggest that regucalcin activates (Ca²⁺–Mg²⁺)-ATPase by the binding to liver plasma membrane lipids, and that the activation is involved in the SH groups which are an active site of the enzyme.     

PKA-mediated effect of MAS receptor in counteracting angiotensin II-stimulated renal Na-ATPase

We showed previously that angiotensin-(1-7) [Ang-(1-7)] reversed stimulation of proximal tubule Na⁺-ATPase promoted by angiotensin II (Ang II) through a d-ala⁷-Ang-(1-7) (A779)-sensitive receptor. Here we investigated the signaling pathway coupled to this receptor. According to our data, Ang-(1-7) produces a MAS-mediated reversal of Ang II-stimulated Na⁺-ATPase by a Gs/PKA pathway because: (1) the Ang-(1-7) effect is reversed by GDPβS, an inhibitor of trimeric G protein and Gs polyclonal antibody. Cholera toxin, an activator of Gs protein, mimicked it; (2) in the presence of Ang II, Ang-(1-7) increased the PKA activity 10-fold; (3) the peptide inhibitor of PKA blocked the Ang-(1-7) effect on Ang II-stimulated Na⁺-ATPase; (4) Ang-(1-7) reverses the Ang II-stimulated PKC activity; (5) cAMP mimicked the Ang-(1-7) effect on the Ang II-stimulated Na⁺-ATPase. Our results provide new understanding about the signaling mechanisms coupled to MAS receptor-mediated renal Ang-(1-7) effects.     

Angiotensin II stimulates renal proximal tubule Na-ATPase activity through the activation of protein kinase C

Recently, our group described an AT₁-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na⁺-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na⁺-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na⁺-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na⁺-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.     

Processing of angiotensin peptides by NG108-15 neuroblastoma x glioma hybrid cell line

The metabolism of angiotensin (Ang) peptides was studied in NG108-15 neuroblastoma x glioma hybrid cells which express Ang II receptors, renin, dipeptidyl carboxypeptidase A (converting enzyme), as well as Ang I and Ang II. In these experiments, 0.2 nM of either 125I-Ang I or 125I-Ang II was incubated with intact cell monolayers and the medium was analyzed for 125I-products by high performance liquid chromatography. The major product generated from the metabolism of labeled Ang I or Ang II was identified as the amino-terminal heptapeptide Ang-(1-7). N-benzyloxycarbonyl-prolyl-prolinal (ZPP), a specific inhibitor of prolyl endopeptidase, inhibited the formation of Ang-(1-7) from Ang I by 35%. Complete inhibition of Ang-(1-7) generation was attained with p-chloromercuriphenyl-sulfonate, which suggests that a sulfhydryl-containing peptidase other than prolyl endopeptidase is also involved in Ang-(1-7) formation. Ang II was observed to be a minor product resulting from Ang I metabolism. Although the converting enzyme inhibitor enalaprilat (MK-422) significantly reduced Ang II formation, it had no effect on the levels of Ang-(1-7). These findings demonstrate a preferential processing of Ang I into Ang-(1-7) which is not dependent on the prior formation of Ang II.     

Elevation of Different Ion-Specific ATPase Activities by L-Thyroxine (T4) in Different Tissues of Tasar Silkworm, Antheraea mylitta (Lepidoptera: Saturniidae) during Developmental Stages

Tissue-specific age-dependent changes were observed in Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in tropical tasar silkworm, Antheraea mylitta Drury. Maximum enzyme activity was recorded in all the tissues on day 12 (before spinning) in control group of animals. In testis, Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities gradually increased from day 2 to day 12 during fifth larval age and level was maintained up to adult eclosion while, in ovary, a marked decline was noted up to day of adult emergence. Further, a significant and sharp rise was found in ATPase activity in silk gland tissue up to day 12 and afterwards a drastic fall was noted on day 15 (end of spinning) during fifth larval age.Administration of T4 to fifth stage larvae (1 hr old) at doses 0.5–2.0 μg/g significantly elevated the Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in larval and pupal gonads in a dose-dependent fashion. But, in moths, the enhancement was very much confined to Na⁺K⁺- and Ca²⁺-ATPase in testes and only Ca²⁺-ATPase in ovaries. Again, in silk glands thyroxine (0.5–2.0 μg/g) caused a significant rise in the all ion-dependent ATPase activities only during the fifth larval stage. Interestingly, higher doses of T4 (4.0 μg/g) caused a significant reduction in Na⁺K⁺-, Ca²⁺- and Mg²⁺-ATPase in all the tissues almost all the days studied so far. However, lower doses of T4 (0.1 and 0.25 μg/g) remained ineffective in altering the different ion-specific ATPase activities. This study suggests, that mammalian thyroxine has a metabolic influence showing biphasic nature of action in tasar silkworm ATPase system.     

Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium

Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca²⁺ binding and accumulating activities as well as (Mg²⁺ + Ca²⁺)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca²⁺ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca²⁺ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca²⁺ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca²⁺ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca²⁺ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca²⁺ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca²⁺ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca²⁺ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca²⁺ accumulation (V (nmol Ca²⁺/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca²⁺ required for half-maximal velocities did not differ significantly with age (K_0.5 for Ca²⁺ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca²⁺ binding also indicated that the velocity of Ca²⁺ binding but not the concentration of Ca²⁺ required for half-maximal binding was altered due to aging. At identical Ca²⁺ concentrations, the combined Ca²⁺ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca²⁺ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca²⁺ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg²⁺ + Ca²⁺)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca²⁺ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca²⁺ transport. Further, the age-related increment in the Ca²⁺ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca²⁺ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca²⁺ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart.     

Vanadate inhibition of the Ca-ATPase from human red cell membranes

1. (1) VO₃⁻ combines with high affinity to the Ca²⁺-ATPase and fully inhibits Ca²⁺-ATPase and Ca²⁺-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca²⁺-dependent phosphoenzyme.

2. (2) VO₃⁻ blocks hydrolysis of ATP at the catalytic site. The sites for VO₃⁻ also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca²⁺-ATPase.

3. (3) The sites for VO₃⁻ show positive interactions in affinity with sites for Mg²⁺ and K⁺. This accounts for the dependence on Mg²⁺ and K⁺ of the inhibition by VO₃⁻. Although, with less effectiveness, Na⁺ substitutes for K⁺ whereas Li⁺ does not. The apparent affinities for Mg²⁺ and K⁺ for inhibition by VO₃⁻ seem to be less than those for activation of the Ca²⁺-ATPase.

4. (4) Inhibition by VO₃⁻ is independent of Ca²⁺ at concentrations up to 50 μM. Higher concentrations of Ca²⁺ lead to a progressive release of the inhibitory effect of VO₃⁻.

The α1-adrenergic receptor in human erythrocyte membranes mediates interaction in vitro of epinephrine and thyroid hormone at the membrane Ca-ATPase

Membrane Ca²⁺-ATPase activity was stimulated in vitro separately by T₄ (10⁻¹⁰ M) and by epinephrine (10⁻⁶ M). In the presence of a fixed concentration of T₄, additions of 10⁻⁸ and 10⁻⁶ M epinephrine reduced the T₄ effect on the enzyme. β-Adrenergic blockade with propranolol (10⁻⁶ M) prevented stimulation by epinephrine of Ca²⁺-ATPase activity, but did not prevent the suppressive action of epinephrine on T₄-stimulable Ca²⁺-ATPase. In contrast α₁-adrenergic blockade with unlabelled prazosin restored the effect of T₄ on Ca²⁺-ATPase activity in the presence of epinephrine. Like propranolol, prazosin prevented enhancement of enzyme activity by epinephrine in the absence of thyroid hormone. Neither prazosin nor propranolol had any effect on the stimulations by T₄ of red cell Ca²⁺-ATPase in the absence of epinephrine. Analysis of radiolabelled prazosin binding to human red cell membranes revealed the presence of a single class of high-affinity binding sites (K_d, 1.2 × 10⁻⁸ M; B_max, 847 fmol/mg membrane protein). Thus, the human erythrocyte membrane contains α₁-radrenergic receptor sites that are capable of regulating Ca²⁺-ATPase activity.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles

The Ca²⁺-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca²⁺-uptake and Ca²⁺-ATPase activities were significantly increased (2–3 fold). The highest activation was obtained at a 50:50 molar ratio of PSYC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca²⁺-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca²⁺-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca²⁺-ATPase were similar in either PC or PC: PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca²⁺-ATPase. Regulation of the Ca²⁺-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca²⁺-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.     

Angiotensin-(3-4) counteracts the Angiotensin II inhibitory action on renal Ca2+-ATPase through a cAMP/PKA pathway

We recently demonstrated that Angiotensin-(3-4) [Ang-(3-4)], an Ang II-derived dipeptide, overcomes inhibition of plasma membrane Ca(2+)-ATPase promoted by nanomolar concentrations of Ang II in basolateral membranes of renal proximal tubule cells, with involvement of a so far unknown AT(2)R-dependent and NO-independent mechanism. The present study investigates the signaling pathway triggered by Ang-(3-4) that is responsible for counteracting the inhibitory effect of Ang II, and attempts to elucidate the functional interaction of the dipeptide with Ang II at the level of AT(2)R. Stimulation by cholera toxin of G(s)α protein structurally linked to AT(2)R--as revealed by their co-immunoprecipitation--mimicked the effect of Ang-(3-4) on Ca(2+)-ATPase activity. Furthermore, addition of dibutyril-cAMP (db-cAMP) mimicked Ang-(3-4), whereas the specific PKA inhibitor, PKAi(5-24) peptide, suppressed the counter-regulatory effect of Ang-(3-4) and the AT(2)R agonist, CGP42112A. Membrane-associated PKA activity was stimulated by Ang-(3-4) or CGP42112A to comparable levels as db-cAMP, and the Ang-(3-4) effect was abrogated by the AT(2)R antagonist PD123319, whereas the AT(1)R antagonist Losartan had no effect. Ang-(3-4) stimulated PKA-mediated phosphorylation of Ca(2+)-ATPase and activated PKA to comparable levels. Binding assays demonstrated that Ang-(3-4) could not displace (3)H-Ang II from HEK 293T cells expressing AT(2)R, but 10(-10) mol/L Ang-(3-4) resulted in the appearance of a probable higher-affinity site (picomolar range) for Ang II. The results presented herein demonstrate that Ang-(3-4), acting as an allosteric enhancer, suppresses Ang II-mediated inhibition of Ca(2+)-ATPase through an AT(2)R/cAMP/PKA pathway, after inducing conformational changes in AT(2)R that results in generation of higher-affinity sites for Ang II.     

Partial ischemia reduces the efficiency of sarcoplasmic reticulum Ca2+ transport in rat EDL

To investigate the hypothesis that prolonged partial ischemia would result in a depression in homogenate sarcoplasmic reticulum (SR) Ca²⁺-sequestering and mechanical properties in muscle, a cuff was placed around the hindlimb of 8 adult Sprague–Dawley rats (267 ± 5.8 g; × ± S.E.) and partially inflated (315 mm Hg) for 2 h. Following occlusion, the EDL was sampled both from the ischemic (I) and contralateral control (C) leg and SR properties compared with the EDL muscles extracted from rats (n = 8) immediately following anaesthetization (CC). Ischemia was indicated by a lower (p < 0.05) concentration (mmol.kg dry wt^–1) of ATP (19.0 ± 0.7 vs. 16.7 ± 0.7) and phosphocreatine (58.1 ± 5.7 vs. 35.0 ± 4.6) in I compared to C. Although Ca²⁺-ATPase activity (mol·g protein^–1.sec^–1 ), both maximal and submaximal, was not different between C and I (19.7 ± 0.4 vs. 18.5 ± 1.3), reductions (p < 0.05) in Ca²⁺-uptake (mmol·g protein^–1.sec^–1 ) of between 18.2 and 24.7% across a range of submaximal free Ca²⁺-levels were observed in I compared to C. Lower submaximal Ca²⁺-ATPase activity and Ca²⁺-uptake were also observed in the EDL in C compared to CC animals. Time dependent reductions (p < 0.05) were found in peak twitch and maximal tetanic tension in EDL from I but not C. It is concluded that partial ischemia, resulting in modest reductions in energy state in EDL, induces a reduction in Ca²⁺-uptake independent of changes in Ca²⁺-ATPase activity. These changes reduce the coupling ratio and the efficiency of Ca²⁺-transport by SR.     

Pathways of [Ca2+]i rise evoked by angiotensin II in MDCK renal tubular cells

Abstract

The effect of angiotensin II (Ang II) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in MDCK renal tubular cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Ang II at concentrations of 5–40?µM induced a [Ca²⁺]_i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Ang II evoked store-operated Ca²⁺ entry that was inhibited by La³⁺ and Gd³⁺. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) or thapsigargin abolished Ang II-induced Ca²⁺ release. Inhibition of phospholipase C with U73122 abolished Ang II-induced [Ca²⁺]_i rise. Three Ang II analogues [(ASN1,VAL5)-Ang II acetate, (SAR1,THR8)-Ang II acetate, (VAL5)-Ang II acetate] failed to induce a [Ca²⁺]_i rise. Together, in MDCK cells, Ang II induced a [Ca²⁺]_i rise via Ca²⁺ entry through store-operated Ca²⁺ channels and phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum. Moreover, Ang II’s amino acid sequence is important in its stimulatory effect on [Ca²⁺]_i.     

Angiotensin II and angiotensin-(1-7) inhibit the inner cortex Na+ -ATPase activity through AT2 receptor

In the present paper, the modulation of the basolateral membrane (BLM) Na⁺-ATPase activity of inner cortex from pig kidney by angiotensin II (Ang II) and angiotensin-(1–7) (Ang-(1–7)) was evaluated. Ang II and Ang-(1–7) inhibit the Na⁺-ATPase activity in a dose-dependent manner (from 10⁻¹¹ to 10⁻⁵ M), with maximal effect obtained at 10⁻⁷ M for both peptides. Pharmacological evidences demonstrate that the inhibitory effects of Ang II and Ang-(1–7) are mediated by AT₂ receptor: The effect of both polypeptides is completely reversed by 10⁻⁸ M PD 123319, a selective AT₂ receptor antagonist, but is not affected by either (10⁻¹²–10⁻⁵ M) losartan or (10⁻¹⁰–10⁻⁷ M) A779, selective antagonists for AT₁ and AT_(1–7) receptors, respectively. The following results suggest that a PTX-insensitive, cholera toxin (CTX)-sensitive G protein/adenosine 3′,5′-cyclic monophosphate (cAMP)/PKA pathway is involved in this process: (1) the inhibitory effect of both peptides is completely reversed by 10⁻⁹ M guanosine 5′-O-(2-thiodiphosphate) (GDPβS; an inhibitor of the G protein activity), and mimicked by 10⁻¹⁰ M guanosine 5′-O-(3-thiotriphosphate) (GTPγS; an activator of the G protein activity); (2) the effects of both peptides are mimicked by CTX but are not affected by PTX; (3) Western blot analysis reveals the presence of the G_s protein in the isolated basolateral membrane fraction; (4) (10⁻¹⁰–10⁻⁶ M) cAMP has a similar and non-additive effect to Ang II and Ang-(1–7); (5) PKA inhibitory peptide abolishes the effects of Ang II and Ang-(1–7); and (6) both angiotensins stimulate PKA activity.     

Angiotensin-(1-7) Attenuates Kidney Injury Due to Obstructive Nephropathy in Rats

Background

Angiotensin-(1–7) [Ang-(1–7)] counteracts many actions of the renin-angiotensin-aldosterone system. Despite its renoprotective effects, extensive controversy exists regarding the role of Ang-(1–7) in obstructive nephropathy, which is characterized by renal tubulointerstitial fibrosis and apoptosis.

Methods

To examine the effects of Ang-(1–7) in unilateral ureteral obstruction (UUO), male Sprague-Dawley rats were divided into three groups: control, UUO, and Ang-(1–7)-treated UUO rats. Ang-(1–7) was continuously infused (24 μg/[kg·h]) using osmotic pumps. We also treated NRK-52E cells in vitro with Ang II (1 μM) in the presence or absence of Ang-(1–7) (1 μM), Mas receptor antagonist A779 (1 μM), and Mas receptor siRNA (50 nM) to examine the effects of Ang-(1–7) treatment on Ang II-stimulated renal injury via Mas receptor.

Results

Angiotensin II (Ang II) and angiotensin type 1 receptor (AT₁R) protein expression was higher in UUO kidneys than in controls. Ang-(1–7) treatment also decreased proapoptotic protein expression in UUO kidneys. Ang-(1–7) also significantly ameliorated TUNEL positive cells in UUO kidneys. Additionally, Ang-(1–7) reduced profibrotic protein expression and decreased the increased tumor growth factor (TGF)-β1/Smad signaling present in UUO kidneys. In NRK-52E cells, Ang II induced the expression of TGF-β1/Smad signaling effectors and proapoptotic and fibrotic proteins, as well as cell cycle arrest, which were attenuated by Ang-(1–7) pretreatment. However, treatment with A779 and Mas receptor siRNA enhanced Ang II-induced apoptosis and fibrosis. Moreover, Ang II increased tumor necrosis factor-α converting enzyme (TACE) and decreased angiotensin-converting enzyme 2 (ACE2) expression in NRK-52E cells, while pretreatment with Ang-(1–7) or A779 significantly inhibited or enhanced these effects, respectively.

Conclusion

Ang-(1–7) prevents obstructive nephropathy by suppressing renal apoptosis and fibrosis, possibly by regulating TGF-β1/Smad signaling and cell cycle arrest via suppression of AT₁R expression. In addition, Ang-(1–7) increased and decreased ACE2 and TACE expression, respectively, which could potentially mediate a positive feedback mechanism via the Mas receptor.     

The stimulatory effect of angiotensin II on Na-ATPase activity involves sequential activation of phospholipases and sustained PKC activity

Angiotensin II (Ang II) stimulates the proximal tubule Na⁺-ATPase through the AT₁ receptor/phosphoinositide phospholipase Cβ (PI-PLCβ)/protein kinase C (PKC) pathway. However, this pathway alone does not explain the sustained effect of Ang II on Na⁺-ATPase activity for 30 min. The aim of the present work was to elucidate the molecular mechanisms involved in the sustained effect of Ang II on Na⁺-ATPase activity. Ang II induced fast and correlated activation of Na⁺-ATPase and PKC activities with the maximal effect (115%) observed at 1 min and sustained for 30 min, indicating a pivotal role of PKC in the modulation of Na⁺-ATPase by Ang II. We observed that the sustained activation of PKC by Ang II depended on the sequential activation of phospholipase D and Ca²⁺-insensitive phospholipase A₂, forming phosphatidic acid and lysophosphatidic acid, respectively. The results indicate that PKC could be the final target and an integrator molecule of different signaling pathways triggered by Ang II, which could explain the sustained activation of Na⁺-ATPase by Ang II.     

Ethanol modulates calmodulin-dependent Ca2+-activated ATPase in synaptic plasma membranes

The effects of ethanol in vitro on calmodulin-dependent Ca²⁺-activated ATPase (CaM–Ca²⁺-ATPase) activity were studied in synaptic plasma membranes (SPM) prepared from the brain of normal and chronically ethanol-treated rats. In SPM from normal animals, ethanol at 50–200 mM inhibited the Ca²⁺-ATPase activity. Lineweaver-Burk analysis indicates that the inhibition was the result of a decreased affinity of the enzyme for calmodulin, whereas the maximum activity of the enzyme was not changed. Arrhenius analysis indicates that the enzyme activity was influenced by lipid transition of the membranes, and ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were resistant to the inhibitory effect of ethanol on the enzyme activity. The resistance to ethanol inhibition was correlated with a higher enzyme affinity for calmodulin and a higher transition temperature, as compared with normal SPM. Since the calmodulin-dependent Ca²⁺-ATPase in synaptic plasma membranes is believed to be the Ca²⁺ pump controlling free Ca²⁺ levels in synaptic terminals, its inhibition by ethanol could therefore lead to altered synaptic activity.Abbreviations used ATPase adenosine triphosphatase - CaM calmodulin - CaM–Ca²⁺-ATPase calmodulin-dependent Ca²⁺-activated ATPase - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - EtOH ethanol - Hepes N—2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SPM synaptic plasma membranes - TFP trifluoperazine - Tris tris(hydroxymethyl)aminomethane - K_m Michaelis constant - T_d transition temperature - V_max maximum velocity     

The plasma membrane (Mg2+)-dependent adenosine triphosphatase from the human erythrocyte is not an ion pump

Summary The plasma membrane (Mg²⁺)-dependent adenosine triphosphatase ((Mg²⁺)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg²⁺)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H⁺ transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca²⁺)-dependent adenosine triphosphatase ((Ca²⁺)-ATPase) activity. Proton efflux associated with (Ca²⁺)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca²⁺)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982)J. Biol. Chem. 257:2350–2356)) and was shown to be stimulated by calmodulin. The ability of the (Mg²⁺)-ATPase to pump²⁸Mg²⁺,³⁵SO ₄ ^2– and⁸⁶Rb⁺ was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system.     

Regulation of cardiac sarcolemmal Ca2+ channels and Ca2+ transporters by thyroid hormone

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca²⁺-channels, Na⁺–Ca²⁺ exchange and Ca²⁺-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca²⁺-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca²⁺ and Ca²⁺ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca²⁺ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [³H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (B_max) without any change in the dissociation constant for receptor-ligand complex (K_d) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake and ouabain-sensitive Na⁺–K⁺ ATPase were decreased whereas the Mg²⁺-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na⁺–K⁺ ATPase activity, whereas Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake, and Mg²⁺-ATPase activities were unchanged. The V_max and K_a for Ca²⁺ of cardiac sarcolemmal Na⁺–Ca²⁺ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca²⁺-transport processes is regulated by thyroid hormones and the modification of Ca²⁺-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.